Section 1.1 and 1.

Gastrulation
 An early phase in the embryonic development
 The single-layered blastula is reorganised into a multilayered structure known as the
gastula.
 Takes place after cleavage and the formation of the blastula
 Is followed by organogenesis, when individual organs develop within the newly formed
germ layers
 Ectoderm gives rise to: epidermis, the nervous system and to the neural crest
 Endoderm: epithelium of the digestive system and respiratory system and organs
associated with the digestive system including liver and pancreas
 Mesoderm: give rise to many cell type including muscle, bone and connective tissue.
Also include the notochord, heart , blood and blood vessels, cartilage of the ribs and
vertebrae and the dermis.
 Some common features of gastrulation across triploblastic organisms include:
 A change in the topological structure of the embryo - from a simply connected surface
to a non-simply connected surface
 The differentiation of cells into one of three of three types (endodermal, mesodermal
and ectodermal)
 The digestive function of a large number of endodermal cells
 Blastocysts begins to transform into two epithelial layers - the epiblast is the outer
layer that consists of columnar cell while inner layer is called the hypoblast which is
composed of cuboidal cells. The layer of these two cells are called bilaminar
blastocyst or bilaminar disc.
 Bilaminar blastocyst also define the primitive dorsal ventral axis. The epiblast is dorsal
and the hypoblast is ventral.
 Start with appearance of the primitive streak, a region of thickened embroynic epiblast
 Streak extends along the embryo, posterior to anterior due to intercalation of
surrounding cells - convergent extension (the process by which the tissue of an
embryo is restructured to converge (narrow) along one axis and extend (elongate)
along a perpendicular axis cellular movement) -- Crucial role in shaping the body plan
during embryogenesis and occurs during gastrulation, neurulation, axis elongation and
organogensesis.
 A depression, the primitive groove, forms within the streak allowing migrating cells to
pass into deeper layers of the embryo
 Hensen's node or "the node" is a thickening of cells at the anterior end of streak with a
depressive, the primitive pit, at its centre
 Epiblast cells migrate inwards through the groove/pit
 Some cells move ventrally, displace hypoblast cells and form endoderm
 Other migrating cells occupy a position between forming endoderm and epiblast, and
form mesoderm
 Epiblast cells that do not migrate form ectoderm
 Primitive streak defines the embryonic axes: extends posterior to anterior, migrating
cells enter from the dorsal side and move ventrally, sits at the midline separating left
and right
 Neural induction - Neural plate induction
 A key developmental structure that serves as the basis for the nervous system
 Opposite the primitive streak in the embryo, ectodermal tissue thickens and flattens to
become the neural plate.
 Hensen's node or primitive knot is the organiser for gastrulation in the vertebrate
embryo
 The ends of neural plate, known as the neural folds, push the ends of the plate up and
together, folding into the neural tube, a structure critical to brain and spinal cord
development. This process as a whole is termed primary neurulation.
 Signalling proteins are also important in neural plate development and aid in
differentiating the tissue destined to become the neural plate, including Bone
Morphogenetic Proteins (BMP) and cadherins (play a role in cell adhesion, forming
adhesion is mediated by extracellular cadherin domains, and are dependent on
calcium ions to function). Expression of these proteins is essential to neural plate
folding and subsequent neural tube formation
 Molecular signals for neural plate induction: signal required for epidermal fate is bone
morphogenetic protein (BMP). Blocking BMP promotes neural fate.
 BMP signalling in ventral ectoderm represses neural fate and promotes epidermal fate.
 BMP signalling must be blocked in dorsal ectoderm for these cells to adopt a neural
fate
 Patterning the neural plate
 Anterior - posterior axis determined by a gradient of Wnt
 Wnt/B-catenin signalling: high at the posterior end and progressively declines from
posterior to anterior
 Wnt inhibitors produced by anterior AME (axial mesendoderm)
 Mediolateral axis determined by a gradient of BMP
 BMP signalling high laterally and declines towards the midline
 BMP inhibitors produced by the AME
