patterns and experiments in developmental biology

207 Poly-L-Lysine-coated coverslips or slides optional India ink optional Modeling clay S OLUTIONS AND C HEMICALS Seawater or appropriate salt mixture artificial seawater—see Appendix A

Publication Date: January 2001

Overview

A laboratory manual for developmental biology offering basic, easy to use, laboratory investigations (18 experiments) spanning various models including echinoderm (Sea Urchin), amphibian (Frog), chick embryo, and fern gametophyte.

As with the earlier editions, the goal of this edition of Patterns and Experiments is to facilitate

and encourage developmental biology and embryology laboratory experiences that bring students gether with fascinating and dynamic developing systems Professional biologists and nonbiologists bothoften relate that the study of some aspect of development of a living organism has been a memorablehighlight in their educational experience How fascinating it is to watch those tiny clusters of cells asone makes that first marathon set of observations of a batch of developing sea urchin embryos Howexciting it is to return to the lab to find a vigorously beating heart in an in vitro cultured chick embryowhere there had been no visible heart and a much simpler form only twenty-four hours earlier

to-My own view of biology and my career plans changed when I had that experience I want to say

to students who will use this manual that I envy you the excitement that comes with those first portunities to experiment with living, developing organisms I hope that a few of you might be inspired

op-to go on op-to careers researching developmental processes and sharing the fascination of developmentwith your own students This is a truly exciting time in developmental biology because we are nowable to investigate directly many of the genetic mechanisms underlying various developmental processes.However, as you begin your study of developmental biology, whether you pursue that study only in thiscourse or study development for many years to come, I would like to offer one bit of advice from theperspective of many years in developmental biology.As intently as you may study certain individual de-velopmental processes, please try not to lose sight of the whole developing organism and the still broaderpicture of the role of development in the perpetuation of species Much of the fascination and beauty

of development is to be found at those levels

This third edition of Patterns and Experiments includes a number of additions and new features.

Several of the additions are to the considerably expanded section on echinoderm development.There are much more detailed directions for caring for sea urchin and sand dollar embryos and larvae( Laboratory 1 and Appendix A) Several colleagues have reported that their students have been frus-trated with their inability to observe development beyond the earliest stages, and I think that these di-rections will make it much easier for students to observe additional parts of development.The simplerand more effective procedure for blastomere separation that has been incorporated into Laboratory 2should make it easier for students to conduct “twinning” experiments like those that have such a richhistory in developmental biology’s past Laboratory 2 also includes a fascinating new experiment on thesomewhat surprising, but very adaptive, capacity of echinoderm embryos and larvae to regenerate lostcilia Also, reorganization of the echinoderm portion of the manual led to creation of a new part( Laboratory 3) that includes investigation of differentiation of an enzyme system This investigation pro-vides students a chance to study specific localized genetic activation in differentiation.Also,“Suggestionsfor Further Investigation of Echinoderm Development” was reorganized and substantially rewritten

There is an important addition to the chick embryo section as well In Laboratory 11, an earlierbrief suggestion about investigating heart duplication has been expanded to a full experiment on heartrudiment separation and heart tube duplication that includes informative new illustrations.

Numerous other updates and additions, including several added illustrations, have been madethroughout the manual Well over one hundred new references have been incorporated into the

“Suggestions for Further Investigation” that appear at the ends of the portions of the manual Each set

of references has been updated, and the majority of the new references are to works that have beenadded to the very dynamic literature of developmental biology since publication of the second edition

of Patterns and Experiments in 1995.

I’ve also added citations to a number of the useful websites, many of which have come into beingsince 1995 as well I’ve tried for a modest mix of specialized websites as well as general ones that pro-vide links to many more of the valuable resources now available on the World Wide Web and which arelikely to incorporate additional links to important sites that surely will be developed in the comingyears

Developmental biology is not a discipline isolated from other aspects of biology This is larly evident, for example, in regard to the worldwide ecological problem of declining populations ofnumerous amphibian species recognized during the 1980s and 1990s Appendix G contains some sug-gestions concerning responsible use of amphibians in teaching that are relevant to this problem Thatappendix also contains suggestions of strategies for teaching developmental biology without sacrificingadult vertebrate animals, which is an option that a number of biologists, including me, prefer to choose

particu-I thank the colleagues and students who have used the earlier editions of this manual and havetaken the time to share some of their experiences in developmental biology They have made insight-ful comments about the manual and have offered helpful suggestions and criticisms.A number of thosesuggestions led to additions to the second edition, and others have influenced the development of thisthird edition I also warmly thank the many colleagues from colleges and universities across the UnitedStates and Canada who have participated over the years in my summer workshops on the DevelopmentalBiology Teaching Laboratory at the University of Maine’s Darling Marine Center Those developmentalbiologists have brought their own individual perspectives and expertise to the workshop sessions, andwe’ve shared some remarkable learning experiences in that beautiful setting I owe them and my DarlingCenter colleagues a great deal

