Biology 3235
Developmental Biology Laboratory
University of Utah
Spring 2000
FERTILIZATION AND EARLY DEVELOPMENT OF ECHINODERMS
For the first 3-4 weeks, we will be studying the fertilization and early development of sea
urchin eggs and embryos. Historically, marine invertebrates, including both sea urchins and
starfish, have been widely used as models of animal development. Sea urchins provide several
advantages for studying fertilization and developmental processes: (1) gametes are readily
obtained in large quantities; (2) eggs are readily fertilized in vitro; (3) embryos are readily
cultured in vitro; (4) eggs and embryos are relatively large, and in some cases, transparent,
simplifying microscopic examination of development; and (5) development is relatively rapid and
synchronous (which is particularly advantageous for biochemical analysis).
In the first week, you will examine commercially-prepared whole mounts of urchin
embryos to familiarize yourself with the normal stages of development and use of the
microscopes. In the remaining 2-3 weeks, we will be using living urchin embryos to investigate
some of the cellular mechanisms underlying fertilization and early development.
With luck, we will have three species of urchins available in the laboratory:
Strongylocentrotus pupuratus (from California), Lytechinus variegatus, and Arbacia punctulata
(both from Florida). This will allow you to compare fertilization and development in these
different species, as well as investigate mechanisms for ensuring the fertilization is
species-specific.
Sea urchin labs-1
Biology 3235
Developmental Biology Laboratory
University of Utah
Spring 2000
I. ECHINODERM EMBRYOLOGY EXERCISE AND “MINI-“ REPORT.
The purpose of this assignment, which is to be completed during the first 2-3 lab periods, is to (1)
familiarize you with the use of the upright microscopes and CCD cameras; and (2) introduce you
to the basic embryology of echinoderms (sea urchins and starfish) in preparation for the “live” labs
that follow. Your assignment:
A.
Use phase- and/or DIC microscopy to examine prepared slides of both sea urchin and
starfish embryos (for instructions on setting up and using microscopes, see the
“Microscopes” pages). Learn to recognize the main stages of echinoderm development, as
illustrated on the “Normal stages” page.
Note: slides contain mixed stages, so you will need to hunt around for the different stages of
development. Some embryos might be damaged during fixation and preparation, so find examples
that look “normal,” based on the illustrations provided.
B.
Document with figures (and captions) each of the following important milestones in
echinoderm development: (a) early cleavage and the formation of the micromeres (collect
images before and after formation of the micromeres); (b) formation of the blastocoel
(earliest stages) (c) ingression of the primary mesenchyme; (d) invagination of the
archenteron; (e) ingression of the secondary mesenchyme, and interactions of the
archenteron with the oral surface; (f) formation of the spicules; (g) a pluteus larva; and (h)
anything else that looks neat!
C.
Document several stages of starfish development that differ from that of sea urchins.
The sea urchin “mini-report” is due before class on 13 January.
Sea urchin labs-2
Biology 3235
Developmental Biology Laboratory
University of Utah
Spring 2000
II. NORMAL STAGES OF SEA URCHIN DEVELOPMENT (Lytechinus variegatus).
Fertilization: The cell cycle of unfertilized sea urchin eggs is arrested following
completion of the second meiotic division. Fertilized eggs can be recognized by
their fertilization envelopes, which are raised by the rapid exocytosis of the
cortical granules within seconds of sperm entry.
Figure 1 Fertilized sea
urchin egg.
Cleavage: First cleavage begins 60-120 minutes after fertilization (depending on
species and temperature), and the subsequent cleavages occur at ~60 minute
intervals. The first two divisions are meridional, occurring along the animalvegetal axis (a four-cell embryo is shown). The third division is equatorial,
dividing the embryo into two tiers (animal and vegetal) of four cells each.
Figure 2 4-cell sea
urchin embryo.
At the fourth division, each of the blastomeres in the animal tier divides equally
along a meridian to form eight mesomeres. Cells of the vegetal tier divide
asymmetrically, giving rise to four large macromeres and four smaller
micromeres at the vegetal pole (arrow).
Figure 3 16-cell with
micromeres.
Figure 4a Early
blastula.
Figure 4b Midblastula.
Blastula: By the 16-cell stage, the blastocoel (Bl)
can be discerned (a 32-64-cell embryo is shown
in panel 1). Embryos hatch during the blastulastage of development. During the late blastulastage of some species, the vegetal pole flattens,
forming the “vegetal plate (VP)”.
Figure 4c Late
blastula.
Gastrulation begins with the ingression of the
primary mesenchyme cells (5a, arrow). Shortly
after, the archenteron begins to invaginate (5b,
arrow) and elongate (5c).
Figure 5a Ingression
of IE mesenchyme.
Figure 5b Invaginating Figure 5c Elongating
archenteron.
archenteron.
