Download Biology 131 Outline of lectures on animal development and Problem

Biology 131 Outline of lectures on animal development and Problem Set 6
In the study of development, we wish to understand how the complexity of the adult
multicellular animal emerges from the relative simplicity of the egg. It has long been established
that that the degree of preformation of the embryo in the egg is slight; the pattern of the embryo
(and the adult) develops gradually by the process of epigenesis.
I. Fertilization
Development is a cyclical process. Adult organisms, when reproducing sexually, make
haploid gametes (egg and sperm) that unite to form a fertilized egg, the zygote. The zygote then
undergoes a series of mitotic cell divisions to form a blastula, which then gastrulates to form the
three layered embryo. Through the process of epigenesis, the form of the embryo gradually
emerges, until an adult finally appears. In many cases, the adult form is preceded by a freeliving larval form, such as the tadpoles of amphibians. In other cases, such as mammals, the
adult develops directly from the embryo.
We first considered how the gametes arise. Early in embryonic development, a special
group of cells, the germ line cells, are set aside. These cells are already fated to become the
gametes of the future adult. The processes of spermatogenesis and oogenesis, each involving
meiosis, lead to the formation of sperm and eggs, respectively in the gonads. The female gamete
is called an oocyte before it is fertilizable and an egg after it becomes fertilizable.
The fertilization process in sea urchins begins when the eggs and sperm are released into
the sea water and the sperm sense a chemical signal that attracts them to the eggs. When a sperm
encounters the egg jelly, it triggers the acrosome reaction, which involves the fusion of the
acrosomal vesicle with the sperm’s plasma membrane and the release of its contents to the
extracellular space, the polymerization of the pool of G-actin into filamentous f-actin, and the
extension of the acrosomal process. This process occurs in many sperm cells that are attracted to
a single egg as they all vie to become the one that fertilizes the egg. Even if thousands of sperm
contact the outer coats of an egg, only one actually fuses with the egg and fertilizes it. If more
than one sperm fertilizes the egg, that condition is called polyspermy and is lethal in sea urchins,
as well as in many other (but not all) species.
The contents of the acrosomal vesicle include enzymes that digest the extracellular layers
around the egg and thus allow the membrane at the tip of the acrosome to come in direct contact
with the egg’s plasma membrane. The close apposition of the two membranes leads to their
fusion, so that the sperm and egg plasma membranes become one. The contents of the sperm,
especially the sperm nucleus, are then drawn into the egg’s cytoplasm.
Meanwhile, the fusion of the two plasma membranes has triggered other events. Within
1 s of fusion, the membrane potential of the egg, which is initially about –70 mV, undergoes a
dramatic depolarization to about +20 mV, a shift of almost 100 mV. This shift is cause primarily
by an increase in the permeability of the plasma membrane to Na+ and the consequent influx of
Na+ down its electrochemical gradient. The depolarization lasts for 1 –2 min. The depolarization
of the membrane potential that occurs when the first sperm fuses with the egg prevents other
sperm from fusing with the egg. The activation potential acts, therefore, as a fast block to
polyspermy; fast because it is established within 1 s of the fusion of the sperm. The fertilization
of sea urchin eggs in an artificial sea water that lacks Na+ also results in polyspermy. The reason
for this is obvious: without Na+ in the external medium, the increase in membrane permeability
to Na+ does not lead to a depolarization, so sperm can continue to fuse with the egg plasma
membrane. The fast electrical block to polyspermy occurs widely in biological groups
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(amphibians, worms, algae) but not in mammals. Perhaps mammalian sperm are too lazy and
sluggish to require a fast block.
