CHAPTER 1

development is continually expanded and enhanced by rev- 5. In many species, the organism that hatches from the egg
olutions in microscopy, computer-aided graphical recon- or is born into the world is not sexually mature. Rather,
structions of three-dimensional objects, and methods of the organism needs to undergo metamorphosis to
applying mathematics to biology. Many of the beautiful become a sexually mature adult. In most animals, the
photographs in this book reflect this increasingly impor- young organism is a called a larva, and it may look sig-
tant component of embryology. nificantly different from the adult. In many species, the
larval stage is the one that lasts the longest, and is used
for feeding or dispersal. In such species, the adult is a
The Cycle of Life brief stage whose sole purpose is to reproduce. In silk-
One of the major triumphs of descriptive embryology was worm moths, for instance, the adults do not have
the idea of a generalizable animal life cycle. Each animal, mouthparts and cannot feed; the larvae must eat
whether earthworm or eagle, termite or beagle, passes enough so that the adult has the stored energy to sur-
through similar stages of development. The stages of devel- vive and mate. Indeed, most female moths mate as soon
opment between fertilization and hatching are collectively as they eclose from their pupa, and they fly only once—
called embryogenesis. to lay their eggs. Then they die.
Throughout the animal kingdom, an incredible variety 6. In many species, a group of cells is set aside to produce
of embryonic types exist, but most patterns of embryogen- the next generation (rather than forming the current
esis are variations on six fundamental processes: fertiliza- embryo). These cells are the precursors of the gametes.
tion, cleavage, gastrulation, organogenesis, metamorpho- The gametes and their precursor cells are collectively
sis, and gametogenesis. called germ cells, and they are set aside for reproduc-
tive function. All the other cells of the body are called
1. Fertilization involves the fusion of the mature sex cells, somatic cells. This separation of somatic cells (which
the sperm and egg, which are collectively called the give rise to the individual body) and germ cells (which
gametes. The fusion of the gamete cells stimulates the contribute to the formation of a new generation) is often
egg to begin development and initiates a new individ- one of the first differentiations to occur during animal
ual. The subsequent fusion of the gamete nuclei (both development. The germ cells eventually migrate to the
of which have only half the normal number of chromo- gonads, where they differentiate into gametes. The
somes characteristic for the species) gives the embryo development of gametes, called gametogenesis, is usu-
its genome, the collection of genes that helps instruct ally not completed until the organism has become phys-
the embryo to develop in a manner very similar to that ically mature. At maturity, the gametes may be released
of it parents. and participate in fertilization to begin a new embryo.
2. Cleavage is a series of extremely rapid mitotic divisions The adult organism eventually undergoes senescence
that immediately follow fertilization. During cleavage, and dies, its nutrients often supporting the early
the enormous volume of zygote cytoplasm is divided embryogenesis of its offspring and its absence allowing
into numerous smaller cells called blastomeres. By the less competition. Thus, the cycle of life is renewed.
end of cleavage, the blastomeres have usually formed
a sphere, known as a blastula.
3. After the rate of mitotic division slows down, the blas- A Frog's Life
tomeres undergo dramatic movements and change their
All animal life cycles are modifications of the generalized
positions relative to one another. This series of exten-
one described above. Figure 1.1 shows the development of
sive cell rearrangements is called gastrulation, and the
the leopard frog, Rana pipiens, and provides a good start-
embryo is said to be in the gastrula stage. As a result of
ing point for a more detailed discussion of a representa-
gastrulation, the embryo contains three germ layers that
tive life cycle.
will interact to generate the organs of the body.
4. Once the germ layers are established, the cells interact
with one another and rearrange themselves to produce Gametogenesis and fertilization
tissues and organs. This process is called organogene-
The end of one life cycle and the beginning of the next are
sis. Many organs contain cells from more than one germ
often intricately intertwined. Life cycles are often controlled
layer, and it is not unusual for the outside of an organ
by environmental factors (tadpoles wouldn't survive if
to be derived from one layer and the inside from anoth-
they hatched in the fall, when their food is dying), so in
er. For example, the outer layer of skin (epidermis)
most frogs, gametogenesis and fertilization are seasonal
comes from the ectoderm, whereas the inner layer (the
events. A combination of photoperiod (hours of daylight)
dermis) comes from the mesoderm. Also during organo-
and temperature informs the pituitary gland of the mature
genesis, certain cells undergo long migrations from their
female frog that it is spring. The pituitary then secretes hor-
place of origin to their final location. These migrating
mones that stimulate her ovary to make the hormone estro-
cells include the precursors of blood cells, lymph cells,
gen. Estrogen then instructs the liver to make and secrete
pigment cells, and sex cells.
yolk proteins, which are then transported through the
 U C V t L U n v i t l M A L AINAIUMY

: 1.1 Developmental history of the leopard frog, Rana

.The stages from fertilization through hatching (birth) are
collectively as embryogcnesis. The region set aside for pro-
- - :n cells is shown in purple. Cametogenesis, which is Fertilization accomplishes several things. First, it allows
fed in the sexually mature adult, begins at different times the haploid nucleus of the egg (the female pronucleus) to
development, depending on the species. (The sizes of the merge with the haploid nucleus of the sperm (the male
wed wedges shown here arc arbitrary and do not corre- pronucleus) to form the diploid zygote nucleus. Second,
> the proportion of the life cycle spent in each stage.) fertilization causes the cytoplasm of the egg to move such
that different parts of the cytoplasm find themselves in
new locations (Figure 1.2D). This cytoplasmic migration
i into the enlarging eggs in the ovary. The yolk is will be important in determining the three embryonic axes
: acted into the bottom portion of the egg, called the of the frog: anterior-posterior (head-tail), dorsal-ventral
al hemisphere, where it will serve as food for the (back-belly), and right-left. Third, fertilization activates
krping embryo (Figure 1.2A). The upper half of the egg those molecules necessary to begin cell cleavage and gas-
fed the animal hemisphere.* Sperm formation also trulation (Rugh 1950).
i on a seasonal basis. Male leopard frogs make sperm
I ; the summer, and by the time they begin hiberna-
r. the fall they have produced all the sperm that will Cleavage and gastrulation
iflable for the following spring's breeding season. During cleavage, the volume of the frog egg stays the same,
•ost species of frogs, fertilization is external. The but it is divided into tens of thousands of cells (Figure
rog grabs the female's back and fertilizes the eggs as 1.2E-H). The cells in the animal hemisphere of the egg
male releases them (Figure 1.2B). Some species lay divide faster than those in the vegetal hemisphere, and the
:eggs in pond vegetation, and the egg jelly adheres to cells of the vegetal hemisphere become progressively larg-
sr.ts and anchors the eggs (Figure 1.2C). Other species er the more vegetal the cytoplasm. Meanwhile, a fluid-filled
tr.eir eggs into the center of the pond without any cavity, the blastocoel, forms in the animal hemisphere (Fig-
DTL So the first important thing to remember about ure 1.21). This cavity will be important for allowing cell
fries is that they are often intimately involved with movements to occur during gastrulation.
xrrjnental factors. Gastrulation in the frog begins at a point on the embryo
surface roughly 180 degrees opposite the point of sperm
entry with the formation of a dimple, called the blastopore.
of the terms animal and vegetal for the upper and lower This dimple (which will mark the future dorsal side of the
; of the early frog embryo reflect the division rates of
The upper cells divide rapidly and become actively mobile embryo) expands to become a ring, and cells migrating
nimated"), while the yolk-filled cells of the lower half through the blastopore become the mesoderm (Figure
: as being immobile (hence like plants, or "vegetal"). 1.3A-C). The cells remaining on the outside become the ecto-
 CHAPTER 1

