.

Republic of the Philippines

NORTHERN ILOILO STATE UNIVERSITY
NISU Main Campus, V Cudilla Sr. Ave, Estancia, Iloilo

Reg. No. 97Q19783

COLLEGE OF ARTS AND SCIENCES
Bachelor of Science in Biology

INSTRUCTIONAL MANUAL
IN
DEVELOPMENTAL BIOLOGY
(Plants and Animals)

Prepared by:

Dr. Annie C. Castillano

Course/Subject Professor
January 3, 2023

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 CHAPTER 1

INTRODUCTION TO DEVELOPMENTAL BIOLOGY

Introduction:

Developmental biology is the study of the process by which organisms grow and
develop. In this field, an understanding of the specialization of cells during embryogenesis
yields information on how to specialize stem cells to specific tissues and organs, which could
lead to the specific cloning of organs for medical purposes.

Another biologically important process that occurs during development is apoptosis -

programmed cell death or "suicide". For this reason, many developmental models are used to
elucidate the physiology and molecular basis of this cellular process. Similarly, a deeper
understanding of developmental biology can foster greater progress in the treatment
of congenital disorders and diseases.

Intended Learning Outcomes:

At the end of this module, students should be able to:

1. Explain the main processes involved in the embryonic development of plants and animals.
2. Explain how plants grow.
3. Explain the embryonic development of plants.
4. Recognize the role played by some plants and animals as models in Developmental
Biology.
5. Perform plant germination.

Learning Content:

1. Nature and Scope

Developmental biology is the study of the process by which animals and plants grow
and develop. Developmental biology also encompasses the biology of regeneration, asexual
reproduction, metamorphosis, and the growth and differentiation of stem cells in the adult
organism.
The main processes involved in the embryonic development of animals are: tissue
patterning (via regional specification and patterned cell differentiation); tissue morphogenesis;
and tissue growth .
The development of plants involves similar processes to that of animals. However plant
cells are mostly immotile so morphogenesis is achieved by differential growth, without cell
movements. Also, the inductive signals and the genes involved are different from those that
control animal development.

 Regional specification refers to the processes that create spatial pattern in a ball or sheet
of initially similar cells. This generally involves the action of cytoplasmic determinants,
located within parts of the fertilized egg, and of inductive signals emitted from signalling
centers in the embryo. The early stages of regional specification do not generate
functional differentiated cells, but cell populations committed to develop to a specific
region or part of the organism. These are defined by the expression of specific
combinations of transcription factors.
 Cell differentiation relates specifically to the formation of functional cell types such as
nerve, muscle, secretory epithelia etc. Differentiated cells contain large amounts of
specific proteins associated with the cell function.
 Morphogenesis relates to the formation of three-dimensional shape. It mainly involves the
orchestrated movements of cell sheets and of individual cells. Morphogenesis is important
for creating the three germ layers of the early embryo

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 (ectoderm, mesoderm and endoderm) and for building up complex structures during organ
development.
 Tissue growth involves both an overall increase in tissue size, and also the differential
growth of parts (allometry) which contributes to morphogenesis. Growth mostly occurs
through cell proliferation but also through changes of cell size or the deposition of
extracellular materials.
Cell differentiation
Cell differentiation is the process whereby different functional cell types arise in
development. For example, neurons, muscle fibers and hepatocytes (liver cells) are well
known types of differentiated cells. Differentiated cells usually produce large amounts of a few
proteins that are required for their specific function and this gives them the characteristic
appearance that enables them to be recognized under the light microscope. The genes
encoding these proteins are highly active. Typically their chromatin structure is very open,
allowing access for the transcription enzymes, and specific transcription factors bind to
regulatory sequences in the DNA in order to activate gene expression. For
example, NeuroD is a key transcription factor for neuronal differentiation, myogenin for
muscle differentiation, and HNF4 for hepatocyte differentiation. Cell differentiation is usually
the final stage of development, preceded by several states of commitment which are not
visibly differentiated. A single tissue, formed from a single type of progenitor cell or stem cell,
often consists of several differentiated cell types. Control of their formation involves a process
of lateral inhibition, based on the properties of the Notch signaling pathway. For example, in
the neural plate of the embryo this system operates to generate a population of neuronal
precursor cells in which NeuroD is highly expressed.
Regeneration
Regeneration indicates the ability to regrow a missing part. This is very prevalent
amongst plants, which show continuous growth, and also among colonial animals such as
hydroids and ascidians. But most interest by developmental biologists has been shown in the
regeneration of parts in free living animals. In particular four models have been the subject of
much investigation. Two of these have the ability to regenerate whole bodies: Hydra, which
can regenerate any part of the polyp from a small fragment, and planarian worms, which can
usually regenerate both heads and tails. Both of these examples have continuous cell
turnover fed by stem cells and, at least in planaria, at least some of the stem cells have been
shown to be pluripotent. The other two models show only distal regeneration of appendages.
These are the insect appendages, usually the legs of hemimetabolous insects such as the
cricket, and the limbs of urodele amphibians. Considerable information is now available about
amphibian limb regeneration and it is known that each cell type regenerates itself, except for
connective tissues where there is considerable interconversion between cartilage, dermis and
tendons. In terms of the pattern of structures, this is controlled by a re-activation of signals
active in the embryo. There is still debate about the old question of whether regeneration is a
"pristine" or an "adaptive" property. If the former is the case, with improved knowledge, we
might expect to be able to improve regenerative ability in humans. If the latter, then each
instance of regeneration is presumed to have arisen by natural selection in circumstances
particular to the species, so no general rules would be expected.
2. Plant Development
Plant development is the process by which structures originate and mature as a plant
grows. Plants constantly produce new tissues and structures throughout their life
from meristems located at the tips of organs, or between mature tissues. Thus, a living plant
always has embryonic tissues. By contrast, an animal embryo will very early produce all of the
body parts that it will ever have in its life. When the animal is born or hatches from its egg, it
has all the body parts and from that point will only grow larger and more mature.
The properties of organization seen in a plant are emergent properties which are more
than the sum of the individual parts. "The assembly of these tissues and functions into an
integrated multicellular organism yields not only the characteristics of the separate parts and
processes but also quite a new set of characteristics which would not have been predictable
on the basis of examination of the separate parts."

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Growth
A vascular plant begins from a single celled zygote, formed by fertilization of an egg
cell by a sperm cell. From that point, it begins to divide to form a plant embryo through the
process of embryogenesis. As this happens, the resulting cells will organize so that one end
becomes the first root, while the other end forms the tip of the shoot. In seed plants, the
embryo will develop one or more "seed leaves" (cotyledons). By the end of embryogenesis,
the young plant will have all the parts necessary to begin its life.
Once the embryo germinates from its seed or parent plant, it begins to produce
additional organs (leaves, stems, and roots) through the process of organogenesis. New roots
grow from root meristems located at the tip of the root, and new stems and leaves grow from
shoot meristems located at the tip of the shoot. Branching occurs when small clumps of cells
left behind by the meristem, and which have not yet undergone cellular differentiation to form
a specialized tissue, begin to grow as the tip of a new root or shoot. Growth from any such
meristem at the tip of a root or shoot is termed primary growth and results in the lengthening
of that root or shoot. Secondary growth results in widening of a root or shoot from divisions of
cells in a cambium.
In addition to growth by cell division, a plant may grow through cell elongation. This
occurs when individual cells or groups of cells grow longer. Not all plant cells will grow to the
same length. When cells on one side of a stem grow longer and faster than cells on the other
side, the stem will bend to the side of the slower growing cells as a result. This directional
growth can occur via a plant's response to a particular stimulus, such as light (phototropism),
gravity (gravitropism), water, (hydrotropism), and physical contact (thigmotropism).
Plant growth and development are mediated by specific plant hormones and plant
growth regulators (PGRs). Endogenous hormone levels are influenced by plant age, cold
hardiness, dormancy, and other metabolic conditions; photoperiod, drought, temperature, and
other external environmental conditions; and exogenous sources of PGRs, e.g., externally
applied and of rhizospheric origin.
Morphological variation
Plants exhibit natural variation in their form and structure. While all organisms vary
from individual to individual, plants exhibit an additional type of variation. Within a single
individual, parts are repeated which may differ in form and structure from other similar parts.
This variation is most easily seen in the leaves of a plant, though other organs such as stems
and flowers may show similar variation. There are three primary causes of this variation:
positional effects, environmental effects, and juvenility.
Evolution of plant morphology
Transcription factors and transcriptional regulatory networks play key roles in plant
morphogenesis and their evolution. During plant landing, many novel transcription factor
families emerged and are preferentially wired into the networks of multicellular development,
reproduction, and organ development, contributing to more complex morphogenesis of land
plants.
Most land plants share a common ancestor, multicellular algae. An example of the
evolution of plant morphology is seen in charophytes. Studies have shown that charophytes
have traits that are homologous to land plants. There are two main theories of the evolution of
plant morphology, these theories are the homologous theory and the antithetic theory. The
commonly accepted theory for the evolution of plant morphology is the antithetic theory. The
antithetic theory states that the multiple mitotic divisions that take place before meiosis, cause
the development of the sporophyte. Then the sporophyte will development as an independent
organism.
3. Embryonic Development of Animals
The sperm and egg fuse in the process of fertilization to form a fertilized egg,
or zygote. This undergoes a period of divisions to form a ball or sheet of similar cells called
a blastula or blastoderm. These cell divisions are usually rapid with no growth so the
daughter cells are half the size of the mother cell and the whole embryo stays about the same

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size. They are called cleavage divisions. Mouse epiblast primordial germ cells undergo
extensive epigenetic reprogramming. This process involves genome-wide DNA
demethylation, chromatin reorganization and epigenetic imprint erasure leading
to totipotency. DNA demethylation is carried out by a process that utilizes the DNA base
excision repair pathway.
Morphogenetic movements convert the cell mass into a three layered structure
consisting of multicellular sheets called ectoderm, mesoderm and endoderm. These sheets
are known as germ layers. This is the process of gastrulation. During cleavage and
gastrulation the first regional specification events occur. In addition to the formation of the
three germ layers themselves, these often generate extraembryonic structures, such as the
mammalian placenta, needed for support and nutrition of the embryo, and also establish
differences of commitment along the anteroposterior axis (head, trunk and tail).
Regional specification is initiated by the presence of cytoplasmic determinants in one
part of the zygote. The cells that contain the determinant become a signaling center and emit
an inducing factor. Because the inducing factor is produced in one place, diffuses away, and
decays, it forms a concentration gradient, high near the source cells and low further
away. The remaining cells of the embryo, which do not contain the determinant, are
competent to respond to different concentrations by upregulating specific developmental
control genes. This results in a series of zones becoming set up, arranged at progressively
greater distance from the signaling center. In each zone a different combination of
developmental control genes is upregulated. These genes encode transcription factors which
upregulate new combinations of gene activity in each region. Among other functions, these
transcription factors control expression of genes conferring specific adhesive and motility
properties on the cells in which they are active. Because of these different morphogenetic
properties, the cells of each germ layer move to form sheets such that the ectoderm ends up
on the outside, mesoderm in the middle, and endoderm on the inside. Morphogenetic
movements not only change the shape and structure of the embryo, but by bringing cell
sheets into new spatial relationships they also make possible new phases of signaling and
response between them.
Growth in embryos is mostly autonomous. For each territory of cells the growth rate is
controlled by the combination of genes that are active. Free-living embryos do not grow in
mass as they have no external food supply. But embryos fed by a placenta or extraembryonic
yolk supply can grow very fast, and changes to relative growth rate between parts in these
organisms help to produce the final overall anatomy.
The whole process needs to be coordinated in time and how this is controlled is not
understood. There may be a master clock able to communicate with all parts of the embryo
that controls the course of events, or timing may depend simply on local causal sequences of
events.
Metamorphosis
Developmental processes are very evident during the process of metamorphosis. This
occurs in various types of animal. Well-known examples are seen in frogs, which usually
hatch as a tadpole and metamorphoses to an adult frog, and certain insects which
hatch as a larva and then become remodeled to the adult form during a pupal stage.
All the developmental processes listed above occur during metamorphosis. Examples
that have been especially well studied include tail loss and other changes in the tadpole of the
frog Xenopus, and the biology of the imaginal discs, which generate the adult body parts of
the fly Drosophila melanogaster.
4. Developmental Model Organisms
Often used model organisms in developmental biology include the following:
 Chordates
 Lancelet Branchiostoma lanceolatum
 Zebrafish Danio rerio
 Medakafish Oryzias latipes
 Fugu Takifugu rubripes
 Frogs Xenopus laevis

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  Chicken Gallus gallus
 Mouse Mus musculus (Mammalian embryogenesis)
 Invertebrates
 Sea urchin
 Round worm Caenorhabditis elegans
 Fruit fly Drosophila melanogaster (Drosophila embryogenesis)
 Plants (Plant embryogenesis)
 Arabidopsis thaliana
 Maize
 Snapdragon

References:

Creative Diagnostics. Developmental Biology. https://www.creative-

diagnostics.com/developmental-biology.htm. 2022.

Psychology Wiki. Introduction to Developmental Biology.

https://psychology.fandom.com/wiki/Introduction_to_developmental_biology#Concepts_in_de
velopmental_biology. 2022

Wikipedia. Developmental Biology.

https://en.wikipedia.org/wiki/Developmental_biology#:~:text=Developmental%20biology%20is
%20the%20study,cells%20in%20the%20adult%20organism. 2022

Assessment:

I. Define the following terms:

1. Developmental biology 6. Regeneration
2. Regional specification 7. Fertilization
3. Cell differentiation 8. Blastula
4. Morphogenesis 9. Cleavage division
5. Tissue growth 10. Metamorphosis

II. Enumerate the following:

1. Main processes involved in the embryonic development of animals (4)
2. Often used model organisms in developmental biology: chordates (3), invertebrates (3),
and plants (3)

III. Explain:
1. The main processes involved in the embryonic development of plants and animals.
2. How plants grow.
3. The embryonic development of plants.

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 CHAPTER 2

IMECHANISMS OF CELLULAR DIFFERENTIATION

Introduction:

Cellular differentiation is the process in which a cell changes from one cell type to
another. Usually, the cell changes to a more specialized type. Differentiation occurs numerous
times during the development of a multicellular organism as it changes from a
simple zygote to a complex system of tissues and cell types. Differentiation continues in
adulthood as adult stem cells divide and create fully differentiated daughter cells during tissue
repair and during normal cell turnover. Some differentiation occurs in response
to antigen exposure.
With a few exceptions, cellular differentiation almost never involves a change in
the DNA sequence itself. Although metabolic composition does get altered quite
dramatically where stem cells are characterized by abundant metabolites with highly
unsaturated structures whose levels decrease upon differentiation. Thus, different cells can
have very different physical characteristics despite having the same genome.

Intended Learning Outcomes:

At the end of this module, students should be able to:

1. Explain the main mechanisms of cellular differentiation.

2. Recognize the importance of epigenetic control in cellular differentiation.
3. Illustrate the role of signaling in epigenetic control.

Learning Content:
1. Mechanisms of Cellular Differentiation
Each specialized cell type in an organism expresses a subset of all the genes that
constitute the genome of that species. Each cell type is defined by its particular pattern
of regulated gene expression. Cell differentiation is thus a transition of a cell from one cell
type to another and it involves a switch from one pattern of gene expression to another.
Cellular differentiation during development can be understood as the result of a gene
regulatory network. A regulatory gene and its cis-regulatory modules are nodes in a gene
regulatory network; they receive input and create output elsewhere in the
network. The systems biology approach to developmental biology emphasizes the importance
of investigating how developmental mechanisms interact to produce predictable patterns
(morphogenesis). However, an alternative view has been proposed recently. Based
on stochastic gene expression, cellular differentiation is the result of a Darwinian selective
process occurring among cells. In this frame, protein and gene networks are the result of
cellular processes and not their cause.

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 While evolutionarily conserved molecular processes are involved in the cellular
mechanisms underlying these switches, in animal species these are very different from the
well-characterized gene regulatory mechanisms of bacteria, and even from those of the
animals' closest unicellular relatives. Specifically, cell differentiation in animals is highly
dependent on biomolecular condensates of regulatory proteins and enhancer DNA
sequences.
Cellular differentiation is often controlled by cell signalling. Many of the signal
molecules that convey information from cell to cell during the control of cellular differentiation
are called growth factors. Although the details of specific signal transduction pathways vary,
these pathways often share the following general steps. A ligand produced by one cell binds
to a receptor in the extracellular region of another cell, inducing a conformational change in
the receptor. The shape of the cytoplasmic domain of the receptor changes, and the receptor
acquires enzymatic activity. The receptor then catalyzes reactions that phosphorylate other
proteins, activating them. A cascade of phosphorylation reactions eventually activates a
dormant transcription factor or cytoskeletal protein, thus contributing to the differentiation
process in the target cell. Cells and tissues can vary in competence, their ability to respond to
external signals.
Signal induction refers to cascades of signalling events, during which a cell or tissue
signals to another cell or tissue to influence its developmental fate. Yamamoto and
Jeffery investigated the role of the lens in eye formation in cave- and surface-dwelling fish, a
striking example of induction. Through reciprocal transplants, Yamamoto and Jeffery found
that the lens vesicle of surface fish can induce other parts of the eye to develop in cave- and
surface-dwelling fish, while the lens vesicle of the cave-dwelling fish cannot.
Other important mechanisms fall under the category of asymmetric cell divisions,
divisions that give rise to daughter cells with distinct developmental fates. Asymmetric cell
divisions can occur because of asymmetrically expressed maternal cytoplasmic
determinants or because of signaling. In the former mechanism, distinct daughter cells are
created during cytokinesis because of an uneven distribution of regulatory molecules in the
parent cell; the distinct cytoplasm that each daughter cell inherits results in a distinct pattern
of differentiation for each daughter cell. A well-studied example of pattern formation by
asymmetric divisions is body axis patterning in Drosophila. RNA molecules are an important
type of intracellular differentiation control signal. The molecular and genetic basis of
asymmetric cell divisions has also been studied in green algae of the genus Volvox, a model
system for studying how unicellular organisms can evolve into multicellular
organisms. In Volvox carteri, the 16 cells in the anterior hemisphere of a 32-cell embryo divide
asymmetrically, each producing one large and one small daughter cell. The size of the cell at
the end of all cell divisions determines whether it becomes a specialized germ or somatic cell.
2. Epigenetic Control
Since each cell, regardless of cell type, possesses the same genome, determination of
cell type must occur at the level of gene expression. While the regulation of gene
expression can occur through cis- and trans-regulatory elements including a
gene's promoter and enhancers, the problem arises as to how this expression pattern is
maintained over numerous generations of cell division. As it turns out, epigenetic processes
play a crucial role in regulating the decision to adopt a stem, progenitor, or mature cell fate.
Importance of epigenetic control
The first question that can be asked is the extent and complexity of the role of
epigenetic processes in the determination of cell fate. A clear answer to this question can be
seen in the 2011 paper by Lister R, et al. on aberrant epigenomic programming
in human induced pluripotent stem cells. As induced pluripotent stem cells (iPSCs) are
thought to mimic embryonic stem cells in their pluripotent properties (not fixed or capable of
differentiating), few epigenetic differences should exist between them. To test this prediction,
the authors conducted whole-genome profiling of DNA methylation patterns in several human
embryonic stem cell (ESC), iPSC, and progenitor cell lines.
Female adipose cells, lung fibroblasts, and foreskin fibroblasts were reprogrammed
into induced pluripotent state. Patterns of DNA methylation in ESCs, iPSCs, somatic cells
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were compared. Lister R, et al. observed significant resemblance in methylation levels
between embryonic and induced pluripotent cells. Around 80% of CG (Cytosine-Guanine)
dinucleotides in ESCs and iPSCs were methylated, the same was true of only 60% of CG
dinucleotides in somatic cells. In addition, somatic cells possessed minimal levels of cytosine
methylation in non-CG dinucleotides, while induced pluripotent cells possessed similar levels
of methylation as embryonic stem cells, between 0.5 and 1.5%. Thus, consistent with their
respective transcriptional activities, DNA methylation patterns, at least on the genomic level,
are similar between ESCs and iPSCs.
However, upon examining methylation patterns more closely, the authors discovered
1175 regions of differential CG dinucleotide methylation between at least one ES or iPS cell
line. By comparing these regions of differential methylation with regions of cytosine
methylation in the original somatic cells, 44-49% of differentially methylated regions reflected
methylation patterns of the respective progenitor somatic cells, while 51-56% of these regions
were dissimilar to both the progenitor and embryonic cell lines. In vitro-induced differentiation
of iPSC lines saw transmission of 88% and 46% of hyper and hypo-methylated differentially
methylated regions, respectively.
Two conclusions are readily apparent from this study. First, epigenetic processes are
heavily involved in cell fate determination, as seen from the similar levels of cytosine
methylation between induced pluripotent and embryonic stem cells, consistent with their
respective patterns of transcription. Second, the mechanisms of reprogramming (and by
extension, differentiation) are very complex and cannot be easily duplicated, as seen by the
significant number of differentially methylated regions between ES and iPS cell lines.
References:

Britanica. The Process of Differentiation. https://www.britannica.com/science/cell-biology/The-

process-of-differentiation. 2022.

Lumen Boundless Biology. Regulating Gene Expression in Cell Development.

https://courses.lumenlearning.com/boundless-biology/chapter/regulating-gene-expression-in-
cell-development/. 2022

Science Direct. Cell Differentiation. https://www.sciencedirect.com/topics/medicine-and-

dentistry/cell-
differentiation#:~:text=Cell%20differentiation%20is%20the%20process,the%20top%20of%20t
he%20hierarchy.. 2022.

Wikipedia. Cellular Differentiation. https://en.wikipedia.org/wiki/Cellular_differentiation. 2022.

Assessment:

I. Define the following terms:

1. Growth factors 4. Epigenetic control/process
2. Signal induction 5. Pluripotent
3. Asymmetric cell division

II. Identify the meaning of the following acronyms:

1. iPSC
2. ESC

III. Explain:
1. The main mechanisms of cellular differentiation.

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 CHAPTER 3

CELL–CELL SIGNALING

Introduction:
In biology, cell signaling (cell signalling in British English) or cell communication is
the ability of a cell to receive, process, and transmit signals with its environment and with
itself. It is a fundamental property of all cells in every living organism such as bacteria, plants,
and animals. Signals that originate from outside a cell (or extracellular signals) can be
physical agents like mechanical pressure, voltage, temperature, light, or chemical signals
(e.g., small molecules, peptides, or gas). Chemical signals can be hydrophobic or hydrophillic.
Receptors play a key role in cell signaling as they are able to detect chemical signals
or physical stimuli. Receptors are generally proteins located on the cell surface or within the
interior of the cell such as the cytoplasm, organelles, and nucleus. Cell surface
receptors usually bind with extracellular signals (or ligands), which causes a conformational
change in the receptor that leads it to initiate enzymatic activity, or to open or close ion
channel activity. Some receptors do not contain enzymatic or channel-like domains but are
instead linked to enzymes or transporters. Other receptors like nuclear receptors have a
different mechanism such as changing their DNA binding properties and cellular localization
to the nucleus.
Each cell is programmed to respond to specific extracellular signal molecules, and is
the basis of development, tissue repair, immunity, and homeostasis. Errors in signaling
interactions may cause diseases such as cancer, autoimmunity, and diabetes.