Another consequence of the fusion of the sperm and egg membranes is the generation of
a wave of Ca2+ that starts at the point of fusion of the two membranes and travels through the
cytoplasm of the egg. Lionel Jaffe, while a Purdue Biology professor, and his students were the
first to demonstrate the existence of this Ca2+ wave, which is shown in the figure below. Those
experiments are famous and have greatly influenced modern developmental biology. The Ca2+
wave causes the cortical vesicles of the egg to fuse with the plasma membrane. This turns out to
be a general rule: the fusion of cytoplasmic vesicles with the plasma membrane requires elevated
levels of Ca2+. Vesicle fusion occurs frequently in biological systems; for example, the secretion
of many hormones by the cells that make the hormones requires vesicle fusion and thus elevated
Ca2+. The elevated levels of Ca2+ also act to initiate the other events of egg activation.
The cortical reaction (the fusion of the cortical vesicles with the plasma membrane)
causes the vitelline envelope to lift off the surface of the egg and harden. The elevated, hardened
vitelline envelope is call the fertilization envelope and is impenetrable by sperm, so it acts as a
second, slower but permanent, block to polyspermy.
The activation of the sea urchin egg by sperm, with the increase in Ca2+ being the major
signal, leads to other events besides the elevation of the vitelline envelope. The egg nucleus and
the sperm nucleus move toward each other and fuse, to form the zygotic nucleus. DNA synthesis
and mitosis follow during the next hour.
Many of these events can be initiated by Ca2+ alone, without sperm. If a frog egg is
pricked with a bloody needle in the presence of extracellular Ca2+, it is possible to activate the
egg and have it develop into an adult frog! The function of the blood is to supply a centriole,
which would normally be contributed by the sperm. The activation of an egg without a sperm is
called parthenogenesis.
II. Cleavage and the formation of the blastula
After fertilization, the first task of the egg (now called a zygote) is to undergo a rapid
series of cell divisions. We discussed the pattern of cleavage of four diverse animals.
A. Frogs
After fertilization, the cortex (a thin region of cytoplasm, 2 –5 µm thick, just underneath
the plasma membrane) rotates with respect to the interior of the egg. The direction of rotation is
determined by the point of sperm entry. The cylindrical symmetry of the egg has been broken
and two axes are now defined: the anterior-posterior (head-tail) axis and the dorsal-ventral axis.
The cell now has bilateral symmetry. First cleavage occurs about 90 min after fertilization (in
Xenopus; the time varies in other amphibian species) and the cleavage plane runs through the
animal and vegetal poles, and bisects the gray crescent. If the blastomeres are separated, each is
capable of producing a normal embryo and adult.
Second division is also through the animal and vegetal poles, but perpendicular to the
plane of first division. Third division is equatorial, resulting in eight unequal cells, as the four on
top are derived from animal cytoplasm and the bottom four from vegetal cytoplasm. Further
rounds of cell division result in the formation the blastula.
The outer layer of cells is already a functional organ, the first to form in embryonic
development. It is a functioning epithelium or skin, called the ectoderm. Epithelia are sheets of
cells that have the job of isolating one compartment from another. The cells of an epithelium
accomplish this by forming specialized connections between themselves called tight junctions. In
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the regions of tight junctions, the cells’ membranes are held in close apposition to each other, so
that little space exists between the membranes. This squeezes out the water and greatly restricts
the movement of water and solutes from one side of the epithelial layer to the other. In this way,
movement of solutes must be across the plasma membranes of the cells, and the cells can
regulate the traffic. In the case of embryonic (and adult) frog skin, the membranes of the outer
epithelial cells facing the outside medium (the pond) contain sodium channels that allow Na+ to
move down its electrochemical gradient into the cells. The Na-K ATPase molecules are
localized to the inner face of the plasma membrane of the epithelial cells, and pump into the
embryo the Na+ that has leaked in. In this way, the frog embryo deals with the problem that the
fluids of the developing embryo require a Na+ concentration of around 100 mM while the pond
water may have very low Na+ levels. This polarization of cell function (ion channels on one
side, pumps on the other) is typical of transporting epithelia; the vertebrate kidney is organized in
much the same way.