FIGURE 1.2 Early development of the frog Xenopus laevis. (A) As the egg matures, il
accumulates yolk (here stained yellow and green) in the vegetal cytoplasm. (B) Frogs
mate by amplexus, the male grasping the female around the belly and fertilizing the
eggs as they are released. (C) A newly laid clutch of eggs. The brown area of each egg
is the pigmented animal hemisphere. The while spot in the middle of the pigment is
where the egg's nucleus resides. (D) Cytoplasm rearrangement seen during first cleav-
age. Compare with the initial stage seen in (A). (E) A 2-cell embryo near the end of its
first cleavage. (F) An 8-cell embryo. (G) Early blastula. Note that the cells get smaller,
but the volume of the egg remains the same. (H) Late blastula. (I) Cross section of a
late blastula, showing the blastococl (cavity). (A-H courtesy of Michael Danilchik and
Kimberly Ray; I courtesy of J. Heasman.)

1
1
 DEVELOPMENTAL ANATOMY

FIGURE 1.3 Continued development of Xcnopus laevis. (A) Gastrulation begins with
an invagination, or slit, in the future dorsal (top) side of the embryo. (B)This slit, the dor-
sal blastopore lip, as seen from the ventral surface (bottom) of the embryo. (C)The slit
oecomes a circle, the blastopore. Future mesoderm cells migrate into the interior of the
embryo along the blastopore edges, and the ectoderm (future epidermis and nerves)
"ligrates down the outside of the embryo. The remaining part, the yolk-filled endoderm,
is eventually encircled. (D) Neural folds begin to form on the dorsal surface. (E) A
groove can be seen where the bottom of the neural tube will be. (F) The neural folds
come together at the dorsal midline, creating a neural tube. (G) Cross section of the
\enopus embryo at the neurula stage. (H) A pre-hatching tadpole, as the protrusions of
the forebrain begin to induce eyes to form. (I) A mature tadpole, having swum away
Tom the egg mass and feeding independently. (Courtesy of Michael Danilchik and
Kimberly Ray.)

Dorsal blastopore lip (C) Yolk plug Blastopore

H I

1
UufiH

(G) Dorsal
(back)
Notochord Neural tube
Somite

Epidermis Mesoderm
Neural groove Open neural tube (ectoderm) Ventral
(belly)

Somites
Brain

Gill area

Expansion of
forebrain to touch
surface ectoderm
(induces eyes to form)
Stomodeum (mouth) Tailbud
10 CHAPTER 1

-**»•
/ ' - • - ^ ^

FIGURE 1.4 Metamorphosis of the frog. (A) Huge changes are obvious when one con-
trasts the tadpole and the adult bullfrog. Note especially the differences in jaw structure
and limbs. (B) Premetamorphic tadpole. (C) Prometamorphic tadpole, showing hindlimb
growth. (D) Onset of metamorphic climax as forelimbs emerge. (E,F) Climax stages. ',-' i^>>
(A © Patrice CeiselA/isuals Unlimited.)

derm, and this outer layer expands to enclose the entire

embryo. The large, yolky cells that remain in the vegetal Organogenesis
hemisphere (until they are encircled by the expanding ecto- Organogenesis begins when the notochord—a rod of
derm) become the endoderm. Thus, at the end of gastrula- mesodermal cells in the most dorsal portion of the
tion, the ectoderm (precursor of the epidermis, brain, and embryo'"—signals the ectodermal cells above it that they
nerves) is on the outside of the embryo, the endoderm (pre-
cursor of the gut and respiratory systems) is on the inside
The notochord consists of cells such as those mentioned on p. 2 of
of the embryo, and the mesoderm (precursor of the connec- the Introduction—i.e., cells that are important for constructing the
tive tissue, blood, heart, skeleton, gonads, and kidneys) is embryo but which, having performed their tasks, die. Although
between them. adult vertebrates do not have notochords, this embryonic organ is
critical for establishing the fates of the ectodermal cells above it, as
we shall sec in Chapters 7-9.

Meiosis I: Separation of homologous chromosomes

Nuclear Homologous Homologous

envelope Chromatin chromosomes chromatids
Nucleus

Interphase Early prophase I Mid prophase I Late prophase I Metaphase I

DNA replicates The nuclear envelope breaks down and homologous chromosomes
(each chromosome being double, with the chromatids joined at the
kinetochore) align in pairs. Chromosomal rearrangements can occur
between the four homologous chromatids at this time
 DEVELOPMENTAL ANATOMY 11