Finally, I wish once again to offer my thanks to Peter Volpe who was my colleague and mentor inpreparation of the first edition of this manual Several of the amphibian development labs, especiallyLaboratories 4, 5, 6, and parts of Laboratory 8 have been only slightly updated and have remained largely

as Peter conceived them, as has Appendix B Some years ago, Peter’s main interests moved into the eas of human development, medical genetics, and biomedical ethics, and he turned his full energy andattention to those pursuits Thus, his direct involvement with this manual ended with the first edition,but his influence remains evident in a number of places The manual’s current title stands as a recog-nition of his original contributions

ar-Leland G Johnson

L A B O R A T O R Y

1

Fertilization and Early Development of

Sea Urchins and Sand Dollars

Echinoid echinoderms (sea urchins and sand dollars, which are also known as irregular urchins)have been the subjects of many investigations of fertilization and early development, and much of ourunderstanding of developmental processes in animals has come from this research Sea urchin and sanddollar gametes are readily obtained just before, and during, the breeding season and their developingembryos can be cultured in seawater or salt solutions that approximate the osmotic and ionic proper-ties of seawater Eggs and embryos of many species are quite translucent, so it is possible to observe anumber of cell activities during early development, using a light microscope

In this laboratory, you will have opportunity to observe development from fertilization through sembly of the pluteus larva, which is the swimming, feeding larval form that is characteristic of many

as-of the echinoid echinoderms

ani-You should be very careful about conditions under which gametes and embryos are maintained.Temperature control is especially important, and your instructor will provide information concerningtemperatures that are appropriate for the species you are studying.

1 Gently blot excess water off an adult urchin and place it on a clean surface with its aboral (opposite to,

or away from, the mouth) surface down Induce shedding of gametes by injecting 1 or 2 ml of 0.5 M KCl throughthe membrane surrounding the mouth opening (perioral membrane) Sand dollars should be injected with a fine-gauge needle inserted at a very shallow angle To enhance effectiveness of the KCl injection, it is advisable to di-vide the injected dose of KCl among two or three sites in the perioral membrane Several websites demonstratethese techniques (see Materials, p 8)

It is very important to avoid possible contamination of eggs with sperm.This can be accomplished by using

a separate syringe and needle for each animal, but that usually isn’t practical An alternative technique is to retainenough KCl solution in the syringe so that some can be expelled after each injection to flush the needle Thenrinse the needle surface with distilled water and dry it with a clean Kimwipe before refilling the syringe and in-jecting the next animal

2 Collect eggs by inverting a female over a beaker or a finger bowl containing seawater.The water level inthe beaker should be such that the female’s gonopores are in the seawater The eggs will flow out of the gono-pores and settle to the bottom of the beaker.After the eggs have been shed, they should be washed by decantingthe supernatant water and replacing it with clean seawater.This washing removes coelomic fluid, broken spines,and body surface debris from the water Eggs should be washed twice if time permits Alternatively, it is possible

to collect shed gametes directly from the body surface with a pipette, which helps avoid contamination by debrisand extraneous fluids Direct collection by pipette often is the best technique to use when only a few gametesare shed, as is sometimes the case with sand dollars

It is best to proceed with fertilization immediately, but if necessary, the eggs of some species can be erated at 5° C for several hours and still respond fairly well in fertilization

refrig-3 Active sperm, unlike eggs, are viable for only a few minutes in seawater.Thus, it is necessary to keep thesperm quiescent by collecting them under “dry” conditions (that is, in an undiluted suspension) A small portion

of the “dry” suspension can be diluted in seawater each time active sperm are needed

When an animal has been identified as a male, wipe away excess moisture from among the spines on theaboral surface Invert the male over a clean, dry petri dish or Syracuse dish.After several large drops of the whitesperm suspension are in the dish, remove the animal and snugly cover the dish with parafilm or aluminum foil.The sperm should be kept concentrated until just prior to use, when they are activated by dilution in seawater.Collected sperm may be stored “dry” at a cool room temperature for an hour or so, but they should be stored

in a 5° C refrigerator if longer storage is required Sperm of some species can be stored in a refrigerator for up

to a day

4 Observe suspensions of eggs and sperm microscopically and record your observations.To observe activesperm, add 1 drop of “dry” sperm to about 100 ml of seawater in a small container Sperm are best observed us-ing phase contrast, a dark-field technique, or some other type of microscopy that increases contrast or otherwiseenhances visibility of very small objects If you don’t have available phase-contrast optics or a dark-field arrange-ment on the microscope that you are using, close down the iris diaphragm of the microscope’s condenser Thiswill add some artificial contrast that will facilitate these observations

The newly shed echinoid egg is surrounded by a transparent jelly coat that has a refractive index similar tothat of seawater If you wish to observe the eggs’ jelly coats, mix a drop of India ink with a small quantity of sea-water and observe eggs in the suspension Since the India ink particles do not penetrate the jelly coat, each eggshould appear to be surrounded by a clear area (the jelly coat) containing no ink particles

Fertilization

1 The fertilization procedure involves mixing drops of a diluted sperm suspension with eggs in seawater

A dilute sperm suspension is prepared by placing 1 drop of the undiluted (“dry”) sperm in a beaker containing