Intended Learning Outcomes:

At the end of this module, students should be able to:

1. Define sending cell, target cell and non-target cell.

2. Compare the forms of cell-cell signaling.
3. Appreciate the roles played by receptors in cell-cell signaling.
4. Illustrate cell-cell signaling in plants and animals using a diagram.

Learning Content:

1. Signaling through Cell-Cell Contact

Gap junctions in animals and plasmodesmata in plants are tiny channels that directly
connect neighboring cells. These water-filled channels allow small signaling molecules,
called intracellular mediators, to diffuse between the two cells. Small molecules and ions
are able to move between cells, but large molecules like proteins and DNA cannot fit through
the channels without special assistance.
The transfer of signaling molecules transmits the current state of one cell to its
neighbor. This allows a group of cells to coordinate their response to a signal that only one of
them may have received. In plants, there are plasmodesmata between almost all cells,
making the entire plant into one giant network.

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Signaling across gap junctions. A cell targets a neighboring cell connected via gap junctions.
Signals travel from one cell to the other by passing through the gap junctions.
In another form of direct signaling, two cells may bind to one another because they
carry complementary proteins on their surfaces. When the proteins bind to one another, this
interaction changes the shape of one or both proteins, transmitting a signal. This kind of
signaling is especially important in the immune system, where immune cells use cell-surface
markers to recognize ―self‖ cells (the body's own cells) and cells infected by pathogens.

Cells typically communicate using chemical signals. These chemical signals, which are
proteins or other molecules produced by a sending cell, are often secreted from the cell and
released into the extracellular space. There, they can float – like messages in a bottle – over
to neighboring cells.

Sending cell: this cell secretes a ligand.

Target cell: this cell has a receptor that can bind the ligand. The ligand binds to the
receptor and triggers a signaling cascade inside the cell, leading to a response.
Non-target cell: this cell does not have a receptor for the ligand (though it may have
other kinds of receptors). The cell does not perceive the ligand and thus does not respond to
it.
Not all cells can ―hear‖ a particular chemical message. In order to detect a signal (that
is, to be a target cell), a neighbor cell must have the right receptor for that signal. When a
signaling molecule binds to its receptor, it alters the shape or activity of the receptor,
triggering a change inside of the cell. Signaling molecules are often called ligands, a general
term for molecules that bind specifically to other molecules (such as receptors).
The message carried by a ligand is often relayed through a chain of chemical
messengers inside the cell. Ultimately, it leads to a change in the cell, such as alteration in
the activity of a gene or even the induction of a whole process, such as cell division. Thus, the
original intercellular (between-cells) signal is converted into an intracellular (within-cell)
signal that triggers a response.

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2. Forms of Cell-Cell Signaling
Autocrine
Autocrine signaling involves a cell secreting a hormone or chemical messenger
(called the autocrine agent) that binds to autocrine receptors on that same cell, leading to
changes in the cell itself. This can be contrasted with paracrine signaling, intracrine signaling,
or classical endocrine signaling.
Paracrine
In paracrine signaling, a cell produces a signal to induce changes in nearby cells,
altering the behavior of those cells. Signaling molecules known as paracrine factors diffuse
over a relatively short distance (local action), as opposed to cell signaling by endocrine
factors, hormones which travel considerably longer distances via the circulatory
system; juxtacrine interactions; and autocrine signaling. Cells that produce paracrine factors
secrete them into the immediate extracellular environment. Factors then travel to nearby cells
in which the gradient of factor received determines the outcome. However, the exact distance
that paracrine factors can travel is not certain.
Paracrine signals such as retinoic acid target only cells in the vicinity of the emitting
cell. Neurotransmitters represent another example of a paracrine signal.
Some signaling molecules can function as both a hormone and a neurotransmitter. For
example, epinephrine and norepinephrine can function as hormones when released from
the adrenal gland and are transported to the heart by way of the blood stream.
Norepinephrine can also be produced by neurons to function as a neurotransmitter within the
brain. Estrogen can be released by the ovary and function as a hormone or act locally via
paracrine or autocrine signaling.
Although paracrine signaling elicits a diverse array of responses in the induced cells,
most paracrine factors utilize a relatively streamlined set of receptors and pathways. In fact,
different organs in the body - even between different species - are known to utilize a similar
sets of paracrine factors in differential development. The highly conserved receptors and
pathways can be organized into four major families based on similar structures: fibroblast
growth factor (FGF) family, Hedgehog family, Wnt family, and TGF-β superfamily. Binding of a
paracrine factor to its respective receptor initiates signal transduction cascades, eliciting
different responses.
Endocrine
Endocrine signals are called hormones. Hormones are produced by endocrine cells
and they travel through the blood to reach all parts of the body. Specificity of signaling can be
controlled if only some cells can respond to a particular hormone. Endocrine signaling
involves the release of hormones by internal glands of an organism directly into the circulatory
system, regulating distant target organs. In vertebrates, the hypothalamus is the neural
control center for all endocrine systems. In humans, the major endocrine glands are
the thyroid gland and the adrenal glands. The study of the endocrine system and its disorders
is known as endocrinology.
Juxtacrine
Juxtacrine signaling is a type of cell–cell or cell–extracellular matrix signaling
in multicellular organisms that requires close contact. There are three types:

1. A membrane ligand (protein, oligosaccharide, lipid) and a membrane protein of two

adjacent cells interact.
2. A communicating junction links the intracellular compartments of two adjacent cells,
allowing transit of relatively small molecules.
3. An extracellular matrix glycoprotein and a membrane protein interact.
Additionally, in unicellular organisms such as bacteria, juxtacrine signaling means
interactions by membrane contact. Juxtacrine signaling has been observed for some growth
factors, cytokine and chemokine cellular signals, playing an important role in the immune
response.
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3. Receptors
Cells receive information from their neighbors through a class of proteins known
as receptors. Receptors may bind with some molecules (ligands) or may interact with physical
agents like light, mechanical temperature, pressure, etc. Reception occurs when the target
cell (any cell with a receptor protein specific to the signal molecule) detects a signal, usually in
the form of a small, water-soluble molecule, via binding to a receptor protein on the cell
surface, or once inside the cell, the signaling molecule can bind to intracellular receptors,
other elements, or stimulate enzyme activity (e.g. gasses), as in intracrine signaling.
Signaling molecules interact with a target cell as a ligand to cell surface receptors,
and/or by entering into the cell through its membrane or endocytosis for intracrine signaling.
This generally results in the activation of second messengers, leading to various physiological
effects. In many mammals, early embryo cells exchange signals with cells of the uterus. In the
human gastrointestinal tract, bacteria exchange signals with each other and with
human epithelial and immune system cells. For the yeast Saccharomyces
cerevisiae during mating, some cells send a peptide signal (mating factor pheromones) into
their environment. The mating factor peptide may bind to a cell surface receptor on other
yeast cells and induce them to prepare for mating.
Cell surface receptors
Cell surface receptors play an essential role in the biological systems of single- and
multi-cellular organisms and malfunction or damage to these proteins is associated with
cancer, heart disease, and asthma. These trans-membrane receptors are able to transmit
information from outside the cell to the inside because they change conformation when a
specific ligand binds to it. There are three major types: Ion channel linked receptors, G
protein–coupled receptors, and enzyme-linked receptors.
Ion channel linked receptors
Ion channel linked receptors are a group of trans-membrane ion-channel proteins
which open to allow ions such as Na+, K+, Ca2+, and/or Cl− to pass through the membrane in
response to the binding of a chemical messenger (i.e. a ligand), such as a neurotransmitter.
When a presynaptic neuron is excited, it releases a neurotransmitter from vesicles into
the synaptic cleft. The neurotransmitter then binds to receptors located on the postsynaptic
neuron. If these receptors are ligand-gated ion channels, a resulting conformational change
opens the ion channels, which leads to a flow of ions across the cell membrane. This, in turn,
results in either a depolarization, for an excitatory receptor response, or a hyperpolarization,
for an inhibitory response.
These receptor proteins are typically composed of at least two different domains: a
transmembrane domain which includes the ion pore, and an extracellular domain which
includes the ligand binding location (an allosteric binding site). This modularity has enabled a
'divide and conquer' approach to finding the structure of the proteins (crystallising each
domain separately). The function of such receptors located at synapses is to convert the
chemical signal of presynaptically released neurotransmitter directly and very quickly into
a postsynaptic electrical signal. Many LICs are additionally modulated by allosteric ligands,
by channel blockers, ions, or the membrane potential. LICs are classified into three
superfamilies which lack evolutionary relationship: cys-loop receptors, ionotropic glutamate
receptors and ATP-gated channels.
G protein–coupled receptors
G protein-coupled receptors are a large group of evolutionarily-related proteins that
are cell surface receptors that detect molecules outside the cell and activate cellular
responses. Coupling with G proteins, they are called seven-transmembrane receptors
because they pass through the cell membrane seven times. Ligands can bind either to
extracellular N-terminus and loops (e.g. glutamate receptors) or to the binding site within
transmembrane helices (Rhodopsin-like family). They are all activated by agonists although a
spontaneous auto-activation of an empty receptor can also be observed.
G protein-coupled receptors are found only in eukaryotes,
including yeast, choanoflagellates, and animals. The ligands that bind and activate these
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receptors include light-sensitive compounds, odors, pheromones, hormones,
and neurotransmitters, and vary in size from small molecules to peptides to large proteins. G
protein-coupled receptors are involved in many diseases.
There are two principal signal transduction pathways involving the G protein-coupled
receptors: cAMP signal pathway and phosphatidylinositol signal pathway. When a ligand
binds to the GPCR it causes a conformational change in the GPCR, which allows it to act as
a guanine nucleotide exchange factor (GEF). The GPCR can then activate an associated G
protein by exchanging the GDP bound to the G protein for a GTP. The G protein's α subunit,
together with the bound GTP, can then dissociate from the β and γ subunits to further affect
intracellular signaling proteins or target functional proteins directly depending on the α subunit
type.
G protein-coupled receptors are an important drug target and approximately 34% of all
Food and Drug Administration (FDA) approved drugs target 108 members of this family. The
global sales volume for these drugs is estimated to be 180 billion US dollars as of 2018. It is
estimated that GPCRs are targets for about 50% of drugs currently on the market, mainly due
to their involvement in signaling pathways related to many diseases i.e. mental, metabolic
including endocrinological disorders, immunological including viral infections, cardiovascular,
inflammatory, senses disorders, and cancer. The long ago discovered association between
GPCRs and many endogenous and exogenous substances, resulting in e.g. analgesia, is
another dynamically developing field of pharmaceutical research.
Enzyme-linked receptors
Enzyme-linked receptors (or catalytic receptors) are trans-membrane receptors that
upon activation by an extracellular ligand, causes enzymatic activity on the intracellular
side. Hence a catalytic receptor is an integral membrane protein possessing
both enzymatic, catalytic, and receptor functions.
They have two important domains, an extra-cellular ligand binding domain and an
intracellular domain, which has a catalytic function; and a single trans-membrane helix. The
signaling molecule binds to the receptor on the outside of the cell and causes a
conformational change on the catalytic function located on the receptor inside the cell.
Examples of the enzymatic activity include:

 Receptor tyrosine kinase, as in fibroblast growth factor receptor. Most enzyme-linked

receptors are of this type.
 Serine/threonine-specific protein kinase, as in bone morphogenetic protein
 Guanylate cyclase, as in atrial natriuretic factor receptor

Intracellular receptors
Steroid hormone receptor
Steroid hormone receptors are found in the nucleus, cytosol, and also on the plasma
membrane of target cells. They are generally intracellular receptors (typically cytoplasmic or
nuclear) and initiate signal transduction for steroid hormones which lead to changes in gene
expression over a time period of hours to days. The best studied steroid
hormone receptors are members of the nuclear receptor subfamily 3 (NR3) that include
receptors for estrogen (group NR3A) and 3-ketosteroids (group NR3C). In addition to nuclear
receptors, several G protein-coupled receptors and ion channels act as cell surface
receptors for certain steroid hormones.

References:
Biology Dictionary. Cell Signaling. https://biologydictionary.net/cell-signaling/. 2022

Khan Academy. Introduction to Cell Signaling. https://www.khanacademy.org/science/ap-

biology/cell-communication-and-cell-cycle/cell-communication/a/introduction-to-cell-signaling.
2022

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Science Direct. Cell Signaling. https://www.sciencedirect.com/topics/neuroscience/cell-
signaling. 2022

Wikipedia. Cell Signaling. https://en.wikipedia.org/wiki/Cell_signaling. 2022

Assessment:

I. Define the following terms:

1. Cell signaling 8. Ligands
2. Receptors 9. Intercellular
3. Gap junctions 10. Intracellular
4. Plasmodesmata 11. Autocrine signaling
5. Sending cell 12. Paracrine
6. Target cell 13. Endocrine signaling
7. Non-target cell 14. Juxtacrine signaling

II. Enumerate the following:

1. Forms of cell-cell signaling (4)
2. Types of cell receptors (5)

III. Compare:
1. The forms of cell-cell signaling.

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 CHAPTER 4

FERTILIZATION IN PLANTS AND ANIMALS

Introduction:

All plants and animals across the world reproduce in some way or another, as a way
of bringing in new generations and slowly ushering in changes in the species. Some forms
of copulation seem similar to humanity's mating processes — most, but not all, mammalian
breeding, for instance — while others seem alien by comparison. For example, some
species can reproduce asexually and, others like the egg-laying duck-billed platypus, buck
the reproductive norms of their scientific classifications. Still, much of the reproduction
across all species begins with the fertilization of an egg, and many of the species in the
Kingdom Animalia raise their young to some extent.

Fertilization is the process by which male and female gametes are fused together,
initiating the development of a new organism. The male gamete or ’sperm’, and the female
gamete, ’egg’ or ’ovum’ are specialized sex cells, which fuse together to begin the formation
of a zygote during a process called sexual reproduction.

Intended Learning Outcomes:

At the end of this module, students should be able to:

1. Identify and explain the stages involved in fertilization.

2. Compare fertilization in plants and in animals.
3. Recognize the role of fertilization in reproduction.
4. Explore and familiarize the reproductive structures of angiosperms using actual specimen
(gumamela flower).

Learning Content:

1. The Fertilization Process

There are three stages to fertilization which ensure that the appropriate egg and sperm
are able to find each other and to warrant that only one sperm enters the egg: chemotaxis,
sperm activation/acrosomal reaction and sperm/egg adhesion.

Ovulation must occur before fertilization can happen; in humans, ovulation occurs once
a month during the menstrual cycle. This cycle releases an egg cell from the ovaries and the
first stage of fertilization begins. In other animals, ovulation can occur in cycles of different
length, or is triggered by the occurrence of sexual intercourse.

In mammals, after ejaculation, the sperm locates the oocyte (the immature egg),
through changes in temperature and chemical gradients. Sperm chemotaxis, a type of
interaction in which sperm cells are guided to the oocyte by the hormone progesterone, which
is secreted by the oocyte, and sperm thermotaxis, which involves the response to changes in
temperature, ensure that the sperm are able to locate the oocyte (usually within
the ampulla of the fallopian tube. While the sperm is in the reproductive tract, it
undergoes capacitation, which increases its movement ability and destabilizes its membrane,
preparing it for the acrosome reaction.

Once the sperm locates the oocyte, it binds to the zona pellucida, which is a thick layer
of jelly-like, extra-cellular matrix consisting of glycoproteins, surrounding the egg. A
specialized molecule on the surface of the sperm binds to a ZP3 glycoprotein in the zona
pellucida, triggering the acrosome reaction. The acrosome reaction releases hyaluronidase,
which digests the hyaluronic acid around the oocyte, allowing the sperm to pass through.

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Upon successful implantation of a sperm, the cortical granules within the oocyte fuse with
the plasma membrane of the cell, and are expelled into the zona pellucida, causing the
surface to become hard and impenetrable. This process is called the cortical reaction and is
responsible for ensuring that only one sperm cell can enter and fertilize the egg.

Once the sperm has successfully penetrated the oocyte, the outer coating and the tail
of the sperm disintegrates. The oocyte undergoes meiosis to produce the haploid ovum. The
two haploid cells, each containing 23 chromosomes, undergo fusion of their genetic material,
ultimately creating a diploid cell containing 46 chromosomes, called a zygote (a cell that is
formed when an egg and a sperm combine, also called a fertilized egg). The zygote then
begins mitosis, the repeated cellular division necessary for the growth of an organism,
forming a blastocyst, which is implanted into the wall of the uterus, beginning the pregnancy.

2. Fertilization in Plants

Fertilization in plants occurs after pollination and germination. Pollination occurs

through the transfer of pollen – which is the male microgametes of seed plants, producing
the sperm – from one plant to the stigma (the female reproductive organ) of another. The
pollen grain takes up water and germination occurs.

The germinated pollen grain sprouts a pollen tube, which grows and penetrates
the ovule (the egg structure of the plant) through a pore called a mycropyle. The sperm are
then transferred through the pollen tube from the pollen.

In flowering plants, a secondary fertilization event takes place. Two sperm are
transferred from each pollen grain, one of which fertilizes the egg cell to form a diploid zygote.
The nucleus of the second sperm cell fuses with two haploid nuclei contained within a second
female gamete called the central cell. This second fertilization forms a triploid cell, which
subsequently swells and develops a fruiting body.

Steps of Sexual Reproduction in Plants

Step 1: Pollination

Pollination is the first required step in sexual reproduction in plants. The male portion
of the plant produces the pollen – typically in the flower. A long filament, called a stamen,
holds the bits of pollen at the end and one of several pollinators take the pollen grains to the
female part of the flower, which is called the pistil.

Pollinators can be insects or birds drawn to the plant by the colorful flowers and
fragrance for the nectar inside. As they enjoy feeding on the plant, the pollen sticks to their
bodies and is then carried away to another flower that may contain a pistil. The wind can
also carry pollen to other flowers, as can water in some plant species.

Some plants have male and female parts on the same flower and can self-pollinate,
while other plants produce male and female parts on separate plants. In any event,
pollination requires the movement of pollen from the stamen to the pistil so the steps of
sexual plant reproduction can continue.

Step 2: Fertilization

If conditions are favorable, fertilization can happen when the pollen arrives at the
female part of the plant. The pistil is comprised of the stigma, style, ovary and ovule. The
pollen travels down the style, which connects the stigma to the ovary of the plant. The ovary
is the female part of the plant where the ovules begin to grow as a result of fertilization.

After fertilization, the flower eventually withers and the ovary begins to grow larger
and form a fruit. This may be an actual fruit, nut or berry depending on the plant species.
The fruit grows and develops seeds inside that are protected by the flesh of the fruit.
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Step 3: Seed Dispersal

The seeds inside the fruit of a plant must be redistributed to make new plants, a
process called seed dispersal. In nature this often happens when the fruit ripens and falls off
the plant onto the ground. The flesh of the fruit rots and exposes the seed inside, which is
fertilized by the organic material from the rotten fruit.

In other instances, the seed dispersal happens when animals brush against certain
plants and get the seeds stuck in their fur and transport it to another spot where it falls off.
This is common with plants that have a burr-type fruit, like burdocks. Wind, rain and other
natural elements may combine to move the seed to a new place. Seed dispersal is essential
for moving seeds away from their parent plants, so they can access the soil, sunshine and
nutrients in order to thrive.

Step 4: Germination

Germination is the actual birth of the new plant. Once the seed has emerged from its
fruit, it will hopefully be in the proper environment to induce a sprout. The process of
sprouting into a new plant is referred to as germination.

This is the final stage of sexual plant reproduction. The new plant grows and forms its
own male and female parts and starts the life cycle all over again.

3. Fertilization in Animals

The fertilization process in animals can occur either internally or externally, a

difference which is largely determined by the method of birth. Animals which
use viviparous and ovoviviparous reproduction (embryos develop within the animal‘s body),
and oviparous animals which lay hard shelled eggs, use internal fertilization.

Internal fertilization involves the union of sperm and eggs within the body of the
(usually female) parent. In order for internal fertilization to occur, the male must implant his
sperm into the female reproductive tracts. Implantation can be achieved by either: copulation
in which sperm transfer is performed by insertion of the penis or other
male intromittent organ and ejaculation into the vagina, or cloaca: or by a cloacal kiss in
which two birds press their cloacae together and sperm transfer takes place. Some animals,
such as mollusks, arachnids, salamanders and certain insects, transfer a spermatophore, a
bundle or capsule containing sperm, which is stored within the cloaca until oviposition takes
place.

Animals which are oviparous, though produce eggs that are lacking, or have thin
egg membranes, reproduce by external fertilization. External fertilization is a reproductive
strategy involving the joining of gametes outside of the body, either in a spawning event,
where gametes from both sexes are rapidly released into an aquatic environment, or may
occur when eggs are laid by a female on a substrate, and are subsequently fertilized by a
male. External fertilization holds certain benefits, such as reducing the chance of contracting
sexually transmitted diseases, protection from violent behavior between organisms, and
increasing the genetic variation within a population.

Internal vs. External Fertilization

Within chordates as a phylum, there are some great examples of both internal
fertilization and external fertilization. The benefits and drawbacks of internal and external
fertilization are pretty much the opposite of one another. For example, one benefit of
external fertilization is that many eggs can be fertilized at one time. However, those eggs
tend to be very vulnerable, which is one of the reasons why making so many might be
necessary.

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 On the other hand, internal fertilization is much more limited. Often only one or a few
eggs can be fertilized at one time, but those eggs might be more protected, either by a shell
or the mother's body. The trade-off here is that more energy is spent per egg, but they are
more likely to survive.

Short-lived aquatic species, like fish and amphibians, tend to reproduce through
external fertilization. Because most of the eggs won't survive, many are produced cheaply in
somewhat of a "shotgun" approach to reproduction.

Internal fertilization enables animals to have longer gestation periods. This is

important for long-lived, large animals like mammals. While fewer offspring are produced,
they often have a much better chance of survival.

References:

Biology Dictionary. Fertilization. https://biologydictionary.net/fertilization/. 2022.

Science Learning Hub. Pollination and Fertilization.

https://www.sciencelearn.org.nz/resources/77-pollination-and-fertilisation. 2022.

Sciencing. Reproduction of Plants and Animals. https://sciencing.com/reproduction-plants-

animals-6404461.html. 2022

Toppr. Introduction to Reproduction. https://www.toppr.com/guides/biology/how-do-

organisms-reproduce/introduction-to-reproduction/. 2022.

Assessment:

I. Define the following terms:

1. Fertilization 9. Pollination
2. Sperm 10. Germination
3. Egg/Ovum 11. Internal fertilization
4. Sperm chemotaxis 12. External fertilization
5. Sperm Thermotaxis 13. Viviparous
6. Zona pellucida 14. Oviparous
7. Copulation
8. Cloacal kiss

II. Enumerate:
1. The Steps of Sexual Reproduction in Plants (4)

III. Compare:
1. The fertilization in plants and in animals.

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 CHAPTER 5

HORMONE REGULATION IN PLANTS

Introduction:

Hormone actions form a signaling network and regulate various systems in plants.
Some of the major plant-growth-regulating compounds include auxin, gibberellin (GA),
cytokinin, ethylene, and abscisic acid (ABA). For the most part, each group contains both
naturally occurring hormones and synthetic substances.

Intended Learning Outcomes:

At the end of this module, students should be able to:

1. Describe phytohormones and explain their functions.

2. Identify and explain some of the common themes in plant hormone signaling.
3. Appreciate the role of phytohormones in regulating plant growth and development.