B. Birds
We will not consider chicken development is great detail, but it is important to be aware
of the variations that occur among animals in the pattern of early development. The egg cell of
birds is the egg’s yolk. This yolky mass is too large to permit the complete cell division like the
sea urchin and frog. The small region of active cytoplasm of the chicken egg lies on the top of
the yolk. First cell division is a furrow that separates the cytoplasm into two parts and the ends
of the cleavage plane are somewhat indistinct. Second division is perpendicular to the first,
creating four quadrants that are open at the outer boundaries. As cleavage continues, complete
cells are created in the center of the active cytoplasm and with further development, a twodimensional layer several cells thick forms. The inner cells of this layer separate from the outer
cells and form a fluid-filled space, the blastocoel.
C.Insects
Our model system here is the fruit fly, Drosophila melanogaster, which illustrates a very
different pattern of cleavage. After fertilization, the zygote nucleus undergoes several mitoses,
but no cytokinesis (separation of the cytoplasm) occurs. When 256 nuclei are present in the
cytoplasm, they begin to migrate to the periphery of the cell. The Drosophila egg has a distinct
anterior-posterior and dorsal ventral polarity. These two axes are formed during oogensis. The
nuclei that arrive at the posterior pole are soon surrounded by membrane and become separate
cells. These cells are called the pole cells and they give rise to the primordial germ cells that
later produce the gametes. The other nuclei continue to divide and when there are about 6000 of
them, they are positioned around the periphery of the cell and they begin to be individually
enveloped by the zygote’s membrane. At the conclusion of this process, the embryo consists of a
single layer of cells surrounding a yolky, fluid-filled cavity, the blastocoel.
III. Gastrulation.
All animal embryos form a blastula, which is a hollow ball of cells. However, the animal
body plan also includes a gut tube that runs the length of the animal with a mouth at one end and
an anus at the other. It is the process of gastrulation that forms the gut tube.
A. Sea Urchin
As the sea urchin blastula continues to develop, the vegetal end of the embryo flattens out to
form the vegetal plate. The cells of the vegetal plate include the descendents of the micromeres
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and it is these descendents that migrate into the blastocoel. They are then called the primary
mesenchyme cells and there are about 40 of them. The primary mesenchyme cells somehow
induce the other cells of the vegetal plate to buckle inward, forming a pore, the blastopore, that
extends into the blastocoel. This tube, called the archenteron, elongates and a second
invagination begins on the ventral side of the embryo. The archenteron grows toward and fuses
with this second invagination so that a complete tube is formed which is open to the sea at both
ends. The blastopore forms the anus and the second site of invagination on the ventral side
forms the mouth. Because the mouth forms second, sea urchins are in the category of
deuterostomes, a category that also includes the vertebrates. Organisms that form the mouth
invagination first during gastrulation are call protostomes, and include the arthropods (insects
and crustaceans), the molluscs and the worms. In the early 19th century based on observation of
animal morphology, Geoffroy Saint-Hilaire declared that very different animals still have the
same body plan. The central nervous system in deuterostomes lies on the dorsal side, but it lies
on the ventral side in protostomes, such as the lobster. Likewise, deuterostomes have their heart
on the ventral side, while protostomes have it on the dorsal side. Saint-Hilaire hypothesized that
vertebrates are essentially upside-down invertebrates, but an explanation for these body patterns
has only recently become known.
B. Frogs
Gastrulation in amphibians is more complex than in sea urchins. The frog blastula is not
a hollow blastocoel surrounded by a single layer of cells. Compared to the sea urchin embryo,
the frog blastocoel is a considerably smaller fraction of the embryo and the surrounding layer is
several cells thick. At the end of blastula formation, the embryo has undergone about 12 rounds
of cell division and consists of several thousand cells. Gastrulation begins by the inward
movement of cells on the dorsal side of the embryo, near the area where the gray crescent formed
on the just-fertilized egg. This migration of cells forms curved line on the surface that is referred
to as the dorsal lip of the blastopore. As more cells migrate into the blastopore, they form a
cavity called the archenteron, which is the future gut. The archenteron extends along the dorsal
side of the embryo, displacing the blastocoel. As the archenteron deepens, the three germ
layers—the ectoderm, the mesoderm and the endoderm—are formed.