 These are morphogen, which are signalling molecule that induces different cell fates/
responses at different concentrations
 AME from anterior to posterior, consists of the anterior endoderm (AE), prechordal
mesendoderm (PME) and chordamesoderm (CM)
 AE gives rise to the foregut, thyroid gland and liver
 PME gives rise to the prechordal plate that underlines the presumptive forebrain region
 CM gives rise to the notochord, a rod shape structure that acts as a signalling centre
involved in neural tube polarisation
 Neurulation
 Nervous system arises from ectoderm
 Part of the dorsal ectoderm, termed the neural plate is specified to become neural
tissue
 Neurulation: process by wich the neural plate forms the neural tube which develops
into the brain and spinal cord
 Primary neurulation: neural plate cells invaginate and pinch off from the surface to
form a hollow tube
 Secondary neurulation: a solid mass of cells sinks into the embryo and is then
hollowed out to form a tube
 Anterior portion of the neural tube is formed by primary neurulation and posterior
portion (in humans from the level of the sacral vertebrae) is formed by secondary
neurulation
 The embryo at this stage is termed the neurula
 Notochord: lies along the anteroposterior (head to tail) axis, is usually closer to the
dorsal than the ventral surface of the animal, and is composed of cells derived from
the mesoderm.
 Plays a key role in signalling and coordinating development. It is found ventral to the
neural tube.
 Can induce the formation of motor neurons in the dorsal tube. Motor neuron formation
generally occurs in the ventral neural tube, while the dorsal tube generally forms
sensory cells.
 The notochord secretes a protein sonic hedgehig homolog (sHg), a key morphogen
regulating organogenesis and having a critical role in signalling the development of
motor neurons. The secretion of sHg by the notochord establishes the ventral pole of
the dorsal-ventral axis in the developing embryo.
 The process begins when the notochord induces the formation of the CNS by
signalling the ectoderm germ layer above it to form the think and flat neural plate.
 The neural plate folds in upon itself to form the neural tube, which will later
differentiate into the spinal cord and the brain, eventually forming the CNS
 Different portions of the neural tube form by two different processes, called primary
and secondary species:
 In primary neurulation, the neural plate creases inward until the edges come in contact
and fuse
 In secondary neurulation, the tube forms by hollowing out of the interior of a solid
precursor
Primary neurulation
 Occurs in response to soluble growth factors secreted by the notochord.
 Ectodermal cells are induced to form neuroectoderm from a variety of signals.
 Ectoderm sends and receives signals of bone morphogenetic protein 4 (BMP4) and
cells which receive BMP4 signal develop into epidermis
 The inhibitory signals chordin, noggin and follistatin are needed to form neural plate.
These inhibitory signals are created and emitted by the Spemann's organiser. Cells
which do not receive BMP4 signalling due to the effects of the inhibitory signals will
develop into the anterior neuroectoderm cells of the neural plate.
 Cells which receive fibroblast growth factor (FGF) in addition to the inhibitory signals
form posterior neural plate cells.
 Formation or shape changing
 Folding or Bending: the process of the flat neural plate folding into the cylindrical tube
is termed primary neurulation. The medial hinge point (MHP) is formed as a result of
the cellular shape changes. The expandng epidermis puts pressure on the MHP and
causes the neural plate to fold resulting in neural folds and the creation of the neural
groove. The notochord plays an integral role in the development of the neural tube.
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 (fig 1)
  Convergence (intercalation) extension: a process the tissue of an embryo is
restructured to converge (narrow) along one axis and extend (elongate) along a
perpendicular axis by cellular movement. Ensures the neural folds come close enough
together to close the neural tube. Requires non-canonical Wnt signalling via the planar
cell polarity (PCP) pathway. Mutations in Wnt-PCP pathway components are
implicated in Neural tube defeats.
 Closure: For a short time, the neural tube is open both cranially and caudally.
 These opening called neuropores close during the fourth week in humans
 Improper closure of the neuropores can result in neural tube defects such as
anencephaly or spinal bifida.