Learning Content:

1. Phytohormones

What are they?

Plant growth and development involves the integration of many environmental and
endogenous signals that, together with the intrinsic genetic program, determine plant form.
Fundamental to this process are several growth regulators collectively called the plant
hormones or phytohormones. This group includes auxin, cytokinin, the gibberellins (GAs),
abscisic acid (ABA), ethylene, the brassinosteroids (BRs), and jasmonic acid (JA), each of
which acts at low concentrations to regulate many aspects of plant growth and development.
With the notable exception of the steroidal hormones of the BR group, plant hormones
bear little resemblance to their animal counterparts. Rather, they are relatively simple, small
molecules such as ethylene gas and indole-3-acetic acid (IAA), the primary auxin in the
majority of plant species. The concept of plant hormones originates from a classical
experiment on phototropism, the bending of plants toward light, carried out by Charles
Darwin and his son Francis in 1880. The Darwins were able to demonstrate that when oat
seedlings were exposed to a lateral light source, a transported signal originating from the
plant apex promoted differential cell elongation in the lower parts of the seedling that resulted
in it bending toward the light source.
What do they do?
Virtually every aspect of plant growth and development is under hormonal control to
some degree. A single hormone can regulate an amazingly diverse array of cellular and
developmental processes, while at the same time multiple hormones often influence a single
process. Well-studied examples include the promotion of fruit ripening by ethylene,
regulation of the cell cycle by auxin, cytokinin is for induction of seed germination, stem
elongation by GA, and the maintenance of seed dormancy by ABA. Historically, the effects
of each hormone have been defined largely by the application of exogenous hormone. More
recently, the isolation of hormone biosynthetic and response mutants has provided powerful
new tools for painting a clearer picture of the roles of the various phytohormones in plant
growth and development.
How do they work?
Plant biologists have been fascinated by the regulatory capacity of phytohormones
since the time of their discovery, and the notion that hormone levels or responses could be
manipulated to improve desired plant traits has long been an area of intense interest. Perhaps
the best-known example of this is the isolation of dwarf varieties of wheat and rice that led to
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the ―green revolution‖ in the second half of the 20th century, which is credited with saving
millions of people around the globe from starvation. These dwarf varieties have shorter stems
than wild-type, making these plants less susceptible to damage by wind and rain. The
molecular isolation of these ―dwarfing genes‖ has revealed that they encode components of
the GA biosynthesis and response pathways.
To elucidate the molecular mechanisms underlying phytohormone action, several
researchers have utilized the genetically facile model plant Arabidopsis thaliana to isolate
mutations that confer altered response to applied hormone. Molecular and biochemical
analysis of the gene products defined by these mutations, coupled with expression studies
aimed at identifying the downstream target genes that mediate hormonal changes in growth
and development, has begun to unlock some of the mysteries behind phytohormone action.
While no hormone transduction pathway is completely understood, we now have a
rudimentary understanding of many of the molecular events underlying hormone action.
2. Common Themes
Regulation by proteolysis (the hydrolysis of proteins to form simple and soluble
products) has emerged as a resounding theme in plant hormone signaling. The ubiquitin-
mediated degradation of key regulatory proteins has been demonstrated, or is at least likely,
for all of the phytohormone response pathways. In the case of auxin, the response pathway is
normally subject to repression by a large family of transcriptional regulators called the
Aux/IAA proteins. These proteins dimerize with members of the auxin response factor (ARF)
family of transcription factors, thus preventing ARFs from activating auxin-responsive genes.
Upon an auxin stimulus, an SCF (SKP1/Cullin/F-box protein) ubiquitin ligase containing the
TIR1 F-box protein ubiquitinates the Aux/IAA proteins, marking them for degradation by the
26S proteasome thereby de-repressing the response pathway. The hormone promotes the
Aux/IAA–TIR1 interaction; however, the molecular mechanisms behind this regulation are
unclear. Most yeast and animal SCF substrates must be post-translationally modified, usually
by phosphorylation (the addition of a phosphoryl (PO3) group to a molecule. In biological
systems, this reaction is vital for the cellular storage and transfer of free energy using energy
carrier molecules), before they are recognized by their cognate F-box protein. Despite
numerous efforts to identify auxin-induced modification of Aux/IAA proteins, no such signal
has been discovered, raising the distinct possibility that auxin uses a novel mechanism to
regulate SCF–substrate interactions.
Ethylene and cytokinin are both perceived by receptors sharing similarity to bacterial
two-component regulators. Common in prokaryotes, but apparently restricted to plants and
fungi in eukaryotes, these modular signaling systems involve a membrane-bound receptor
containing an intracellular histidine kinase (HK) domain. Ligand binding activates the kinase,
resulting in autophosphorylation (a biochemical process in which a phosphate is added to a
protein kinase by itself) and initiation of a series of phosphotransfer reactions that culminates
with the activation of a response regulator protein that functions as the effector component of
the pathway. Cytokinin signaling appears to largely follow this paradigm. Ethylene response,
however, appears more complex.
Ethylene is perceived by a family of five receptors. ETR1 and ERS1 contain a
consensus HK domain, however, the HK domains of ETR2, ERS2, and EIN4 are degenerate
and lack elements necessary for catalytic activity. This fact, together with studies of ―kinase-
dead‖ mutants of ETR1, suggests that HK activity is not required for ethylene response.
Mutations that abolish ethylene binding in any of the five receptor genes are dominant and
confer ethylene insensitivity, indicating that the receptors function as negative regulators of
the ethylene pathway.
Genetic and molecular studies have positioned these receptors upstream of the Raf-
like MAP kinase, CTR1, which interacts with the receptors and also acts as a negative
regulator. The integral membrane protein, EIN2, and the transcription factors EIN3 and EIL1
are positive regulators of ethylene signaling downstream of CTR1. Current models propose
that hormone binding inactivates the receptors, thus resulting in down-regulation of CTR1
activity. Since the identification of CTR1, biologists have speculated that a MAP kinase
cascade may be involved. Only recently, however, have putative MAP kinase kinase and
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MAP kinase components of the ethylene pathway been identified. Interestingly, these kinases
appear to positively regulate ethylene response, suggesting that CTR1 must inhibit their
function. If so, this would represent a novel twist on the traditional MAP kinase signaling
paradigm. Precisely how the ethylene signal is transduced to the EIN3 and EIL1 transcription
factors remains unclear. However, the recent finding that ethylene stabilizes these
transcription factors, which are targeted for degradation by an SCF complex in the absence of
ethylene, clearly indicates a role for the ubiquitin pathway. One of the known targets for EIN3
is the ERF1 transcription factor, which activates several genes involved in a subset of
ethylene responses.
3. Signal Integration and Combinatorial Control
Long ago, plant physiologists noted the apparent antagonistic interactions between
some of the phytohormones, such as between auxin and cytokinin in the regulation of root–
shoot differentiation and between GA and ABA in germination. Other processes are
synergistically regulated by multiple hormones. While it has long been obvious that hormones
do not function in discrete pathways, but rather exhibit extensive cross-talk and signal
integration with each other and with environmental and developmental signaling pathways,
the molecular basis for such coordinated regulation has been unclear. Several recent findings
have begun to elucidate the molecular details of some of these events.
One example of such signal integration was recently described for the ethylene and JA
pathways. Genetic studies had previously implicated both hormones as important regulators
of pathogen defense responses, as well as of the wounding response and other stress-related
pathways. Additionally, microarray analysis has identified a large number of genes that are
responsive to both hormones. The ERF1 transcription factor was recently found to be an
intersection point for these two signaling pathways. Like ethylene, JA rapidly
induces ERF1 expression, and treatment with both hormones synergistically activates ERF1.
Induction of ERF1 by both hormones alone or in combination is dependent upon both
signaling pathways, and constitutive overexpression of ERF1 rescues the defense-response
defects of both ethylene- and JA-insensitive mutants. These findings suggest
that ERF1 represents one of the first signaling nodes identified in the complex web of
hormonal cross-talk.
The auxin and BR pathways also appear to converge and mutually regulate some
developmental processes. Both hormones promote cell expansion, and microarray studies
have revealed that as many as 40% of all BR-induced genes are also up-regulated by auxin.
BR is perceived by the cell surface receptor kinase BRI1. The SHAGGY/GSK3-type kinase
BIN2 acts as a negative regulator of the pathway downstream of the receptor. In the absence
of a BR signal, BIN2 phosphorylates the transcription factors BES1 and BZR1, targeting them
for proteolysis by the 26S proteasome. Upon a BR stimulus, BIN2 is inactivated, allowing
BES1 and BZR1to accumulate in the nucleus, where they are presumably involved in
regulating BR-responsive genes.
Using combined genetic, physiological, and genomic approaches, Nemhauser and
colleagues (2004) were able to demonstrate that auxin and BR
regulate Arabidopsis hypocotyl (embryonic stem) elongation in a synergistic and
interdependent fashion. Elevating endogenous auxin levels rendered plants more sensitive to
BR application in hypocotyl elongation assays, and this response was dependent upon both
the auxin and BR signaling pathways. Genetic studies suggest that the convergence of these
two pathways occurs at a late point in hormone signaling, perhaps at the promoters of the
many genes responsive to both hormones. In support of this notion, bioinformatic analysis
identified distinct sequence elements that were enriched specifically in the promoters of
auxin-induced, BR-induced, and auxin/BR-induced genes.
References:

Britanica. The Hormones of Plants. https://www.britannica.com/science/hormone/The-

hormones-of-plants. 2022

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Intech Open Book Series. Hormonal Regulation in Plant Development and Stress Tolerance.
https://www.intechopen.com/chapters/56091. 2022

NCBI Resources. Hormonal Regulation of Plant Growth and Development.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC516799/. 2022

Assessment:

I. Define the following terms:

1. Plant hormones or Phytohormones
2. Phototropism
3. Proteolysis
4. Phosphorylation
5. Autophosphorylation

II. Identify the functions of the following:

1. Auxin
2. Cytokinin
3. Gibberellins
4. Abscisic acid

III. Enumerate:
1. The types of plant hormones or phytohormones (7)

IV. Explain:
1. The common themes in plant hormone signaling.

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 CHAPTER 6

ROOT DEVELOPMENT

Introduction:

Upon germination, cells of the meristem begin to divide and the root expands axially as
a result of cell expansion. As the root continues to grow, the number of cells in the meristem
increases and the rate of cell production increases. This increase in the number of cells can
account for the doubling in the rate of root elongation between 6 and 10 days after
germination.

Intended Learning Outcomes:

At the end of this module, students should be able to:

1. Explain the root growth in plants.

2. Recognize the roles of certain genes in various aspects of root development.
3. Illustrate distal patterning, radial patterning, epidermal patterning, cell division and cell
expansion using a diagram.

Learning Content:

1. Root Growth

Early root growth is one of the functions of the apical meristem located near the tip of
the root. The meristem cells more or less continuously divide, producing more meristem, root
cap cells (these are sacrificed to protect the meristem), and undifferentiated root cells. The
latter become the primary tissues of the root, first undergoing elongation, a process that
pushes the root tip forward in the growing medium. Gradually these cells differentiate and
mature into specialized cells of the root tissues.
Growth from apical meristems is known as primary growth, which encompasses all
elongation. Secondary growth encompasses all growth in diameter, a major component
of woody plant tissues and many non-woody plants. For example, storage roots of sweet
potato have secondary growth but are not woody. Secondary growth occurs at the lateral
meristems, namely the vascular cambium and cork cambium. The former forms secondary
xylem and secondary phloem, while the latter forms the periderm.
In plants with secondary growth, the vascular cambium, originating between the xylem
and the phloem, forms a cylinder of tissue along the stem and root. The vascular cambium
forms new cells on both the inside and outside of the cambium cylinder, with those on the
inside forming secondary xylem cells, and those on the outside forming secondary phloem
cells. As secondary xylem accumulates, the "girth" (lateral dimensions) of the stem and root
increases. As a result, tissues beyond the secondary phloem including the epidermis and
cortex, in many cases tend to be pushed outward and are eventually "sloughed off" (shed).
At this point, the cork cambium begins to form the periderm, consisting of
protective cork cells. The walls of cork cells contain suberin thickenings, which is an extra
cellular complex biopolymer. The suberin thickenings functions by providing a physical
barrier, protection against pathogens and by preventing water loss from the surrounding
tissues. In addition, it also aids the process of wound healing in plants. It is also postulated
that suberin could be a component of the apoplastic barrier (present at the outer cell layers of
roots) which prevents toxic compounds from entering the root and reduces radial oxygen loss
from the parenchyma during waterlogging. In roots, the cork cambium originates in
the pericycle, a component of the vascular cylinder.
The vascular cambium produces new layers of secondary xylem annually. The xylem
vessels are dead at maturity but are responsible for most water transport through the vascular
tissue in stems and roots.

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 Tree roots usually grow to three times the diameter of the branch spread, only half of
which lie underneath the trunk and canopy. The roots from one side of a tree usually supply
nutrients to the foliage on the same side. Some families however, such
as Sapindaceae (the maple family), show no correlation between root location and where the
root supplies nutrients on the plant.
2. Molecular Genetics of Root Development
Many genes have been shown to be involved in various aspects of root development
such as distal patterning, radial patterning, epidermal patterning, cell division and cell
expansion.
Distal Patterning
Numerous physiological experiments have demonstrated that the formation of entire
root systems can be stimulated by auxins. The analysis of mutants impaired in root formation
provides more evidence for the role of auxins in formation of the entire root as well as in the
specification of its distal elements.
If primary root development strongly depends on the phytohormone auxin, it seems
reasonable to expect a similar important role for auxins in lateral root initiation. Indeed,
genetic analysis of the formation of lateral roots has also revealed numerous links to auxins.
Exogenous auxin addition leads to supernumerary roots, and superroot mutants (also referred
to as aberrant lateral root formation1 and hookless3) display a similar phenotype. Auxin-
resistant mutants have a reduced number of lateral roots. The auxin-inducible NAC1
transcription factor, whose activity determines the number of lateral root primordia, acts
downstream of tir1-dependent auxin responses. Lateral root formation is not stimulated by
IAA in aberrant lateral root formation4 (alf4) mutants, suggesting that the corresponding gene
also acts downstream of an initial IAA-related stimulus for lateral root formation. Lastly,
the solitary root mutant with a specific defect in lateral root formation carries a dominant
mutation in a gene encoding an AUX-IAA protein family member, suggesting a specific role
for this particular member in the induction of lateral roots. The many mutants and
corresponding genes provide an entry to dissect the mechanisms by which auxins appear to
promote lateral root development. It will be interesting to determine the extent of overlap and
specific differences between inductive processes in lateral roots to those operating in the
embryo.
Radial Patterning
The radial organization of the root is generated by stereotyped division of initial cells
and subsequent acquisition of cell fate. In a transverse root section, there are four radially
symmetric layers (from outside in, epidermis, cortex, endodermis, pericycle) that surround
the bilaterally symmetric vascular tissue (consisting of phloem, xylem and procambium). The
vascular tissue and surrounding pericycle are termed the stele. Mutations that disrupt the
radial pattern have been useful in identifying genes that play important roles in establishing
and maintaining the pattern. Many of these mutations were isolated by screening for roots
that were no longer able to grow in an indeterminate fashion. From these screens, mutations
that disrupt patterning of the ground tissue and vascular cylinder have been identified.
In the wooden leg (wol) mutant, protoxylem is the only tissue in the vascular cylinder,
compare with wild. Normally phloem and procambium are established through a set of
asymmetric cell divisions of their initials. These divisions require the WOODEN LEG (WOL)
gene, which is expressed in the vascular tissue of the root beginning at the early stages of
embryogenesis. The WOL gene encodes a novel two-component histidine kinase. There is
evidence that the WOL gene product is able to bind cytokinin raising the possibility that this
phytohormone plays a direct role in regulating the patterning of the vasculature.
Epidermal Patterning
The root epidermis is composed of two cell types whose identity is regulated by
positional information. Trichoblasts develop into hair cells and are located in the cleft
between underlying cortical cells when (viewed in transverse section) while atrichoblasts
remain hairless and are located over single cortical cell. Laser ablation experiments and
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clonal analysis indicates that positional cues direct cell identity and there is some evidence
that these positional cues are located in the cell wall.
Once hair cells have been specified, the hairs are initiated from the outer side of the
hair cell nearest the meristem. The polar localisation of the hair cell is dependent on an
ethylene and auxin pathway and coincides with the establishment of a high Ca 2+ gradient in
the hair tip. Tip growth is established and hairs elongate. While a large number of mutants
with defects in hair growth have been identified, to date few have been characterised in detail.
ROOT HAIRLESS1 is a nuclear protein required for hair initiation. ROOT HAIR DEFECTIVE3
is a small G-protein required to control the direction of tip growth. KOJAK/ AtCSLD3 is
required for the synthesis of cell wall polysaccharide. LEUCINE RICH EXPANSIN1 (LRX1) is
cell surface protein required to maintain cell shape. TINY ROOT HAIR3 is a potassium carrier
required during initiation. Together these molecular and genetic analyses are contributing to
the construction of a model for hair cell growth.
Cell Division
New cells are produced in the meristem, in which distinct zones of cell division
activities are evident. The central ―quiescent center‖ cells divide infrequently, and cell division
rates increase progressively up the root until a maximum is reached at a point that is
dependent on root age and growth conditions.
Trichoblasts are shorter than atrichoblasts throughout their development through the
meristem, elongation and differentiation zones. The difference in cell length is visible already
near the promeristem and is subsequently maintained through differentiation. Because the
two cell types differ in length from early development through to the mature stages, the rates
of cell division are identical in both cell types through the meristematic zone. Nevertheless the
difference in cell size between the two cell types is being constantly regulated. While the
majority of cell divisions in the epidermis are transverse, adding more cells to each file,
occasional longitudinal divisions occur that result in the formation of a pair of cells side by
side (twins) resulting in a file duplication. Both daughter cells are the same length but after a
few rounds of division the cells in the trichoblast position that are derived from one of the
twins becomes shorter than those derived from the other twin in the atrichoblast position. For
cells in the trichoblast position to be shorter than those in the atrichoblast position the rates of
cell division must be greater in the trichoblasts than atrichoblasts. Therefore upon switching
positions the cell division parameters are altered, resulting in the formation of cells with the
appropriate dimensions. This indicates that relative cell size and cell division rates are strictly
regulated in the epidermis.
The longitudinal divisions that produce twin cells giving rise to file duplications take
place predominantly in cells in the trichoblast position. The restriction of these longitudinal
divisions to the trichoblasts is controlled by a subset of the genes that regulate cell type
specification in the epidermis. Transparent testa glabra (ttg) mutants develop hairs on every
cell in the epidermis but also undergo longitudinal divisions in both atrichoblast and trichoblast
positions, indicating that TTG is also required for the suppression of longitudinal divisions in
the atrichoblasts. On the other hand every epidermal cell in glabra2 (gl2) mutants develop
hairs but no longitudinal divisions occur in the atrichoblasts, indicating that the specification of
cell fate and longitudinal divisions can be uncoupled. Together these data indicate that a
subset of the genes regulating cell specification also regulate the plane of cell division in the
epidermis.

Cell Expansion
Because there is no cell movement during plant development, cell expansion is one of
the key parameters that determine the ultimate form of plant organs. Different types of cell
expansion occur in different regions of the root. To generate cell files, initial cells and their
immediate progeny go through a continuous process of expansion and division. At first the
expansion of these dividing root cells is relatively slow and non-polar, such that their size
remains fairly constant. During this time, continuing cell divisions in the meristematic zone
displace these cells upward in the cell file. A transition to anisotropic expansion then occurs
which, when combined with cell division, results in cells with longer radial dimensions than
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longitudinal. This is particularly apparent in the epidermal cells. During this phase the ultimate
root radius is established. A second transition to highly polarized longitudinal expansion
occurs in the middle of the elongation zone. By this time cell division has almost stopped. This
expansion is rapid and highly oriented in the longitudinal axis.
All evidence indicates that the plant cell wall regulates both the extent and orientation
of cell expansion. The primary load-bearing molecule in the cell wall is thought to be cellulose
microfibrils, which are arranged like hoops around a barrel. Thus, it is not surprising that many
of the genes identified in mutant screens for altered root cell expansion appear to play a role
in cellulose synthesis. For example, rsw1 is a temperature-sensitive mutation that at the
restrictive temperature has swollen cells, reduced crystalline cellulose and a lack of rosettes
from which cellulose is produced. The RSW1 gene encodes a distant relative of the catalytic
subunit of bacterial cellulose synthases with predicted eight membrane-spanning domains.
References:

IBiology. Root Development: Genetics, Form and Function. https://www.ibiology.org/plant-

biology/root-development/. 2022

Science Direct. Root Development. https://www.sciencedirect.com/topics/medicine-and-

dentistry/root-development. 2022

Study.Com. Root System Growth: The Root Cap, Primary Roots and Lateral Roots.
https://study.com/academy/lesson/root-system-growth-the-root-cap-primary-roots-lateral-
roots.html. 2022

Wikipedia. Root. https://en.wikipedia.org/wiki/Root. 2022

Assessment:

I. Define the following terms:

1. Elongation
2. Primary growth
3. Secondary growth
4. Trichoblasts
5. Atrichoblasts

II. Enumerate the following:

1. Aspects of Root Development (5)
2. Radially symmetric layers (4)

III. Explain:
1. The root growth in plants.

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 CHAPTER 7

SHOOT DEVELOPMENT

Introduction:

A plant shoot system mainly includes the stems, leaves, flowers and fruits. The new
growth from seed germination that grows upward is a shoot where leaves will develop.

Some plant parts, such as stems and roots, continue to grow throughout a plant‘s life:
a phenomenon called indeterminate growth. Other plant parts, such as leaves and flowers,
exhibit determinate growth, which ceases when a plant part reaches a particular size.

Intended Learning Outcomes:

At the end of this module, students should be able to:

1. Explain how stem, leaf and flower develop.

2. Compare the development of the lateral shoot and the main shoot.
3. Explain and appreciate the natural coordination of the shoot and the root during
development.
4. Perform an activity pertaining to shoot reproduction of fruit trees using the latest technique.

Learning Content:

1. Stem, Leaf and Flower Development

In comparison to roots, in which cell division, elongation, and maturation occur in

distinct regions, growth in stems occurs in several regions. Rapid cell division occurs at the
shoot apical meristem of the shoot tip. Some of the cells produced from these divisions give
rise to leaf primordia, which ultimately develop into leaves. Division, elongation, and cell
differentiation (maturation) also occurs at the intercalary meristems of internodes, the stem
segments between nodes, the points where leaves attach. As a result, multiple internodes
can lengthen simultaneously. Under certain circumstances, the axillary buds at each node
can become active and produce axillary shoots, which branch from the main stem.

Changes in gene expression can cause a shoot apical meristem to develop into a floral
meristem, which leads to flowering. The ABCDE model (ABC model) of floral development
directs the formation of each of the four whorls of a complete flower. From outside to
inside, these whorls are the calyx (sepals), corolla (petals), androecium (stamens), and
gynoecium (carpels). Each unit of these whorls (sepal, petal, etc.) developmentally and
evolutionarily originated from the leaf.