IV. Genomic Competence
It is obvious that the cells of an embryo begin to specialize—differentiate—early in
development. Clearly, a nerve cell doesn’t need to make the same proteins that a blood cell or a
skin cell makes. Early embryologists recognized that this raises an important question: do
differentiated cells retain the full complement of genes that they inherited from the zygotic
nucleus, or do they lose genes that they don’t need? In other words, is the genome from a
differentiated cell capable supporting full development? We already know part of the answer.
The first two blastomeres of the frog embryo and the first four blastomeres of the sea urchin
embryo are capable of developing into an entire normal embryo if they are separated from each
other; thus the nuclei of those blastomeres must retain in functional form all of the organism’s
genes. But what about the nuclei of later embryonic cells, when some overt differentiation has
occurred? Weismann, in the 1880s, thought that the nuclei of differentiated cells lost some of
their unnecessary genes early in development. The first experimental test of this idea was carried
out by Spemann. He used a hair loop to constrict a fertilized amphibian egg so that its nucleus
was isolated into one half of the constricted cell. The half of the egg containing the nucleus
divided and continued the normal program of development. The half without a nucleus did
nothing but was still attached to the other half by a thin cytoplasmic bridge. After the developing
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half had formed an embryo with considerable differentiation, a nucleus from one of the embryo’s
cell escaped through the cytoplasmic bridge into the enucleated half egg. That half then began to
divide and differentiate, forming a normal embryo. This proved that Weismann’s idea was
wrong: nuclei from differentiated cells in an embryo had not lost any genomic information and
could support the development of an embryo when placed in the environment of the egg.
The question of whether adult cell nuclei retain full competence to support embryonic
development remains unclear. The cloning of the sheep, Dolly, was said to have been done
using an adult cell as a nuclear donor.
V. Embryonic Induction and Spemann’s Organizer
Spemann and his colleague, Hilde Mangold, carried out a classic series of experiments in
the 1920s. They used the early amphibian embryo to show that a group of cells on the future
dorsal side of the frog embryo, just above where the dorsal lip of the blastopore would form, has
special properties. They surgically removed this small group of cells from one embryo in the
late blasula stage and transplanted them to the ventral of another embryo of the same stage.
Startlingly, they found that the transplanted cells caused a second blastopore to form near the site
of the transplant, and then a second anterior-posterior axis. Two embryos developed from the
original embryo, with two developing nervous systems, two heads, and two tails. The
transplanted cells had the capacity to organize a second embryonic axis. The organizer could
send a signal to adjacent cells causing them to change their fate.
The Organizer has a further inductive role during development. Some descendents of the
organizer migrate into the embryo during gastrulation and give rise to a tube-like structure that
runs along the dorsal side of the embryo. This structure is called the notochord and is one of the
distinguishing features of vertebrate development. The key role of the notochord is to send an
inductive signal to the ectodermal epithelial cells adjacent to it that causes those cells to form a
neural tube, the precursor to the spinal cord and brain.
VI. Neurulation
After the cells of the epithelium have received the inductive signal from the notochord,
they begin the process of neurulation. While the process is similar in all vertebrates, we will
consider frog neurulation particularly. The induced epithelial cells flatten out to form the neural
plate and the cells at the margin of the neural plate undergo an apical constriction so that the
opposite edges of the neural plate begin to fold toward each other. They continue to move
toward each other until they meet and fuse at the midline of the dorsal side of the embryo. This
results in formation a hollow tube, the neural tube on the interior of the embryo from cells that
were once part of the skin. On top of the neural tube (that is, between the neural tube and the
skin) are an important group of cells called the neural crest. These cells were originally at the
margins of the neural folds. The neural crest cells migrate away from the developing spinal cord
later in development and give rise to all of the peripheral nervous system as well as non-visual
pigment cells and other structures.