are not going to become epidermis. Instead, these dorsal tongue muscle of the frog develops. Meanwhile, the tad-
ectoderm cells form a tube and become the nervous sys- pole's lengthy intestine—a characteristic of herbivores—
tem. At this stage, the embryo is called a neurula. The neu- shortens to suit the more carnivorous diet of the adult frog.
ral precursor cells elongate, stretch, and fold into the The gills regress and the lungs enlarge. The speed of meta-
embryo, forming the neural tube (Figure 1.3D-F); the morphosis is carefully keyed to environmental pressures.
future epidermal cells of the back cover the neural tube. In temperate regions, for instance, Rana metamorphosis
Once the neural tube has formed, it and the notochord must occur before ponds freeze in winter. An adult leop-
induce changes in their neighbors, and organogenesis con- ard frog can burrow into the mud and survive the winter;
tinues. The mesodermal tissue adjacent to the neural tube its tadpole cannot.
and notochord becomes segmented into somites (Figure As metamorphosis ends, the development of the germ
1.3G,H), the precursors of the frog's back muscles, spinal cells begins. Gametogenesis can take a long time. In Rana
vertebrae, and dermis (the inner portion of the skin). The pipiens, it takes 3 years for the eggs to mature in the
embryo develops a mouth and an anus, and it elongates female's ovaries. (Sperm take less time; Rana males are
into the familiar tadpole structure (Figure 1.31). The neu- often fertile soon after metamorphosis.) To become mature,
rons make their connections to the muscles and to other the germ cells must be competent to complete meiosis.
neurons, the gills form, and the larva is ready to hatch from Meiosis (Figure 1.5) is one of the most important evolu-
its egg jelly. The hatched tadpole will feed for itself as soon tionary processes characteristic of eukaryotic organisms.
as the yolk supplied by its mother is exhausted. It makes fertilization possible and is critical in recombin-
ing genes from the two parents. Genetics, development,
See VADE MECUM
and evolution throughout the animal kingdom are predi-
The amphibian life cycle
cated on meiosis. We will discuss meiosis more thorough-
ly in Chapter 16, but the most important things to remem-
ber about meiosis are:
Metamorphosis and gametogenesis
1. The chromosomes replicate prior to cell division, so that
Metamorphosis of the fully aquatic tadpole larva into an
each gene is represented four times.
adult frog that can live on land is one of the most striking
2. The replicated chromosomes (each called a chromatid)
transformations in all of biology. In amphibians, metamor-
are held together by their kinetochores (centromeres),
phosis is initiated by hormones from the tadpole's thyroid
and the four homologous chromatids pair together.
gland. (The mechanisms by which thyroid hormones
accomplish these changes will be discussed in Chapter 15.)
In frogs, almost every organ is subject to modification, and FIGURE 1.5 Summary of meiosis. The DNA replicates during
the resulting changes in form are striking and very obvi- interphase. During first meiotic prophase, the nuclear envelope
ous (Figure 1.4). The hmdlimbs and forelimbs the adult will breaks down and the homologous chromosomes (each chromo-
use for locomotion differentiate as the tadpole's paddle tail some is double, with its two chromatids joined at the kinetochore)
align together. Chromosome rearrangements ("crossing over") can
recedes. The cartilaginous tadpole skull is replaced by the occur at this stage. After the first metaphase, the kinetochore
predominantly bony skull of the young frog. The horny remains unsplit and the pairs of homologous chromosomes are
teeth the tadpole uses to tear up pond plants disappear as sorted into different cells. During the second meiotic division, the
the mouth and jaw take a new shape, and the fly-catching kinlochore splits and the sister chromatids are moved into sepa-
rate cells, each with a haploid set of chromosomes.

Meiosis II: Separation of the chromatids

Telophase I Anaphase II Telophase II

The two original homo- The kinetochore splits Each new cell has
logous chromosomes one copy of each
are segregated into chromosome
different cells
12 CHAPTER 1

3. The first meiotic division separates the chromatid pairs mammals—originate from eggs. Ex ovo omnia ("All from
from one another. the egg") was the motto on the frontispiece of Harvey's On
4. The second meiotic division splits the kinetochore such the Generation of Living Creatures, and this precluded the
that each chromatid becomes a chromosome. spontaneous generation of animals from mud or excre-
5. The result is four germ cells, each with a haploid ment. This statement was not made lightly, for Harvey
nucleus. knew that it went against the views of Aristotle, whom
Having undergone meiosis, the mature sperm and egg Harvey still venerated. (Aristotle had thought that men-
nuclei can unite in fertilization, restoring the diploid chro- strual fluid formed the material of the embryo, while the
mosome number and initiating the events that lead to semen gave it form and animation.) Harvey also was the
development and the continuation of the circle of life. first to see the blastoderm of the chick embryo (the small
region of the egg containing the yolk-free cytoplasm that
gives rise to the embryo), and he was the first to notice that
"How Are You?" "islands" of blood tissue form before the heart does. Har-
The fertilized egg has no heart. It has no eye. No limb is vey also suggested that the amniotic fluid might function
found in the zygote. So how did we become what we are? as a "shock absorber" for the embryo.
What part of the embryo forms the heart? How do the cells As might be expected, embryology remained little but
that form the eye's retina migrate the proper distance from speculation until the invention of the microscope allowed
the cells that form the lens? How do the tissues that form a detailed observations. In 1672, Marcello Malpighi pub-
bird's wing relate to the tissues that form fish fins or the lished the first microscopic account of chick development.
human hand? What organs are affected by mutations in Here, for the first time, the neural groove (precursor of the
particular genes? These are the types of questions asked neural tube), the muscle-forming somites, and the first cir-
by developmental anatomists. culation of the arteries and veins—to and from the yolk—
Several strands weave together to form the anatomical were identified (Figure 1.6).
approaches to development. The first strand is compara-
tive embryology, the study of how anatomy changes dur-
ing the development of different organisms. The second Epigenesis and preformation
strand, based on the first, is evolutionary embryology, the With Malpighi begins one of the great debates in embry-
study of how changes in development may cause evolu- ology: the controversy over whether the organs of the
tionary change and of how an organism's ancestry may embryo are formed dc novo ("from scratch") at each gen-
constrain the types of changes that are possible. The third eration, or whether the organs are already present, in
strand of the anatomical approach to developmental biol- miniature form, within the egg (or sperm). The first view,
ogy is teratology, the study of birth defects. called epigenesis, was supported by Aristotle and Harvey.
The second view, called preformation, was reinvigorated
with Malpighi's support. Malpighi showed that the unin-
Comparative embryology cubated* chick egg already had a great deal of structure,
The first known study of comparative developmental and this observation provided him with reasons to ques-
anatomy was undertaken by Aristotle in the fourth centu- tion epigenesis. According to the preformationist view, all
ry BCE. In The Generation of Animals (ca. 350 BCE), he noted the organs of the adult were prefigured in miniature with-
some of the variations on the life cycle themes: some ani- in the sperm or (more usually) the egg. Organisms were
mals are bom from eggs (oviparity, as in birds, frogs, and not seen to be "constructed" but rather "unrolled."
most invertebrates); some by live birth (viviparity, as in The preformationist hypothesis had the backing of eigh-
placental mammals); and some by producing an egg that teenth-century science, religion, and philosophy (Gould
hatches inside the body (ovoviviparity, as in certain rep- 1977; Roe 1981; Pinto-Correia 1997). First, if all organs were
tiles and sharks). Aristotle also identified the two major prefigured, embryonic development merely required the
cell division patterns by which embryos are formed: the growth of existing structures, not the formation of new
holoblastic pattern of cleavage (in which the entire egg is ones. No extra mysterious force was needed for embryon-
divided into smaller cells, as it is in frogs and mammals) ic development. Second, just as the adult organism was
and the meroblastic pattern of cleavage (as in chicks, prefigured in the germ cells, another generation already
wherein only part of the egg is destined to become the existed in a prefigured state within the germ cells of the
embryo, while the other portion—the yolk—serves as first prefigured generation. This corollary, called embdit-
nutrition for the embryo). And should anyone want to ment (encapsulation), ensured that the species would
know who first figured out the functions of the placenta
and the umbilical cord, it was Aristotle. "As pointed out by Maitre-Jan in 1722, the eggs Malpighi examined
There was remarkably little progress in embryology for may technically be called "unincubated," but as they were left sit-
the two thousand years following Aristotle. It was only in ting in the Bolognese sun in August, they were not unheated. Such
1651 that William Harvey concluded that all animals—even eggs would be expected to have developed into chicks.
 DEVELOPMENTAL ANATOMY 13