The four whorls of a flower are the calyx (sepals), corolla (petals), androecium (stamens), and
gynoecium (carpels). The ovules are considered part of the carpels. Each structure results
from expression of a different combination of genes, which are identified in parentheses:
carpel (C+E), stamen (B+C+E), petal (A+B+E), ovule (C+D+E), and sepal (A+E).
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 The ABCDE model refers to five groups of homeotic genes, each represented by one
letter. Homeotic genes are those that control the development and organization of body
parts (body plan). Each of the five groups of genes that encode the transcription factors
(proteins required for gene expression), which turn on the genes for development of each
whorl. Genetic analysis of mutants especially those found in the Arabidopsis thaliana and in
the snapdragon (Antirrhinum) support the ABCDE model of flowering. Each group of genes
has a distinct role as follows:

 A genes are needed for sepal and petal development

 B genes are needed for petal and stamen development.
 C gene are needed for stamen and carpel (including ovules) development.
 D genes are need for ovule development (part of the carpels).
 E genes are needed to produce any of the floral whorls.

The ABCDE model of floral development. Different floral structures are represented by the top
row of bars. (Note that each of the first four segments of this bar represents a whorl. Ovules
are considered part of carpels, which make up the innermost whorl, the gynoecium.) The
remaining bars each represent a different group of homeotic gene. Expression of these genes
in different combinations produces each structure.

Thus, each whorl results from the expression of a different gene combination as
follows:

 Expression of A and E genes produce sepals.

 Expression of A, B, and E genes produce petals.
 Expression of B, C, and E genes produce stamens.
 Expression of C and E genes produce carpels. To produce ovules, which are
considered part of the carpels, D genes must also be expressed (C+D+E = ovules).

In summary, the formation of a flower requires a cascade of sequential gene activity

that gradually converts a mass of undifferentiated cells (the shoot apical meristem) into the
parts of a flower. The genes encode transcription factors that act as master switches, turning
on (or off) downstream genes that ultimately make each part of the flower in its appropriate
location.

2. Branching of the Shoot

The shoots of most vascular plants branch according to a consistent plan, with each
new axis arising in the angle between a leaf and a stem—that is, in a leaf axil. In some plants,
buds may also form from the older parts of shoot or root remote from the main apices; these
buds, termed adventitious, do not conform to the general plan.
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 A lateral shoot apex is initiated on the flanks of the main apex but at some distance
below the point of emergence of the youngest leaf primordium. As in the origin of a leaf,
generally the outer cell layers contribute to the surface tissues of the new apex by maintaining
a consistent pattern of divisions. In some species a tunica of more than one cell layer quickly
forms, so that the new apex appears as a miniature version of the main one; alternatively, the
differentiation may not become apparent until the new primordium has attained considerable
bulk. In all cases, the new apex must reach a minimal volume before it in turn can begin to
form its own lateral primordia and to organize true axillary buds. As this volume is attained,
zonation appears. As in the main apex, the formation of new primordia is associated with the
annular zone.

From this point on, the development of the lateral shoot is the same as that of the main
shoot, except that growth may not be as rapid because the main apex, or leading bud,
dominates and absorbs much of the available nutrient. The early growth of the axillary bud
proceeds quite vigorously until a certain number of leaf primordia has been formed; then
apical activity slows. Cell division gradually stops, and with it the associated syntheses; thus
there is no increase in the DNA of the nuclei of the meristem after the last division. The bud,
in effect, passes into a state of dormancy, even though the external conditions for growth are
propitious. This phenomenon is known as correlative bud inhibition, since it is determined by
the activity of the leading bud of the shoot. If the leading bud is removed, the inhibited lateral
buds resume growth, and with it the associated syntheses.

3. Coordination of Shoot and Root Development

Although the structural organization of the vascular plant is comparatively

loose, development of the various parts is well coordinated. Control is dependent upon the
movement of chemical substances, including both nutrients and hormones.

The enlargement of aerial parts is accompanied by increased demands for water,

minerals, and mechanical support that are met by coordinated growth of the root system.
Several factors apparently are concerned with control, because shoot and root affect each
other reciprocally. The root depends on the shoot for organic nutrients, just as the shoot
depends on the root for water and inorganic nutrients and the flow of ordinary nutrients must,
therefore, play some part. More specific control, however, may be provided by the supply of
nutrients required in very small amounts. The root depends on the shoot for certain vitamins,
and variation in the supply, reflecting the metabolic state of the aerial parts, may also
influence root growth. In addition, hormonal factors affecting cell division pass upward from
the root into the stem; although the exact role of the hormones has not yet been established
with certainty, they may provide one way by which the root system can influence the activity
of the shoot apex.

The control of secondary thickening is another important example of growth

coordination. As the size of the shoot system increases, the need for both greater mechanical
support and increased transport of water, minerals, and manufactured food is met by an
increase in stem girth through the activity of the vascular cambium. Generally, the cambium of
trees in temperate zones is most active in the spring, when buds open and shoots extend,
creating a demand for nutrients. Cell division begins near the bud in each shoot and then
spreads away from it. The terminal bud stimulates the cambium to divide rapidly through the
action of two groups of plant hormones: auxins and gibberellins.

The inhibition of lateral buds illustrates a reaction opposite to that occurring in the
control of cambial activity. Lateral buds are inhibited in general because axillary shoots grow
more slowly or not at all, while the terminal bud is active. This so-called apical dominance is
responsible for the characteristic single trunk growth seen in many conifers and in
herbaceous plants such as the hollyhock. Weaker dominance results in a bushy growth form
with repeated branching. The fact that lateral, or axillary, buds become more active when the
terminal bud is removed suggests that hormonal control is involved.

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 The flow of auxin from the shoot tip is, in part, responsible for inhibiting axillary buds.
The nutritional status of the plant also plays a role, apical dominance being strongest when
mineral supply and light are inadequate. Because axillary buds are released from inhibition
when treated with cell-division promoting substances (cytokinins), it has been suggested that
these substances are also concerned in regulating axillary-bud activity.

References:

Biology Libre Text. Shoot Development.

https://bio.libretexts.org/Bookshelves/Botany/Botany_(Ha_Morrow_and_Algiers)/Unit_3%3A_
Plant_Physiology_and_Regulation/18%3A_Development/18.05%3A_Shoot_Development.
2022

Britanica, The Shoot System and its Derivatives. https://www.britannica.com/science/plant-

development/The-shoot-system-and-its-derivatives. 2022

Wikipedia. Shoot. https://en.wikipedia.org/wiki/Shoot. 2022

Assessment:

I. Define the following terms:

1. Determinate growth
2. Indeterminate growth
3. Leaf primordial
4. Internode
5. Node
6. Homeotic genes

II. Enumerate the following:

1. Main Parts of the Shoot System (4)
2. Whorls of a Complete Flower (4)

III. Compare:
1. The development of the lateral shoot and the main shoot.

IV. Explain the following:

1. How stem, leaf and flower develop.
2. The natural coordination of the shoot and the root during development.

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 CHAPTER 8

DEVELOPMENTAL PLASTICITY IN PLANTS

Introduction:

Developmental plasticity can be defined as the developmental changes that follow

the perception and integration of environmental information. Although developmental plasticity
plays a major role in the adaptation of both animals and plants to heterogeneous conditions, it
is thought to be of particular importance in plants. A reason for this could be the limitation of
motility.

Phenotypic plasticity is often described as the responses of organisms to

environmental conditions or stimuli. Although depicting the essence of the phenomenon, this
definition is extremely broad and overly inclusive. For example, the ‗environment‘, as
perceived by ecologists, usually consists of the external surroundings and factors, whereas
many other biologists consider the ‗environment‘ in terms of the effects of neighboring cells or
morphogenic hormones.

Plant plasticity refers to a plant's ability to adapt to and cope with changes in its
environment. In contrast to animals, which are able to actively move away to avoid
challenges such as a predators or a changing climate, plants have acquired the ability to
biosynthesize an unprecedented array of structurally complex bioactive natural
compounds with specialized roles in order to cope with environmental challenges.

The greatest chemical complexity is found in toxic defence compounds which

plants deploy to deter herbivores and pests. As a result, plants have become the world
champions in carrying out complex chemistry. Plant plasticity is indeed phenomenal and
we can find plants that grow in the most unfavorable conditions.

Intended Learning Outcomes:

At the end of this module, students should be able to:

1. Explain and appreciate the role of physiological, morphological, and anatomical plasticity in
plants‘ adaption to environmental changes.
2. Explain the effect of different light environments to the phenotypic plasticity of leaves.
3. Define shade tolerance and compare the shade tolerance of some species of plants.
4. Compare the ecotypes of some species of plants.
5. Illustrate the plasticity of some invasive and native species of plants using role playing.

Learning Content:

1. Physiological, Anatomical, and Morphological Plasticity

Physiological, morphological, and anatomical plasticity may have a different role in

plant adaption to environmental changes. In particular, plasticity for physiological and life-
history traits may allow plants to grow and reproduce in spatially or temporally variable
environments. Physiological plasticity is more linked to an enhanced capacity to colonize
gaps and open areas because it ensures adjustments of gas exchange in response to
environmental factor changes in a short term. This aspect evidences the importance of
physiological plasticity in plant acclimatization to adverse environments where morphological
and anatomical plasticity play a secondary role. In fact, plants growing in stress conditions
tend to have a conservative leaf morphological pattern to avoid the production of structures
too expensive to be sustained. Moreover, morphological plasticity is more linked to an
enhanced plant capacity to grow in forest understories by having an important role in resource
acquisition.

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2. Plant Response to Light

One of the main morphological traits which changes in response to light variations is
the specific leaf area (SLA). The plasticity of SLA implies the morphogenetic control of
leaves which tends to increase leaf area in the shade in order to intercept more light.
SLA reflects leaf thickness and the relative proportion of assimilatory, conductive, and
mechanical tissues. In particular, the increased total lamina thickness in sun leaves compared
to shade leaves is mainly due to the greater palisade parenchyma, spongy parenchyma, and
epidermal tissue thickness, suggesting that leaf internal structure may play an important role
in light capture.

Sun leaves with respect to shade leaves, on an average, have a higher photosynthetic
rate on a leaf area The photosynthetic capacity is generally higher in leaves under high light
conditions. The higher photosynthetic rates of sun leaves are supported by higher stomatal
conductance and stomatal density to maximize CO 2 absorption. On the contrary, leaf trait
adjustments to low light increase the capacity of light absorption at the expense of the
photosynthetic capacity minimizing carbon loss through respiration.

Leaf trait variations in response to the light gradient within the canopy change in
different species and forest types. In forests with dense foliage, the upper layers absorb the
majority of the incoming radiation. In broadleaf and conifer forests of the temperate zone, on
an average, 3–10% of the incident photon flux density (PFD) penetrates the tree canopy.
Nevertheless, taxonomically different species co-occurring in the same habitat often share
common morphological and physiological traits, reflecting a convergent evolution in response
to environmental factors.
3. Shade Tolerance

Shade tolerance is one of the most important ecological factors with respect to
interspecific competition among forest trees of the temperate climate zone. The shade
tolerance of a given plant is defined as the minimum light under which a plant can survive;
however, only a fraction of plants can reproduce under these conditions. In general, sun and
shade leaves are thinner in tolerant species than in intolerant species. The overall trend in
literature suggests that early successional and light demanding species are more plastic than
late-successional and shade-tolerant species. Nevertheless, there is increasing evidence that
indicates that adjustments are not necessarily related to the successional status of the
species.

Numerous studies focus on acclimation of the photosynthetic properties of plant

species to different light conditions; nevertheless, results sometime disagree. For
example, Coffea arabica is an evergreen perennial tree from the African forest understory
which is considered an obligatory shade species. Matos et al. show that plasticity of
physiological and biochemical traits in C. arabica is more important for acclimation to
intracanopy light variations than morphological and anatomical trait plasticity. The authors
concluded that shade tolerance was not a consistent predictor of anatomical plasticity and
that, for many traits, differences between the light treatments were magnified in the second
year, showing that anatomical and physiological adjustment to shade is a long-term process.
Differences in forest structure determine changes in the light gradient and,
consequently, in the presence of shade-tolerant and intolerant species. In particular, the
deciduous forests may consist of an upper layer with shade-intolerant tree species and a
lower layer with shade-tolerant tree species, while in mixed forests, deciduous and evergreen
species with contrasting ecological potentials can occupy different layers.
Despite the assumption that shade leaves develop in response to reduced light, other
factors may also be involved, such as temperature and water stress. It has been

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hypothesized that plants cannot tolerate combined shade and drought, as a result of a
morphological trade-off.
4. Ecotypes

Long-term selection can lead to the development of morphological and physiological

adaptations to the local environment generating ecotypic differentiation in functional traits.
Genotypes adapted to local environmental conditions are referred to as ecotypes. When
environments within the distribution area of a species differ, it is unlikely that any single
phenotype confers high fitness in all situations. The distinction between phenotypic plasticity
and local adaptation of an ecotype is based primarily upon genetic analysis and
transplantation experiments. In particular, spatial genetic differentiation along climatic
gradients has been documented for many species as well as for ecotype formations.

Adaptation of species to geographic environmental variations often depends on genetic

variations among seed sources. Nahum et al. showed that Pistacia lentiscus ecotypes
growing in diverse habitats along a climatic gradient in Israel do not have any pattern of
ecologically related genetic differentiation and morphological and physiological differences
are probably due to phenotypic plasticity. Thus, adaptive plasticity can expand environmental
tolerance contributing to a wide distribution of P. lentiscus around the Mediterranean region.
Emery et al. show that ecotypes of Stellaria longipes, an herbaceous perennial species
growing along an altitudinal gradient on Plateau Mountain (Alberta) from the alpine tundra
(i.e., higher altitude), has a lower plasticity than the ecotype from the prairie (i.e., lower
altitude).
5. Competition between Invasive and Native Species

The increase of air temperature and carbon dioxide (CO2) concentration over recent
decades has determined novel environmental conditions which might act as a potent agent of
natural selection among species favoring more phenotypically plastic species and resulting in
a competition between invasive species (an introduced plant brought about by human
action) over co-occurring native species (presence is due to natural process, not by human
action). The phenotypic plasticity of co-occurring native and invasive species in the broadleaf
forest developing in the Natural Reserve Siro Negri (45°12′39′′ N, 9°3′26′′ E, Italy) attests to
the considered species responsiveness to light variations. Q. robur (a native species) and R.
pseudoacacia (an invasive species) have a similar physiological plasticity. Nevertheless, the
significantly higher morphological and anatomical plasticity of R. pseudoacacia than Q.
robur confirms its past capability of colonizing the forest and then growing successfully into
both the dominant and dominated layers (data Catoni et al. not published).

Introduced species frequently exhibit little genetic variations in the new environment
due to the genetic bottleneck and drift experienced by the small founding populations. Even
with genetic variations, local adaptation may not arise if selection is weak or unpredictable, or
if considerable gene flow occurs among populations. Despite these limitations, local
adaptation often contributes to the success of plants introduced in the new environments.
Nevertheless, despite the effort over the last decades, the evolutionary mechanisms leading
to invasiveness remain unclear.

An increased plasticity in plants reduces relative fitness (non-adaptive plasticity).

Reseach showed that plants originating from more southern Eurasian localities were more
similar to the invasive plants in North America than to plants from northern Eurasian localities.
Variability in growth characteristics across the north-south gradient within the native range
could result from long-term adaptation to prevailing environmental conditions, particularly day-
length. Moreover, variability for some growth characteristics (i.e., dry weight and number of
lateral branches, root dry weight), both between and within Eurasian populations, indicates a
plastic growth response to the local environmental conditions.

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References:

Hindawi. Plant Phenotypic Plasticity in Response to Environmental Factors.

https://www.hindawi.com/journals/abot/2014/208747/. 2022

Novoplansky, Ariel. Developmental Plasticity in Plants: Implications of Non-Cognitive

Behavior. http://www.esalq.usp.br/lepse/imgs/conteudo_thumb/Developmental-plasticity-in-
plants-implications-of-noncognitive-behavior-1.pdf. 2022

Science Direct. Phenotypic Plasticity. https://www.sciencedirect.com/topics/agricultural-and-

biological-sciences/phenotypic-plasticity. 2022

Wikipedia. Phenotypic Plasticity. https://en.wikipedia.org/wiki/Phenotypic_plasticity. 2022

Assessment:

I. Define the following terms:

1. Developmental plasticity
2. Phenotypic plasticity
3. Plant plasticity
4. Shade tolerance
5. Ecotypes
6. Invasive species
7. Native species
8. Non-adaptive plasticity

II. Explain the following:

1. The role of physiological, morphological, and anatomical plasticity in plants‘ adaption to
environmental changes.
2. The effect of different light environments to the phenotypic plasticity of leaves.

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 CHAPTER 9

INDUCED PLURIPOTENT STEM CELLS (iPS)

Introduction:

Induced pluripotent stem cell (iPS cell), immature cell that is generated from an
adult (mature) cell and that has regained the capacity to differentiate into any type of cell in
the body. Induced pluripotent stem cells (iPS cells) differ from embryonic stem cells (ES
cells), which form the inner cell mass of an embryo but also are pluripotent, eventually giving
rise to all the cell types that make up the body. Induced pluripotent cells were first described
in 2006 by Japanese physician and researcher Shinya Yamanaka and colleagues. The first
experiments were performed by using mouse cells. The following year, however, Yamanaka
successfully derived iPS cells from human adult fibroblast cells. Until that time, human stem
cells could be obtained only by isolating them from early human embryos. Hence, an
important feature of iPS cells is that their generation does not require an embryo, the use of
which is fraught with ethical issues.

Intended Learning Outcomes:

At the end of this module, students should be able to:

1. Explain how induced pluripotent stem cells (iPSCs) are generated.

2. Explain and appreciate the roles played by some genes in the production of iPSCs.
3. Explain the similarities between the generated iPSCs and the naturally isolated pluripotent
stem cells.
4. Explain the safety of iPSCs in clinical application.
5. Appreciate the clinical application of iPSCs.

Learning Content:

1. Production

First generation (mouse)

Induced pluripotent stem cells were first generated by Shinya Yamanaka's team
at Kyoto University, Japan, in 2006. They hypothesized that genes important to embryonic
stem cell (ESC) function might be able to induce an embryonic state in adult cells. They
chose twenty-four genes previously identified as important in ESCs and used retroviruses to
deliver these genes to mouse fibroblasts. The fibroblasts were engineered so that any cells
reactivating the ESC-specific gene, Fbx15, could be isolated using antibiotic selection.
Upon delivery of all twenty-four factors, ESC-like colonies emerged that reactivated the
Fbx15 reporter and could propagate indefinitely. To identify the genes necessary for
reprogramming, the researchers removed one factor at a time from the pool of twenty-four. By
this process, they identified four factors, Oct4, Sox2, cMyc, and Klf4, which were each
necessary and together sufficient to generate ESC-like colonies under selection for
reactivation of Fbx15.
Second generation (mouse)
In June 2007, three separate research groups, including that of Yamanaka's,
a Harvard/University of California, Los Angeles collaboration, and a group at MIT, published
studies that substantially improved on the reprogramming approach, giving rise to iPSCs that
were indistinguishable from ESCs. Unlike the first generation of iPSCs, these second
generation iPSCs produced viable chimeric mice and contributed to the mouse germline,
thereby achieving the 'gold standard' for pluripotent stem cells.
These second-generation iPSCs were derived from mouse fibroblasts by retroviral-
mediated expression of the same four transcription factors (Oct4, Sox2, cMyc, Klf4).
However, instead of using Fbx15 to select for pluripotent cells, the researchers used Nanog, a

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gene that is functionally important in ESCs. By using this different strategy, the researchers
created iPSCs that were functionally identical to ESCs.
Human induced pluripotent stem cells
Generation from human fibroblasts
Reprogramming of human cells to iPSCs was reported in November 2007 by two
independent research groups: Shinya Yamanaka of Kyoto University, Japan, who pioneered
the original iPSC method, and James Thomson of University of Wisconsin-Madison who was
the first to derive human embryonic stem cells. With the same principle used in mouse
reprogramming, Yamanaka's group successfully transformed human fibroblasts into iPSCs
with the same four pivotal genes, Oct4, Sox2, Klf4, and cMyc, using a retroviral system, while
Thomson and colleagues used a different set of factors, Oct4, Sox2, Nanog, and Lin28, using
a lentiviral system.
Generation from additional cell types
Obtaining fibroblasts to produce iPSCs involves a skin biopsy, and there has been a
push towards identifying cell types that are more easily accessible. In 2008, iPSCs were
derived from human keratinocytes, which could be obtained from a single hair pluck. In 2010,
iPSCs were derived from peripheral blood cells, and in 2012, iPSCs were made from renal
epithelial cells in the urine.
Other considerations for starting cell type include mutational load (for example, skin
cells may harbor more mutations due to UV exposure), time it takes to expand the population
of starting cells, and the ability to differentiate into a given cell type.
2. Genes Used to Produce iPSCs
The generation of induced pluripotent cells is crucially dependent on the transcription
factors used for the induction.
Oct-3/4 and certain products of the Sox gene family (Sox1, Sox2, Sox3, and Sox15)
have been identified as crucial transcriptional regulators involved in the induction process
whose absence makes induction impossible. Additional genes, however, including certain
members of the Klf family (Klf1, Klf2, Klf4, and Klf5), the Myc family (c-myc, L-myc, and N-
myc), Nanog, LIN28, Glis1, have been identified to increase the induction efficiency.

 Oct-3/4 (Pou5f1) Oct-3/4 is one of the family of octamer ("Oct") transcription factors, and
plays a crucial role in maintaining pluripotency. The absence of Oct-3/4 in Oct-3/4+ cells,
such as blastomeres and embryonic stem cells, leads to
spontaneous trophoblast differentiation, and presence of Oct-3/4 thus gives rise to the
pluripotency and differentiation potential of embryonic stem cells. Various other genes in
the "Oct" family, including Oct-3/4's close relatives, Oct1 and Oct6, fail to elicit induction,
thus demonstrating the exclusiveness of Oct-3/4 to the induction process. However a
team headed by Hans Schöler (who discovered the Oct4 gene back in 1989) showed that
Oct4 overexpression during reprogramming causes epigenetic changes deteriorating the
quality of iPSCs. Comparing to OSKM (Oct4, Sox2, Klf4 and c-Myc) new SKM (Sox2, Klf4
and c-Myc) reprogramming generates iPSCs with developmental potential equivalent
to embryonic stem cell, as determined by their ability to generate all-iPSC mice
through tetraploid embryo complementation.
 Sox family: The Sox family of transcription factors is associated with maintaining
pluripotency similar to Oct-3/4, although it is associated with multipotent and unipotent
stem cells in contrast with Oct-3/4, which is exclusively expressed in pluripotent stem
cells. While Sox2 was the initial gene used for induction by Yamanaka et al., Jaenisch et
al., and Thomson et al., other transcription factors in the Sox family have been found to
work as well in the induction process. Sox1 yields iPS cells with a similar efficiency as
Sox2, and genes Sox3, Sox15, and Sox18 also generate iPS cells, although with
decreased efficiency.
 Klf family: Klf4 of the Klf family of transcription factors was initially identified by
Yamanaka et al. and confirmed by Jaenisch et al. As a factor for the generation of mouse
iPS cells and was demonstrated by Yamanaka et al. as a factor for generation of human
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 iPS cells. However, Thomson et al. reported that Klf4 was unnecessary for generation of
human iPS cells and in fact failed to generate human iPS cells. Klf2 and Klf4 were found
to be factors capable of generating iPS cells, and related genes Klf1 and Klf5 did as well,
although with reduced efficiency.
 Myc family: The Myc family of transcription factors are proto-oncogenes implicated in
cancer. Yamanaka et al. and Jaenisch et al. demonstrated that c-myc is a factor
implicated in the generation of mouse iPS cells and Yamanaka et al. demonstrated it was
a factor implicated in the generation of human iPS cells. However, Thomson et al.,
Yamanaka et al. usage of the "myc" family of genes in induction of iPS cells is troubling for
the eventuality of iPS cells as clinical therapies, as 25% of mice transplanted with c-myc-
induced iPS cells developed lethal teratomas. N-myc and L-myc have been identified to
induce instead of c-myc with similar efficiency.
 Nanog: In embryonic stem cells, Nanog, along with Oct-3/4 and Sox2, is necessary in
promoting pluripotency. Therefore, it was surprising when Yamanaka et al. reported that
Nanog was unnecessary for induction although Thomson et al. has reported it is possible
to generate iPS cells with Nanog as one of the factors.
 LIN28: LIN28 is an mRNA binding protein expressed in embryonic stem cells and
embryonic carcinoma cells associated with differentiation and proliferation. Thomson et al.
demonstrated that LIN28 is a factor in iPSC generation in combination with OCT4, SOX2,
and NANOG.
 Glis1: Glis1 is transcription factor that can be used with Oct-3/4, Sox2 and Klf4 to induce
pluripotency. It poses numerous advantages when used instead of C-myc.