100 ml of seawater Mix with a clean pipette to obtain a uniform, faintly cloudy suspension The “dry” sperm pension is quite viscous so it is sometimes difficult to control the amount transferred to the beaker of seawater.The final diluted sperm suspension should be only slightly cloudy, not milky, in appearance, because use of an ex-cessively dense sperm suspension can lead to polyspermy Polyspermy, the entry of more than one sperm into anegg, results in abnormal, arrested development Since sperm activation requires several minutes, the dilute spermsuspension should be allowed to stand for 5 to 8 minutes before use

sus-Transfer several drops of washed eggs to a container with about 100 ml of clean seawater.A thinly scatteredlayer of eggs on the bottom of a beaker or finger bowl is an appropriate egg density Eggs, when they have set-tled, should cover no more than about one-third of the area of the bottom of the container.Then, add 2 or 3 drops

of the dilute sperm suspension to the beaker or dish containing the eggs Mix the sperm and eggs by stirring verygently with a clean pipette

2 Transfer a sample of the suspension of eggs and sperm to a slide and observe it with a compound

mi-croscope.The most conspicuously observable event is the formation of the fertilization membrane (fertilization

envelope), which is a visible indication that the union of sperm and egg has occurred If fewer than two-thirds ofthe eggs display fertilization membranes after 2 or 3 minutes, add several more drops of dilute sperm suspensionand stir gently Repeat if necessary

The fertilization membrane gradually rises away from the surface of the egg, beginning in the area of spermpenetration and spreading outward around the entire egg Fertilization membrane elevation is usually completewithin 1 to 2 minutes Elevation of the fertilization membrane is associated with the exocytosis of the contents

of cortical granules that are located just below the surface of the egg The fertilization membrane, which initially

is thin and soft, hardens within a few minutes after its elevation.The translucent hyaline layer that forms just over

the surface of the egg also develops within a few minutes

These processes are quite temperature dependent, and their timing varies among species, but table 1.1 showsthe approximate sequence of events following the addition of sperm to eggs Note that several of the listedprocesses cannot be observed with a light microscope

3 Since the fertilization membrane is elevated rather quickly, you may miss its formation and wish to useanother technique to observe the process directly One means of direct observation consists of placing a drop ofeggs and a drop of sperm side by side on a slide With the microscope focused on the eggs, the 2 drops can beconnected by pushing them together with a needle This technique will permit you to observe sperm swarmingaround the eggs

Another method that facilitates direct observation involves placing a pair of 1-mm thick threads of ing clay parallel to one another on a microscope slide.After laying down the clay, place a drop of unfertilized eggs

model-on the slide between the two strips of modeling clay and add a coverslip Focus model-on a group of eggs and, withoutmoving the slide, add a drop of sperm at one edge of the coverslip You should be able to observe the arrival ofsperm in your field of view and the elevation of fertilization membranes on the eggs as you watch them.You mayalso be lucky enough to observe an egg in which you can see the fertilization cone form at the point where spermentry is occurring

If you can produce dark-field or Rheinberg illumination on your microscope, you will find these dark-fieldtechniques especially helpful in making these observations

Caring for Embryos and Larvae

1 Once you are satisfied that most of the eggs have fertilization membranes, leave the beaker undisturbeduntil the zygotes have largely finished settling to the bottom.Then pour off the supernatant water and add cleanseawater This step eliminates many of the extra sperm that can degenerate later and foul the water around thedeveloping embryos

TABLE 1.1 Timing of Some Fertilization Events

0 seconds Insemination 30– 40 seconds Exocytosis of cortical granules 35– 50 seconds Initiation of fertilization membrane elevation

(5–10 seconds following cortical granule exocytosis)

60– 70 seconds Completion of cortical granule exocytosis 65– 80 seconds Completion of fertilization membrane elevation

2 minutes Hyaline layer formed

5 minutes Fertilization membrane hardened

FIGURE 1.1 Cleavage stages in the sand dollar, Dendraster excentricus (a) Zygote shortly after fertilization The fertilization

membrane (fertilization envelope) has been elevated Pigment granules in the jelly coat are visible outside the fertilization membrane.

(b) 2-cell stage (c) 4-cell stage (d ) 8-cell stage, lateral view (e) 16-cell stage, vegetal view Note the presence of four micromeres that were produced during the fourth cleavage (f ) 32-cell stage, lateral view Note the cluster of micromeres in the vegetal region at the right of this photo (g) 64-cell stage, lateral view (h) Blastula shortly before hatching (The egg cell in a is approximately 120 ␮m

in diameter, and all other photos are printed at the same magnification.)

Photos by R B Emlet.

