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Frog & Sea Urchin Development
BIOL212
1
Spring 2012
Frog & Sea Urchin Development Lab
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Be able to recognize the various stages of animal development
Compare and contrast the structural components of the blastula and gastrula embryo.
Describe the purpose of gastrulation.
Compare the process of gastrulation in the sea urchin and the frog.
Describe the processes of fertilization and cleavage division in the sea urchin.
Observe formation of the fertilization membrane as it expands to surround the sea urchin.
Describe how the amount and distribution of cytoplasm within a fertilized egg influence
the patterns of cleavage.
Describe how cleavage occurs in an echinoderm and a frog.
Compare the differences in development with and without the presence of a yolk
Describe the structure of a typical blastula.
Relate the structure of the frog blastula to the establishment of the dorso-ventral and
antero-posterior axes of the embryo
Describe the process of neurulation.
Relate the development of the brain and the spinal column to the development of the
neural tube and neural folds.
Know differences related to external and internal early development
Some species have one or more larval stages
Relate commonalities to the Unity of Life
Introduction:
All living things must be able to reproduce and develop. In animals, gametes produced
by the process of meiosis unite during fertilization to form a single diploid cell, the zygote. The
processes of cell division, cell movement, cellular differentiation, and morphogenesis result in
the development of a multicellular embryo that will grow to form an adult.
The hereditary material within fertilized eggs, or zygotes, of animal organisms holds the
key to both structure and function. DNA guides, through time, the structural and functional
development of a single cell into an embryo and its morphogenesis into an adult. Even aging is a
result of genetic programming.
Biologists are continuing to understand more of the genetic control of development.
Advanced technologies as well as molecular and recombinant DNA techniques have made it
possible to discover more and more about the genomes of organisms and to map the entire
genomes of some. Common patterns of gene function are beginning to emerge, suggesting that
some day, perhaps, we will be able to explain the mysteries of animal development. Once again
DNA exhibits its programming functions—DNA holds the secrets not only for “what” it produces,
but also for “how” and “when” it produces its products.
While Animal development is a complex process of sequential activation of genes, which
in turn code for proteins that allow for the growth and differentiation of the organism, the early
stages of animal development are, or at least appear, very similar across species. We have
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already seen the difference in Protostomes and Deuterostomes depending on whether the
blastopore becomes the mouth or the anus. The presence or absence of a yolk imposes
necessary differences on the common theme. You will examine prepared slides as well as live
animal examples (if available) to get a sense of the changes that occur during early
development. During this laboratory you will have the opportunity to explore the development of
sea urchin and frog embryos.
Advance Preparation:
Review your notes on animal development from lecture and Sections 46.3 (p. 1005 –
1008), 4.5 (p. 1011 – 1015) and Chap. 47 (p. 1021 – 1044)
Structures and terms to know: gametes, ovum, sperm, fertilization, zygote, cleavage,
blastula, blastomeres, blastocoel, blastopore, mesenchyme cells, archenteron, gastrula,
invagination, gastrulation, filopodia, animal pole, vegetal pole, dorsal, ventral, notochord,
neural fold, neural plate, neural crest, neural tube, coelom, yolk sac, yolk stalk, somites,
organogenesis, morphogenesis, holoblastic and meroblastic cleavage, totipotent,
determination, differentiation, convergent extension, mouth, anus, fate map, ectoderm,
endoderm, mesoderm, extra-embryonic membranes (amnion, chorion, yolk sac, allantois)
polyspermy, vitelline membrane, Neurulation, homeobox
Laboratory Procedures:
You will find microscope slides of the stages of development of frogs and sea urchins
and (hopefully) some live specimens to examine. For each organism you view under the
microscope, you should draw and label the specimen. When you label the drawings, be
sure to include all the structures or cells that you can identify on the specimen and the
total magnification you used. To assist in tracking the stages, there will also be models
available and attachments to these instructions.
I. Slides
1. Prepared slides
Microscope Slides: Frog [Order Anura (Merrem 1820)] and Sea Urchin [Strongylocentrotus sp.
(Brandt, 1835)] Developmental Stages (see overhead: there may be a more detailed list.)