3. Identity

Induced pluripotent stem cells are similar to natural pluripotent stem cells, such
as embryonic stem (ES) cells, in many aspects, such as the expression of certain stem cell
genes and proteins, chromatin methylation patterns, doubling time, embryoid
body formation, teratoma formation, viable chimera formation, and potency and
differentiability, but the full extent of their relation to natural pluripotent stem cells is still being
assessed.
Gene expression and genome-wide H3K4me3 and H3K27me3 were found to be
extremely similar between ES cells and iPS cells. The generated iPSCs were remarkably
similar to naturally isolated pluripotent stem cells (such as mouse and human embryonic stem
cells, mESCs and hESCs, respectively) in the following respects, thus confirming the identity,
authenticity, and pluripotency of iPSCs to naturally isolated pluripotent stem cells:

 Cellular biological properties

o Morphology: iPSCs were morphologically similar to ESCs. Each cell had round shape,
large nucleolus and scant cytoplasm. Colonies of iPSCs were also similar to that of
ESCs. Human iPSCs formed sharp-edged, flat, tightly packed colonies similar to
hESCs and mouse iPSCs formed the colonies similar to mESCs, less flat and more
aggregated colonies than that of hESCs.
o Growth properties: Doubling time and mitotic activity are cornerstones of ESCs, as
stem cells must self-renew as part of their definition. iPSCs were mitotically active,
actively self-renewing, proliferating, and dividing at a rate equal to ESCs.
o Stem cell markers: iPSCs expressed cell surface antigenic markers expressed on
ESCs. Human iPSCs expressed the markers specific to hESC, including SSEA-3,
SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, and Nanog. Mouse iPSCs expressed
SSEA-1 but not SSEA-3 nor SSEA-4, similarly to mESCs.
o Stem Cell Genes: iPSCs expressed genes expressed in undifferentiated ESCs,
including Oct-3/4, Sox2, Nanog, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, and
hTERT.
o Telomerase activity: Telomerases are necessary to sustain cell division unrestricted by
the Hayflick limit of ~50 cell divisions. hESCs express high telomerase activity to
sustain self-renewal and proliferation, and iPSCs also demonstrate high telomerase

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 activity and express hTERT (human telomerase reverse transcriptase), a necessary
component in the telomerase protein complex.
 Pluripotency: iPSCs were capable of differentiation in a fashion similar to ESCs into fully
differentiated tissues.
o Neural differentiation: iPSCs were differentiated into neurons, expressing βIII-tubulin,
tyrosine hydroxylase, AADC, DAT, ChAT, LMX1B, and MAP2. The presence
of catecholamine-associated enzymes may indicate that iPSCs, like hESCs, may be
differentiable into dopaminergic neurons. Stem cell-associated genes were
downregulated after differentiation.
o Cardiac differentiation: iPSCs were differentiated into cardiomyocytes that
spontaneously began beating. Cardiomyocytes expressed TnTc, MEF2C, MYL2A,
MYHCβ, and NKX2.5. Stem cell-associated genes were downregulated after
differentiation.
o Teratoma formation: iPSCs injected into immunodeficient mice spontaneously
formed teratomas after nine weeks. Teratomas are tumors of multiple lineages
containing tissue derived from the three germ
layers endoderm, mesoderm and ectoderm; this is unlike other tumors, which typically
are of only one cell type. Teratoma formation is a landmark test for pluripotency.
o Embryoid body: hESCs in culture spontaneously form ball-like embryo-like structures
termed "embryoid bodies", which consist of a core of mitotically active and
differentiating hESCs and a periphery of fully differentiated cells from all three germ
layers. iPSCs also form embryoid bodies and have peripheral differentiated cells.
o Chimeric mice: hESCs naturally reside within the inner cell mass (embryoblast)
of blastocysts, and in the embryoblast, differentiate into the embryo while the
blastocyst's shell (trophoblast) differentiates into extraembryonic tissues. The hollow
trophoblast is unable to form a living embryo, and thus it is necessary for the
embryonic stem cells within the embryoblast to differentiate and form the embryo.
iPSCs were injected by micropipette into a trophoblast, and the blastocyst was
transferred to recipient females. Chimeric living mouse pups were created: mice with
iPSC derivatives incorporated all across their bodies with 10–90% chimerism.
o Tetraploid complementation: iPS cells from mouse fetal fibroblasts injected into
tetraploid blastocysts (which themselves can only form extra-embryonic tissues) can
form whole, non-chimeric, fertile mice, although with low success rate.
 Epigenetic reprogramming
o Promoter demethylation: Methylation is the transfer of a methyl group to a DNA base, typically
the transfer of a methyl group to a cytosine molecule in a CpG site (adjacent cytosine/guanine
sequence). Widespread methylation of a gene interferes with expression by preventing the
activity of expression proteins, or by recruiting enzymes that interfere with expression. Thus,
methylation of a gene effectively silences it by preventing transcription. Promoters of
pluripotency-associated genes, including Oct-3/4, Rex1, and Nanog, were demethylated in
iPSCs, demonstrating their promoter activity and the active promotion and expression of
pluripotency-associated genes in iPSCs.
o DNA methylation globally: Human iPS cells are highly similar to ES cells in their patterns of
which cytosines are methylated, more than to any other cell type. However, on the order of a
thousand sites show differences in several iPS cell lines. Half of these resemble the somatic
cell line the iPS cells were derived from, the rest are iPSC-specific. Tens of regions which
are megabases in size have also been found where iPS cells are not reprogrammed to the ES
cell state.
o Histone demethylation: Histones are compacting proteins that are structurally localized to DNA
sequences that can affect their activity through various chromatin-related modifications. H3
histones associated with Oct-3/4, Sox2, and Nanog were demethylated, indicating the
expression of Oct-3/4, Sox2, and Nanog.

4. Safety

 The major concern with the potential clinical application of iPSCs is their propensity to form
tumors. Much the same as ESC, iPSCs readily form teratoma when injected into immunodeficient
mice. Teratoma (a type of germ cell tumor that may contain several different types of tissue such
as hair, muscle and bone) formation is considered a major obstacle to stem-cell based
regenerative medicine by the FDA.
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 A more recent study on motor functional recovery after spinal cord injuries in mice showed that
after human-induced pluripotent stem cells were transplanted into the mice, the cells differentiated
into three neural lineages in the spinal cord. The cells stimulated regrowth of the damaged spinal
cord, maintained myelination, and formed synapses. These positive outcomes were observed for
over 112 days after the spinal cord injury, without tumor formation. Nevertheless, a follow-up study
by the same group showed distinct clones of human-induced pluripotent stem cells eventually
formed tumors.
 Since iPSCs can only be produced with high efficiency at this time using modifications, they are
generally predicted to be less safe and more tumorigenic than hESC. All the genes that have been
shown to promote iPSC formation have also been linked to cancer in one way or another. Some of
the genes are known oncogenes, including the members of the Myc family. While omitting Myc still
allows for IPSC formation, the efficiency is reduced up to 100 fold.
 A non-genetic method of producing iPSCs has been demonstrated using recombinant proteins,
but its efficiency was quite low. However, refinements to this methodology yielding higher
efficiency may lead to production of safer iPSCs. Other approaches such as using adenovirus or
plasmids are generally thought to be safer than retroviral methods.
 An important area for future studies in the iPSC field is directly testing iPSC tumorigenicity using
methods that mimic the approaches that would be used for regenerative medicine therapies. Such
studies are crucial since iPSCs not only form teratoma, but also mice derived from iPSCs have a
high incidence of death from malignant cancer. A 2010 paper was published in the journal Stem
Cells indicating that iPS cells are far more tumorigenic than ESC, supporting the notion that iPS
cell safety is a serious concern.
 Concern regarding the immunogenicity of IPS cells arose in 2011 when Zhou et al. performed a
study involving a teratoma formation assay and demonstrated that IPS cells produced an immune
response strong enough to cause rejection of the cells. When a similar procedure was performed
on genetically equivalent ES cells however, Zhou et al. found teratomas, which indicated that the
cells were tolerated by the immune system. In 2013, Araki et al. attempted to reproduce the
conclusion obtained by Zhou et al. using a different procedure. They took cells from a chimera that
had been grown from IPSC clones and a mouse embryo, this tissue was then transplanted
into syngenic mice. They conducted a similar trial using ES cells instead of IPSC clone and
compared the results. Findings indicate that there was no significant difference in the
immunogenic response produced by the IPS cells and the ES cells. Furthermore, Araki et al.
reported little or no immunogenic response for both cell lines. Thus, Araki et al. was unable to
come to the same conclusion as Zhou et al.

References:

Britanica. Induced Pluripotent Stem Cell. https://www.britannica.com/science/induced-pluripotent-

stem-cell. 2022

Science Direct. Induced Pluripotent Stem Cells. https://www.sciencedirect.com/topics/biochemistry-

genetics-and-molecular-biology/induced-pluripotent-stem-cell. 2022

Wikipedia. Induced Pluripotent Stem Cell. https://en.wikipedia.org/wiki/Induced_pluripotent_stem_cell.

2022

Assessment:

I. Define the following terms:

1. Induced pluripotent stem cell (iPS cell)
2. Teratoma

II. Enumerate the following:

1. Genes used to produce iPSCs (6)
2. Similarities between ESC and iPSC (3)

III. Explain the following:

1. How induced pluripotent stem cells (iPSCs) are generated.
2. The roles played by some genes in the production of iPSCs.
3. The similarities between the generated iPSCs and the naturally isolated pluripotent stem cells.
4. The safety of iPSCs in clinical application.

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 CHAPTER 10

EARLY DROSOPHILA DEVELOPMENT

Introduction:

During Drosophila development, cellular membranes do not form until after the
thirteenth nuclear division. Prior to this time, all the nuclei share a common cytoplasm, and
material can diffuse throughout the embryo. In these embryos, the specification of cell types
along anterior-posterior and dorsal-ventral axes is accomplished by the interactions of
cytoplasmic materials within the single, multinucleated cell. Moreover, the initiation of the
anterior-posterior and dorsal-ventral differences is controlled by the position of the egg within
the mother's ovary. Whereas the sperm entry site may fix the axes in ascidians and
nematodes, the fly's anterior-posterior and dorsal-ventral axes are specified by interactions
between the egg and its surrounding follicle cells.

Intended Learning Outcomes:

At the end of this module, students should be able to:

1. Explain the stages of development in Drosophila.

2. Explain and appreciate the roles of the maternal effect genes, segmentation genes, and
homeotic genes in the anterior-posterior axis patterning in Drosophila.
3. Illustrate the dorsal-ventral axis patterning in Drosophila using a diagram.

Learning Content:

1. Life cycle

Drosophila display a holometabolous method of development, meaning that they

have three distinct stages of their post-embryonic life cycle, each with a radically different
body plan: larva, pupa and finally, adult. The machinery necessary for the function and
smooth transition between these three phases develops during embryogenesis (the
developmental stage of an animal embryo). During embryogenesis, the larval stage fly will
develop and hatch at a stage of its life known as the first larval instar. Cells that will produce
adult structures are put aside in imaginal discs. During the pupal stage, the larval body breaks
down as the imaginal discs grow and produce the adult body. This process is
called complete metamorphosis. About 24 hours after fertilization, an egg hatches into a
larva, which undergoes three molts taking about 5.5 to 6 days, after which it is called a pupa.
The pupa metamorphoses into an adult fly, which takes about 3.5 to 4.5 days. The entire
growth process from egg to adult fly takes an estimated 10 to 12 days to complete at 25 °C.

The mother fly produces oocytes that already have anterior-posterior and dorsal-
ventral axes defined by maternal activities.

Embryogenesis in Drosophila is unique among model organisms in that cleavage

occurs in a multinucleate syncytium (strictly a coenocyte). Early on, 256 nuclei migrate to the
perimeter of the egg, creating the syncytial blastoderm. The germ line segregates from the
somatic cells through the formation of pole cells at the posterior end of the embryo. After
thirteen mitotic divisions and about 4 hours after fertilization, an estimated 6,000 nuclei
accumulate in the un-separated cytoplasm of the oocyte before they migrate to the surface
and are encompassed by plasma membranes to form cells surrounding the yolk sac
producing a cellular blastoderm.

Like other triploblastic metazoa, gastrulation leads to the formation of three germ
layers: the endoderm, mesoderm, and ectoderm.

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2. Anterior-Posterior Axis Patterning in Drosophila

One of the best understood examples of pattern formation is the patterning along the
future head to tail (antero-posterior) axis of the fruit fly Drosophila melanogaster. There are
three fundamental types of genes that give way to the developmental structure of the fly:
maternal effect genes, segmentation genes and homeotic genes. The development
of Drosophila is particularly well studied, and it is representative of a major class of animals,
the insects or insecta.

Maternal effect genes

The building-blocks of anterior-posterior axis patterning in Drosophila are laid out

during egg formation (oogenesis), well before the egg is fertilized and deposited. The
maternal effect genes are responsible for the polarity of the egg and of the embryo. The
developing egg (oocyte) is polarized by differentially localized mRNA molecules.

Maternal effect genes encode for proteins that get translated upon fertilization to
establish concentration gradients that span the egg. Bicoid and Hunchback are the maternal
effect genes that are most important for patterning of anterior parts (head and thorax) of
the Drosophila embryo. Nanos and Caudal are maternal effect genes that are important in the
formation of more posterior abdominal segments of the Drosophila embryo.

Gap genes and Segmentation Genes

The other important function of the gradients of Bicoid, Hunchback, and Caudal
proteins is in the transcriptional regulation of other zygotically expressed proteins. Many of
these are the protein products derived from members of the "gap" family of developmental
control genes. Giant, huckebein, hunchback, knirps, Krüppel and tailless are all gap genes.
Their expression patterns in the early embryo are determined by the maternal effect gene
products. The gap genes are part of a larger family called the segmentation genes. These
genes establish the segmented body plan of the embryo along the anterior-posterior axis. The
segmentation genes specify 14 parasegments that are closely related to the final anatomical
segments. The gap genes are the first layer of a hierarchical cascade of the segmentation
control genes.

3. Dorsal-Ventral Axis

Formation of the dorsal-ventral axis (dorsal end – back side, ventral end – front side) is
dependent on the ventral nuclear concentration of a maternally synthesized transcription
factor called Dorsal. The determination of the dorsal side of the embryo occurs
during oogenesis when the oocyte nucleus moves along microtubules from the posterior to
the anterior-dorsal margin of the oocyte. The nucleus expresses a protein
called Gurken which is secreted locally and thus only activates follicle cells in the dorsal
region by interacting with the Torpedo receptor. This inhibits the production of Pipe protein
and thus follicular cells expressing Pipe are on the ventral side. Pipe activates an extracellular
protease cascade in the perivitelline space between the follicle cells and the egg which results
in the cleavage of the Toll-ligand Spätzle and activation of the Toll signaling cascade on the
ventral side. Dorsal protein is present throughout embryonic cytoplasm but bound to Cactus
which prevents it from translocating to the nucleus. Toll signaling results in the degradation of
Cactus which allows Dorsal to enter the nuclei on the ventral side of the blastoderm. Overall,
a difference in the localization of the oocyte nucleus becomes a difference in the signaling
state of the surrounding follicle cells which then signal to the resulting blastoderm nuclei.

Once in the nucleus, Dorsal activates different genes depending upon its nuclear
concentration. This process sets up a gradient between the ventral and dorsal side of the
blastoderm embryo with the repression or induction of Dorsal target genes being differentially
regulated. At the ventral end of the embryo, blastoderm nuclei exposed to high concentrations

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of dorsal protein induce the transcription of the transcription factors. This results in the
formation of the mesoderm.
References:

Current Biology. Patterning the Drosophila Embryo. https://www.cell.com/current-

biology/comments/S0960-9822(02)00406-2. 2022

NCBI. Early Drosophila Development. https://www.ncbi.nlm.nih.gov/books/NBK10081/. 2022

Study.com. Embryonic Development and Life Cycles of Invertebrates

https://study.com/academy/lesson/embryonic-development-life-cycles-of-invertebrates-
vertebrates.html#:~:text=Invertebrate%20Embryonic%20Development,to%20develop%20furt
her%20into%20adulthood. 2022

Wikipedia. Drosophila Embryogenesis.

https://en.wikipedia.org/wiki/Drosophila_embryogenesis. 2022.

Assessment:

I. Identify/describe the following:

1. Holometabolous
2. Embryogenesis
3. After fertilization, how long does it take for Drosophila‘s egg to hatch into a larva?
4. How long does it take for the entire growth process of Drosophila to complete at 25 °C?
5. Oogenesis
6. Antero-posterior axis
7. Maternal effect genes
8. Segmentation genes

II. Enumerate the following:

1. Distinct Stages of Drosophila Development (3)
2. Genes responsible for developmental structure of the fly (3)

III. Explain the following:

1. The stages of development in Drosophila.
2. The roles of the maternal effect genes, segmentation genes, and homeotic genes in the
anterior-posterior axis patterning in Drosophila.

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 CHAPTER 11

EARLY ASCIDIAN DEVELOPMENT

Introduction:

Ascidians, or sea squirts, are marine invertebrate chordates with several

characteristics that make them useful for studying processes of cell fate specification. These
characteristics have attracted many researchers over the past decade, and ascidians have
emerged as one of the most popular model organisms in developmental biology.

Gastrulation and neurulation involve cellular rearrangements that are comparable to

those seen in vertebrates, except that ascidian embryos are composed of just a few hundred
cells whereas comparable vertebrate embryos contain many thousands of cells. The resultant
motile larva is analogous to the amphibian tadpole. This larva ultimately undergoes
metamorphosis into a sessile, filter-feeding adult.

Intended Learning Outcomes:

At the end of this module, students should be able to:

1. Explain the ascidian embryogenesis.

2. Explain how the 3 main embryonic axes of the ascidians are established and appreciate
their existence.
3. Explain how endoderm, mesoderm and ectoderm of the ascidians are formed and
appreciate the developmental fates of these.

Learning Content:

1. Embryogenesis

Ascidian embryogenesis proceeds rapidly and involves a small number of cells. The
first cleavage occurs 1 hr 45 min after fertilization in Halocynthia roretzi at 13°C and 1 hr after
in Ciona intestinalis at 18°C. After two more synchronous (occurring at the same
time/simultaneous) and four asynchronous (not simultaneous) cleavages, the embryo reaches
the 110-cell stage (9 hr in Halocynthia, 5 hr in Ciona), when gastrulation starts. During
gastrulation, a single layer of endoderm and mesoderm cells in the vegetal hemisphere
invaginates into the interior as the ectoderm layer migrates toward the vegetal pole to form a
surface around the embryo. The spatial relationship between the cells derived from the
vegetal hemisphere is dramatically changed during morphogenetic cell movements.
Neurulation begins soon after gastrulation (13 hr in Halocynthia, 7 hr in Ciona). In ascidian
neurulation, the neural plate is curled up dorsally to form a tube-like structure known as the
neural tube. The formation of the tube-like structure progresses from posterior to anterior.
Once it is completely closed, the tail becomes distinguishable (18 hr in Halocynthia, 9 hr
in Ciona). With tail bud formation, the embryo enters a relatively long-lasting stage, the tail
bud stage, during which the tail continues to elongate until the embryo is ready to hatch (35 hr
in Halocynthia, 18 hr in Ciona). Inside the embryo, the notochord cells converge and extend
during neurulation and at the early tail bud stage. After the notochord cells are all intercalated
and stacked in a single row, they further extend individually along the anterior–posterior (A–P)
axis and provide a driving force for tail elongation. Cellular mechanisms of convergence and
extension are conserved between ascidians and vertebrates.

The anatomy of the ascidian larva is similar to that of its amphibian counterpart. Both
possess a notochord flanked by blocks of muscle and paralleled by a hollow neural tube
located dorsally. The major tissues that constitute the ascidian tadpole are the epidermis,
nervous system, notochord, muscle, mesenchyme, trunk lateral and ventral cells, and
endoderm.

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2. Establishment of Embryonic Axes

The first axis to be established in ascidian development is the animal–vegetal (A–V)

axis. The animal pole is defined by the position in the egg where the polar bodies form, and
the vegetal pole by the position opposite the animal pole. Although the polar bodies become
visible after fertilization, the A–V axis is already established during oogenesis.

The anterior-posterior (A–P) axis is established perpendicular to the A–V axis after
fertilization. The second phase of ooplasmic movements translocates the vegetally localized
cytoplasm/cortex, which contains mitochondria, cortical endoplasmic reticulum, and
postplasmic/PEM RNAs, to the future posterior pole, in a region called the posterior vegetal
cytoplasm/cortex (PVC). This second reorganization, unlike the first, is driven by microtubules
emanated from the centrosome supplied by the sperm. How the mechanism by which the
cytoplasm/cortex is reorganized is switched from microfilament-dependent during the first
phase to microtubule-dependent during the second is not known. The establishment of the A–
P axis depends on the PVC, which exhibits a posteriorizing activity. The removal or
transplantation of the PVC to the future anterior region of the normal egg causes duplication
or deficiency of the anterior structure in the original posterior or anterior region, respectively.
These results suggest that the anterior fate may arise by the absence of the posteriorizing
activity. Some of the localized postplasmic/PEM RNAs within the PVC represent the
posteriorizing activity. PEM, first identified as a postplasmic/PEM RNA, regulates the
posterior-specific cleavage pattern, while Macho-1 plays an essential role in the formation of
the posterior-specific tissues such as the primary muscle and mesenchyme. Knockdown of
other postplasmic/PEM RNAs by morpholino antisense oligo (MO) injection has indicated that
the postplasmic/PEM RNAs function over and influence a broader region of the embryo than
just individual tissues. This mechanism by which an axis is formed by the inheritance of
maternal localized factors is conserved in many other organisms as well. In ascidians, the
conventional A–P axis does not precisely correspond to that of larvae, because cells do not
stay stationary during gastrula cell movements.

When the A–P axis forms, the third axis, the mediolateral axis, is automatically
established. The left–right asymmetry in the ascidian embryo is morphologically recognizable
during tail elongation and in the brain positioning. The tail bends leftward, while elongating in
most cases in Halocynthia.