2 Put a loose-fitting aluminum-foil cover over the beaker and set it aside or put it in an appropriate stant temperature chamber until you are ready to make the observations described in the following Developingembryos and larvae must be maintained at a temperature appropriate for the particular species (See Appendix A,

con-p 201)

3 When blastulae eventually hatch, you will be able to see them swimming near the surface of the water

in the upper part of the beaker Sometimes shining a flashlight or microscope illuminator through the culture canhelp you to spot the swimmers Once a substantial number of blastulae have hatched, pour the swimmers fromthe upper part of the culture into a clean beaker Avoid pouring over the unhatched or nonswimming embryosthat remain at the bottom of the original culture.These should be discarded so that they do not foul the water inthe culture when they die and degenerate.While 100-ml beakers are very useful for some lab manipulations, longer-term cultures should be maintained in 250-ml or larger beakers

4 Aerate the cultures twice daily by repeatedly and gently pipetting air to the bottom of the cultures.However, you need to be careful to avoid sucking embryos in and out the pipette.This means that once you haveexpelled air from the pipette into the culture, you should keep the pipette bulb compressed until you have liftedthe tip of the pipette above the water’s surface Cultures also may be aerated with a very slow stream of air bub-bles from an air line or an air pump, but this needs to be done very cautiously because vigorous bubbling candamage swimming embryos and larvae

5 If you wish to extend the time that you maintain cultures and feed the developing larvae, you will tually need to exchange the water in the cultures A convenient technique for water exchange is described on

even-p 13 in Laboratory 2 For some hints on feeding larvae, see Appendix A (even-p 208)

Embryonic Development

1 Cleavage of echinoid echinoderm embryos (fig 1.1) is holoblastic; that is, the entire cell is divided at tokinesis during each cleavage division The first cleavage, which is meridional, produces a two-cell embryo Thesecond cleavage division is also meridional and yields a four-cell embryo In the third cleavage, the plane of divi-sion is at right angles to the first two cleavages and the product of the division is an eight-cell embryo with up-per and lower quartets of cells During the fourth cleavage, the four blastomeres of the upper (animal) quartet di-

cy-vide equally to form a single tier of eight medium-sized cells called mesomeres However, the divisions of the other four (vegetal) blastomeres are very unequal, producing a middle tier of four larger macromeres and a lower tier

of four much smaller micromeres that lie at the vegetal pole of the embryo.As cleavage proceeds, the embryo comes organized as a single-layered, hollow ball of cells surrounding a cavity that is known as the blastocoel.The embryo at this stage of development is called a blastula.

be-2 An embryo will hatch as a blastula, and just before hatching, the blastula begins to rotate within its tilization membrane as a result of ciliary activity Hatching involves enzymatic digestion of the membrane, whichbecomes fainter in appearance as it thins Eventually, the membrane opens at one side, allowing the blastula toroll out The time from fertilization to hatching varies among species and also is strongly influenced by the tem-perature at which development takes place Once embryos have hatched, they are harder to observe because theyswim more or less continuously Some individuals eventually become “beached” near the edge of drops on slides,but it is also possible to take active steps to slow or stop them If Poly-L-Lysine-coated coverslips or slides are avail-able,embryos will settle out of a drop onto the coated surface and be held still while you observe them.Alternatively,embryos and larvae can be anesthetized.To do this, mix about 8 drops from the culture with 1 drop of saturatedMgCl2solution in a small container before transferring the embryos or larvae to a slide for observation

fer-3 Gastrulation is a set of processes by which embryonic cells are repositioned as the basic body ganization of the larva is established It is somewhat difficult to observe details of gastrulation because em-bryos swim actively during these stages of development, but patient viewing of several embryos will permityou to see at least some of the interesting cellular activities that are involved Review a description of gas-trulation in your textbook or in references provided in the lab before observing various gastrulation processesfor yourself

or-Just before gastrulation begins, one side of the blastula wall flattens and thickens to form a prominent etal plate Cells of the vegetal plate play major roles in gastrulation Gastrulation begins with the separation of the

veg-primary mesenchyme cells (fig 1.2a) from the vegetal plate portion of the blastula wall and their subsequent ward movement (ingression) These cells are among the descendants of the micromeres that originally were es-

in-tablished during the fourth cleavage The primary mesenchyme cells move over the inner surface of the blastulawall to new positions where they form clusters of cells Cells in these clusters will begin to assemble the crys-talline spicules of the larval skeleton

(a) (b) (c)

FIGURE 1.2 Gastrulation in sea urchin embryos (a) Mesenchyme blastula of a sea urchin embryo Primary mesenchyme cells have

entered the blastocoel and are beginning to migrate over the blastocoel’s inner surface Note the vegetal plate made up of relatively

taller cells (b) Scanning electron micrograph (SEM) of an external view of a gastrulating sea urchin embryo showing the invagination

of the vegetal plate at the beginning of archenteron formation Cilia have been removed from the surface of this embryo (c) Composite

of SEM photos showing the interior of an embryo during archenteron invagination Note the primary mesenchyme cells migrating over the surface of the blastocoel wall.

(a) is a differential interference contrast photo by R B Emlet; (b and c) are SEM photos by John B Morrill (c) is from Morrill and Santos, 1985, in R H Showman and R M Sawyer, eds., The Cellular and Molecular Biology of Invertebrate Development, Univ S.C Press.