2. Live specimens
Check overhead – if we have any live specimens, this and instructions will be announced!
(If not available at this lab period, we will try to have them at the next lab period.)
Part A: Fertilization and Early Development in Sea Urchins.
Understanding how DNA regulates cell division, differentiation, and morphogenesis
begins with observing the developing embryo. Sea urchins have long been the developmental
biologist’s favorite organism of study because they are relatively simple to obtain and culture in
the laboratory. The cells of the developing sea urchin are also fairly transparent, providing us
with a limited ability to “look inside” the embryo.
Early development of the sea urchin is under the genetic control not only of the zygote’s
DNA but of messenger RNA (mRNA) stored in the egg during its development. These messages
include maternal mRNAs synthesized from maternal DNA prior to the meiotic events of
oogenesis—mRNAs made from DNA that may not be included in the egg itself. Thus, the story
of development begins before fertilization. The unfertilized sea urchin egg is surrounded by a
vitelline membrane that lies just above the surface of the cell’s plasma membrane. Within the
cytoplasm, yolk granules (sea urchin eggs are microlecithal—they have very little yolk) can be
observed. In addition to other cytoplasmic determinants and stored mRNAs, small cortical
granules, composed of proteins and mucopolysaccharides, lie just beneath the plasma
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membrane (in the outer rim or cortex of the egg). When a single sperm enters the egg plasma
membrane, the membrane potential quickly changes as a wave of depolarization spreads from
the site of sperm entry. This reaction is often referred to as a “fast block to polyspermy,” since no
additional sperm can gain entry following the change in membrane potential. Release of Ca2~
ions from the egg’s endoplasmic reticulum, in response to G protein, causes the cortical
granules in the egg’s cortex to fuse with the plasma membrane. The cortical granules discharge
their contents into the space between the plasma membrane and the vitelline membranes. The
excess mucopolysaccharide now present in the perivitelline space lowers the water potential of
that area, and water flows in. This causes the perivitelline space to increase in diameter, making
it appear as if the vitelline membrane is lifting off the surface of the zygote. Addition of proteins
to the vitelline membrane hardens (or “tans”) it as it is transformed into a fertilization membrane.
Formation of the fertilization membrane (often called the “slow block to polyspermy”) offers
additional protection against multiple sperm entry. Hundreds of sperm can usually be observed
still attached to the old vitelline membrane, now the fertilization membrane. These will be
removed by the action of enzymes released from the cortical granules.
Following fertilization, the stored mRNAs are responsible for directing protein synthesis
during the early stages of development—cleavage, blastula formation, and gastrulation. In
addition to the mRNA and protein already present in the fertilized egg, newly synthesized protein
products are responsible for establishing regional differences in the egg cytoplasm. These
proteins or cytoplasmic determinants are responsible for establishing the cytoarchitecture of the
unfertilized egg, reorganizing the cytoplasmic elements in response to fertilization, controlling
the direction of the first cleavage divisions, and establishing the axis of the embryo.
In this exercise, you will have the opportunity to observe the earliest stages of sea urchin
development— beginning with fertilization. You will also have available later-stage embryos for
observation of gastrulation and formation of pluteus larvae.
Prepared Slides--Embryological Stages (and structures)
Draw and label each of the slides indicated below, and answer the following questions.
Sperm (sperm cells)
a) How does the size of the sperm cells compare with the size of the unfertilized egg cells
(be as specific as possible: indicate the magnification of the scope that you had to use.)
Egg, unfertilized (egg cells)
b) Is this cell haploid or diploid?
Egg, fertilized (zygote, fertilization membrane)
c) Is this cell haploid or diploid?
d) How does the appearance of this cell compare with the appearance of the unfertilized egg
cell?
e) What structure causes the change in appearance?
Part B: Cleavage
In order for a fertilized egg or zygote to become a multicellular organism, the zygote must
divide by mitosis. During early development, this process of division is known as cleavage. The
fertilized egg is not a uniform sphere. Differential concentrations of cytoplasm and yolk (if
present) can affect the cleavage process. The upper portion of the egg, usually richest in
cytoplasm, is known as the animal pole, and the lower portion of the egg, containing more yolk,
as the vegetal pole. The first plane of cleavage is vertical, bisecting both the animal and vegetal
poles. Depending upon the amount of yolk in the egg, the planes of cleavage may pass all the
way through the zygote (holoblastic cleavage, typical of cells with small to medium amounts of
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yolk; sea urchin and frog) or through only a part of the zygote (meroblastic cleavage, typical of
cells with large amounts of yolk; chicken).