Formation of the skeletal rods (by the primary mesenchyme cells,
6a) begins to shape the embryo into the characteristic shape of the
pluteus larva (6b).
Figure 6a Formation
of skeletal rods.
Figure 6b Pluteus.
From Wray, in “Embryology: constructing the organism” by Gilbert.
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Biology 3235
Developmental Biology Laboratory
University of Utah
Spring 2000
III. BASIC TECHNIQUES FOR FERTILIZING AND CULTURING SEA URCHIN EMBRYOS.
A. Collecting sea urchin gametes. The instructors will demonstrate how to obtain gametes,
fertilize, and culture sea urchin embryos. Briefly, the steps are as follows:
1.
Sea urchins can be induced to release their gametes by injecting 0.5 M KCl into the body
cavity through the soft membrane surrounding the mouth (“Aristotle´s lantern”). Gametes
will “ooze” from pores on the upper surface of the urchin.
2.
The sex of the urchins can be determined by the color of gametes. Sperm is white or
cream-colored, and eggs are yellow orange (Strongylocentrotus), red (Arbacia), or
translucent (Lytechinus).
3.
Collect eggs by inverting female urchins over a dish or beaker of
artificial sea water (ASW) with the upper surface in the water. Eggs
fall in streams to the bottom. Wash once or twice by carefully
decanting and replacing the ASW, or by transferring a dense
suspension of eggs to a new dish of ASW with a Pasteur pipette.
Never handle unfertilized eggs with a pipette that might have been
used with sperm!! Eggs can be stored for a day or two on ice or in the
refrigerator.
4.
Collect sperm “neat” by blotting away fluid on the surface of male urchins, and inverting
each over a clean petri dish. Use a clean pipet to collect the sperm into screw cap
centrifuge tubes (label tubes with species and date!). Sperm can be stored refrigerated for
several days if it is not allowed to dry out. Once diluted in ASW, sperm is active for <60'.
B. Fertilizing sea urchin eggs.
1.
Transfer eggs into fresh sea water in a small dish.
2.
In a separate dish, dilute a small drop of "neat" sperm into a small amount of sea water,
making a cloudy suspension. Diluted sperm must be used within a few minutes.
3.
Add a few drops of the diluted sperm to the eggs and stir immediately.
4.
Fertilization success can be monitored by elevation of the fertilization envelope. Transfer a
drop of the sperm-egg mixture to a microscope slide, gently cover with a coverslip, and
examine by phase or bright field microscopy for the presence of a raised fertilization
envelope.
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Biology 3235
Developmental Biology Laboratory
University of Utah
Spring 2000
Figure A shows an unfertilized egg (just prior to fertilization). Note the sperm (arrows) bound to
the egg surface. Figure B shows a fertilized egg in the process of raising the vitelline envelope
(VE). Note the many sperm bound to the VE surface as it elevates. Figure C shows a fertilized
egg after complete elevation of the vitelline envelope, now referred to as the “fertilization
envelope (FE).” Note the lack of sperm bound to the FE surface.
Poor fertilization success may result from too few sperm. In this case, additional sperm can be
added. However, adding too many sperm may result in “polyspermy.”
C. Concentrating eggs or embryos (for washing or sampling).
Eggs or early embryos can be concentrated in the center of a culture dish by gently swirling the
disk, and then abruptly stopping. As the ASW continues to swirl, the embryos will be carried to
the center of the disk by the fluid motion. Concentrating in this way allows you to sample larger
numbers in a smaller volume.
Eggs and embryos can also be concentrated and collected by gently spinning them in a handcranked centrifuge for a few seconds. This technique is also successful for concentrating or
washing swimming blastulae, although you will have to sample the embryos or decant the ASW
quickly, before the blastulae have a chance to disperse again.
D. Caring for sea urchin embryos.
1.
To culture embryos, allow the fertilized eggs to settle to the bottom of the dish, and wash
them by carefully decanting the sea water, and refilling with fresh seawater (alternatively,
remove the seawater with a transfer pipet). It also is possible to concentrate most of the
eggs into a small volume and transfer them to a new dish containing fresh seawater.
2.
Incubate at the correct temperature: 13-15E for Strongylocentrotus, 15-17E for Arbacia,
room temperature for Lytechinus.
3.
Once they have “hatched,” blastulae actively swim using cilia on their surface, and it is no
longer possible to decant solution without losing embryo. Thus, we do not routinely
change the sea water. Keep cultures covered to cut down evaporation. You can mark the
original fluid level and replace evaporation losses with distilled water.
E. Fixing embryos for microscopy.
Sea urchin eggs or embryos can be fixed for microscopy in either 100% methanol (for DNA
staining) or in 3.7% formaldehyde in ASW (for other staining procedures, including phalloidinstaining of F-actin and immunofluorescence microscopy of MTs).
1.
Prepare a microcentrifuge with ~ 1 ml of the desired fixative.