FIGURE 1.3 (a) Midgastrula stage of sea urchin development As the archenteron lengthens, its wall thins.

Some of the secondary mesenchyme cells at the tip of the archenteron have filopodia extending to the

blastocoel wall (This gastrula is approximately 120 ␮m long.) (b) A sea urchin pluteus larva Propelled by

cilia, a pluteus swims with its mouth and arms directed upward This pluteus measures approximately

200 ␮m from apex to arm tip.

(a) is a differential interference photo by Jeff Hardin, Dept of Zoology, University of Wisconsin; (b) is a light micrograph by

John B Morrill.

mouth

anus skeletal rod

Movements of the primary mesenchyme cells involve rather complex individual cell behavior, but the shape

changes that initiate development of the archenteron ( primitive gut) depend upon the collective activity of a

number of cells.Archenteron development begins with the invagination (inward sinking or “in-pocketing”) of the

vegetal plate (fig 1.2b and c).You should be able to observe some parts of this invagination process Once it has

been established by invagination, the archenteron lengthens, and its wall thins appreciably.The final phase of

ex-tension involves activity of the secondary mesenchyme cells, a group of cells that become evident at the

archen-teron’s tip when it has extended about halfway across the blastocoel Some secondary mesenchyme cells extend

long, thin projections known as filopodia that reach out to touch various sites on the inside of the blastocoel wall (fig 1.3a) It is often possible to observe extended filopodia Some of the contacts made by filopodia are tempo-

rary, and the cells retract these filopodia, but other filopodia remain attached if they have contacted the region of

the oral ectoderm where the mouth will form These attached filopodia contract, pulling the tip of the

archen-teron over into contact with the oral ectoderm, and the larval mouth develops in the contact area

The oral surface becomes flattened, giving the embryo an angular appearance that characterizes the prism

stage of development The angularity of prism-stage embryos contrasts distinctly with the spherical shape of theblastula and gastrula stages Skeletal spicules are clearly evident in prism-stage embryos

Over the next day or two, you will be able to observe the differentiation of the pyramid-shaped, four-armedpluteus larva First, two arms (the postoral arms), and slightly later, two more (the anterolateral arms) are extended

(fig 1.3b) These arms, along with the main portion of the body of the pluteus, are supported by skeletal rods

whose development began with formation of the skeletal spicules If you have a microscope with polarizing tics, it would be interesting for you to examine the developing skeleton using polarized light

op-A pluteus larva swims with its arms directed upward and its beating cilia set up currents that sweep smallfood particles into its mouth Observe as much detail of gut structure, skeleton organization, and other features ofthe pluteus larva as time permits For example, differentiation of esophagus, stomach, and intestine can easily beseen Watch for muscular contractions in the digestive tract

Pluteus larvae will swim for some time but usually will not develop beyond the four-armed stage unless theyare fed Unfed larvae eventually starve, fall to the bottom of the culture container, and degenerate Further devel-opment can be observed only if larvae are fed, and feeding usually requires availability of cultures of appropriatemarine algae (See comments on feeding pluteus larvae in Appendix A.)

E QUIPMENT AND G LASSWARE

Beakers, various sizes, but 100-ml and 250-ml beakers are especially useful

Clean syringes and hypodermic needles

Clean Pasteur pipettes

Petri dishes

Aluminum foil or parafilm

Microscope slides and coverslips

Depression slides

Compound microscope

Dark-field stop for microscope, if available (see p 206)

Rheinberg filter for microscope, if available (see p 207)

Polarizing optics, if available (see p 207)

Poly-L-Lysine-coated coverslips or slides (optional)

India ink (optional)

Modeling clay

S OLUTIONS AND C HEMICALS

Seawater or appropriate salt mixture (artificial seawater—see Appendix A)

0.5 M KCl solution

Saturated MgCl2solution (optional)

A NIMALS

Sea urchins or sand dollars

Some Useful Information Sources

V IDEO —A D OZEN E GGS

This video includes several nice video sequences of sea urchin development photographed byRachel Fink, Mount Holyoke College, and Seth Ruffins and Charles Ettensohn, Carnegie Mellon University.The video was produced under the auspices of the Society for Developmental Biology and is availablefrom: Sinauer Associates, Inc., P O Box 407, 23 Plumtree Road, Sunderland, MA 01375-0407

This website prepared by Jeff Hardin of the University of Wisconsin contains a number of instructive

illustrations and micrographs as well as a lot of useful information about sea urchin development

CD-ROM—S EA U RCHIN E MBRYOLOGY

This CD-ROM gives access to the same material available on the urchin website listed previously

It is inexpensive and is available from Hopkins Marine Station, Stanford University, Dept of Biol Sci.,Pacific Grove, CA 93950-3094

CD-ROM—V ADE M ECUM : A N I NTERACTIVE G UIDE TO D EVELOPMENTAL B IOLOGY

This CD-ROM, prepared by Mary Tyler and R N Kozlowski of the University of Maine, containsmuch information about development and techniques, including a good deal of information about echin-oderm development It is available from Sinauer Associates, Inc., P O Box 407, 23 Plumtree Road,Sunderland, MA 01375-0407

of those many studies have greatly expanded knowledge in several areas of biology.