3. Specification of Developmental Fates

Endoderm Formation

Endoderm formation relies on the inheritance of localized maternal factors by cells of

the vegetal hemisphere. In ascidians, maternal β-catenin sits at the top of a hierarchy so far
that leads to endoderm formation.

Ascidian endoderm is subdivided into anterior and posterior parts with regard to their
origins. The anterior endoderm is originated from the A4.1, the A–V blastomeres. The
posterior endoderm, on the other hand, is derived from the B4.1, the P–V blastomeres.
Endoderm serves as the source of mesoderm-inducing signals. The ascidian endoderm
induces adjacent cells into mesoderm, such as the notochord and mesenchyme.

Mesoderm Formation

Mesoderm in ascidians is induced in the equatorial region (marginal zone of the

vegetal hemisphere) mainly by a signal from the vegetal cells. As observed in the case of the
notochord and mesenchyme induction, mesoderm in many organisms is induced in different
cell types by the same signal. This finding is because regionalization within the mesoderm-
arising territory takes place before the induction and provides different competence to
respond to the inducing signal.
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 In organisms that are known for their invariant embryogenesis with a relatively small
number of embryonic cells, such as C. elegans and ascidians, cell fates are often induced
and determined in a cell cycle before the fates are restricted.

In ascidian notochord or mesenchyme induction, cells are fated to become both

notochord and nerve cord, or mesenchyme and muscle, respectively.

Ectoderm Formation

Recent studies with chicks (Gallus gallus) and Xenopus have challenged the default
model for neural specification in which a default neural state is preserved within the ectoderm.
In ascidians, the FGF9/16/20 signal also induces neural tissues.

The ascidian CNS develops from three different lineages: the brain (sensory vesicle)
mainly from the animal a-line, the dorsal-most row of ependymal cells in the nerve cord from
the animal b-line, and the lateral and ventral rows from the vegetal A-line.

Talking about animals in general, cells in each germ layer differentiate into tissues
and embryonic organs. The ectoderm gives rise to the nervous system and the epidermis.
The mesoderm gives rise to the muscle cells and connective tissue in the body. The
endoderm gives rise to the gut (gastro-intestinal system) and many internal organs.

References:

Anatomy Journal. Ascidian Embryonic Development: An Emerging Model System for the
Study of Cell Fate Specification in Chordates.
https://anatomypubs.onlinelibrary.wiley.com/doi/full/10.1002/dvdy.21108. 2022

Cell.com. The Ascidian as a Model Organism in Developmental and Evolutionary Biology.

https://www.cell.com/fulltext/S0092-8674(01)00481-0. 2022

JSTOR. Evolution of Ascidian Development. https://www.jstor.org/stable/1313057. 2022

Assessment:

I. Talking about animals in general, identify the developmental fates of the following germ
layers:
1. Ectoderm
2. Mesoderm
3. Endoderm

II. Enumerate the following:

1. Processes involved in Ascidian Embryogenesis (3)
2. Major tissues that constitute the ascidian tadpole (7)
3. Embryonic Axes of the Ascidian (3)

III. Explain the following:

1. The ascidian embryogenesis.
2. How the 3 main embryonic axes of the ascidians are established.
3. How endoderm, mesoderm and ectoderm of the ascidians are formed.

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 CHAPTER 12

EARLY AMPHIBIAN DEVELOPMENT

Introduction:

In typical amphibian development, eggs are laid in water and larvae are adapted to an
aquatic lifestyle. Frogs, toads, and newts all hatch from the eggs as larvae with external gills
but it will take some time for the amphibians to interact outside with pulmonary respiration.
Eggs are fertilized by sperm from a male frog. The resulting zygote goes through embryonic
development to become a free-living tadpole, which then metamorphosis into an adult frog—
for instance, by losing its tail through programmed cell death, or apoptosis.

Frogs start off as eggs and go through four more stages in their life cycle, becoming
five different things in the process: eggs, tadpoles, tadpoles with legs, froglets, and adult
frogs.

Intended Learning Outcomes:

At the end of this module, students should be able to:

1. Explain cleavage in amphibians.

2. Explain the major functions of the amphibians‘ blastocoel.
3. Explain the fate mapping in Xenopus.
4. Appreciate the whole process of gastrulation in amphibians.

Learning Content:

1. Cleavage in Amphibians

Cleavage or the first few cellular divisions of a zygote (fertilized egg) in most frog
and salamander embryos is radially symmetrical and holoblastic (complete cleavage), just like
echinoderm cleavage. The amphibian egg, however, contains much more yolk. This yolk,
which is concentrated in the vegetal hemisphere, is an impediment to cleavage. Thus, the first
division begins at the animal pole and slowly extends down into the vegetal region.

An amphibian embryo containing 16 to 64 cells is commonly called

a morula (plural: morulae). At the 128-cell stage, the blastocoel becomes apparent, and the
embryo is considered a blastula. The blastocoel probably serves two major functions in
frog embryos: (1) it permits cell migration during gastrulation, and (2) it prevents the cells
beneath it from interacting prematurely with the cells above it.

2. Amphibian Gastrulation

The study of amphibian gastrulation is both one of the oldest and one of the newest
areas of experimental embryology. Even though amphibian gastrulation has been extensively
studied for the past century, most of our theories concerning the mechanisms of these
developmental movements have been revised over the past decade. The study of amphibian
gastrulation has been complicated by the fact that there is no single way amphibians
gastrulate. Different species employ different means toward the same goal. In recent years,
the most intensive investigations have focused on the frog Xenopus laevis.

The fate map of Xenopus

Fate mapping has shown that cells of the Xenopus blastula have different fates
depending on whether they are located in the deep or the superficial layers of the embryo.
In Xenopus, the mesodermal precursors exist mostly in the deep layer of cells, while the
ectoderm and endoderm arise from the superficial layer on the surface of the embryo. Most of

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the precursors for the notochord and other mesodermal tissues are located beneath the
surface in the equatorial (marginal) region of the embryo. In urodeles (salamanders such
as Triturus and Ambystoma) and in some frogs other than Xenopus, many more of the
notochord and mesoderm precursors are among the surface cells.

Cell movements during amphibian gastrulation

Invagination and involution

Invagination is the process of a surface folding in on itself to form a cavity, pouch or

tube. In developmental biology, invagination is a mechanism that takes place
during gastrulation. This mechanism or cell movement happens mostly in the vegetal pole.
Invagination consists of the folding of an area of the exterior sheet of cells towards the inside
of the blastula.

Involution, on the other hand, is a type of cell movement during gastrulation which
involves the in-turning or inward movement of an expanding outer layer so that it spreads
over the internal surface of the remaining external cells.

Amphibian gastrulation is first visible when a group of marginal endoderm cells on the
dorsal surface of the blastula sinks into the embryo. The outer (apical) surfaces of these cells
contract dramatically, while their inner (basal) ends expand. The apical-basal length of these
cells greatly increases to yield the characteristic ―bottle‖ shape.

In salamanders, these bottle cells appear to have an active role in the early
movements of gastrulation.

The major factor in the movement of cells into the embryo appears to be the involution
of the subsurface marginal cells, rather than the superficial ones. The movements of the
vegetal endoderm place the prospective pharyngeal endoderm adjacent to the roof of the
blastocoel. This places the prospective pharyngeal endoderm immediately ahead of the
migrating mesoderm. The cells then migrate along the basal surface of the blastocoel roof.

The superficial layer of marginal cells is pulled inward to form the endodermal lining of
the archenteron, merely because it is attached to the actively migrating deep cells. While
experimental removal of the bottle cells does not affect the involution of the deep or
superficial marginal zone cells into the embryo, the removal of the deep involuting marginal
zone (IMZ) cells and their replacement with animal region cells (which do not normally
undergo involution) stops archenteron formation.

The convergent extension of the dorsal mesoderm

The involuting marginal zone (IMZ) cells are originally several layers thick. Shortly
before their involution through the blastopore lip, the several layers of deep IMZ cells
intercalate radially to form one thin, broad layer. This intercalation further extends the IMZ
vegetally. At the same time, the superficial cells spread out by dividing and flattening. When
the deep cells reach the blastopore lip, they involute into the embryo and initiate a second
type of intercalation. This intercalation causes a convergent extension along the mediolateral
axis that integrates several mesodermal streams to form a long, narrow band. This is
reminiscent of traffic on a highway when several lanes must merge to form a single lane. The
anterior part of this band migrates toward the animal cap. Thus, the mesodermal stream
continues to migrate toward the animal pole, and the overlying layer of superficial cells
(including the bottle cells) is passively pulled toward the animal pole, thereby forming the
endodermal roof of the archenteron. The radial and mediolateral intercalations of the deep
layer of cells appear to be responsible for the continued movement of mesoderm into the
embryo.

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 The adhesive changes driving convergent extension appear to be directed by two cell
adhesion molecules, paraxial protocadherin and axial protocadherin. The former is initially
found throughout the dorsal mesoderm, but then is turned off in the precursors of the
notochord. At that time, axial protocadherin becomes expressed in the notochordal tissue. An
experimental dominant negative form of paraxial protocadherin (which is secreted instead of
being bound to the cell membrane) prevents convergent extension. Moreover, the expression
domain of paraxial protocadherin separates the trunk mesodermal cells, which undergo
convergent extension, from the head mesodermal cells, which do not.

Migration of the involuting mesoderm

As mesodermal movement progresses, convergent extension continues to narrow and

lengthen the involuting marginal zone. The IMZ contains the prospective endodermal roof of
the archenteron in its superficial layer (IMZS) and the prospective mesodermal cells, including
those of the notochord, in its deep region (IMZ D).

During the middle third of gastrulation, the expanding sheet of mesoderm converges
toward the midline of the embryo. This process is driven by the continued mediolateral
intercalation of cells along the anterior-posterior axis, thereby further narrowing the band.
Toward the end of gastrulation, the centrally located notochord separates from the somitic
mesoderm on either side of it, and the notochord cells elongate separately. This may in part
be a consequence of the different protocadherins in the axial and paraxial mesoderms. This
convergent extension of the mesoderm appears to be autonomous, because the movements
of these cells occur even if this region of the embryo is experimentally isolated from the rest of
the embryo.

During gastrulation, the animal cap and noninvoluting marginal zone (NIMZ) cells
expand by epiboly to cover the entire embryo. The dorsal portion of the NIMZ extends more
rapidly toward the blastopore than the ventral portion, thus causing the blastopore lips to
move toward the ventral side. While those mesodermal cells entering through the dorsal lip of
the blastopore give rise to the dorsal axial mesoderm (notochord and somites), the remainder
of the body mesoderm (which forms the heart, kidneys, blood, bones, and parts of several
other organs) enters through the ventral and lateral blastopore lips to create the mesodermal
mantle. The endoderm is derived from the IMZS cells that form the lining of the archenteron
roof and from the subblastoporal vegetal cells that become the archenteron floor.

References:

JSTOR. Early Amphibian Development. https://www.jstor.org/stable/1310156. 2022

IBiology. Early Frog Development: How to Make a Tadpole. https://www.ibiology.org/development-

and-stem-cells/frog-development/. 2022

NCBI. Early Amphibian Development. https://www.ncbi.nlm.nih.gov/books/NBK10113/. 2022

Assessment:

I. Define the following:

1. Holoblastic
2. Morula
3. Invagination
4. Involution

II. Enumerate the following:

1. Stages of Amphibian‘s Life Cycle (5)
2. Major Functions of Blastocoel in Frog (2)

III. Explain the following:

1. Cleavage in amphibians.
2. The major functions of the amphibians‘ blastocoel.
3. The fate mapping in Xenopus.
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 CHAPTER 13

NEURULATION

Introduction:

Neurulation refers to the folding process in vertebrate embryos, which includes the
transformation of the neural plate into the neural tube. The embryo at this stage is
termed neurula.

The extent to which these modes of construction are used varies among vertebrate
classes. Neurulation in fishes is exclusively secondary. In birds, the anterior portions of the
neural tube are constructed by primary neurulation, while the neural tube caudal to the
twenty-seventh somite pair (i.e., everything posterior to the hindlimbs) is made by secondary
neurulation. In amphibians, such as Xenopus, most of the tadpole neural tube is made by
primary neurulation, but the tail neural tube is derived from secondary neurulation. In mice
(and probably humans, too), secondary neurulation begins at or around the level of somite 35.

Intended Learning Outcomes:

At the end of this module, students should be able to:

1. Describe the processes involved in the primary neurulation.

2. Compare primary neurulation and secondary neurulation.
3. Explain what happens if the neural tube of humans fails to close.
4. Appreciate the role played by secondary neurulation in humans.

Learning Content:

1. Primary Neurulation
Primary neural induction
It was Hans Spemann who first popularized the term ―primary neural induction‖ in
reference to the first differentiation of ectoderm into neural tissue during neurulation. It was
called "primary" because it was thought to be the first induction event in embryogenesis. The
Nobel prize-winning experiment was done by his student Hilda Mangold. Ectoderm from the
region of the dorsal lip of the blastopore of a developing salamander embryo was
transplanted into another embryo and this "organizer" tissue ―induced‖ the formation of a full
secondary axis changing surrounding tissue in the original embryo from ectodermal to neural
tissue. The tissue from the donor embryo was therefore referred to as the inducer because it
induced the change.
Shape change
As neurulation proceeds after induction the cells of the neural plate become high-
columnar and can be identified through microscopy as different from the surrounding
presumptive epithelial ectoderm (epiblastic endoderm in amniotes). The cells move laterally
and away from the central axis and change into a truncated pyramid shape. This pyramid
shape is achieved through tubulin and actin in the apical portion of the cell which constricts as
they move. The variation in cell shapes is partially determined by the location of the nucleus
within the cell, causing bulging in areas of the cells forcing the height and shape of the cell to
change. This process is known as apical constriction. The result is a flattening of the
differentiating neural plate which is particularly obvious in salamanders when the previously
round gastrula becomes a rounded ball with a flat top.
Folding
The process of the flat neural plate folding into the cylindrical neural tube is
termed primary neurulation. As a result of the cellular shape changes, the neural plate
forms the medial hinge point (MHP). The expanding epidermis puts pressure on the MHP and
causes the neural plate to fold resulting in neural folds and the creation of the neural groove.
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The neural folds form dorsolateral hinge points (DLHP) and pressure on this hinge cause the
neural folds to meet and fuse at the midline. The fusion requires the regulation of cell
adhesion molecules.
The notochord plays an integral role in the development of the neural tube. Prior to
neurulation, during the migration of epiblastic endoderm cells towards the hypoblastic
endoderm, the notochordal process opens into an arch termed the notochordal plate and
attaches overlying neuroepithelium of the neural plate. The notochordal plate then serves as
an anchor for the neural plate and pushes the two edges of the plate upwards while keeping
the middle section anchored. Some of the notochodral cells become incorporated into the
center section neural plate to later form the floor plate of the neural tube. The notochord plate
separates and forms the solid notochord.
The folding of the neural tube to form an actual tube does not occur all at once.
Instead, it begins approximately at the level of the fourth somite at Carnegie stage 9 (around
Embryonic day 20 in humans). The lateral edges of the neural plate touch in the midline and
join together. This continues both cranially (toward the head) and caudally (toward the tail).
The openings that are formed at the cranial and caudal regions are termed the cranial and
caudal neuropores. In human embryos, the cranial neuropore closes approximately on day
24 and the caudal neuropore on day 28. Failure of the cranial (superior) and caudal (inferior)
neuropore closure results in conditions called anencephaly and spina bifida, respectively.
Patterning
According to the French Flag model where stages of development are directed by
gene product gradients, several genes are considered important for inducing patterns in the
open neural plate, especially for the development of neurogenic placodes. These placodes
first become evident histologically in the open neural plate. After sonic hedgehog (SHH)
signalling from the notochord induces its formation, the floor plate of the incipient neural tube
also secretes SHH. After closure, the neural tube forms a basal or floor plate and a roof
or alar plate. The basal plate forms most of the ventral portion of the nervous system,
including the motor portion of the spinal cord and brain stem; the alar plate forms the dorsal
portions, devoted mostly to sensory processing.
Complexities of the model
Neural tube closure is not entirely understood. Closure of the neural tube varies by
species. In mammals closure occurs by meeting at multiple points which then close up and
down. In birds neural tube closure begins at one point of the midbrain and moves anteriorly
and posteriorly.
2. Secondary Neurulation
Primary neurulation develops into secondary neurulation when the caudal neuropore
undergoes final closure. The cavity of the spinal cord extends into the neural cord. In
secondary neurulation, the neural ectoderm and some cells from the endoderm form
the medullary cord. The medullary cord condenses, separates and then forms cavities. These
cavities then merge to form a single tube. Secondary neurulation occurs in the posterior
section of most animals but it is better expressed in birds. Tubes from both primary and
secondary neurulation eventually connect at around the sixth week of development.
In humans, the mechanisms of secondary neurulation plays an important role given its
impact on the proper formation of the human posterior spinal cord. Errors at any point in the
process can yield problems. For example, retained medullary cord occurs due to a partial or
complete arrest of secondary neurulation that creates a non-functional portion on the vestigial
end.
Early brain development
The anterior portion of the neural tube forms the three main parts of the brain:
the forebrain (prosencephalon), midbrain (mesencephalon), and
the hindbrain (rhombencephalon). These structures initially appear just after neural tube
closure as bulges called brain vesicles in a pattern specified by anterior-posterior patterning
genes, including Hox genes, other transcription factors such as Emx, Otx, and Pax genes, and

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secreted signaling factors such as fibroblast growth factors (FGFs) and Wnts. These brain vesicles
further divide into subregions.
The prosencephalon gives rise to the telencephalon and diencephalon, and
the rhombencephalon generates the metencephalon and myelencephalon. The hindbrain, which is the
evolutionarily most ancient part of the chordate brain, also divides into different segments
called rhombomeres. The rhombomeres generate many of the most essential neural circuits needed
for life, including those that control respiration and heart rate, and produce most of the cranial
nerves. Neural crest cells form ganglia above each rhombomere. The early neural tube is primarily
composed of the germinal neuroepithelium, later called the ventricular zone, which contains
primary neural stem cells called radial glial cells and serves as the main source of neurons produced
during brain development through the process of neurogenesis (the process by which new neurons
are formed in the brain).
Non-neural ectoderm tissue
Paraxial mesoderm surrounding the notochord at the sides will develop into the somites (future
muscles, bones, and contributes to the formation of limbs of the vertebrate ).
Neural crest cells
Masses of tissue called the neural crest that are located at the very edges of the lateral plates
of the folding neural tube separate from the neural tube and migrate to become a variety of different
but important cells.
Neural crest cells will migrate through the embryo and will give rise to several cell populations,
including pigment cells and the cells of the peripheral nervous system.
Neural tube defects
Failure of neurulation, especially failure of closure of the neural tube are among the most
common and disabling birth defects in humans, occurring in roughly 1 in every 500 live births. Failure
of the rostral end of the neural tube to close results in anencephaly, or lack of brain development, and
is most often fatal. Failure of the caudal end of the neural tube to close causes a condition known
as spina bifida, in which the spinal cord fails to close.

References:

BioNinja. Neurulation. https://ib.bioninja.com.au/options/option-a-neurobiology-and/a1-neural-

development/neurulation.html. 2022

Lecturio. Gastrulation and Neurulation. https://www.lecturio.com/concepts/gastrulation-and-

neurulation/. 2022

NCBI. Formation of the Neural Tube. https://www.ncbi.nlm.nih.gov/books/NBK10080/. 2022

Wikipedia. Neurulation. https://en.wikipedia.org/wiki/Neurulation. 2022

Assessment:

I. Define the following:

1. Neurulation
2. Neurula
3. Primary Neurulation
4. Cranial and caudal neuropores
5. Anencephaly
6. Spina bifida
7. Neurogenesis

II. Enumerate:
1. The main parts of the brain (3)

III. Explain:
1. The processes involved in the primary neurulation.
2. Primary neurulation and secondary neurulation.
3. What happens if the neural tube of humans fails to close.

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 CHAPTER 14

VERTEBRATE LIMB DEVELOPMENT

Introduction:

The limbs develop from small protrusions (limb buds) that arise from the body wall of
the embryo. Positioning and patterning the limb involves cellular interactions both between
the ectoderm surrounding the limb bud and between the mesenchymal cells that form the
core of the limb bud.

As the limb grows out the cells acquire a positional value that relates to their position in
the bud with respect to all three axes, proximo-distal, antero-posterior, and dorso-
ventral. These positional values largely determine how the cells will develop such as what
sort of cartilaginous elements they will form. The positional value of the cells is acquired in the
progress zone at the tip of the growing bud.

Intended Learning Outcomes:

At the end of this module, students should be able to:

1. Explain the forelimb and hindlimb positioning in the chick and mouse embryos.
2. Explain the forelimb and hindlimb polarity in the chick and mouse embryos.
3. Explain the forelimb and hindlimb initiation in the chick and mouse embryos.
4. Explain how the pattern of tissues along the proximo-distal, dorso-ventral, and antero-
posterior axes of the limb bud are specified.
5. Appreciate the nature of the vertebrate body.
6. Perform an activity about human embryogenesis.

Learning Content:

1. Limb Positioning

Vertebrate limbs form at distinct and reproducible locations along the main body axis.
Forelimbs always form at the cervico-thoracic vertebrae boundary and hindlimbs at the
lumbar-sacral boundary. The relative position at which these boundaries are found varies
greatly between vertebrates and this has contributed greatly to the differences in the extent of
body extension observed across evolution.

Classical fate-mapping and tissue transplantation experiments in the chick embryo

have revealed that cells in distinct regions of the lateral plate mesoderm (LPM) are in position
to form the limbs as early as the 2-somite stage. Candidates for specifying the position of the
limbs include Hox family genes, which are expressed in gastrulating cells, and later along the
antero-posterior axis of the LPM. Hox genes are expressed in the order in which they are
found on the chromosome in the 3′-5′ direction – a process called spatial and temporal co-
linearity. Indeed, it has long been suspected that Hox proteins are important determinants of
forelimb position, because the functional inactivation of Hoxb5 in the mouse repositions the
forelimbs anteriorly.

A recent study in mice has indicated that Oct4 indirectly controls forelimb position by
repressing posterior 5′ Hox genes (Hox10-Hox13 paralogues), because the inactivation
of Oct4 precociously activates the posterior programme of embryo development and results in
posterior truncations, which, in less severe cases, can cause the hindlimb to form next to the
forelimb. Conversely, when the duration of Oct4 expression is extended, more-posterior
development of the embryo is delayed, which elongates the trunk. These effects are
associated with corresponding changes in the timing of Hox gene expression along the
antero-posterior axis of the embryo.

The position of presumptive hindlimb cells is determined later than forelimb cells.
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2. Limb Polarity

Once cells are in position to form limbs along the antero-posterior axis of the embryo,
they become polarized along two developmental axes. 180° tissue rotation experiments in the
chick embryo have shown that the antero-posterior polarity and dorso-ventral polarity of the
limb is determined in the Lateral Plate Mesoderm at pre-limb bud stages: at the 9- to 10- and
13-somite stages, respectively. The establishment of antero-posterior polarity results in the
formation of the polarizing region (or zone of polarizing activity, ZPA) – a limb part
composed of posterior limb bud mesoderm cells that pattern the antero-posterior axis. A
distant cis-regulatory sequence containing multiple enhancers called the ZPA regulatory
sequence (ZRS) controls Shh expression.