VII. Summary of lecture on polarity in Drosophila
The existence of gradients of cytoplasmic determinants in animal eggs has been
recognized for many decades. It is clear that the animal-vegetal axis of the frog and sea urchin
eggs represents something more profound than just a gradient of yolk, pigment, and nuclear
position. There are materials localized to animal and vegetal poles that are necessary for the
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specification of parts of the embryo, and a complete, normal embryo cannot be formed without
this gradient system. Despite having been recognized long ago, we had no idea as to the
molecular identity of these cytoplasmic determinants until fairly recently.
Molecular biologists have discovered remarkable genetic connections between very
diverse animal species. Certain genes called homeotic genes (homeo=alike) are amazingly
similar in structure and function in all animals; they serve as molecular architects and direct the
building of bodies according to definite detailed plans. Like so many breakthroughs in genetics,
this one came from the humble fruit fly, Drosophila melanogaster, a laboratory favorite because
it reproduces rapidly, has only 4 chromosomes, and readily exhibits mutations induced by
inbreeding and x-rays. Fruit flies are highly specialized insects with 2 wings and 3 body
segments. Their ancestors had 4 wings and many body segments. The fruit fly embryo starts out
with a series of 16 equal-sized segments. Various segments merge to make the 3 segments
we recognize as the head, thorax, and abdomen. Our understanding of the genetics of fruit flies
relies on the fact that the flies are easy to grow and breed in large quantities, and the short
generation time. It only takes about nine days from the egg of one generation to the egg of the
next generation.
In the 1940s, American biologist Edward B. Lewis began studying the homeotic genes
that affect segmentation in Drosophila. He found that mutations in a cluster of genes, called the
bithorax complex, caused duplication of a body segment with an extra pair of wings. These
mutations were weird and hard to explain because hundreds of different genes participate in the
formation of a body segment and wings. Yet here were single mutations creating new body parts
and eliminating others. These genes were acting as master switches, turning on and off arrays of
other genes involved in body shape, and controlling the number, pattern, position, and fusion of
segments and appendages. In the late 1970s, German biologists Christiane Nüsslein-Volhard and
Eric F. Wieschaus sequenced the homeotic genes controlling the development of the fruit fly's
body. They observed that in each of these genes a particular DNA segment 180 bases long was
virtually identical. This DNA sequence, called the homeobox, translates into a protein sequence
60 amino acids in length. This protein sequence binds to DNA and switches on and off the
process of transcription, the expression of genes into proteins. By controlling the transcription in
all cells, homeobox (Hox) genes act as master switches determining cell fates, growth, and
development.
About 1980, Christiane Nüsslein-Volhard (and her colleagues) began a systematic effort
to understand the origin of polarity in fly eggs. Her approach was to induce mutations in the
flies and then look for embryos that had altered patterns of development. Among the mutations
that were characterized were ones that affected the development of either the anterior-posterior
axis or the dorsal-ventral axis of the embryo. One mutation that disrupted the anterior-posterior
axis was called bicoid. The phenotype of bicoid was that it failed to develop head and thorax
structures at the anterior end; instead, the head end developed like the caudal (tail) end, so the
embryo was bicaudal (from whence came the name bicoid). It was shown that some product of
the bicoid gene was localized to the anterior pole of the egg. If cytoplasm from the anterior pole
of a normal egg was sucked out, the resultant embryo resembled the bicoid mutant. If anterior
pole cytoplasm from a normal egg was sucked into a micropipette and then injected into the
anterior pole of a bicoid mutant egg, some rescue of the embryo was achieved. That is, the
injected bicoid egg developed into an embryo that was more nearly normal; it had some head and
thoracic structures. If cytoplasm from the anterior pole of a normal egg was injected into the
middle of a bicoid mutant egg, the resultant embryo developed head structures in the wrong
location, in the middle of the embryo.