2.
Carefully transfer eggs or embryos to the fix in a minimal volume of ASW.
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Biology 3235
3.
Developmental Biology Laboratory
University of Utah
Spring 2000
Fix at room temperature for 30-60 minutes. Follow additional instructions as per
individual experimental protocols.
IV. INVESTIGATING FERTILIZATION OF SEA URCHIN EGGS.
Fertilization of haploid eggs by haploid sperm reconstitutes the diploid state, forming the
“zygote.” Successful fertilization requires: (1) specific recognition of egg by sperm, (2) binding of
sperm to the egg, and (3) fusion of the plasma membranes of the two gametes. In organisms that
undergo external fertilization, a number of mechanisms (both behavioral and physiological) have
been evolved to ensure that sperm find eggs of the same species. The following series of exercises
are provided as an example of some of the experiments you might perform to investigate the
mechanisms of fertilization.
A. Observing and quantifying fertilization:
Procedure:
1.
Prepare 8-12 microcentrifuge tubes containing ~ 1ml of 3.7% formaldehyde in ASW.
Label the tubes for time intervals of 5-20 seconds, out to ~120 seconds after fertilization.
2.
Prepare rather dense suspensions of eggs and sperm (denser than would normally be used
to start a culture) in two separate petri dishes. There should be enough eggs to take 8-12
timed samples, and the sperm suspension should be distinctly milky.
3.
Quickly mix the sperm and eggs, and immediately remove and fix an aliquot for the first
(T=0 seconds) timepoint. Use a pasteur or transfer pipet to withdraw the sample, and add
it quickly to the first tube of formaldehyde. Do not return a pipette that has contacted
formaldehyde to the dish of eggs, and keep any glassware that has contacted
formaldehyde away from live material.
4.
Continue taking samples at intervals of 5-20 seconds. Mix each sample with the
formaldehyde as quickly as possible. This is easiest if you work in pairs, with one student
collecting samples while the other serves as the timekeeper.
5.
Allow the samples to fix for a few minutes in formaldehyde, and then wash several times in
fresh ASW. Some loss during washing can be tolerated, if you started with a sufficient
number of eggs in each sample.
6.
Place a drop of each sample on a microscope slide, gently cover with a coverslip, and use
phase microscopy to examine the cells. How does the appearance of the egg change after
fertilization? How fast is the fertilization envelope raised? Can you quantify the rate and
success of fertilization by observing the raising of the fertilization envelope? How does the
number of sperm bound to eggs change at different times after fertilization?
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Biology 3235
Developmental Biology Laboratory
University of Utah
Spring 2000
Questions you might address with additional experiments:
Is sea urchin fertilization species specific? Try repeating the above experiment with eggs
and sperm from different species (with luck, Arbacia sp. or Lytechinus sp. will be
available during the second and third weeks of lab).
Are Ca2+ ions required for fertilization? Wash eggs into Ca2+-free seawater (several
washes with gentle centrifugation to settle the eggs). Dilute sperm into Ca2+-free seawater.
Fertilize, and monitor fertilization, as above. Follow until a parallel group of fertilized eggs
in ASW undergo cleavage. Finally, what effect does Ca2+ influx have on sea urchin eggs?
How does the sperm trigger egg activation? Treat eggs with the 1 mM A23187 (a Ca2+
ionophore) in ASW. What effect does raising the internal [Ca2+] have on the egg?
Is the sperm required for cleavage and development? (1) transfer unfertilized eggs into
hypertonic ASW (0.5 M NaCl in ASW); (2) Let stand 20–30 min. (3) wash the eggs and
return to normal sea water. Record the formation of fertilization envelopes, and check for
evidence of cleavage 1-2 hrs later. Note: The fraction of eggs that activate and develop
may be quite small, so you may need to look at a good number of eggs to find interesting
ones. Transfer activated or any “interesting” eggs to a new dish of ASW, and follow their
development.
Are Na+ ions necessary for fertilization? Perform the same experiment as in #2 using
seawater in which most of the Na+ has been replaced with another impermeant cation.
Fertilize, and monitor fertilization, as above. Follow until a parallel group of fertilized eggs
in ASW undergo cleavage.
B. Activation of sperm and the acrosome reaction:
Binding of sperm and egg requires specific interactions between receptors on the gamete
surfaces. Initially, the sperm’s egg receptor is sequestered in the “acrosomal vacuole.” Contact of
sperm with components of the egg jelly coat induces rapid exocytosis of the acrosomal vacuole, in
a process called the acrosome reaction. In some species, exocytosis of the acrosomal vacuole is
accompanied by the rapid extension or extrusion of actin filaments to form the “acrosomal
process.” The jelly components that trigger the acrosome reaction are soluble in ASW, and can be
extracted from the jelly by simply soaking eggs in ASW overnight. In the next few exercises, you
can use this “egg water” to examine the effect of eggs on sperm.