In the mouse, the products of Hox genes specify the antero-posterior polarity of the
developing forelimb field just as they specify antero-posterior position along the main body
axis. Deletion of all Hox9 paralogous group genes in the mouse embryo results in the loss of
posterior polarity. Conversely, deletion of all Hox5 paralogous group genes results in the loss
of anterior polarity, and Shh expression becomes detectable at the anterior margin of the limb
bud.

The antero-posterior polarity of the presumptive hindlimb is also specified at early

stages and does not appear to involve Hox genes, but instead involves regionalized
transcription factors.

3. Limb Pattern Specifications

a. Proximo-distal specification

Recent experiments in the chick support a ‗signal-time model‘ in which signals specify
proximal limb segments (i.e. humerus), and then intrinsic timing specifies distal segments (i.e.
wrist/digits). When distal mesoderm from an early chick wing bud was grafted beneath the
apical ectodermal ridge of a host wing bud, the grafted cells maintained their intrinsic timing of
cell proliferation which marks the specification of distal positional values. Therefore, it appears
that signals control the transition from proximal to intermediate specification (Hoxa10 and
Hoxd10 to Hoxa11 and Hoxd11) and that timing controls the transition from intermediate to
distal specification (Hoxa11 and Hoxd11 to Hoxa13 and Hoxd13).

How the pattern of tissues along the dorso-ventral axis of the limb bud is specified has
not been investigated in as much detail as the other axes.

b. Antero-posterior specification

Several types of tissue-grafting experiments performed in the chick embryo have

resulted in a positional information model of antero-posterior specification, based on graded
signalling by the polarizing region. The polarizing region was discovered in experiments in
which grafts of posterior chick wing mesoderm were made to the anterior margin of the wing
bud of a host embryo. This resulted in the normal digit pattern (1, 2 and 3) being
symmetrically duplicated (i.e. 3, 2, 1, 1, 2 and 3).

Evidence that Shh may not operate as a graded morphogen in the specification of
antero-posterior positional values has been obtained by genetic lineage-tracing experiments
in mouse forelimbs and hindlimbs, revealing that the two most-posterior digits (4 and 5 out of
digits 1-5) are derived from the polarising region itself.

In the mouse limb, the use of a Gli1 reporter transgene showed that only polarizing
region cells directly receive Shh signalling during the 2-3 h it is required for specification.
Therefore, it is suggested that digits 1 to 3 (and possibly 4 and 5) are specified by secondary
relay signals emanating from the polarising region. It is unclear whether Bmps are involved in
the specification of antero-posterior positional values in the mouse limb, because the genetic
removal of Bmp2, Bmp4 and Bmp7 function does not appear to cause overt transformations
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of digit identity. However, it has been noted that digits 2 to 5 of the mouse limb have very
similar anatomies in terms of phalange number and proportion.

References:

Journals.Biologists. Establishing the Pattern of the Vertebrate Limb.

https://journals.biologists.com/dev/article/147/17/dev177956/225797/Establishing-the-pattern-
of-the-vertebrate-limb. 2022

Journals.Biologists. Growing Models of Vertebrate Limb Development.

https://journals.biologists.com/dev/article/136/2/179/65397/Growing-models-of-vertebrate-
limb-development. 2022

PubMed. Vertebrate Limb Development and Malformations.

https://pubmed.ncbi.nlm.nih.gov/10473037/#:~:text=The%20limbs%20develop%20from%20s
mall,core%20of%20the%20limb%20bud. 2022

Assessment:

I. Define the following:

1. Limb buds
2. Polarizing region (or zone of polarizing activity, ZPA)

II. Enumerate the following:

1. Developmental axes of the vertebrate limb (3)
2. Limb pattern specifications (2)

III. Explain:
1. The forelimb and hindlimb positioning in the chick and mouse embryos.
2. The forelimb and hindlimb polarity in the chick and mouse embryos.
3. The forelimb and hindlimb initiation in the chick and mouse embryos.
4. How the pattern of tissues along the proximo-distal, dorso-ventral, and antero-posterior
axes of the limb bud are specified.

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 CHAPTER 15

HEART DEVELOPMENT

Introduction:
Heart development (also known as cardiogenesis) refers to the prenatal
development of the heart. This begins with the formation of two endocardial tubes which
merge to form the tubular heart, also called the primitive heart tube. The heart is the first
functional organ in vertebrate embryos.
The heart tube elongates on the right side, looping and becoming the first visual sign
of left-right asymmetry of the body. Septa form within the atria and ventricles to separate
the left and right sides of the heart.

Intended Learning Outcomes:

At the end of this module, students should be able to:

1. Explain how heart begins to form.

2. Explain what happens during cardiac looping.
3. Explain how each of the parts/chambers of the heart developed.
4. Explain the development of human embryonic heart.
5. Recognize the role of the human heart to sustain life.
6. Illustrate certain factors that affect human heartbeat rate.

Learning Content:

1. Early Development

Endocardial tubes
At around 18 to 19 days after fertilization, the heart begins to form. Early in the fourth
week, around day 22 the developing heart starts to beat and to pump circulating blood. The
heart begins to develop near the head of the embryo in the cardiogenic area. Following cell
signalling, two strands or cords begin to form in the cardiogenic region. These are the
endocardial tubes. At the same time that the tubes are forming other major heart
components are also being formed. The two tubes migrate together and fuse to form a single
primitive heart tube, the tubular heart which quickly forms five distinct regions. From head to
tail, these are the truncus arteriosus, bulbus cordis, primitive ventricle, primitive atrium, and
the sinus venosus. Initially, all venous blood flows into the sinus venosus, and contractions
propel the blood from tail to head, or from the sinus venosus to the truncus arteriosus. The
truncus arteriosus will divide to form the aorta and pulmonary artery; the bulbus cordis will
develop into the right ventricle; the primitive ventricle will form the left ventricle; the primitive
atrium will become the front parts of the left and right atria and their appendages, and the
sinus venosus will develop into the posterior part of the right atrium, the sinoatrial node and
the coronary sinus.
Heart tube position
The central part of cardiogenic area is in front of the oropharyngeal membrane and the
neural plate. The growth of the brain and the cephalic folds push the oropharyngeal
membrane forward, while the heart and the pericardial cavity move first to the cervical region
and then into the chest. The curved portion of the horseshoe-shaped area expands to form
the future ventricular infundibulum and the ventricular regions, as the heart tube continues to
expand. The tube starts receiving venous drainage in its caudal pole and will pump blood out
of the first aortic arch and into the dorsal aorta through its polar head.
The myocardium thickens and secretes a thick layer of rich extracellular
matrix containing hyaluronic acid which separates the endothelium. Then mesothelial cells
form the pericardium and migrate to form most of the epicardium. Then the heart tube is
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formed by the endocardium, which is the inner endothelial lining of the heart, and the
myocardial muscle wall which is the epicardium that covers the outside of the tube.

2. Heart Folding

The heart tube continues stretching and by day 23, cardiac looping begins. The
cephalic portion curves in a frontal clockwise direction. The atrial portion starts moving in a
cephalically and then moves to the left from its original position. This curved shape
approaches the heart and finishes its growth on day 28. The conduit forms the atrial and
ventricular junctions which connect the common atrium and the common ventricle in the early
embryo. The arterial bulb forms the trabecular portion of the right ventricle. A cone will form
the infundibula blood of both ventricles. The arterial trunk and the roots will form the proximal
portion of the aorta and the pulmonary artery. The junction between the ventricle and the
arterial bulb will be called the primary intra-ventricular hole. The tube is divided into cardiac
regions along its craniocaudal axis: the primitive ventricle, called primitive left ventricle, and
the trabecular proximal arterial bulb, called the primitive right ventricle. This time no septum is
present in heart.

3. Heart Chambers and Other Parts

Sinus venosus
In the middle of the fourth week, the sinus venosus receives venous blood from the
poles of right and left sinus. Each pole receives blood from three major veins: the vitelline
vein, the umbilical vein and the common cardinal vein. The sinus opening moves clockwise.
This movement is caused mainly by the left to right shunt of blood, which occurs in the
venous system during the fourth and fifth week of development.
When the left common cardinal vein disappears in the tenth week only the oblique vein
of the left atrium and the coronary sinus remain. The right pole joins the right atrium to form
the wall portion of the right atrium. The right and left venous valves fuse and form a peak
known as the septum spurium. At the beginning, these valves are large, but over time the left
venous valve and the septum spurium fuse with the developing atrial septum. The upper right
venous valve disappears, while the bottom venous valve evolves into the inferior valve of
the vena cava and the coronary sinus valve.
Heart wall
The main walls of the heart are formed between day 27 and 37 of the development of
the early embryo. The growth consists of two tissue masses actively growing that approach
one another until they merge and split light into two separate conduits. Tissue masses
called endocardial cushions develop into atrioventricular and conotroncal regions. In these
places, the cushions will help in the formation of auricular septum, ventricular conduits, atrio-
ventricular valves and aortic and pulmonary channels.
Atria
At the end of the fourth week, a crest grows that leaves the cephalic part. This crest is
the first part of the septum primum. The two ends of the septum extend into the interior of
the endocardial cushions in the atrioventricular canal. The opening between the bottom edge
of the septum primum and endocardial cushions is the ostium primum (first opening). The
extensions of the upper and lower endocardial pads grow along the margin of the septum
primum and close the ostium primum. Coalescence of these perforations will form the ostium
secundum (second opening), which allows blood to flow freely from the right atrium to the left.
When the right of the atrium expands due to the incorporation of the pole of the sinus,
a new fold appears, called the septum secundum. At its right side it is fused with the left
venous valve and the septum spurium. A free opening will then appear, called the foramen
ovale. The remains of the upper septum primum, will become the valves of the foramen ovale.
The passage between the two atrial chambers consists of a long oblique slit through which
blood flows from the right atrium to the left.

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Ventricles
Initially, a single pulmonary vein develops in the form of a bulge in the back wall of the
left atrium. This vein will connect with the veins of the developing lung buds. As development
proceeds, the pulmonary vein and its branches are incorporated into the left atrium and they
both form the smooth wall of the atrium. The embryonic left atrium remains as the trabecular
left atrial appendage, and the embryonic right atrium remains as the right atrial appendage.
Atrioventricular canal
At the end of the fourth week, two atrioventricular endocardial cushions appear. Initially
the atrioventricular canal gives access to the primitive left ventricle, and is separated from
arterial bulb by the edge of the ventricular bulb. In the fifth week, the posterior end terminates
in the center part of the upper endocardial cushion. Because of this, blood can access both
the left primitive ventricle and the right primitive ventricle. As the anterior and posterior pads
project inwardly, they merge to form a right and left atrioventricular orifice.
Atrioventricular valves

When forming intra-atrial septa, atrioventricular valves will begin to grow. A

muscular interventricular septum begins to grow from the common ventricle to the
atrioventricular endocardial cushions. The division begins in the common ventricle where a
furrow in the outer surface of the heart will appear the interventricular foramen eventually
disappears. This closure is achieved by further growth of the muscular interventricular
septum, a contribution of trunk crest-conal tissue and a membranous component.

4. Pacemaker and Conduction System

The rhythmic electrical depolarization waves that trigger myocardial contraction is
myogenic, which means that they begin in the heart muscle spontaneously and are then
responsible for transmitting signals from cell to cell. Myocytes that were obtained in the
primitive heart tube, start beating as they connect together by their walls in a syncytium.
Myocytes initiate rhythmic electrical activity, before the fusion of the endocardial tubes. The
heartbeat begins in the region of the pacemaker which has a spontaneous depolarization time
faster than the rest of myocardium.
The primitive ventricle acts as initial pacemaker. But this pacemaker activity is actually
made by a group of cells that derive from the sinoatrial right venous sinus. These cells form
an ovoid sinoatrial node (SAN), on the left venous valve. After the development of the SAN,
the superior endocardial cushions begin to form a pacemaker also known as
the atrioventricular node. With the development of the SAN, a band of specialized conducting
cells start to form creating the bundle of His that sends a branch to the right ventricle and one
to the left ventricle. Most conduction pathways originate from the cardiogenic mesoderm but
the sinus node may be derived from the neural crest.
The human embryonic heart begins beating approximately 21 days after fertilization, or
five weeks after the last normal menstrual period (LMP), which is the date normally used to
date pregnancy in the medical community. The electrical depolarizations that trigger
cardiac myocytes to contract arise spontaneously within the myocyte itself. The heartbeat is
initiated in the pacemaker regions and spreads to the rest of the heart through a conduction
pathway. Pacemaker cells develop in the primitive atrium and the sinus venosus to form
the sinoatrial node and the atrioventricular node respectively. Conductive cells develop
the bundle of His and carry the depolarization into the lower heart. Cardiac activity is visible
beginning at approximately 5 weeks of pregnancy.
The human heart begins beating at a rate near the mother’s, about 75-80 beats per
minute (BPM). The embryonic heart rate (EHR) then accelerates linearly for the first month of
beating, peaking at 165-185 BPM during the early 7th week, (early 9th week after the LMP).
This acceleration is approximately 3.3 BPM per day, or about 10 BPM every three days, an
increase of 100 BPM in the first month.

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References:

Karger. The Transitional Heart: From Early Embryonic and Fetal Development to Neonatal
Life. https://www.karger.com/Article/Fulltext/501906. 2022

TeachMeAnatomy. Development of the Cardiovascular System.

https://teachmeanatomy.info/the-basics/embryology/cardiovascular-system/. 2022

Wikipedia. Heart Development.

https://en.wikipedia.org/wiki/Heart_development#:~:text=Heart%20development%20(also%20
known%20as,functional%20organ%20in%20vertebrate%20embryos.. 2022

Assessment:

I. Define/describe the following:

I. Cardiogenesis
2. Endocardial tubes
3. Around 18 to 19 days after fertilization
4. Around day 22 of the developing heart

II. Enumerate the following:

1. Parts of the animal heart that form during gestation (6)

III. Explain:
1. How heart begins to form.
2. What happens during cardiac looping.
3. How each of the parts/chambers of the heart developed.
4. The development of human embryonic heart.

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 CHAPTER 16

GUT DEVELOPMENT

Introduction:

The development of the digestive system begins as a simple blind-ended gut tube. The
accessory digestive organs form as out pouchings from the primitive gut tube, whereas
formation of the intestines require them to first protrude out into the umbilical cord before
retracting back into the abdominal cavity.

Understanding the embryology and the origins of the foregut, midgut, and hindgut, as
well as their accompanied derivatives will provide more insight behind the intricate
vascularization and the mesenteries of the abdominal cavity.

Intended Learning Outcomes:

At the end of this module, students should be able to:

1. Explain how the primitive gut is formed.

2. Describe the 4 main sections of the primitive gut.
3. Recognize the role of mesenteries in the gut tube development.
4. Identify the derivatives of the foregut, midgut and hindgut and appreciate their contributions
to the total development of the gut or gastro-intestinal tract.

Learning Content:

1. Gut tube

The primitive gut is formed when a portion of the yolk sac becomes incorporated into
the embryo, which occurs due to the cephalocaudal and lateral folding of the embryo. The
portions that remain outside the embryo are the yolk sac and the allantois. The primitive gut
forms a blind-ended tube on both the cephalic and caudal ends of the embryo, forming
the foregut and the hindgut, respectively. The middle part forms the midgut, but remains
temporarily connected to the yolk sac via the vitelline duct (yolk stalk).

In the abdominal cavity, the gut tube and its derivatives are suspended from the dorsal
and ventral body wall by mesenteries; these are double layers of peritoneum that enclose
organs and connect them to the body wall. Mesenteries between two organs or one organ
and the body wall are known as peritoneal ligaments. Together, mesenteries and peritoneal
ligaments serve as a conduit for vessels, nerves, and lymphatics to and from the abdominal
organs.

A. Foregut
The foregut gives rise to the:
 pharynx
 lower respiratory system
 esophagus
 stomach
 proximal half of the duodenum
 liver
 biliary apparatus
 pancreas

The derivatives of the foregut, except for the pharynx and the lower respiratory system,
are mostly supplied by the celiac artery (trunk).

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Esophagus

During the third week of gestation, a respiratory diverticulum (lung bud) forms as an
outgrowth from the ventral wall of the proximal foregut. While the lung bud continues to
expand, it becomes separated from the foregut, which forms the esophagus. Initially, the
esophagus is short, but becomes rapidly elongated as a result of the growth and relocation of
the heart and lungs.

Stomach

During the fourth week of gestation, the rudimentary stomach appears as a fusiform-
shaped dilation of the distal foregut. Subsequently, its appearance and position drastically
changes; the latter can be better understood by visualizing a longitudinal axis and an antero-
posterior axis around which the stomach rotates.

The stomach rotates 90 degrees clockwise around its longitudinal axis, resulting in its
left side facing anteriorly and its right side posteriorly. This explains why the left vagus nerve
innervates the anterior wall, as it once innervated the left side of the stomach, whereas
the right vagus nerve innervates the posterior wall, as it once innervated the right side.

The stomach also rotates around its antero-posterior axis, resulting in the caudal end
(pyloric part) to move upward and to the right and the cranial end (cardiac part) slightly
downward and to the left. Thus, the stomach assumes its final position, with its pylorus located
superiorly to the left and its cardia inferiorly to the right.

Duodenum

During the fourth week of gestation, the duodenum begins to develop from two
sources: the caudal part of the foregut and the cranial part of the midgut, where the junction
lies just distal to the origin of the bile duct. The developing duodenum forms a C-shaped loop
that initially projects ventrally. However, once the stomach rotates, the duodenum rotates to
the right and becomes pressed against the posterior abdominal wall, thus becoming
retroperitoneal. Due to its dual origin, the duodenum is supplied by branches of the celiac
trunk and the superior mesenteric artery.

Liver and biliary apparatus

The development of the liver begins during the fourth week of gestation, with the
appearance of the hepatic diverticulum (liver bud) as an outgrowth from the ventral wall of
distal foregut. The stalk connecting the diverticulum and the foregut narrows and forms the
bile duct, whereas the gallbladder and the cystic duct form as a ventral outgrowth from the bile
duct. Initially, the bile duct opens anteriorly into the duodenum, but ends up posteriorly due to
the rotational changes of the duodenum.

As mentioned, the hepatic diverticulum extends into ventral mesentery. This positions
the future liver between the foregut and ventral abdominal wall, and divides the ventral
mesentery into the lesser omentum and the falciform ligament. During the following weeks,
the liver occupies a large portion of the upper abdominal cavity. Further development and
segmentation of the liver is determined by the oxygenated blood flowing from the umbilical
vein into the liver. Similar to the stomach, cellular proliferation occurs much faster on one
side, resulting in a larger right lobe and a smaller left lobe.

Pancreas

The pancreas is formed by two separate pancreatic buds that later join. Specifically,
the dorsal pancreatic bud forms as an outgrowth from the dorsal wall of the duodenum,
whereas the ventral pancreatic bud forms as an outgrowth from the ventral wall of the
duodenum along with the bile duct. When the duodenum rotates and takes its C-shape form,
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it pulls the ventral pancreatic bud dorsally and inferior to the ventral pancreatic bud.
Subsequently, the pancreatic buds along with their rudimentary duct systems fuse to give rise
to the pancreas.

The ventral pancreatic bud forms the uncinate process and the inferior part of the head
of the pancreas. The dorsal pancreatic bud forms the superior part of the head, the neck, and
the body of the pancreas. The distal part of the dorsal pancreatic duct and the entire ventral
pancreatic duct form the main pancreatic duct.

The proximal part of the dorsal pancreatic duct either becomes obliterated or persists
as the accessory pancreatic duct. Thus, the pancreas and its duct systems assume their final
shape; the main pancreatic duct joins the bile duct and enter the duodenum at the major
papilla, whereas the accessory duct (if present) enters the duodenum at the minor papilla.

B. Midgut

The midgut gives rise to the:

 distal half of the duodenum
 jejunum
 ileum
 cecum
 appendix
 ascending colon
 proximal two-thirds of the transverse colon

The derivatives of the midgut are supplied by the superior mesenteric artery.
During the fifth week of gestation, the midgut undergoes a rapid elongation that occurs much
faster than that of the abdominal cavity, resulting in the formation of the primary intestinal loop.
At its apex, the loop remains in open communication with the yolk sac via the vitelline duct,
while the superior mesenteric artery runs along the axis of the loop. The cranial limb of the
loop will develop into the inferior half of the duodenum, the jejunum, and proximal half of the
ileum. The caudal limb of the loop will develop into the distal half of the ileum, the cecum, the
ascending colon, and the proximal two-thirds of the transverse colon.

By the sixth week, the continuing elongation of the midgut, combined with the pressure
exerted by the dramatic growth of the abdominal organs, force the primary intestinal loop to
protrude into the umbilicus (physiological herniation). Concurrently, the loop rotates 90
degrees counterclockwise around the axis of the superior mesenteric artery, resulting in the
cranial limb to move caudally and to the embryo‘s right, and the caudal limb to move cranially
and to the embryo‘s left. While this rotation takes place until the eighth week of gestation, the
lengthening jejunum and ileum develop into a series of folds known as the jejunal-ileal loops,
whereas the expanding cecum gives rise to a wormlike diverticulum, the vermiform appendix.

During the tenth week, the herniated midgut retracts into the abdomen. The
mechanism responsible for this retraction is not fully understood, but may involve the increase
in size of the abdominal cavity. As the intestinal loops reenter the abdomen, it rotates an
additional 180 degrees counterclockwise around the axis of the superior mesenteric artery,
thus having travelled for a total of 270 degrees. As a result, the cecum, being initially
positioned under the liver, becomes displaced inferior, pulling down the proximal hindgut to
form the ascending colon.

By the eleventh week, the intestines have completely retracted into the abdomen.
The dorsal mesenteries of the ascending and descending colon shorten and fold, anchoring
these organs to the dorsal body wall, where they become secondarily retroperitoneal. The
jejunum, ileum, cecum, and the transverse and sigmoid colon remain suspended by a short
mesentery from the dorsal body wall, thus becoming intraperitoneal.

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C. Hindgut

The hindgut gives rise to the distal third of the:

 transverse colon
 descending colon
 sigmoid colon
 rectum
 upper two-thirds of the anal canal

The derivatives of the hindgut are supplied by the inferior mesenteric artery.
During the early weeks of development, the terminal portion of the hindgut enters the
posterior portion of the cloaca, whereas the allantois enters the anterior portion; the cloaca
and the allantois form the future anorectal canal and the urogenital sinus, respectively. The
cavity of the cloaca is lined by endoderm, whereas its ventral boundary is lined by surface
ectoderm; the latter is known as the cloacal membrane.

By the seventh week of gestation, the urorectal septum (a layer of mesoderm) grows
between the allantois and the hindgut. It divides the cloaca into the urogenital sinus and the
anorectal canal, while its tip forms the future perineal body. The urogenital sinus forms the
future bladder, parts of the urethra, and the phallus, whereas the anorectal canal develops
into the rectum and most of the anal canal. Specifically, while the upper two-thirds of the
anorectal canal is derived from the endoderm of the hindgut, the lower one-third is derived
from the surface ectoderm of the cloaca.