So what is the molecular nature of the bicoid gene product that is localized in the anterior
pole of the egg? Once the gene was identified, it became possible to make probes for the bicoid
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mRNA and the Bicoid protein. Staining of bicoid eggs with a probe for bicoid mRNA showed
that the RNA was absent from nearly all of the cytoplasm but was present in high concentration
in a small zone at the anterior pole. Other experiments have shown that the bicoid mRNA is
associated with cytoskeletal structrures, particularly microtubules, thus rendering it immobile.
Little Bicoid protein is present in the unfertilized egg, so the localized mRNA is not being
translated.
Prior to cellularization of the embryo, the bicoid mRNA begins to be translated. The
protein then diffuses away from the anterior pole. In general, proteins have a limited lifetime in
cells because they are degraded by enzymes. A gradient of the bicoid protein is established
because there is a highly localized source at one end (the localized mRNA) and a sink (the
degradation of the protein). The gradient of Biocoid protein is much more gradual than the
gradient of the localized mRNA. As cellularization proceeds, the Bicoid protein is incorporated
into the cells and there is an anterior-posterior gradient of Bicoid protein within the newlyformed cells. If the antibodies to the Bicoid protein are used to map its distribution, the anteriorposterior gradient can be clearly seen.
What does the Bicoid protein do? It turns out that it is of a class of proteins called
transcription factors. These proteins bind to regulatory regions of DNA and control the
transcription of genes. The Bicoid protein is a transcriptional activator of a gene called
hunchback. If the concentration of Bicoid protein is above some critical level in a cell, then the
hunchback gene is transcribed into mRNA and the cell makes the hunchback protein. The
gradient of hunchback mRNA (and thus the Hunchback protein) is quite sharp because it
responds to a threshold level of the Bicoid protein gradient: if Bicoid protein concentration is
above the threshold, hunchback gene is transcribed, if Bicoid protein is below threshold,
hunchback is not transcribed. Of course, in biology, thresholds are rarely absolute, so the
gradient of hunchback isn’t quite as sharp as this discussion would make it appear. Nevertheless,
the gradient of the mRNA that is laid down in the egg during oogensis lead to a different gradient
of the protein from another gene during embryonic development. The proper establishment of
these gradients is necessary for the normal development of the embryo.
VIII. Evolutionary History of Animals
Of 35 different animal phyla with distinct body plans, 30 are bilaterians. Bilaterian
fossils made their appearance suddenly between 535 to 525 million years ago (mya) during the
Cambrian explosion (Burgess Shale in British Columbia, Canada, and of the Chengjiang
formation in Yunnan, China). All bilaterian body plans can be traced back to the Cambrian
explosion. Before these animals, Precambrian ancestors must have existed, but they left very few
adult bilaterian fossils. Amazingly, all the body plans characteristic of the 35 phyla that exist
today were already present 525 mya. Individual species thrived and developed many new
variations, but the overall body plans were maintained.
The emergence of different forms was facilitated by the evolution of a robust body
organization, which allowed for variation in shape and growth. The embryo had become
flattened and was composed of three layers, now including the mesoderm. The inner cells,
derived from the endoderm, formed the gut tube. Ectodermal sense organs and a mouth
developed at the front, specialized to feed. The mesoderm allowed for formation of muscle and
circulatory systems. These animals now could acquire an elongated shape with a clear front and
back, top and bottom, and bilateral symmetry. In other words, this primordial animal must have
already had all the genes in the most important signaling cascades and the gradient system
creating the dorsal–ventral axis.
When genes between different organisms are compared, one striking observation is how
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similar they are in sequence and often also in molecular function when it comes to the patterning
of body plans. This is especially surprising in the case of developmental genes that regulate how
body plans and organs are formed. These genes are sometimes so similar that they can readily
replace each other in two animals that look very different.