(A) (B)

% V-J

(Q

(D) Auditory Myelencephalon Metencephalon

vesicle

Aortic arches

Atrium Mesencephalon
Heart <
Truncus Diencephalon
arterious
Choroid fissure
Ventricle Lens r e
Sensory retina r 7
FIGURE 1.6 Depictions of chick developmental anatomy. (A) Liver Pigmented retina
Dorsal view (looking "down" at what will become the back) of a rudiment
2-day chick embryo, as depicted by Marcello Malpighi in 1672. Olfactory pit
(B) Ventral view (looking "up" al the prospective belly) of a chick Forelimb Telencephalon
embryo at a similar stage, seen through a dissecting microscope bud
and rendered by F. R. Lillie in 1908. (C) Eduard d'Alton's depic- Vitelline vein
tion of a later stage 2-day chick embryo in Pander (1817). (D) Somite
Modern rendering of a 3-day chick embryo. Details of the anato- Vitelline artery
my will be discussed in later chapters. (A from Malpighi 1672; Hindlimb
B from Lillie 1908; C from Pander 1817, courtesy of Ernst Mayr bud
Library of the Museum of Comparative Zoology, Harvard; D after
Carlson 1981.)

cell theory arose in the mid-1800s), nor did they view

mankind's tenure on Earth as potentially infinite- Rather,
remain constant. Although certain microscopists claimed said Bonnet (1764), "Nature works as small as it wishes,"
to see fully formed human miniatures within the sperm or and the human species existed in that finite time between
egg, the major proponents of this hypothesis—Albrecht Creation and Resurrection. This view was in accord with
von HaJler and Charles Bonnet—knew that organ systems the best science of its time, conforming to the French math-
develop at different rates, and that structures need not be ematician-philosopher Rene Descartes' principle of the infi-
in the same place in the embryo as they are in the newborn. nite divisibility of a mechanical nature initiated, but not
The preformationists had no cell theory to provide a interfered with, by God. It also conformed to Enlighten-
lower limit to the size of their preformed organisms (the ment views of the Deity. The scientist-priest Nicolas Male-
14 CHAPTER 1

branche saw in preformationism the fusion of the rule-giv- instructions for forming the organism are already -.- --,. -
ing God of Christianity with Cartesian science (Churchill in the fertilized egg.*
1991; Pinto-Correia 1997).*
The embryological case for epigenesis w a s revived at
Naming the parts: The primary germ layers
the same time by Kaspar Friedrich Wolff, a German embry-
ologist working in St. Petersburg. By carefully observing and early organs
the d e v e l o p m e n t of chick embryos, Wolff demonstrate d The end of preformationism did not come until the 152/s
that the embryonic parts develop from tissues that have w h e n a combination of new staining techniques, impnn
n o counterpart in the adult organism. The heart and blood microscopes, a n d institutional reforms in German uni
vessels (which, according to preformationism, h a d to b e sities created a revolution in descriptive embryology,
present from the beginning to ensure embryonic growth) n e w techniques enabled microscopists to d o c u m e n t
could b e seen t o develop a n e w in each embryo. Similarly, epigenesis of anatomical structures, a n d the i n s t i t u t k r
the intestinal tube w a s seen to arise by the folding of an reforms provided audiences for these reports and stiiders
originally flat tissue. This latter observation was explicitly to carry on the work of their teachers. Among the most taK
detailed by Wolff, w h o proclaimed in 1767 that "When the
ented of this new g r o u p of microscopically inclined n i
formation of the intestine in this m a n n e r has b e e n duly
tigators were three friends, b o r n withi n a year of eac
w e i g h e d , almost n o d o u b t can remain , I believe, of the
other, all of w h o m came from the Baltic region and s t a w
truth of epigenesis." To explain h o w an organism is creat-
ied in northern Germany. The work of Christian Panda
ed anew each generation, however, Wolff had to postulate
an u n k n o w n force—the vis essentialis ("essential force")— Karl Ernst v o n Baer, a n d Heinrich Rathke t r a n s f o n s
which, acting according to natural laws in the same w a y embryology into a specialized branch of science.
as gravity or m a g n e t i s m , w o u l d organize embryonic Pander studied the chick embryo for less than tw:
development. (before becoming a paleontologist), b u t in those 15 mor_i~
h e discovered the ger m layers + —three distinct regions if
A reconciliation between preformationism a n d epigen- the embryo that give rise to the differentiated celL
esis w a s attempted by the German philosopher Immanuel a n d specific organ systems (Figure 1.7).
Kant (1724-1804) a n d his colleague, biologist J o h a n n • The ectoderm generates the outer layer of the e m b r r t
Friedrich Blumenbach (1752-1840). Attempting to construct It produces the surface layer (epidermis) of the skin a
a scientific theory of racial descent, Blumenbach postulat- forms the brain a n d nervou s system.
ed a mechanical, goal-directed force h e called Bildungstrieb
• The e n d o d e r m becomes the innermost layer of ti
("developmental force")- Such a force, he said, was not the-
embryo and p r o d u c e s the epithelium of the digest*
oretical, b u t could b e shown to exist b y experimentation.
tube a n d its associated organs (including the lungs).
A hydra, when cut, regenerates its amputated parts by rear-
ranging existing elements (see Chapter 15). Some purpose- • The mesoderm becomes sandwiched between th e e d o
ful organizing force could be observed in operation, and derm and endoderm. It generates the blood, heart, k i i -
this Bildungstrieb w a s a p r o p e r t y of the organism itself, ney, gonads, bones, muscles, a n d connective tissues.
t h o u g h t to be inherited t h r o u g h the g e rm cells. Thus,
These three layers are found in the e m b r v o s of t
development could proceed through a predetermined force
triploblastic ("three-layer") animals. Some phyla, such,
inherent in the matter of the embryo (Cassirer 1950; Lenoir
the poriferans (sponges) and ctenophores (comb jeffies)
1980). Moreover, this force was believed to be susceptible to
lack a true m e s o d e r m a n d are considered d i p l o b l a s t i r
change, as demonstrated by the left-handed variant of snail
coiling (where left-coiled snails can p r o d u c e right-coiled animals.
progeny). In this hypothesis, wherein epigenetic develop- Pander and Rathke also m a d e observations that weigh*
ment is directed b y preformed instructions, we are not far ed the balance in favor of epigenesis. Rathke follow e i I
from the v i e w held by m o d e r n biologists that most of the intricate development of the vertebrate skull, excr;:;:
terns, a n d respiratory systems, showing that these betas
increasingly complex. He also showed that their compie
ity took on different trajectories in different classes of TI
''Preformation was a conservative theory, emphasizing the lack of
tebrates. For instance, Rathke was the first to idenfcr
change between generations. Its principal failure was its inability to
account for the variations revealed by the limited genetic evidence
of the time. It was known, for instance, that matings between white
and black parents produced children of intermediate skin color, an *But, as we shall see, not all the instructions there. Late in iias
impossibility if inheritance and development were solely through book, we will see that temperature, diet, predators, symbionE,
either the sperm or the egg. In more controlled experiments, the crowding, and other environmental agents normally regulate
German botanist Joseph Kolreutcr (1766) produced hybrid tobacco expression in the embryo and can cause particular phenoc-T -;
plants having the characteristics of both species. Moreover, by mat- occur.
ing the hybrid to either the male or female parent, Kolrcuter was +
From the same root as germination, the Latin germen mearts
able to "revert" the hybrid back to one or the other parental type "sprout" or "bud." The names of the three germ layers a t:
after several generations. Thus, inheritance seemed to arise from a
Greek: ectoderm from ektos ("outside") plus derma ("skin"k
mixture of parental components.
derm from mesos ("middle"); and endoderm from endtm
 DEVELOPMENTAL ANATOMY 15