A second cleavage division typically occurs at a right angle to the first, producing four
cells. The third cleavage division cuts horizontally to form eight cells, four on the top and four on
the bottom. The cells produced during these cleavage divisions are known as blastomeres. If the
blastomeres in the top “tier” lie directly above those in the bottom tier, the pattern of cleavage is
said to be radial, a pattern characteristic of echinoderms and chordates (deuterostomes). In the
sea urchin, the fourth cleavage results in the formation of 16 blastomeres of three different sizes.
Eight medium-size mesomeres are the product of the division of the four blastomeres in the
animal hemisphere. The lower four blastomeres (vegetal hemisphere) produce four large
macromeres and four small micromeres. As a consequence of this cleavage pattern,
cytoplasmic determinants are distributed in an unequal manner, laying the groundwork for future
development.
Prepared Slides--Embryological Stages (and structures)
Draw and label each of the slides indicated below, and answer the following questions.
Early Cleavage (morula)
f) How many cells make up the morula of the embryo at this stage?
g) How does the size of this morula compare with the size of the fertilized egg cell?
h) What is the effect of cleavage on cell size? On embryo size?
Late Cleavage (morula)
i) How many cells make up the morula of the embryo at this stage?
j) How does the size of this morula compare with the size of the morula at early cleavage?
k) Is the mass of a four-cell or an eight-cell embryo any larger than the mass of the zygote?
Part C: Formation of the Blastula
Repeated cleavages will result in formation of a hollow ball of cells called a blastula. The
cavity inside the blastula is called the blastocoel. Even as early as this blastula stage, groups or
layers of cells are already destined to become particular organs or organ systems; these layers
of cells are known as presumptive germ layers. The major germ layers and their derivatives
are listed in Table 1.
Table 1: Germ Layers and Their Derivatives
Germ Layer
Ectoderm
Mesoderm
Endoderm
Derivative
Brain, spinal cord, and neural crest cells; Skin
Skeleton, circulatory system, excretory system, and parts of organs belonging to
other systems; Notochord and spinal disks in some organisms; ganglia
Gut and associated out-pocketings
In the sea urchin, blastomeres that will give rise to cells of germ layers are already laid out at the
16-cell stage. A fate map can be assigned. Mesomeres will give rise to ectodermal structures
including the cilia that develop on the blastula’s surface. Macromeres will give rise to
endodermal structures. Micromeres will be responsible for formation of the body cavity, many
internal organs, and the skeletal elements (spicules) of the embryo.
Lab Prepared Slides--Embryological Stages (and structures)
Draw and label each of the slides indicated below, and answer the following questions.
Blastulae (Label blastocoel, blastomeres)
l) How does the size of the blastula compare with the size of the morula?
m) How has the position of the cells change?
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n) Why does the center appear to be lighter?
Locate a later blastula stage. The cells of the blastula are now so small that it will be difficult to
distinguish individual cells. Again, the outer rim appears to be dark. At this time, the cells of the
blastula are covered by cilia that allow the blastula to spin and move. Draw your observations.
o) Why?
Part D: Gastrulation
As a blastula, most of the cells of the embryo (including those destined to become cells
of internal organs and tissues) are on the outside of the hollow ball, and it is obvious that some
of the sheets of cells must move, or migrate, to the inside of the blastula. This process of cell
movement is called gastrulation. In some organisms, such as the starfish and sea urchin,
gastrulation is accomplished invagination, or simply by buckling or pushing inward, forming a
depression or blastopore. Endoderm and mesoderm reach the inside of the embryo in this
manner. In other organisms, such as the frog or the chick, cells migrate to the interior by way of
the blastopore, the portion of the embryo that will eventually contribute to the development of the
anus. Mesoderm, endoderm, and chordamesoderm (notochord material) migrate to the inside.