The Hox-genes, as well as other genes determining the position of sense organs and the
heart were probably also included in the early repertoire of developmental genes. The succession
of the Hox-genes, which pattern the anterior–posterior axis, has been preserved in all animals.
Dpp and sog create a gradient that determines the dorsal–ventral axis in arthropods. Vertebrates
develop a corresponding dorsal–ventral gradient with BMP (homologous to Dpp) and chordin
(homologous to sog). Many molecular feedback mechanisms of this system have been preserved,
which suggests that they already operated in the ancestors of protostomes and deuterostomes.
But in vertebrates and arthropods, they are arranged in opposing orientation. The BMP signal is
ventral in vertebrates and dorsal in arthropods (Dpp).
Hox genes evidently duplicated twice during the evolution of invertebrates into
vertebrates. Instead of one cluster of about 10 genes on 1 chromosome like the fruit fly, the
mouse has 4 clusters of about 10 genes each, on 4 different chromosomes. Hox genes in mice
and humans are very similar in number and chromosomal arrangement. It is remarkable that only
about 40 genes out of a total of about 100,000 control most of the development, architecture, and
appearance of the body plan of complex mammalian species.
As different as the adult fly and mouse appear, their homeotic genes had a common
evolutionary origin, shown by the marked similarity in homeobox sequences. Fly and mouse had
a common ancestor half a billion years ago, but the homeobox sequence has hardly changed
during that long time period. The same Hox genes that determine the belly side of invertebrates
establish the back side of vertebrates. Saint-Hilaire's idea that vertebrates have the body plan of
upside-down insects now has an explanation.
Hox genes provide spectacular insight into the evolution of the eye. Different kinds of
eyes in a variety of animals, for instance, octopuses, flies, and humans, posed a puzzle for
evolutionary biologists. Ernst Mayr concluded that eyes may have evolved independently 40
different times. In 1994, however, Swiss biologist Walter Gehring and his team found that the
Hox gene responsible for induction of the Drosophilia eye is virtually identical to the one that
induces the mouse eye. This Hox gene switches on eye formation in the myriad of creatures that
see. Hence, it appears that all eyes, no matter how differently constructed they appear now, had a
common evolutionary origin.
Problem Set 5
1. You have decided that you represent the apex of evolution and you conclude that it is your
duty to humanity to clone yourself. Before trying your hand at human cloning, you decide to
practice with mice. The first problem that you face is the choice of cells to act as donors of
nuclei for transplanting into enucleated eggs. You decide to try red blood cells, reasoning
that they are easy to isolate and can be easily seen under the microscope because of their
color. You take blood from a black strain of mice and suck red blood cells into a fine
micropipette to rupture the plasma membrane and isolate the nucleus in the micropipette.
Because of the dense red color of the hemoglobin, you can’t actually see the nucleus, but you
assume it is there. You then inject the nucleus into an enucleated, activated egg from a white
strain of mice and incubate it to await development. Much to your disappointment, the
injected egg never divides. You try the experiment repeatedly, with the same discouraging
result. You mention your frustration to a colleague, who smiles condescendingly at you and
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2.
3.
4.
5.
6.
7.
suggests that you really ought to know something about blood cell biology. You do a little
library work and discover your silly error. What was it?
It was asserted in class that the outer epidermal cells of the early frog embryo take up Na+
from the surrounding pond water by having sodium channels in the apical (outward facing)
membranes of those cells. Assuming that the cells have a membrane potential of –58 mV
and a cytoplasmic Na+ concentration of 10 mM, will Na+ enter the cell passively if the
concentration of Na+ in the pond water is 5 mM? Hint: use the Nernst equation to find what
pond Na+ would be in equilibrium with a membrane potential of –58 mV and internal Na+ of
10 mM.