1. First, observe the effect of egg water on the sperm by phase microscopy:
a.
Place a small drop of “virgin” sperm on a slide, and gently cover with a coverslip.
Use phase or DIC microscopy to observe sperm motility and the morphology at
40× or 100×.
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Spring 2000
b.
While continuously observing, add a small drop of “egg water” (prepared by
shaking eggs in ASW for a few minutes, or soaking eggs in ASW overnight) to
one edge of the coverslip.
c.
What effect does the egg water have on sperm motility and morphology?
2. Agglutination of sperm by egg jelly. You may also assay the binding of sperm to the egg
jelly by observing the agglutination (clumping) of sperm in egg water:
a.
Place a fairly concentrated drop of sperm into a well slide.
b.
Add a drop or two of egg water, and mix briefly.
c.
Observe macroscopically (by eye) and/or microscopy. What effect does the egg
water have on the consistency of the sperm suspension?
d.
Alternatively, mix small amounts of a diluted sperm suspension (try a few different
dilutions) and egg water in a clear glass test tube, and observe changes in the
consistency of the suspension.
Is the agglutination of sperm by eggs species specific? Try the agglutination assay using
egg water and sperm from different species (set up a matrix of all possible combinations of
egg water and sperm). You might try and set a more quantitative assay, by using serial
dilutions (1:2, 1:4, 1:8, 1:16, etc) of egg water and constant sperm number.
3. Observing the sperm acrosome reaction by phase and fluorescence
microscopy: The acrosome reaction normally is triggered by contact of the
sperm with components of the jelly layer surrounding the egg. However, it
can also be triggered by “egg water” (sea water in which eggs have been
allowed to soak overnight). Although the acrosome process in S. pupuratus is
not as dramatic as some of those mentioned in lecture, with care, it can be
(barely) seen by phase-contrast microscope (using 100× oil immersion
objectives) or by staining reacted sperm with fluorescent phalloidin (which
stains F-actin).
a.
Get two clean 15 ml conical centrifuge tubes. Into one tube, add 1 ml of ASW.
Into the other, add 1 ml of “egg water.” Label the tubes “ASW” and “Egg water,”
respectively.
b.
Using a new, clean pipette for each, add ~15 microliter of concentrated sperm
suspension to each tube (< 5 mm in the tip of a Pasteur pipette will do). Do not
get egg water in the sperm stock! Mix gently.
c.
Incubate 5~10 min at room temperature. Note any difference in the appearance of
the sperm suspensions in ASW vs. egg water.
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Biology 3235
Developmental Biology Laboratory
University of Utah
Spring 2000
d.
Add 0.1 ml of fix (3.7% formaldehyde in ASW) to each tube. Mix gently. Allow to
fix for 5~10 minutes.
e.
Add 1.0 ml of fluorescent phalloidin in permeabilization buffer (1:200 phalloidin in
80 mM KPipes pH 6.8, 1 mM MgCl2, 5 mM EGTA, 0.2%TX-100). Mix gently.
f.
Incubate 10~15 minutes at room temperature. Again, note any difference in the
appearance of the sperm suspensions in ASW vs. egg water.
g.
Spread a drop or two of each sperm suspension onto separate polylysine-coated
coverslips (set the coverslips on a piece of parafilm, which is hydrophobic and will
prevent the sperm from spreading off the coverslips). Allow to sit for ~ 1 minute.
h.
Carefully pick up each coverslip with forceps, and gently rinse off the excess sperm
and phalloidin by gently swishing it for 10~20 seconds in a beaker containing 100
ml of TBSN (Tris-buffered saline with NP-40) followed by 10~20 seconds in
diH2O (taking care to remember which side the sperm are on!). Note: you can
stain the sperm nucleus by including 5-10 micrograms/ml Hoechst 33258 in the
final TBSN and water washes.
i.
Mount each coverslip sperm-side down on a drop of glycerol/anti-fade mounting
solution (90% glycerol, 50 mM Tris pH 8.0, 25 mg/ml propylgallate) on a
microscope slide. Gently tap or press the coverslip into the mountant, carefully
wipe away excess mountant, and seal the coverslip to the slide with fingernail
polish. Set in the hood to dry for >15 minutes.
j.
Examine the sperm by phase-contrast and fluorescence microscopy, using the 100×
oil immersion objective (see instructors). Can you recognize acrosome-reacted
sperm by phase-contrast? by fluorescence microscopy? Collect and compare phase
and fluorescence images of both ASW- and egg water-treated sperm.
Questions you might answer with additional experiments:
How soon after addition of egg water can the acrosome reaction be detected? Set up a
time course, following the procedure outlined above but varying the incubation time in
step 3.