It is quite easy to obtain sea urchin and sand dollar gametes, and the requirements for culturingembryos, at least through early phases of development, are fairly simple Thus, a number of relativelysimple experiments on these cells and embryos can be done in the teaching laboratory In this labora-tory, you will have opportunity to conduct several of these experiments

SPERM CLUMPING

Early in the twentieth century, F R Lillie studied sea urchin gametes extensively Among his manyobservations was the discovery of a sperm clumping response induced by extracts from eggs’ jelly.Whenunfertilized eggs are separated from the seawater in which they have been standing for an extendedperiod and the supernatant (or “egg water”) is added to a sperm suspension, the sperm form clusters.Later, the clusters disperse, but sperm that have reacted in this way are no longer capable of partici-pating in fertilization interactions These observations became the basis for the fertilizin-antifertilizinhypothesis of sperm-egg interaction developed by Lillie and others.This hypothesis suggested that “eggwater” contains a soluble agglutinating factor from the jelly surrounding mature sea urchin eggs thatmight play a role in fertilization or possibly in polyspermy prevention

The fertilizin-antifertilizin hypothesis was later reexamined when the responses of sperm in “eggwater” were analyzed further (Collins 1976; see “Suggestions for Further Investigation of EchinodermDevelopment” on p 26) It was observed that “egg water” induces sperm to undergo the acrosomereaction, including extrusion of the acrosomal process, and that the sperm clumping seen in “egg wa-ter” isn’t an agglutination process such as that caused by antibodies or other cross-linking factors Rather,sperm actively swim into rosettelike clusters where their acrosomal processes adhere It is now clearthat the inability of sperm previously subjected to “egg water” to participate in fertilization is due tothe condition of their acrosomes rather than to an agglutinating factor from egg jelly coats that blocksbinding sites

1 The jelly coat surrounding sea urchin eggs will slowly dissolve in seawater, so seawater in which eggshave been stored for a few hours can be used as “egg water” in making these observations To more quickly pre-pare an “egg water” solution, vigorously shake a sample of unfertilized eggs in about 30 ml of seawater in a cov-ered test tube (Shaking helps to dissolve the jelly coats.) Filter the test tube’s contents and collect the filtrate(“egg water”) Place a drop of “egg water” on a slide or in a watch glass Prepare a milky, diluted sperm suspen-sion and add one drop of it to the “egg water.” Within 1 minute, the sperm suspension will take on a granular, orflocculent, appearance Microscopically, it can be seen that the sperm are clustered

2 You can test the functional capacity of the previously clumped sperm by tipping the suspension off theslide into a sample of fresh eggs on a depression slide Check for fertilization membrane formation and, if you doobserve any, determine the fertilization percentage

3 If the fertilization percentage is very low, what conclusions might you draw? What are some alternativeexplanations of the results? What additional experimental step could you take to provide control results that wouldhelp to clarify interpretation of the results? Do the additional test What are the results? What conclusions mightyou draw?

Materials

E QUIPMENT

Basic equipment and supplies for sea urchin and sand dollar experiments as listed in Laboratory 1

Test tube and stopper or cover for the tube during shaking

Filter paper and funnel

S OLUTIONS AND C HEMICALS

Seawater or appropriate salt mixture (artificial seawater—see Appendix A)

tion without sperm contact is called parthenogenesis, and artificial parthenogenesis in sea urchin eggs

has been investigated by many biologists since it was first studied by Oscar and Richard Hertwig ing the 1880s Many experimental treatments have been found to cause some, or even many, of the ac-tivation responses to occur in sea urchin eggs

dur-In this experiment, you will have opportunity to investigate the effects of one of these treatments—immersion in seawater made hypertonic to egg cells by addition of 30 grams of sodium chloride perliter This procedure is one recommended by the great early twentieth-century American develop-mental biologist Ethel Browne Harvey, who, near the end of her career, summarized and reinvesti-gated many of the experimental procedures used to investigate sea urchin development up to the1950s

Techniques

1 Transfer a sample of freshly shed eggs to hypertonic seawater (HSW) by pipette and allow them to tle to the bottom of the beaker or dish

set-2 After 20 minutes, wash the eggs by pouring off as much of the HSW as you can and resuspending them

in normal strength seawater (Since eggs of different species, and even different batches of eggs of a single species,respond differently, you might consider trying several additional treatment times.)