When a sea urchin egg (diameter 100 µm) divides into two blastomeres, both blastomeres are
spherical and contain half of the original volume. What is the diameter of each blastomere?
If Hensen’s Node is the avian equivalent of Spemann’s Organizer, what would you predict
the effect of transplanting Hensen’s Node from one chick embryo to the side of a primitive
streak of a second chick embryo?
Suppose one were to transplant a piece of epidermis from the ventral side of blastula stage
pigmented frog embryo to the dorsal side of an albino (pigmentless) frog embryo. The
ventral epidermis would normally just form skin. Since the transplanted epidermis is
pigmented, one can follow the fate of the transplanted cells in the albino embryo. What
would you predict that the pigmented cells will become, skin or nervous tissue? Why?
From memory, make drawings of the process of gastrulation and neurulation of the Xenopus
embryo. Show both internal and external events.
Read the following abstract from a research paper in Science (1993, Vol. 259, pp. 11341138). To what structure in the frog embryo are the micromeres analogous? Try to draw what
the host embryo that receives the micromere transplant would look like.
A complete second gut induced by transplanted micromeres in the sea urchin
embryo, by A Ransick and EH Davidson, Division of Biology, California Institute
of Technology, Pasadena 91125.
Founder cells for most early lineages of the sea urchin embryo are probably
specified through inductive intercellular interactions. It is shown here that a
complete respecification of cell fate occurs when 16-cell stage micromeres
from the vegetal pole of a donor embryo are implanted into the animal pole of
an intact recipient embryo. Animal pole cells adjacent to the transplanted
micromeres are respecified from presumptive ectoderm into vegetal plate
founder cells. These induced vegetal plate cells express the entire battery of
genes characteristic of the endogenous vegetal plate cells. The ectopic vegetal
plate invaginates during gastrulation to form a second archenteron which
differentiates properly into a tripartite gut, as shown by the spatial pattern of
expression of an endoderm-specific marker gene. Thus, transplanted
micromeres can signal neighboring cells to induce them to change their fate.
8. A news article reports that scientists may hatch a dinosaur from a chicken egg by the end
of the century by modifying the genes of birds, the closest living relatives to dinosaurs
(http://www.acfnewsource.org/science/dino_rebirth.html). Ancestors of birds were the
first dinosaurs approximately 150 million years ago. Fossils from birds in China have
small teeth very similar to an alligator today. By studying the genes of birds, scientists
have been able to isolate specific genes and inhibit or allow certain genes to grow
specific structures that were once prominent in the avian population. In order for bird to
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begin flight, teeth were quite heavy, so through natural selection, teeth were lost in
generations to make room for the fittest of birds. Scientists have also been able to grow
eyes on a normally blind cavefish, reconstruct the primitive back design in rodents, and
create leg extensions from a bug’s antenna. From a biological perspective, are these
stories credible? Justify your ideas.
9. Starting with Milestone 1, the Spemann - Mangold organizing principle online at
http://www.nature.com/milestones/development/milestones/full/milestone1.html look at
other Milestones until you find one with experimental results that can be presented in a
graph. Give the URL and then answer the following questions:
Does the investigation include a categorical variable? Explain.
Does this investigation establish a cause-and-effect relationship between a treatment and a
response variable? Justify your answer.
Identify ONE treatment variable:
Identify ONE response variable:
Sketch an appropriate graph for the findings about this ONE response variable:
Label the x and y axes to show what they represent
Explain what the bars or data points represent.
10. In the lecture, you were not given the whole story of the establishment of the anteriorposterior development of the early Drosophila embryo. Another important player is the
product of the nanos gene. Use the Society for Developmental Biology's "Interactive Fly"
web site (http://www.sdbonline.org/fly/aimain/1aahome.htm) to determine the role of
Nanos protein in the establishment of the anterior-posterior gradient of hunchback. If you
encounter unfamiliar terms like "zinc finger domain", use the index of your textbook or
look on the web for assistance.
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