Anterior Posterior Pattern

 Signals that control anteroposterior neural development include FGF and retinoic acid,
which act in the hindbrain and spinal cord
 The hindbrain, for example, is patterned by Hox gene, which are expressed in
overlapping domains along the anteroposterior axis under the control of retinoic acid.
 The 3' genes in the Hox cluster are induced by retinoic acid in the hindbrain, whereas
the 5' Hox genes are not induced by retinoic acid and are expressed more posteriorly
in the spinal cord.
 Retinoic acid is a metabolite of vitamin A (retinol) that mediates the functions of vitamin
A required for growth and development
 During early embryonic development, retinoic acid generated in a specific region of the
embryo helps determine position along the embryonic anterior/posterior axis by serving
as an intercellular signalling molecular that guides development if the posterior portion
of the embryo.
 Retinoic acid acts through Hox genes which ultimately control anterior/posterior
patterning in early developmental stages

Wnt signalling pathway

 A group of signal transduction pathways made of proteins that pass signals into a cell
through cell surface receptors.
 Three Wnt signalling pathways have been characterised: the canonical Wnt pathway,
the noncanonical planar cell polarity pathway, and the noncanonical Wnt/calcium
pathway
 All three pathways are activated by binding a Wnt-protein ligand to a Frizzled family
receptor.
 The canonical Wnt pathway leads to regulation of gene transcription.
 The noncanonical planar cell polarity pathway regulates the cytoskeleton that is
responsible for the shape of the cell. Convergent extension movements during
gastrulation and neurulation that drive elongation of the embryo along its AP axis
required PCP signaling
 The noncanonical Wnt/calcium pathway regulates calcium inside the cell.
 Wnt signalling begins when a Wnt protein binds to the N-terminal extra-cellular
cysteine -rich domain of a Frizzled (Fz) family receptor

Canonical Wnt pathway

 causes an accumulation of beta catenin in the cytoplasm and its eventual tranlocation
into the nucleus to act as a transcriptional coactivator of transcription factors that
belong to the TCF/LEF family
 Without Wnt signalling, beta catenin would not accumulate in the cytoplasm since a
destruction complex would normally degrade it
 The destruction complex includes the following proteins: Axin, adenomatosis polyposis
coli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and
casein kinase 1 alpha (ck1a) - it degrades beta catenin by targeting it for ubiquination,
which subsequently sends it to the proteasome to be digested.
 However as Wnt binds Fz and LPR5/6, the destruction complex function becomes
disrupted. This is due to Wnt causing the translocation of the negative Wnt regulator,
Axin, and the destruction complex to the plasma membrane.
 Phosphorylation by other proteins in the destruction complex subsequently binds Axin
to the cytoplasmic tail of LRP5/6.
 Axin becomes de-phosphorylated and its stability and levels decrease
 Dsh (a cytoplasmic phosphoprotein that acts directly downstream of Fz receptor) then
becomes activated via phosphorylation and its DIX and PDZ domains inhibit the GSK3
activity of the destruction complex.
 This allows beta catenin to accumulate and localise to the nucleus and subsequently
induce a cellular response via gene transduction alongside the TCF/LEF transcription
factor.

Noncanoncial pathways/ Planar cell polarity pathway

 Does not involve beta catenin
 LRP-5/6 which is used as its co-receptor in canoncial pathways, is not involved and is
thought to use another co-receptor
 The pcp pathway is activated via the binding if Wnt to Fz and its co-receptor.
 The receptor then recruits Dsh, which uses its PDZ and DIX domain to form a complex
with Dishevelled-associated activator of morphogenesis 1(DAAM1)
 DAAM1 then activates the small G-protein Rho through a guanine exchange factor
 Rho activates Rho-associated kinase (ROCK) which is one of the major regulator of
the cytoskeleton
 Dsh also forms a complex with rac1 and mediates profilin binding to actin
 Rac1 activates JNK and can also lead to actin polymerisation
 Profilin binding to actin can result in restructuring of the cytoskeleton and gastrulation


Doral Ventral patterning
 The dorsal part of the neural tube contains the alar plate, which is associated primarily
with sensation
 The ventral part of the neural tube contains the basal plate, which is primarily
associated with motor control.