Zygote Blastula Gastrula

r
Ectoderm (outer layer) Mesoderm (middle layer)
J.
Endoderm (internal layer) Germ cells

Central Respir-
Outer nervous Neural Digestive atory
surface system crest Dorsal Paraxial Intermediate Lateral Head tube Pharynx tube Male Female

0gk
Epidermal Neuron Pigment Noto- Bone Tubule Red Facial Stomach Thvroid Lung
cells of skin of cell chord tissue cell of the blood muscle cell cell cell
brain (melan- kidney cells (alveolar
ocyte) cell)

FIGURE 1.7 The dividing cells of the fertilized egg form three
pharyngeal arches (Figure 1.8). He showed that these same
distinct embryonic germ layers. Each of the germ layers gives rise
to myriad differentiated cell types (only a few representatives are embryonic structures became gill supports in fish and the
shown here) and distinct organ systems. The germ cells (precur- jaws and ears (among other things) in mammals. Pander
sors of the sperm and egg) are set aside early in development and demonstrated that the germ layers did not form their
do not arise from any particular germ layer. respective organs autonomously (Pander 1817). Rather,
each germ layer "is not yet independent enough to indi-
cate what it truly is; it still needs the help of its sister trav-
elers, and therefore, although already designated for dif-
ferent ends, all three influence each other collectively until
each has reached an appropriate level." Pander had dis-

(B)
Upper jaw Braincase Gill arches

FIGURE 1.8 Evolution of pharyngeal

arch structures in the vertebrate head. (A)
Pharyngeal arches (also called branchial
Hvomandibula
arches) in the embryo of the salamander
Ambystoma mexicanum. The surface ecto-
derm has been removed to permit visuali-
(C) (D) Squamosal zation of the arches (highlighted in color)
Squamosal (temporal bone) as they form. (B) In adult fish, pharyngeal
arch cells form the hyomandibular jaws
Nasal Maxilla Quadrate and gill arches. (C) In amphibians, birds,
Nasal and reptiles (a crocodile is shown here),
Premaxilla these same cells form the quadrate bone
of the upper jaw and the articular bone of
the lower jaw. (D) In mammals, the qua-
drate has become internalized and forms
the incus of the middle car. The articular
Middle ear bone retains its contact with the quadrate,
(incus, becoming the malleus of the middle ear.
Dentary Articular malleus) (A courtesy of P. Falck and L. Olsson; B-D
Maxilla Mandible after Zangerl and Williams 1975.)
16 CHAPTER 1

Ectoderm of head and left halves and which instructs the ectoderm above it
to become the nervous system (Figure 1.9). He also discov-
Area pellucida ered the mammalian egg, that long-sought cell that every-
one believed existed but no one before von Baer had ever
seen.*
In 1828, von FJaer reported, "I have two small embrj'os
preserved in alcohol, that I forgot to label. At present I am
unable to determine the genus to which they belong. They
may be lizards, small birds, or even mammals." Figure 1.10
allows us to appreciate his quandary. All vertebrate
Unsegmented embryos (fish, reptiles, amphibians, birds, and mammals)
mesoderm begin with a basically similar structure. From his detailed
study of chick development and his comparison of chick
embryos with the embryos of other vertebrates, von Baer
Hensen's
node derived four generalizations. Now often referred to as "von
Baer's laws," they are stated here with some vertebrate
Primitive examples.
streak
1. The general features of a large group of animals appear
earlier in development than do the specialized features of a
smaller group. All developing vertebrates appear very
Somite (source of Neural groove
muscles, spine, ribs) similar right after gastrulation. It is only later in devel-
Epidermal opment that the special features of class, order, and
ectoderm

*von Baer could hardly believe that he had at last found what so
many others—Harvey, de Graaf, von Haller, Prevost, Dumas, and
even Purkinje—had searched for and failed to find. "I recoiled as if
struck by lightening ... I had to try to relax a while before I could
work up enough courage to look again, as I was afraid I had been
deluded by a phantom. Is it not strange that a sight which is expect-
Lateral plate" Intermediate Notochord F.ndoderm ed, and indeed hoped for, should be frightening when it eventually
mesoderm mesoderm (gut, lungs) materializes?"
(source of (source of
heart, blood kidneys, gonads)
vessels)
FIGURE 1.9 Notochord in chick development. The notochord
separates vertebrate embryos into right and left halves and
instructs the ectoderm above it to become the nervous system.
(A) Dorsal view of the 24-hour chick embryo. (B) Cross section
through the trunk region shows the notochord and developing
neural tube. By comparing this illustration and Figure 1.6, you
can see the remarkable changes between days 1, 2, and 3 of
chick egg incubation. (A after Patten 1951.)

covered the tissue interactions that we now call induction.