3 Check for fertilization membrane elevation, and if fertilization membranes are present, determine the centage of eggs that have them.

per-4 After 10 minutes, wash the eggs again as in Step 2

5 Check again for fertilization membranes and percentage of eggs possessing them

Analysis of Results

1 If fertilization membranes have been found, continue to observe the culture at intervals and be alert forsigns of further development If you detect developmental progress such as cleavage divisions, compare timingwith control embryos

2 Follow your culture long enough to observe hatching and gastrulation should they occur in any embryosdeveloping from parthenogenetically activated eggs However, parthenogenetically activated development onlyrarely proceeds beyond completion of a few cleavages

Materials

E QUIPMENT

Basic equipment and supplies for sea urchin and sand dollar experiments as listed in Laboratory 1

S OLUTIONS AND C HEMICALS

Seawater or appropriate salt mixture (artificial seawater—see Appendix A)

One of the most famous experiments in the early history of developmental biology was performed

on the sea urchin embryo In 1892, Hans Driesch vigorously shook two-cell-stage sea urchin embryos

in a test tube of seawater until some of the blastomeres (cleavage cells) separated from one another.Driesch followed the development of these isolated individual cells and found that, in at least somecases, such cells could give rise to quite normally proportioned, though undersized, larvae Driesch con-cluded that each of the blastomeres at the two- and four-cell stages has the capability to develop into

a whole embryo

Though the developmental capacity of early cleavage cells may not be quite so dramatic or tensive as Driesch thought it to be, his results are still regarded as an important milestone in the in-vestigation of animal development Driesch’s procedure is tricky to repeat, but in this laboratory, youwill have an opportunity to investigate the results of blastomere separation using another technique.The technique that we will use may not be quite so rough as Driesch’s vigorous shaking, but it isharsh, and appropriate controls should be added to your experimental design if you have the opportu-nity to do so Whether or not you carry them out, think about appropriate sets of control observationsfor each of the steps in the technique that follows

ex-Techniques

1 Place a sample of sea urchin eggs in a 10 mM solution of p-aminobenzoic acid (PABA) in seawater Thenproceed with fertilization by adding a sperm suspension to the eggs, using the same techniques that you used inLaboratory 1 PABA prevents the hardening of the fertilization membrane that would normally occur within a fewminutes after its formation and facilitates later removal of the membrane

2 After zygotes have settled to the bottom, gently pour off the PABA solution and replace it with free seawater containing 50 ␮⌴ EGTA

(c)

(b)

FIGURE 2.1 Development of twinned embryos of the sea urchin, Lytechinus pictus (a) Twinned embryos after two

cleavage divisions (These cells are slightly flattened Each is about 60 ␮m in diameter.) (b) Twinned early blastulae (magnification same as in a) (c) A dark-field photo of a control pluteus (right ) and a small pluteus that developed from

an embryo twinned at the two-cell stage (The control pluteus is about 200 ␮m long.)

Photos by L G Johnson.

3 Pour off the supernatant after the zygotes once again have settled and add fresh calcium-free seawatersolution Repeat this washing step.

4 Use a simultaneous control culture to monitor developmental progress and sample your experimentalculture only when cytokinesis of the first cleavage is proceeding in the control culture

5 Following completion of the first cleavage division, pour the embryos through a 70-␮m mesh Nitex ter Check a drop sample from your culture to see what percentage of the embryos have been separated into in-dividual blastomeres If most are still in the two-cell configuration, it may be necessary to repeat this filtering step

fil-If this second pass through the filter does not result in blastomere separation, it may be necessary to use a filterwith a smaller mesh size

6 Once blastomeres have been successfully separated, allow the cells to settle and pour off the supernatant.Resuspend in normal seawater and proceed to the Analysis of Results section that follows

Analysis of Results

1 Observe cleavage in your culture over the next few hours if you are able to do so Watch for differingnumbers of cells in the embryos After two more cleavage divisions,“twinned” embryos will consist of four cells,while embryos whose blastomeres were not separated will contain eight cells On the basis of what you knowabout results of the fourth cleavage in control sea urchin embryos, what cell-size classes might you expect in

“twinned” embryos completing a third division subsequent to blastomere separation at the two-cell stage?

2 If you can return to check your experimental culture when the embryos have hatched and are ming, try to determine whether you have embryos of more than one size in your culture If you can, measure sev-eral embryos in your culture and make comparative measurements of embryos from a control culture

swim-3 If you can maintain your cultures long enough to do so, look for pluteus larvae in your experimental ture Check for size differences among the plutei that you find and compare them with plutei from control cul-tures (fig 2.1)

cul-Materials

E QUIPMENT

Basic equipment and supplies for sea urchin and sand dollar experiments as listed in Laboratory 1

Nylon filters with appropriate mesh size (see Appendix A)

Cylinders to hold filters, for example, 50-ml syringes with tip end cut off or 7.5-cm (3-inch) or 10-cm (4-inch)pieces of PVC pipe with an inside diameter of about 2.5 cm (1 inch)

S OLUTIONS AND C HEMICALS

Seawater or appropriate salt mixture (artificial seawater—see Appendix A)

0.5 M KCl solution

10 mM p-aminobenzoic acid (PABA) in seawater (use the sodium salt of PABA, not the free acid)

Calcium-free seawater containing 50 ␮⌴ EGTA

Hypertonic seawater (seawater with 30 g NaCl added per liter)

L IVING M ATERIAL

Sea urchins or sand dollars

Exchanging Seawater in Cultures

Once embryos have hatched and are swimming, it is very difficult to exchange the water in theculture simply by pouring.The following technique permits removal of a large percentage of the waterfrom a culture while leaving the swimming embryos or larvae behind It also can be used when themedium around early, nonswimming embryos must be changed and there is not time to allow them tosettle to the bottom of the container

1 Submerge the filter-covered (35 to 40 ␮m mesh size) end of a piece of PVC pipe in the culture It maytake a moment for the filter to be wetted by the seawater ( You can pre-wet the filter using a squirt bottle filledwith seawater.)