Can acrosome-reacted sperm subsequently fertilize eggs? Prepare acrosome-reacted and
ASW-treated sperm (from the same sperm concentrate) as in steps 1-3. Then use each to
fertilize fresh batches of eggs, scoring the efficiency of fertilization by observing the %
fertilization envelopes raised after 30, 60, and 90 seconds.
Is the acrosome reaction species specific? Mix egg water and sperm from different
species (we hope to have Arbacia sp. and Lytechinus sp. available next week). Assay the
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Biology 3235
Developmental Biology Laboratory
University of Utah
Spring 2000
acrosome reaction by fluorescence microscopy, and/or by ability of the treated sperm to
fertilize con-specific eggs.
C. Does pronuclear migration require microtubules (MTs) and/or actin filaments (F-actin)?
Fertilization introduces the haploid male genome into the haploid egg. Upon entering the egg, the
sperm nucleus decondenses, and the sperm DNA is packaged into chromatin. The male
“pronucleus” then migrates towards the center of the egg, where it meets (and in some cases,
fuses with) the female (egg) pronucleus. Pronuclear migration can be followed by staining
fertilized eggs at intervals with the fluorescent DNA dye, Hoechst 33258.
Procedure:
1.
Fertilize a large number of eggs as described. Soon after fertilization, split the fertilized
eggs into four aliquots. Transfer one batch of eggs to 10 micrograms/ml cytochalasin B in
ASW; transfer another to 10 micrograms/ml nocodazole in ASW; transfer a third batch to
0.1% DMSO in ASW, and culture the remaining embryos in ASW.
2.
Fix embryos at various intervals after fertilization (i.e. 5, 10, 30, 60 minutes postfertilization to follow pronuclear migration; 32-64 cell stage; or blastula stage; etc). Fix
embryos by adding 5-8 drops of embryos to a microcentrifuge tube containing ~ 1 ml of
3.7% formaldehyde in ASW plus 0.2% TX-100 (alternatively, fix by adding 5-8 drops of
embryos to ~1 ml of 100% methanol).
3.
Allow to fix for 5-10 minutes at room temperature.
4.
Allow embryos to settle to bottom of tube (use hand centrifuge, if needed). Carefully
remove and discard as much of the fix as possible.
5.
Gently resuspend embryos in ~1ml of 100% methanol (MeOH) containing 50
micrograms/ml Hoechst (or another appropriate chromatin dye). Incubate for 30-60
minutes with occasional agitation.
6.
Remove and discard the Hoechst/methanol (the embryos should now sediment without
requiring centrifugation). Wash 2×10 minutes with ~1 ml of 100% MeOH (without dye).
7.
Carefully remove as much of the MeOH as possible, and add ~1 ml of benzyl
alcohol:benzylbenzoate clearing solution (BA:BB). Do NOT mix! Incubate ~ 15 minutes,
allowing the eggs to settle to the bottom of the tube (use hand centrifuge, if necessary). As
the embryos clear, they will become virtually invisible, so you won’t be able to see them.
8.
Remove and discard about ½ of the BA:BB. Gently resuspend the eggs in the remaining
BA:BB, and place 1-3 drops on a clean microscope slide. Carefully remove excess BA:BB
solution, and seal the coverslip with fingernail polish.
9.
Observe by fluorescence microscopy, using the UV filter set.
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Biology 3235
Developmental Biology Laboratory
University of Utah
Spring 2000
What effects do cytochalasin and nocodazole have on migration of the male pronucleus?
V. EARLY DEVELOPMENT OF SEA URCHINS
In the next several exercises, you have the opportunity to investigate some of the mechanisms of
development and pattern formation in sea urchin embryos.
A.
Normal development of sea urchin embryos. First, you should learn to recognize the
normal developmental stages of development of living sea urchin embryos.
Procedure:
1.
Place a drop of embryos from cultures started at various times and/or maintained at
different temperatures onto a clean microscope slides, and gently cover with a coverslip. If
there is enough liquid to spread instantly under the entire cover slip, the embryos should
not be damaged.
Note: From blastula on, embryos swim. In older cultures, those on the bottom are dead. If you
can see specks moving above the bottom, that is where to sample.
2.
Use phase, and/or DIC microscopy to examine the embryos at 20×, 40×, or 100×. Identify
the normal stages of development, from cleavage onward. Do not get sea water on the
microscope objectives!
You might also try making time-lapse recordings of normal development:
3.
Make a small chamber from a microscope slide and coverslip, separated by two small
spacers of double-sided sticky tape.
4.
Use a micropipet to add just enough embryo suspension to fill the chamber. If you gently
expel the embryos along one open edge of the chamber, they will be drawn in by capillary
action.
5.
Use the CCD cameras to collect a time-lapse series (see the instructors for help setting up
the camera and computers).
Questions you might address:
How is the timing of normal development affected by temperature? As you accumulate
observations, it may be useful to make a table showing developmental stage as a function of time
and temperature. You can use this to plan the temperature at which you keep experiments and
your schedule for checking results.