No tissue is able to construct organs by itself; it must inter-
act with other tissues. (We will discuss the principles of
induction more thoroughly in Chapter 3.) Thus, Pander
showed that preformation could not be true, since the
organs come into being through interactions between sim- Human Opossum Chicken Salamander Fish
pler structures. (axolotl) (gar)
FIGURE 1.10 Similarities and differences among vertebrate
The four principles of Karl Ernst von Baer embryos as they proceed through development. Each species'
embryos begin with a basically similar structure, although they
Karl Ernst von Baer extended Pander's studies of the chick acquire this structure at different ages and sizes. As they develop,
embryo. He discovered the notochord, the rod of dor- the species become less like each other. (Adapted from Richard-
salmost mesoderm that separates the embryo into right son ct al. 1998; photograph courtesy of M. Richardson.)
 DEVELOPMENTAL ANATOMY 17

finally species emerge. All vertebrate embryos have gill Keeping Track of Moving Cells:
arches, a notochord, a spinal cord, and primitive kid-
neys.
Fate Maps and Cell Lineages
2. Less general characters develop from the more general, until By the late 1800s, the cell had been conclusively demon-
finally the most specialized appear. All vertebrates initial- strated to be the basis for anatomy and physiology. Embry-
have the same type of skin. Only later does the skin ologists, too, began to base their field on the cell. But unlike
A: velop fish scales, reptilian scales, bird feathers, or the those who studied the adult, developmental anatomist
ir, claws, and nails of mammals. Similarly, the early found that cells do not stay still in the embryo. Indeed, one of
development of limbs is essentially the same in all ver- the most important conclusions of developmental
tebrates. Only later do the differences between legs, anatomists is that embryonic cells do not remain in one
wings, and arms become apparent. place, nor do they keep the same shape (Larsen and
McLaughlin 1987).
3. The embryo of a given specks, instead of passing through the
adult stages of lower animals, departs more and more from Early embryologists recognized that there are two major
diem* The visceral clefts of embryonic birds and mam- types of cells in the embryo: epithelial cells, which are
mals do not resemble the gill slits of adult fish in detail. tightly connected to one another in sheets or tubes; and
Rather, they resemble the visceral clefts of embryonic fish mesenchymal cells, which are unconnected to one anoth-
and other embryonic vertebrates. Whereas fish preserve er and operate as independent units. Morphogenesis is
and elaborate these clefts into true gill slits, mammals brought about through a limited repertoire of variations in
convert them into structures such as the eustachian cellular processes within these two types of arrangements
rubes (between the ear and mouth). (Table 1.1):
4. Therefore, the early embryo of a higher animal is never like a • Direction and number of cell divisions. Think of the faces of
lower animal, but only like its early embryo. Human two dog breeds—say, a German shepherd and a poodle.
embryos never pass through a stage equivalent to an The faces are made from the same cell types, but the
adult fish or bird. Rather, human embryos initially share number and orientation of the cell divisions are differ-
characteristics in common with fish and avian embryos. ent. Think also of the legs of a German shepherd com-
Later, the mammalian and other embryos diverge, none pared with those of a dachshund. The skeleton-forming
of them passing through the stages of the others. cells of the dachshund have undergone fewer cell divi-
sions than those of taller dogs (see Figure 1.21).
nxi Baer also recognized that there is a common pattern
to all vertebrate development: each of the three germ lay- • Cell shape changes. Cell shape change is a critical part of
s ; :;nerally gives rise to the same organs, whether the not only of development but also of cancer. In develop-
rrganism itself is a fish, a frog, or a chick. ment, change in the shapes of epithelial cells often cre-
Comparative embryonic anatomy remains an active ates tubes out of sheets (as when the neural tube forms);
field of research today, although it is now done in an evo- and a shape change from epithelial to mesenchymal is
:: nary context. What embryonic interactions, for critical when cells migrate away from the epithelium (as
sistance, cover the squirrel's tail with fur but provide scales when muscle cells are formed). This same type of epithe-
r. the rat's tail? The author's own research concerns how lial-to-mesenchymal change allows cancer cells to
-uriles get their shells—a skeletal feature generally com- migrate and spread from the primary tumor to new sites.
rvsed of 59 bones that no other vertebrate possesses. What • Cell movement. Cell migration is critical to get cells to their
I: • c relationship of these 59 bones to the bones found in appropriate places. The germ cells have to migrate into
Iligators and prehistoric marine reptiles? What changes the developing gonad, and the primordial heart cells
• :he "typical" development of the vertebrate skeleton meet in the middle of the vertebrate neck and then
allowed these unique bones to form? Jack Horner and migrate to the left part of the chest.
Hans Larsson are looking at the similarities between the • Cell growth. Cells can change in size. This is most appar-
developmental anatomy of chick and dinosaur embryos ent in the germ cells: the sperm eliminates most of its
and have found that the embryonic chick, unlike the cytoplasm and becomes smaller, whereas the develop-
dinosaur, regresses its tail. They are conducting experi- ing egg conserves and adds cytoplasm, becoming com-
ments to block this regression, and actually hope to obtain paratively huge. Many cells undergo an "asymmetric"
a chick that more closely resembles its dinosaur ancestors cell division that produces one big cell and one small
Homer and Gorman 2009).
cell, each of which may have a completely different fate.
• Cell death. Death is a critical part of life. The cells that in
the womb constitute the webbing between our toes and
fingers die before we are bom. So do the cells of our tails.
von Baer formulated these generalizations prior to Darwin's theo- The orifices of our mouth, anus, and reproductive glands
-' evolution. "Lower animals" would be those having simpler all form through cells dying at particular times and
sr-atotnies. places.
18 CHAPTER 1

TABLE 1.1 Summary of major morphogenic processes regulated by mesenchymal and epithelial cells
Process Action Morphology Example

MESENCHYMAL CELLS
Condensation Mesenchyme becomes Cartilage mesenchyme
epithelium

Cell division Mitosis produces more

cells (hyperplasia) <*V Limb mesenchyme

Cell death Cells die

^
£* Interdigital
rrf". mesenchyme

Migration Cells move at particular Heart mesenchyme

times and places
cw \ /*•

Matrix secretion and Synthesis or removal of Cartilage mesenchyme

degradation extracellular layer •ss^

Growth Cells get larger -SET Fat cells

(hypertrophy)