Spinal cord
 Sensory nerves with cell bodies in the dorsal root ganglion, enter the dorsal root
ganglion, enter the dorsal side of the spinal cord
 Somatic motor neurons that innervate skeletal muscle, have cell bodies in the ventral
horn of the spinal cord and their axons leave the cord in the ventral root
 Autonomic neurons that innervate cardiac and smooth muscle are positioned in
between

 The ventral half of the neural plate is controlled by the notochord, which acts as the
organiser
 Ventral neural tube is patterned by Sonic Hedgehog (sHg) from the notochord, which
acts as the inducing tissue
  Notochord, derived sHg signals to the floor plate, and induces sHg expression in the
floor plate
 Floor plate-derived sHg subsequently signals to other cells in the neural tube, and is
essential for proper specification of ventral neuron progenitor domains
 Loss of sHg from the notochord and/or floor plate prevents proper specification of
these progenitor domains
 sHg acts as a morphogen, it induces cell differentiation dependent on its concentration
 At low concentration, it forms ventral interneurons
 High concentration it induces motor neuron development
 Highest concentration it induces floor plate differentiation
 Failure of sHg modulated differentiation causes holoprosencephaly
 Dorsal neural tube is patterned by BMPs from the epidermal ectoderm flanking the
neural plate.
 These induce sensory interneurons by activating Sr/Thr kinases and altering SMAD
transcription factor levels

Homeobox Genes Direct anterior-posterior patterning: OTX, GBX in brain

 In response to Wnt gradient established in the neural plate, future forebrain and
midbrain cells express Otx while future hindbrain expresses Gbx
 Engrailed is expressed at midbrain-hindbrain boundary (MHB) which acts as an
organising centre (the isthmic organiser
 The homeodomain transcription factors regulate expression of other molecules
including fibroblast growth factor (FGF8) which is secreted by the organiser
 FGF diffuses into midbrain and hindbrain and in presence of different homeodomain
proteins induces different types of neurons

Signals from the Isthmic organiser pattern neurons in midbrain and hindbrain
 FGF diffuses from the isthmic organiser into midbrain and hindbrain
 In presence of Otx in the midbrain, dopaminergic neurons form
 In presence of Gbx in the hindbrain, serotonergic neurons form

Regionalisation of neural tube

  Four neural tube subdivisions each eventually develop into distinct regions of the
CNS by the division of neuroepithelial cells: the forebrain (prosencephalon), the
midbrain (mesencephalon), the hindbrain (rhombencephalon) and the spinal cord
 The prosencephalon further goes on to develop into the telencephalon (cerebrum)
and the diencephalon (the optic vesicles and hypothalamus)
 Mesencephalon stays as the midbrain,
 The rhombencephalon develops into the metencephalon (the pons and cerebellum
-- responsible for coordinating movements, posture and balance) and the
myelencephalon (the medulla oblongata -- whose neurons generate the nerves
that regulate respiratory, gastrointestinal and cardiovascular movements)

Homeobox Genes:
 Each hindbrain rhombomere and each region of the spinal cord expresses a different
complement of Hox genes
 Mutual repression between Hox genes, establishes and maintains expression patterns
 The hox expression pattern determines the specific type of neuron that develops in a
given region

Differentiation of spinal motor neurons

 Hox genes determine subtype of motorneuron/muscle innervated
 Lateral motor columns (LMC): within each LMC are multiple motor pools, each
destined to innervate a specific hindlimb or forelimb muscle. Hox genes regulate the
expression of additional transcription factors that distinguish one motor pool from
another
Section 1.2
Cell fate determination
 Signalling between cells involving two groups of genes
 Proneural genes: basic helix-loop-helix (bHLH) transcription factors: Achaete-scute
complex
 Neurogenic genes: surface ligand-receptor pair: delta (ligand) and notch (receptor) and
downstream bHLH transcription factors