When does the developing embryos first become asymmetric? Is this asymmetry developmentally
important?
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B. Using chemical inhibitors to investigate early development.
The following few exercises use inhibitors of specific biochemical or cellular functions to
address their importance in early development. Chemical inhibitors will either be available as dry
powders, concentrated stocks in solvent such as DMSO, or pre-diluted in ASW to twice the
concentration to be used. In the former cases, you will need to make solutions of the appropriate
concentration in ASW. In the latter case, it is sufficient to mix equal parts of inhibitor solution and
a suspension of embryos in ASW (It is adequate to count drops or "squirts" with Pasteur
pipettes).
Many of the inhibitors are expensive or available in limited only quantities. For this reason,
you should set up your cultures in the smallest volumes practical. Multi-well “Linbro” plates or
small Petri plates will be available for this purpose. Linbro plates (multi-well plates) or small petri
plates are useful for this purpose. Remember to set up parallel cultures of untreated embryos or
embryos treated with the appropriate concentration of diluent (DMSO, for example) as controls.
Examine and record the progress of both control and experimental cultures. Remember
that by the late blastula stage, embryos hatch from the fertilization membrane and begin
swimming. Be sure to check for embryos suspended above the bottom of the culture.
1. Are transcription (RNA synthesis) or translation (protein synthesis) required for cleavage
and/or early development? Many eggs contain “maternal” stores of mRNA or proteins used
during early development of the zygote. You can ask when zygotic transcription and/or protein
synthesis are required by inhibiting these biochemical processes with either Actinomycin (an
inhibitor of RNA polymerase) or cycloheximide (a protein synthesis inhibitor), respectively.
Procedure:
a.
Fertilize a fairly large number of eggs.
b.
Within a few minutes after fertilization, transfer a fraction of the eggs to ASW containing
100 or 500 micrograms/ml actinomycin. Treat another batch of eggs with 100 or 500
micrograms/ml cycloheximide (emetine or puromycin can be substituted for
cycloheximide). Remember to keep a parallel untreated culture as a control.
c.
Monitor cleavage and development of the treated and control embryos until the controls
have reached the late gastrula-stage (or longer).
You might also try adding actinomycin or cycloheximide during later development.
2. Do early urchin embryos have cell-cycle checkpoints that monitors DNA replication?
Many early embryonic cells lack the cell cycle controls found in somatic cells. Inhibitors of DNA
replication can be used to investigate whether sea urchin embryos contain a checkpoint
monitoring DNA replication.
Procedure:
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a.
Fertilize a large batch of embryos, as previously described.
b.
Culture one batch of embryos in ASW containing aphidicolin (an inhibitor of DNA
polymerase).
c.
Culture another batch of embryos in ASW containing hydroxyurea (an inhibitor of
ribonucleotide reductase; why would this block DNA replication?).
d.
Compare the development of embryos treated with inhibitors with that of untreated
control embryos.
2. Are actin filaments (F-actin) or microtubules (MTs) required for cleavage and early
development? You may test the requirement for F-actin and/or MTs by treating with either
cytochalasin B (an inhibitor of actin assembly) or nocodazole (a MT inhibitor), respectively.
Procedure:
a.
Fertilize a fairly large number of eggs.
b.
Soon after fertilization, transfer ~1/4 of the embryos to ASW containing 10
micrograms/ml cytochalasin, ~1/4 to ASW containing 10 micrograms/ml nocodazole, and
~1/4 to ASW containing 0.1% DMSO (cytochalasin and nocodazole are both dissolved in
DMSO; the final DMSO concentration in those samples is 0.1%). Maintain the final ~1/4
of the embryos in ASW as controls.
c.
Monitor and record cleavage and early development through several (3-4?) division
cycles.
After several division cycles, you may want to fix some of the embryos from each culture and
stain them with the fluorescent DNA-specific dye Hoechst 33258 (bisbenzimide).
d.
Add ~ 1 ml of 100% methanol containing 5 micrograms per ml Hoechst 33258 to the
appropriate number of microcentrifuge tubes.
e.
Add a few drops of concentrated embryos.
f.
Allow to fix and stain for > 1 hr. Then clear the embryos in BA:BB, mount in BA:BB,
and examine the chromatin organization by fluorescence microscopy. What effect do these
inhibitors have on cleavage and chromatin organization?
You might also ask whether MTs and/or actin are required for the morphogenetic movements of
gastrulation, by treating embryos at later stages with either nocodazole or cytochalasin.
3. Is signaling by GSK-3-related kinases required for patterning of the sea urchin embryo?
Recent studies in Drosophila and Xenopus reveal a critical role for glycogen-synthase kinase 3
(GSK-3) or related kinases in wnt signaling pathways important for embryonic patterning. GSK-3
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University of Utah
Spring 2000
and related kinases are inhibited by Li+ ions, explaining classical observations that treatment with
LiCl can perturb normal pattern formation.