With the change in position of the germ layers, the blastocoel of echinoderms (starfish and sea
urchin) and chordates (frog) is eventually obliterated and a new cavity, the archenteron, is
formed within the gastrula. The archenteron is the primitive gut of the embryo.
Note that during sea urchin development, secondary mesenchyme cells help to “pull” the
endoderm of the newly forming archenteron (gut) toward the opposite side of the embryo, where
it will fuse with the outer layer, or ectoderm, to form the mouth. (Hence the term “deuterostome”
or second mouth, the anus forming at the blastopore or first opening.)
Prepared Slides--Embryological Stages (and structures)
Draw and label each of the slides indicated below, and answer the following questions.
Gastrulae (Label gastrula, archenteron, endoderm, ectoderm, blastocoel)
p) What is the fate of the pore that has formed by the invagination of gastrulation?
q) What is the fate of the archenteron?
r) What is the fate of the bastocoel?
Pluteus Larvae (archenteron, mouth, anus)
s) What is the symmetry of this larvae?
t) How does this compare to the symmetry of the adult?
Part E: Neurulation in Vertebrates
During this stage of development, a strip of neural ectoderm on the outside of the dorsal
surface of the embryo turns upward to form a neural tube. The underlying mesoderm and
notochord tissue induce formation of the neural tube. The folds of tissue forming the tube are the
neural folds and the groove between them is the neural groove. Eventually the anterior end of
the neural tube will expand to form the brain; the spinal cord will develop posterior to the brain.
Aggregations of mesoderm (mesodermal somites) behind the brain and alongside the
spinal cord will form vertebrae (back bones) that protect and enclose the spinal cord.
Mesodermal somites also give rise to dorsal skeletal muscles and to the dermis of the skin. The
presence of somites is an indication of the segmented nature of vertebrate embryos. Biologists
have recently shown that a series of genes control the development of segmentation in all
segmented embryos, whether in fruit flies, mice, or in humans. These genes, called homeotic
genes, often work in a cascade with other genes that control the basic head-to-tail and anteriorto-posterior architecture of the embryo.
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Within each homeotic gene, a special sequence or homeobox of 180 base pairs codes
for a protein that is 60 amino acids long. This is a DNA regulatory protein that can “turn on” other
genes involved in segmentation and segment identity (whether wings, antennae, legs, and so
forth, are attached). The same homeobox is found in all homeotic genes of segmented
organisms—its base sequence has been conserved throughout evolution.
The gut will tubulate during this later stage of development, and the ventral unsegmented
mesoderm will split to form a mesodermally lined coelom.
Procedure
1. Examine and draw a cross section of a frog late neurula. Identify and label the neural tube,
notochord, gut cavity, epidermis, and mesoderm lining the coelom.
u) What happens to the shape of the embryo after the sides of the neural tube close?
NO PRELAB (Besides the text and lecture note reading and define morula.)
Post lab: Use your observations from today to answer the following questions. These questions
should be completed on a separate piece of paper (not in your lab notebook) and turned into
your instructor at the start of the next lab period unless another date is indicated.
1.) Explain why you would not expect a sea urchin's egg to have a large yolk.
2.) Explain why the zygote and early blastosphere (morula) are about the same size.
3.) Does the sea urchin unfertilized egg have an animal pole and vegetal pole?
4.) How can you tell the animal from the vegetal pole?
5.) Fill in the following table to summarize the major events of early development.
Formation of
the Blastula
Early
Gastrula
Late
Gastrula
Neurula
Processes
occurring
Type of
Structure(s)
formed
Characteristics
of structure
Significance of
stage
6.) Compare and contrast major developmental events in the sea urchin and frog by
completing the following table.
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Sea Urchin
Frog
Type of egg
Type/pattern of cleavage
Distinguishing
characteristics of blastula
How gastrulation occurs
Events of
neurulation
Distinguishing
characteristics of later
development
7.) What are cytoplasmic determinants? How do they affect early development, including
cleavage of the zygote?
8.) How can maternal DNA affect the development of an embryo if a particular maternal
allele is not included in the egg produced as a result of meiosis?
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