EPITHELIAL CELLS
Epithelium becomes <3>|j • Qja Mullerian duct
mesenchyme (entire Sj%<3J degeneration
Dispersal structure) v ,3 ®>
Epithelium becomes
mesenchyme (part of " Chick hypoblast
Delamination
structure)

Shape change or growth Cells remain attached as Neurulation

morphology is altered
EEEEE

Cell migration (intercalation) Rows of epithclia merge

to form fewer rows '133 Vertebrate gastrulation

Cell division Mitosis within row

or column CZCHXZJ C 3 G S Z E 0 3 Vertebrate gastrulation

Matrix secretion and Synthesis or removal :'..::' Vertebrate organ

degradation of extracellular matrix formation

Migration Formation of free edges J- Chick ectoderm

Changes in the composition of the cell membrane or secreted allow the migration of their neighboring cells. Extracel-
products. Cell membranes and secreted cell products lular matrices made by other cell types will prohibit the
influence the behavior of neighboring cells. For instance, migration of the same set of cells. In this way, "paths and
extracellular matrices secreted by one set of cells will guiderails" are established for migrating cells.
 DEVELOPMENTAL GENETICS 35

(B) FIGURE 2.3 Nucleosome and chromatin struc-

ture. (A) Model of nucleosome structure as seen
Nucleosome by X-ray crystallography at a resolution of 1.9 A.
Histone Histones H2A and H2B are yellow and red,
octamer respectively; H3 is purple and H4 is green. The
DNA helix (gray) winds around the protein core.
The histone "tails" that extend from the core are
HI the sites of acetylation and methylation, which
histones may disrupt or stabilize, respectively, the forma-
tion of nucleosome assemblages. (B) Histone HI
DNA "wrap" Linker DNA can draw nucleosomes together into compact
forms. About 140 base pairs of DNA encircle each
histone octamer, and about 60 base pairs of DNA
link the nucleosomes together. (C) Model for the
arrangement of nucleosomes in the highly com-
pacted solenoidal chromatin structure. Histone
(C) "tails" protruding from the nucleosome subunits
Histone core Histone octamer allow for the attachment of chemical groups. (D)
DNA of nucleosome
Methyl groups condense nucleosomes more tight-
ly, preventing access to promoter sites and thus
preventing gene transcription. Acetyla-
tion loosens nucleosome packing,
exposing the DNA to RNA poly-
merase and transcription fac-
H2B tail tors lhat will activate the
genes. (A after Davev et al.
2002)
H2Atail

H3 tail

H4 tail

H4tail
H3 tail
(D)
Condensed nucleosomes:
Histone tails largely methylated

> Methyl groups

bind H3 and H4

Uncondensed nucleosomes:
Histone tails largely unmethylated Acetyl groups
and acetylated bind H2, H3 and H4

genes is that eukaryotic genes are contained within a com-

Differential Gene Transcription plex of DNA and protein called chromatin. The protein
So how does the same genome give rise to different cell component constitutes about half the weight of chromatin
types? To understand this, one needs to understand the and is composed largely of histones. The nucleosome is
anatomy of the genes. One of the fundamental differences the basic unit of chromatin structure (Figure 2.3). It is com-
distinguishing most eukaryotic genes from prokaryotic posed of an octamer of histone proteins (two molecules
36 CHAPTER 2

Transcriptional Silent FIGURE 2.4 Histone melhylations on his-

elongation heterochromatin tone H3. The tail of histone H3 (its amino-
most sequence, at the beginning of the pro-
Transcriptional Transcriptional
tein) sticks out from the nucleosome and is
memory activationcapable of being methylated or acetylatcd.
Historic
octamer Here, lysines can be methylated and recog-
nized by particular proteins. Methylated lysine
residues at positions 4, 38, and 79 are associ-
DNA J EAFT) fpC^J (^HPl^ (BPTT ated with gene activation, whereas methylated
lysines at positions 9 and 27 are associated
27 4 H3 tail with repression. The proteins binding these
sites (not shown to scale) are represented
above the methyl group. (After Kouzarides and
Berger 2007.)

Enzymes known as histone acetyltransferases place acetyl

groups on histones (especially on lysines in H3 and H4),
destabilizing the nucleosomes so that they come apart eas-
ily. As might be expected, then, enzymes that remove acetyl
each of histones H2A, H2B, H3, and H4) wrapped with groups—histone deacetylases—stabilize the nucleosomes
two loops containing approximately 140 base pairs of and prevent transcription.
DNA (Kornberg and Thomas 1974). Histone HI is bound Histone methylation, the addition of methyl groups to
to the 60 or so base pairs of "linker" DNA between the histones by histone methyltransferases, can either acti-
nucleosomes (Weintraub 1984). There are 14 points of con- vate or further repress transcription, depending on the
tact between the DNA and the histones (Luger et al. 1997). amino acid being methylated and the presence of other
methyl or acetyl groups in the vicinity (see Strahl and Allis
2000; Cosgrove et al. 2004). For instance, acetylation of the
Anatomy of the gene: Active and
"tails" of H3 and H4 along with methylation of the lysine
repressed chromatin at position 4 of H3 (i.e., H3K4; remember that K is the
Whereas classical geneticists have likened genes to "beads abbreviation for lysine) is usually associated with active-
on a string," molecular geneticists liken genes to "string ly transcribed chromatin. In contrast, a combined lack of
on the beads," an image in which the beads are nucleo- acetylation of the H3 and H4 tails and methylation of the
somes. Most of the time, the nucleosomes are wound into lysine in the ninth position of H3 (H3K9) is usually asso-
tight "solenoids" that are stabilized by histone HI (Figure ciated with highly repressed chromatin (Norma et al. 2001).
2.3C). This Hl-dependent conformation of nucleosomes Indeed, lysine methylations at H3K9, H327, and H4K20 are
inhibits the transcription of genes in somatic cells by pack- often associated with highly repressed chromatin. Figure
ing adjacent nucleosomes together into tight arrays that 2.4 shows a schematic drawing of a nucleosome, with the
prevent transcription factors and RNA polymerases from histone H3 tail having on it some residues whose modifica-
gaining access to the genes (Thoma et al. 1979; Schlissel tion can regulate transcription.
and Brown 1984). It is generally thought, then, that the As might be expected, if methyl groups at specific places
"default" condition of chromatin is a repressed state, and on the histones repress transcription, then getting rid of
that tissue-specific genes become activated by local inter- these methyl moieties should permit transcription. This
ruption of this repression (Weintraub 1985). has been shown in the activation of those genes responsi-
ble for specifying the posterior halves of vertebrate bod-
HISTONES AS AN ACTIVATION SWITCH The histones are crit- ies. These genes, called Hox genes, encode transcription
ical because they are responsible for maintaining the factors that are critical in giving cells their identities along
repression of gene expression. This repression can be local- the anterior-posterior axis. In early development, Hox
ly strengthened (so that it becomes very difficult to tran- genes are repressed by H3K27 trimethylation (the lysine
scribe those genes in the nucleosomes) or relieved (so that at position 27 having three methyl groups). However, in
transcribing them becomes relatively easy) by modifying differentiated cells, a demethylase that is specific for
the histones (Figure 2.3D). Repression and activation are H3K27me3 is recruited to these promoters and enables the
controlled to a large extent by modifying the tails of his- gene to be transcribed (Agger et al. 2007; Lan et al. 2007).
tones H3 and H4 with two small organic groups: methyl The effects of methylation in controlling gene transcrip-
(CH3) and acetyl (COCH3) residues. In general, histone tion are extensive. So far, we have documented transcrip-
acetylation—the addition of negatively charged acetyl tional regulation by histone methylation. Later in this chap-
groups to histones—neutralizes the basic charge of h/sine ter we will discuss the exciting research on the control of
and loosens the histones. This activates transcription. transcription by DNA methylation.
 DEVELOPMENTAL GENETICS 37