Procedures:
a.
Fertilize eggs following standard protocols.
b.
Treat developing embryos with 0.5 M LiCl in ASW. The most dramatic effects have been
reported for embryos treated during mid-cleavage (e.g., starting around 16-32 cell stage).
In general, limited exposures (up to a few hours) work better than continuous culture in
LiCl. Maintain a parallel culture in regular ASW, as a control.
c.
Examine the resulting embryos and larvae. What effect does Li+ exposure have on the
animal-vegetal/dorsoventral patterning of the embryo?
You may also want to try other concentrations/durations of Li+ exposure, or determine the
window of susceptibility of the embryos to Li+ effects.
4. Are sulfated components of the extracellular matrix required for gastrulation in sea urchin
embryos? Synthesis of sulfated polysaccharides of the extracellular matrix can be inhibited by
culturing embryos in sulfate-free ASW.
Procedure:
a.
Wash unfertilized eggs 2-3 times in sulfate-free ASW.
b.
Fertilize the washed, sulfate-free eggs with sperm diluted in sulfate-free ASW (you might
also try centrifuging the sperm and resuspending them in sulfate-free ASW).
c.
Culture in sulfate-free ASW until gastrulation, and examine by phase and/or DIC
microscopy.
Can primary mesenchyme ingress and migrate in the absence of sulfate? Does the archenteron
invaginate normally in the absence of sulfated polysaccharides?
In the next few exercises, you can investigate the role of cytoplasmic organization and the
pattern of cleavage in early development.
C. Does the cytoplasmic organization of the sea urchin egg specify cell fate during
development? Centrifugation can be used to rearrange the cytoplasmic constituents of unfertilized
or fertilized eggs, allowing you to test the importance of cytoplasmic organization in early
development.
Sea urchin labs-14
Biology 3235
Developmental Biology Laboratory
University of Utah
Spring 2000
1.
Fill several 1.5 ml microcentrifuge tubes about half full with 0.85M sucrose. Add
unfertilized or fertilized eggs in sea water to the top of the sucrose “cushions.” Mix the
interface a little by stirring gently with the end of a Pasteur pipet.
2.
Centrifuge the eggs for times ranging from 2–3 minutes up to 10–12 minutes.
3.
Carefully wash out the sucrose, and culture the eggs in fresh ASW.
4.
Immediately examine and record the range of effects (anywhere from slight banding or
“stratification” of the cytoplasm, through distortion of the spherical shape, to
fragmentation into denser and lighter pieces). Samples can be fixed for more leisurely
examination.
5.
Observe and compare the development of centrifuged eggs with untreated controls (how
would you control for the effects of sucrose?).
Can centrifuged unfertilized eggs subsequently be fertilized?
D. Are blastomeres of early urchin embryos developmentally equivalent? Early developmental
biologists often classified development into two patterns: (1) mosaic development, or (2)
regulative development. Embryos following a “mosaic” pattern of development often exhibit
invariant cell lineages and little cell migration. In these embryos, specific cells invariably gave rise
to specific organs or body parts. Thus, ablation or removal of individual cells (or small groups of
cells) in the early embryo result in loss of specific structures in the later embryo or adult. In
contrast, embryos following the “regulative” pattern of development often exhibit extensive cell
migration during development, and are able to regulate their pattern to compensate for the loss of
individual cells. In extreme cases, complete and normal embryos and adults are able to develop
from individual blastomeres, demonstrating the developmental equivalence of the early embryonic
cells. Some of the earliest blastomere separation experiments were performed in sea urchin and
starfish embryos by Wilhelm Roux.
A simple procedure for separating sea urchin blastomeres:
Note: The success of this procedure may vary with the species of sea urchin used. You might
want to try isolating blastomeres from more than one species.
1.
Fertilize a fairly large sample of eggs. Inspect within 1–2 minutes, to confirm a high
percentage of fertilization membranes.
2.
Within 2–3 minutes after fertilization (before the fertilization envelope hardens),
vigorously pipet the eggs up and down with a Pasteur pipet. Direct the stream from the
pipet against the bottom of the dish as you expel the eggs. The aim here is to break the
fertilization envelope on a significant fraction of the eggs. Check this by comparing the
fraction of eggs with raised fertilization envelopes before and after pipetting. If not
reduced significantly, repeat the pipetting (even more vigorously).
Sea urchin labs-15
Biology 3235
Developmental Biology Laboratory
University of Utah
Spring 2000
3.
Let the eggs settle, concentrate them in the center of the dish by gentle swirling, and
collect as many eggs as possible in minimum volume of sea water.
4.
Gently layer the eggs over a much large volume of Ca2+-free ASW in a small beaker or
tall tube. Allow the eggs to settle (you might even wash a second time in Ca2+-free ASW).