Degeneration of the cloacal membrane connects the upper and lower parts of the
anorectal canal, while the location of this juncture is demarcated by the pectinate line, an
irregular folding of the mucosa. This is also where the epithelial lining transitions from
columnar to stratified squamous. The vasculature of the anorectal canal is consistent with its
dual origin: the upper two-thirds (superior to the pectinate line) is supplied by the superior
rectal arteries, branches of the inferior mesenteric artery, whereas its lower one-third (inferior
to the pectinate line) is supplied by the inferior rectal arteries, branches of the internal
pudendal arteries.

References:

DukeMedicine. Gut Development. https://embryology.oit.duke.edu/GI/GI.html. 2022

KENHUB. Development of the Digestive System.

https://www.kenhub.com/en/library/anatomy/development-of-digestive-system. 2022

Lumen. Development of the Digestive System. https://courses.lumenlearning.com/boundless-

ap/chapter/development-of-the-digestive-system/. 2022

MedUmich. Primitive Gut Tube.

https://www.med.umich.edu/lrc/coursepages/m1/embryology/embryo/10digestivesystem.htm.
2022

Osmosis. Development of the Gastrointestinal System.

https://www.osmosis.org/learn/Development_of_the_gastrointestinal_system. 2022

Assessment:

I. Enumerate the following:

1. Derivatives of foregut (8)
2. Derivatives of midgut (7)
3. Derivatives of hindgut (5)

II. Explain:
1. How the primitive gut is formed.
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 Republic of the Philippines
NORTHERN ILOILO STATE UNIVERSITY
NISU Main Campus, V Cudilla Sr. Ave, Estancia, Iloilo

COLLEGE OF ARTS AND SCIENCES

Bachelor of Science in Biology
Name: ________________________ Year & Section: __________ Score: _____

Laboratory Activity No. 1

SEED GERMINATOR

A. Objectives:

1. To identify the key component for soil-free seed germination.

2. To perform soil-free seed germination.
3. To appreciate the process of plant development.

B. Materials:

 Quick-germinating seeds, such as radish

 Water
 Paper towel or coffee filter
 Scissors
 Pencil
 Large petri dish with lid
 Lidless, straight-sided plastic container wide enough to set the petri dish inside, on its
edge, as shown in the photo
 Two rubber bands big enough to fit around the open container
 Metric ruler with millimeter markings
 Magnifying glass

C. Procedures:

1. Soak the seeds overnight in water.

2. Set aside the top of the petri dish. Cut the paper towel (or coffee filter) to fit inside.
3. With a ruler and pencil, draw a straight line across the middle of the paper towel. Lay the
marked-up paper in the bottom of the dish so the line sits horizontally across the center.
4. Pour a little water into the dish to wet the paper towel. Smooth out any bubbles and tip
out any extra water not absorbed by the paper. Later, when you stand the dish on its
edge, the wet paper should remain stuck to the inside of the dish.
5. Place 6 to 10 seeds on the paper towel, evenly spaced along the reference line. Then put
the lid on the petri dish.
6. Stretch the rubber bands, set close to one another, around the center of the straight-
sided plastic container (see photo below). Stand the petri dish between the rubber bands,
and adjust the setup so it‘s secure, standing on edge, upright in the container. With
gentle handling, the seeds should stick to the moistened paper towel. If they move, put
them back in their places on the line.

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 7. Pour water into the container to a depth of about 1 inch (2 to 3 cm). The water should
seep into the petri dish and contact the paper towel, keeping it moist as the seedlings
begin to sprout.
8. Put your seed germinator in a warm place (room temperature or slightly higher), away
from direct sunlight.
9. Check on your seeds once or twice a day, and notice what changes or emerges. It‘s fine
to open the seed germinator just handle it carefully so the seeds don‘t move.
10. Use a magnifying glass to examine the growing structures in more detail.
11. Measure the growth of the roots and shoots over time. Collect data to graph average root
length vs. time, and average shoot length vs. time. Note that it‘s helpful to measure time
in total elapsed hours, rather than days.

D. Guide Questions:

Answer the following:

1. Which emerge first, the shoots with green tips or the white roots?
_________________________________________________________________________

2. Do each seed‘s roots and shoots sprout in the same direction or in different directions?
_________________________________________________________________________

3. Which grows faster, the shoots or the roots?

_________________________________________________________________________

4. In this activity/experiment, what is the key component for soil-free seed germination?
_________________________________________________________________________

5. Using a graphing paper, graph the average root length versus time and the average shoot
length versus time.

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 Republic of the Philippines
NORTHERN ILOILO STATE UNIVERSITY
NISU Main Campus, V Cudilla Sr. Ave, Estancia, Iloilo

COLLEGE OF ARTS AND SCIENCES

Bachelor of Science in Biology
Name: ________________________ Year & Section: __________ Score: _____

Laboratory Activity No. 2

SEED GERMINATION USING A SEED GERMINATOR

A. Objectives:

1. To identify the basic requirements for the seeds to germinate in a seed germinator.
2. To identify the factors affecting the germination of seeds in a seed germinator.
3. To develop questions about seed germination using a seed germinator.
4. To create a digital poster of the final output of the laboratory activity.

B. Materials:

 Photos/photographs of the germinating seeds inside the closed petri dish

 Photos/photographs of the seed germinator
 Photos/photographs of the group members conducting Laboratory Activity #1 and #2
 Printed digital poster (12 Feet x 24 Feet)

C. Procedures:

1. List down the basic requirements for the seeds to germinate in a seed germinator.
2. List down the factors affecting the germination of seeds in a seed germinator.
3. Develop 4 questions about seed germination using a seed germinator and indicate
their answers.
4. Create a printed digital poster of the final output of the laboratory activity.
5. Present the overall output in the class.

D. Result:

Printed digital poster indicating the following:

1. Title of the laboratory activity.

2. The four (4) questions with answers.
3. Photos/photographs of the germinating seeds inside the closed petri dish.
4. Photos/photographs of the seed germinator.
5. Photos/photographs of the group members conducting Laboratory Activity #1 and #2.

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 Republic of the Philippines
NORTHERN ILOILO STATE UNIVERSITY
NISU Main Campus, V Cudilla Sr. Ave, Estancia, Iloilo

COLLEGE OF ARTS AND SCIENCES

Bachelor of Science in Biology
Name: ________________________ Year & Section: __________ Score: _____

Laboratory Activity No. 3

PLANT REPRODUCTION

A. Objectives:

1. To identify the characteristics of flowers that attract pollinators.

2. To explore and familiarize the specialized features of plants used for asexual and
sexual reproduction.
3. To appreciate plant reproduction.

B. Materials:

 Gumamela (Hibiscus rosa-sinensis) flower

 Cutter/Small knife
 Hand lens (optional)

C. Procedures:

1. Use the guide below to familiarize the reproductive system of a gumamela.

Anther – Forms pollen grains.

Filament – Supports the anther.
Ovule – Found inside the ovary and after fertilization develop into seeds.
Ovary – The lower, often times enlarged part of the pistil, which contains the egg cells
and produces the seeds. The ovary becomes the fruit.
Petals – Leaf-like, often colorful part of the plant that surrounds the reproductive parts
of the flower and make the flower conspicuous to pollinators. Petals collectively form
the corolla.
Pistil – The female part of the flower, which is comprised of three parts – stigma, style,
and ovary.
Pollen – Fine powder dust that contains the sperm from a male plant.

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 Sepals – Green leaf-like structures that protect the flower bud. Collectively sepals are
referred to as the calyx. Sometimes sepals are colorful like the petals.
Stamens – The male parts of the flower that produces pollen grains. Stamens consist
of a filament and an anther.
Stigma – Where pollen grains land on the pistil.
Style – Connects the stigma and ovary. Pollen grains travel to the ovary via the style.

D. Result:

Answer the following questions:

1. What are the two main types of plant reproduction?

_________________________________________________________________________

2. What are the 3 main natural forces that aid pollination?

_________________________________________________________________________

3. Name the 3 specialized plant structures used for asexual reproduction.

_________________________________________________________________________

4. What is a pollinator?
_________________________________________________________________________

5. Cite 4 characteristics of flowers that attract pollinators.

_________________________________________________________________________

E. Practicum:

Identification of the parts of the reproductive system of a gumamela using an actual

specimen.

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 Republic of the Philippines
NORTHERN ILOILO STATE UNIVERSITY
NISU Main Campus, V Cudilla Sr. Ave, Estancia, Iloilo

COLLEGE OF ARTS AND SCIENCES

Bachelor of Science in Biology
Name: ________________________ Year & Section: __________ Score: _____

Laboratory Activity No. 4

STEM GROWTH AND DEVELOPMENT USING THE LATEST TECHNIQUE

A. Objectives:

1. To apply the latest technique (shown in the short video) of planting the stem of
mango/sugar apple or custard apple or sweet sop.
2. To monitor and observe the growth and development of the stem of indian mango/sugar
apple or custard apple or sweet sop.

B. Materials:

 Matured stem of Indian Mango (Mangifera indica) or

 Matured stem of Sugar Apple/Custard Apple/ Sweet Sop (Annona squamosal)
 Garden Soil
 Pot
 Gardening tool
 Egg
 Aloe vera
 Water
 Sprayer for water

C. Procedures:

1. List down and follow the procedures cited in the video.

2. Monitor the growth of your plant.
3. Record your observations.

D. Result:

Answer the following:

1. How long does it take for the roots to fully grow?

2. How long does it take for the leaves to fully grow?

3. How long does it take for the flower/s to develop?

4. How long does it take for the fruit/s to develop?

5. Create a table where you can record your observations.

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 Republic of the Philippines
NORTHERN ILOILO STATE UNIVERSITY
NISU Main Campus, V Cudilla Sr. Ave, Estancia, Iloilo

COLLEGE OF ARTS AND SCIENCES

Bachelor of Science in Biology
Name: ________________________ Year & Section: __________ Score: _____

Laboratory Activity No. 5

FETAL DEVELOPMENT
A. Objectives:

1. To measure the given pictures of fetuses of various ages and make a data table.
2. Construct and interpret graphs of fetal size versus time.

B. Materials:
 Provided pictures of fetuses
 Ruler with millimeter markings
 Pencil

C. Background:

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Fertilization –

 Most likely to occur within a few days of ovulation

 Occurs in the fallopian tube (usually)
 Enzymes at the head of a sperm break down the thick outer layer of the egg
 Sperm (haploid) + egg (haploid) à zygote (diploid)

Pregnancy –

 Takes about 38 weeks or 9 months

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  Development takes place in the uterus
 Divided into three trimesters

The First Trimester –

 After about 24 hours the zygote starts to divide in a process called cleavage
 During cleavage the zygote is traveling from the fallopian tubes to the uterus – cells
are not growing
 After about 6 days, the zygote is a hollow ball of cells called a blastocyst – it
implants itself into the built up uterine lining
 Zygote produces hormones that tell the mother to keep producing estrogen and
progesterone – keeps the pregnancy going
 Placenta develops as a way for nutrients and waste to pass between mother and
child – blood does not mix
 Harmful substances such as nicotine, alcohol, bacteria and even caffeine can
pass through the placenta and impact the development of the baby – often
resulting in mental retardation such as fetal alcohol syndrome
 Blastocyst develops into three primary tissue layers (endoderm, mesoderm and
ectoderm)
 Third week – 2mm – blood vessels begin to develop
 Fourth week – limbs and organs form

The Second Trimester –

 Period of rapid growth

 Mother feels movement

The Third Trimester –

 More rapid growth

 Less movement – it gets crowded in there

D. Procedures and Results:

1. Measure the body length (rump to top of head), thigh length (rump to knee), and calf length
(knee to foot) of each pictured fetus using the markings on the 38 week fetus as a guide. Each
picture is only 4/10 as big as the actual fetus, so figure out a conversion factor to use to get
the actual sizes of the fetuses. Record your results in a data chart provided.

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2. Graph your data. Time is the independent variable and Actual Total Fetal Length is the
dependent variable. Make sure to give the graph a title, to label each axis, and to give
units. Graphs must be done in pencil.

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3. Prepare graph based on the data given below concerning fetal weight. Make sure to give
the graph a title, to label each axis, and to give units. Graphs must be done in pencil.

Weeks of Fetal Development Fetal Mass (grams)

4 0.5
8 1
12 15
16 100
20 300
24 650
28 650
32 1700
36 2400
38 3300

Answer the questions below:

1. During which weeks of development is the baby called an embryo? _________________

2. During which weeks of development is the baby called a fetus? __________________

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 Republic of the Philippines
NORTHERN ILOILO STATE UNIVERSITY
NISU Main Campus, V Cudilla Sr. Ave, Estancia, Iloilo

COLLEGE OF ARTS AND SCIENCES

Bachelor of Science in Biology
Name: ________________________ Year & Section: __________ Score: _____

Laboratory Activity No. 6

HOMEOSTASIS

A. Objectives:

1. To observe an example of homeostasis working to control the body‘s pulse rate (heart
rate).

B. Materials:
 Stop watch (you may use a phone app or online version)
C. Background:

Homeostasis means maintaining a relatively constant state of the body‘s internal

environment. The term is used to describe a pattern of response to restore the body to normal
stable level. When a stimulus (environment change) is met by a response that reverses the
trend, it is negative feedback. As a result, the internal environment is returned to normal
levels.

Pulse rate is constantly checked by receptors (sensors) throughout the body. A

stimulus such as exercise causes an elevated pulse rate. The body will respond over time to
return the pulse rate to normal.

D. Procedures and Results:

1. Start by determining which person in your group will exercise and which will watch the
stopwatch and record data.

2. Let him/her sit down and measure his/her pulse rate in the radial artery (wrist). Take three
separate measurements, each for thirty seconds. You will then calculate the beats per minute
by multiplying the values obtained times 2. Finally, find the average for the three values of
your resting heart rate. Record each measurement in Table 1.

Table 1: Resting Pulse Rate

Resting Pulse Rate Resting Pulse Rate Resting Pulse Rate Average Resting
1 2 3 Pulse Rate

3. The person whose heart rate was measured will now exercise vigorously for three minutes
(jog in place). Start the stop-watch when exercising begins. Measure the pulse rate
immediately after finishing the three minutes of activity. Measure the same way as before by
counting heartbeats for 30 seconds then multiply by 2. Record the value in Table 2. Rest for 5
minutes. Measure the pulse rate again after 5 minutes of rest. Record pulse rate after this 5
minute rest period also in Table 2. Repeat steps for the 2nd and 3rd trial.

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Table 2: Pulse Rate After Exercise and Rest (3 Trials)

Pulse Rate After 3 Minutes Pulse Rate After 5 Minutes

of Exercise of Rest
Trial 1
Trial 2
Trial 3

Answer the following questions:

1. What is your average resting pulse rate? Most adults are between 60-90 beats/min.
Compare yours.

__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
______________________________________________________________________

2. What happens to your pulse rate with vigorous exercise?

__________________________________________________________________________
________________________________________________________________________

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References:
I.
Creative Diagnostics. Developmental Biology. https://www.creative-
diagnostics.com/developmental-biology.htm. 2022.
Psychology Wiki. Introduction to Developmental Biology.
https://psychology.fandom.com/wiki/Introduction_to_developmental_biology#Concepts_in_de
velopmental_biology. 2022
Wikipedia. Developmental Biology.
https://en.wikipedia.org/wiki/Developmental_biology#:~:text=Developmental%20biology%20is
%20the%20study,cells%20in%20the%20adult%20organism. 2022
II.
Britanica. The Process of Differentiation. https://www.britannica.com/science/cell-biology/The-
process-of-differentiation. 2022.
Lumen Boundless Biology. Regulating Gene Expression in Cell Development.
https://courses.lumenlearning.com/boundless-biology/chapter/regulating-gene-expression-in-
cell-development/. 2022
Science Direct. Cell Differentiation. https://www.sciencedirect.com/topics/medicine-and-
dentistry/cell-
differentiation#:~:text=Cell%20differentiation%20is%20the%20process,the%20top%20of%20t
he%20hierarchy.. 2022.
Wikipedia. Cellular Differentiation. https://en.wikipedia.org/wiki/Cellular_differentiation. 2022.
III.
Biology Dictionary. Cell Signaling. https://biologydictionary.net/cell-signaling/. 2022
Khan Academy. Introduction to Cell Signaling. https://www.khanacademy.org/science/ap-
biology/cell-communication-and-cell-cycle/cell-communication/a/introduction-to-cell-signaling.
2022
Science Direct. Cell Signaling. https://www.sciencedirect.com/topics/neuroscience/cell-
signaling. 2022
Wikipedia. Cell Signaling. https://en.wikipedia.org/wiki/Cell_signaling. 2022
IV.
Biology Dictionary. Fertilization. https://biologydictionary.net/fertilization/. 2022.
Science Learning Hub. Pollination and Fertilization.
https://www.sciencelearn.org.nz/resources/77-pollination-and-fertilisation. 2022.
Sciencing. Reproduction of Plants and Animals. https://sciencing.com/reproduction-plants-
animals-6404461.html. 2022
Toppr. Introduction to Reproduction. https://www.toppr.com/guides/biology/how-do-
organisms-reproduce/introduction-to-reproduction/. 2022.
V.
Britanica. The Hormones of Plants. https://www.britannica.com/science/hormone/The-
hormones-of-plants. 2022
Intech Open Book Series. Hormonal Regulation in Plant Development and Stress Tolerance.
https://www.intechopen.com/chapters/56091. 2022
NCBI Resources. Hormonal Regulation of Plant Growth and Development.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC516799/. 2022
VI.
IBiology. Root Development: Genetics, Form and Function. https://www.ibiology.org/plant-
biology/root-development/. 2022
Science Direct. Root Development. https://www.sciencedirect.com/topics/medicine-and-
dentistry/root-development. 2022
Study.Com. Root System Growth: The Root Cap, Primary Roots and Lateral Roots.
https://study.com/academy/lesson/root-system-growth-the-root-cap-primary-roots-lateral-
roots.html. 2022
Wikipedia. Root. https://en.wikipedia.org/wiki/Root. 2022
VII.
Biology Libre Text. Shoot Development.
https://bio.libretexts.org/Bookshelves/Botany/Botany_(Ha_Morrow_and_Algiers)/Unit_3%3A_
Plant_Physiology_and_Regulation/18%3A_Development/18.05%3A_Shoot_Development.
2022

77 | P a g e
Annie C. Castillano
Britanica, The Shoot System and its Derivatives. https://www.britannica.com/science/plant-
development/The-shoot-system-and-its-derivatives. 2022
Wikipedia. Shoot. https://en.wikipedia.org/wiki/Shoot. 2022
VIII.
Hindawi. Plant Phenotypic Plasticity in Response to Environmental Factors.
https://www.hindawi.com/journals/abot/2014/208747/. 2022
Novoplansky, Ariel. Developmental Plasticity in Plants: Implications of Non-Cognitive
Behavior. http://www.esalq.usp.br/lepse/imgs/conteudo_thumb/Developmental-plasticity-in-
plants-implications-of-noncognitive-behavior-1.pdf. 2022
Science Direct. Phenotypic Plasticity. https://www.sciencedirect.com/topics/agricultural-and-
biological-sciences/phenotypic-plasticity. 2022
Wikipedia. Phenotypic Plasticity. https://en.wikipedia.org/wiki/Phenotypic_plasticity. 2022
IX.
Britanica. Induced Pluripotent Stem Cell. https://www.britannica.com/science/induced-
pluripotent-stem-cell. 2022
Science Direct. Induced Pluripotent Stem Cells.
https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/induced-
pluripotent-stem-cell. 2022
Wikipedia. Induced Pluripotent Stem Cell.
https://en.wikipedia.org/wiki/Induced_pluripotent_stem_cell. 2022
X.
Current Biology. Patterning the Drosophila Embryo. https://www.cell.com/current-
biology/comments/S0960-9822(02)00406-2. 2022
NCBI. Early Drosophila Development. https://www.ncbi.nlm.nih.gov/books/NBK10081/. 2022
Study.com. Embryonic Development and Life Cycles of Invertebrates
https://study.com/academy/lesson/embryonic-development-life-cycles-of-invertebrates-
vertebrates.html#:~:text=Invertebrate%20Embryonic%20Development,to%20develop%20furt
her%20into%20adulthood. 2022
Wikipedia. Drosophila Embryogenesis.
https://en.wikipedia.org/wiki/Drosophila_embryogenesis. 2022.
XI.
Anatomy Journal. Ascidian Embryonic Development: An Emerging Model System for the
Study of Cell Fate Specification in Chordates.
https://anatomypubs.onlinelibrary.wiley.com/doi/full/10.1002/dvdy.21108. 2022
Cell.com. The Ascidian as a Model Organism in Developmental and Evolutionary Biology.
https://www.cell.com/fulltext/S0092-8674(01)00481-0. 2022
JSTOR. Evolution of Ascidian Development. https://www.jstor.org/stable/1313057. 2022
XII.
JSTOR. Early Amphibian Development. https://www.jstor.org/stable/1310156. 2022
IBiology. Early Frog Development: How to Make a Tadpole.
https://www.ibiology.org/development-and-stem-cells/frog-development/. 2022
NCBI. Early Amphibian Development. https://www.ncbi.nlm.nih.gov/books/NBK10113/. 2022
XIII.
BioNinja. Neurulation. https://ib.bioninja.com.au/options/option-a-neurobiology-and/a1-neural-
development/neurulation.html. 2022
Lecturio. Gastrulation and Neurulation. https://www.lecturio.com/concepts/gastrulation-and-
neurulation/. 2022
NCBI. Formation of the Neural Tube. https://www.ncbi.nlm.nih.gov/books/NBK10080/. 2022
Wikipedia. Neurulation. https://en.wikipedia.org/wiki/Neurulation. 2022
XIV.
Journals.Biologists. Establishing the Pattern of the Vertebrate Limb.
https://journals.biologists.com/dev/article/147/17/dev177956/2257/Establishing-the-pattern-of-
the-vertebrate-limb. 2022
Journals.Biologists. Growing Models of Vertebrate Limb Development.
https://journals.biologists.com/dev/article/136/2/179/65397/Growing-models-of-vertebrate-
limb-development. 2022
PubMed. Vertebrate Limb Development and Malformations.
https://pubmed.ncbi.nlm.nih.gov/10473037/#:~:text=The%20limbs%20develop%20from%20s
mall,core%20of%20the%20limb%20bud. 2022
78 | P a g e
Annie C. Castillano
XV.
Karger. The Transitional Heart: From Early Embryonic and Fetal Development to Neonatal
Life. https://www.karger.com/Article/Fulltext/501906. 2022
TeachMeAnatomy. Development of the Cardiovascular System.
https://teachmeanatomy.info/the-basics/embryology/cardiovascular-system/. 2022
Wikipedia. Heart Development.
https://en.wikipedia.org/wiki/Heart_development#:~:text=Heart%20development%20(also%20
known%20as,functional%20organ%20in%20vertebrate%20embryos.. 2022
XVI.
DukeMedicine. Gut Development. https://embryology.oit.duke.edu/GI/GI.html. 2022
KENHUB. Development of the Digestive System.
https://www.kenhub.com/en/library/anatomy/development-of-digestive-system. 2022
Lumen. Development of the Digestive System. https://courses.lumenlearning.com/boundless-
ap/chapter/development-of-the-digestive-system/. 2022
MedUmich. Primitive Gut Tube.
https://www.med.umich.edu/lrc/coursepages/m1/embryology/embryo/10digestivesystem.htm.
2022
Osmosis. Development of the Gastrointestinal System.
https://www.osmosis.org/learn/Development_of_the_gastrointestinal_system. 2022

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