HISTONE REGULATION OF TRANSCRIPTIONAL ELONGATION lation is the 5' untranslated region, often called the 5'
In addition to regulating the initiation of the transcription- UTR or leader sequence. The 5' UTR can determine the
al complex (i.e., getting RNA polymerase on the promot- rate at which translation is initiated.
er), nucleosomes also appear to regulate the progression • The first exon, which contains 90 base pairs coding for
of RNA polymerase and the elongation of the mRNA. amino acids 1-30 of human P-globin protein.
Indeed, recent evidence suggests that it is relatively com-
• An intron containing 130 base pairs with no coding
mon for RNA polymerase to be poised at the promoters,
sequences for p-globin. However, the structure of this
ready to go. For transcription to occur, these nucleosomes
intron is important in enabling the RNA to be processed
need to be modified, and it is possible that the acetylation
into mRNA and exit from the nucleus.
of histone H3 at positions 9 and 14, coupled with the
trimethylation of that histone at position 4, is critical for • An exon containing 222 base pairs coding for amino
allowing elongation of the message (Guenther et al. 2007; acids 31-104.
Li et al. 2007). • A large intron—850 base pairs—having nothing to do
with globin protein structure.
• An exon containing 126 base pairs coding for amino
Anatomy of the gene: Exons and introns acids 105-146 of the protein.
The second difference between prokaryotic and eukaryot- • A translation termination codon, TAA. This codon
ic genes is that eukaryotic genes are not co-linear with their becomes UAA in the mRNA. The ribosome dissociates
peptide products. Rather, the single nucleic acid strand of at this codon, and the protein is released.
eukaryotic mRNA comes from noncontiguous regions on • A 3' untranslated region (3' UTR) that, although tran-
the chromosome. Between exons—the regions of DNA that scribed, is not translated into protein. This region
code for a protein*—are intervening sequences called includes the sequence AATAAA, which is needed for
introns that have nothing whatsoever to do with the amino polyadenylation, the insertion of a "tail" of some
acid sequence of the protein. The structure of a typical 200-300 adenylate residues on the RNA transcript, about
eukaryotic gene can be illustrated by the human p-globin 20 bases downstream of the AAUAAA sequence. This
gene (Figure 2.5). This gene, which encodes part of the poly A tail (1) confers stability on the mRNA, (2) allows
hemoglobin protein of the red blood cells, consists of the the mRNA to exit the nucleus, and (3) permits the
following elements: mRNA to be translated into protein.
• A promoter region, which is responsible for the binding • A transcription termination sequence. Transcription
of RNA polymerase and for the subsequent initiation of continues beyond the AATAAA site for about 1000
transcription. The promoter region of the human p-glo- nucleotides before being terminated.
bin gene has three distinct units and extends from 95 to
26 base pairs before ("upstream from")* the transcrip- The original transcription product is called nuclear RNA
tion initiation site (i.e., from -95 to -26). (nRNA), sometimes called heterogeneous nuclear RNA
(hnRNA) or pre-messenger RNA (pre-mRNA). Nuclear RNA
• The transcription initiation site, which for human P-glo- contains the cap sequence, the 5' UTR, exons, introns, and
bin is ACATTTG. This site is often called the cap the 3' UTR (Figure 2.6). Both ends of these transcripts are
sequence because it represents the 5' end of the RNA, modified before these RNAs leave the nucleus. A cap con-
which will receive a "cap" of modified nucleotides soon sisting of methylated guanosine is placed on the 5' end of
after it is transcribed. The specific cap sequence varies
the RNA in opposite polarity to the RNA itself. This means
among genes.
there is no free 5' phosphate group on the nRNA. The 5'
• The translation initiation site, ATG. This codon (which cap is necessary for the binding of mRNA to the ribosome
becomes AUG in mRNA) is located 50 base pairs after and for subsequent translation (Shatkin 1976). The 3' ter-
the transcription initiation site in the human P-globin minus is usually modified in the nucleus by the addition
gene (although this distance differs greatly among dif- of a polyA tail. The adenylate residues in this tail are put
ferent genes). The sequence of 50 base pairs intervening together enzymatically and are added to the transcript;
between the initiation points of transcription and trans- they are not part of the gene sequence. Both the 5' and 3'
modifications may protect the mRNA from exonucleases
that would otherwise digest it (Sheiness and Darnell 1973;
Gedamu and Dixon 1978). The modifications thus stabi-
"Tho term exon refers to a nucleotide sequence whose RNA "exits" lize the message and its precursor.
the nucleus. It has taken on the functional definition of a protein-
encoding nucleotide sequence. Leader sequences and 3' UTR As the nRNA leaves the nucleus, its introns are removed
sequences are also derived from exons, even though they are not and the remaining exons spliced together. In this way the
translated into protein. coding regions of the mRNA—i.e., the exons—are brought
' By convention, upstream, downstream, 5', and 3' directions are together to form a single transcript, and this transcript is
specified in relation to the RNA. Thus, the promoter is upstream of translated into a protein. The protein can be further mod-
the gene, near its 5' end. ified to make it functional (see Figure 2.6).