5.
Monitor a control sample after about one hour (room temperature).When first cleavage is
about complete in the control sample, check a sample from the Ca2+-free sea water. A
fraction of embryos in the Ca2+-free ASW should exhibit a "figure 8" configuration, in
which the two blastomeres are spherical and just touching at one point.
6.
Collect as many of the figure 8 embryos as possible, transfer them to a new dish, and pipet
them vigorously.
(Alternatively, transfer a sample of embryos to Ca2+-free seawater in a screw cap centrifuge tube,
and shake vigorously.)
7.
Examine the embryos. Isolated blastomeres will appear as single cells about ½ the volume
of a normal egg. Collect as many as possible, and transfer to a dish with at least 10–20
volumes of regular sea water.
Compare the development of isolated blastomeres to the complete embryos present in the same
culture (use size to distinguish embryos from the isolated blastomeres from their normal sibs).
You may also want to try separating blastomeres of four-cell embryos, to determine whether they
are developmentally equivalent. You may be able to obtain embryos containing 1, 2, or 3 of the
four blastomeres, and follow their development.
An alternate procedure for separating sea urchin blastomeres:
1.
Place unfertilized eggs in a screw-cap tube half filled with seawater.
2.
Add 1 drop of dilute sperm suspension into the tube, and cap. Invert once to mix.
3.
Wait exactly one minute.
4.
Shake vigorously for exactly two minutes. Shaking should remove the vitelline envelopes
of the fertilized eggs (this must be done before the envelope hardens).
5.
Transfer the contents to a petri plate of sea water, and monitor cleavage.
6.
Just after completion of first cleavage either:
A. squirt vigorous jets of seawater into the dish...
B. pipet the eggs up and down in a Pasteur pipet, squirting them against the dish..., or
Sea urchin labs-16
Biology 3235
Developmental Biology Laboratory
University of Utah
Spring 2000
C. transfer to a capped tube and shake vigorously.
7.
Transfer back to petri plate and select isolated blastomeres by size.
You might also try placing the de-enveloped eggs into Ca2+-free sea water (which loosens cell-cell
contacts) until completion of first division, and then try squirting or shaking them as above.
Eggs from different species of urchins respond differently to the blastomere isolation procedures.
If S. pupuratus blastomeres don’t dissociate readily, try with Arbacia or Lytechinus.
E. Is the asymmetric division at the fourth cleavage required for normal development? During
the fourth division cycle (from eight to sixteen cells), the vegetal tier of blastomeres divides
asymmetrically to produce a large quadrant of macromeres and four smaller micromeres at the
vegetal pole. In some species of urchins, the asymmetry of the fourth division can be disrupted by
treatment with low concentrations of the ionic detergent sodium dodecyl sulfate (SDS),
eliminating formation of the micromeres. This technique can be used to assay the role of the
micromeres in development and organogenesis.
Procedure:
1.
Fertilize a fairly large number of eggs.
2.
Just after the second division, transfer some of the embryos to ASW containing 0.05%,
0.1%, and 0.25% SDS.
3.
Culture in SDS until fifth cleavage. Then, carefully wash the embryos.
4.
Monitor development through gastrulation and formation of the pluteus larvae.
F. The role of cell adhesion in early development. Differential cell adhesion appears to play a
very important role in morphogenesis of many embryos. In some cases, embryos can be
disaggregated and the embryonic cells are able to re-aggregate and undergo some semblance of
normal development and/or differentiation.
Procedures for disaggregating sea urchin embryos:
1.
Raise embryos to the blastula stage (embryos which have emerged from the fertilization
envelope and are slowly swimming around the dish).
2.
Wash the embryos at least three times in Ca2+-free ASW, by spinning them down in the
hand-cranked centrifuge, rapidly decanting the ASW, and resuspending them in ASWCa2+. Do not let the embryos sit very long as a tight clump; decant the original solution
and resuspend in the new solution as quickly as possible.
3.
Transfer embryos (in ASW-Ca2+) to a small petri dish and coax them to fall apart by
pipetting them up and down.
Sea urchin labs-17
Biology 3235
Developmental Biology Laboratory
University of Utah
Spring 2000
4.
When most of the embryos are disaggregated, let them settle, remove as much ASWCa2+as possible, and replace with regular ASW. Repeat this wash several times (you can
also use the hand-centrifuge to pellet and wash the blastomeres).
5.
Once returned to regular sea water, try culturing the cells at high density (such that cells
nearly cover the surface of the dish). It also might help to put the blastomere cultures on
an orbital shaker, and rotate them gently (to increase collisions). Monitor re-aggregation
of the blastomeres. How far can they develop?
Is re-aggregation species specific? Try dis-aggregating embryos of different species, and coculture the individual blastomeres (cells from the different species might be distinguished by their
pigmentation).
Sea urchin labs-18