PVC pipe

turkeybaster

embryo culture

filter

FIGURE 2.2 Diagram showing the use of a turkey baster

and a filter-covered pipe to remove liquid from a beaker containing a suspension of embryos or larvae The filter should have a mesh size between 35 ␮m and 40 ␮m (see Appendix A).

2 Squeeze the bulb of an ordinary kitchen turkey baster and insert the baster’s tip into the piece of pipe(fig 2.2).

3 Gently draw seawater into the baster by slowly releasing pressure on the bulb This removes fluid from

the culture while the filter at the end of the pipe blocks passage of embryos or larvae so they are left behind

4 Discard the water in the baster and repeat Steps 1–3 until you have removed most of the water from theculture container

5 Gently pour replacement water into the culture to the desired level

DEVELOPMENTAL EFFECTS OF REDUCED CALCIUM ION CONCENTRATION

Calcium ions are important in some cellular responses to external signals, in the intracellular ulation of various processes, and also are required for some cell movements Therefore, it is not sur-prising that calcium ions must be present at or near the normal seawater concentration for develop-ment to proceed normally In this experiment, you will have an opportunity to experiment with theeffects of reduced calcium ion availability

reg-Techniques

1 Concentrate a culture of embryos between the hatching blastula and very early mesenchyme blastulastages by drawing off almost all of the seawater in the beaker using a filter-covered piece of PVC pipe and a turkeybaster (see p 13) Be especially careful not to use strong suction as you near the end of this process because em-bryos can be damaged if they are drawn forcefully against the filter

2 Fill the beaker with free seawater.Then reduce the volume again and refill the beaker with free seawater once more

calcium-3 Observe samples of embryos from control (ordinary seawater) and experimental cultures at intervals overthe next several days Make morphological comparisons and developmental rate comparisons between controlembryos in ordinary seawater and embryos developing in the calcium-free seawater Watch especially for differ-ences in gastrulation movements

Materials

E QUIPMENT

Basic equipment and supplies for sea urchin and sand dollar experiments as listed in Laboratory 1

PVC pipe with filter-covered end—mesh size 35 to 40 ␮m

Turkey baster

S OLUTIONS AND C HEMICALS

Seawater or appropriate salt mixture (artificial seawater—see Appendix A)

Calcium-free seawater containing 50 ␮⌴ EGTA

as an experimental system for investigating synthesis and assembly processes involved in cilia ment In this experiment, you will have opportunity to observe the process of deciliation following verybrief hypertonic shock and to follow the progress of cilia regeneration

1 Concentrate a culture of swimming blastula or later-stage embryos by drawing off almost all of the water in the beaker using a filter-covered piece of PVC pipe and a turkey baster (see p 13) Be especially carefulnot to use strong suction as you near the end of this process because embryos can be damaged if they are drawnforcefully against the filter

sea-2 Transfer some of the embryos to a second beaker They will serve as controls during the process Thenadd normal seawater to the control beaker and add hypertonic seawater to the original beaker

3 The total period of exposure to hypertonic seawater should be only about 30 seconds so you need to

begin removing the hypertonic seawater almost immediately Remove almost all of the water in the beaker using

a filter pipe and turkey baster Immediately fill the beaker with normal seawater and begin your observations

Alternative Method

1 Concentrate a culture of swimming blastula or later-stage embryos by drawing off almost all of the water in the beaker using a filter-covered piece of PVC pipe and a turkey baster (see p 13) Be especially carefulnot to use strong suction as you near the end of this process because embryos can be damaged if they are drawnforcefully against the filter

sea-2 Transfer some of the embryos to a second beaker containing seawater.They will serve as controls duringthe process Fill the beaker with normal seawater

3 Submerge the tip of another filter-covered piece of PVC pipe in a small quantity of normal seawater in abeaker Slowly and gently pour the remainder of concentrated culture of swimming blastulae through the pipe sothat the embryos are held against the filter inside the pipe

4 Expose the embryos to hypertonic seawater by slowly lifting the filter-covered pipe and immersing itsfilter-covered end in hypertonic seawater for 30 seconds

5 Slowly lift the filter-covered pipe out of the hypertonic seawater, invert it over a beaker containing a tle seawater, and gently pour seawater on the filter, thereby washing the embryos out of the pipe into the beaker.Immediately fill the beaker with normal seawater

lit-Analysis of Results

1 Make a gross comparison between the experimental and control cultures Do you see evidence of a ference in swimming behavior? Do you observe a tendency for the experimental embryos to settle out of theculture?

dif-2 Collect samples from the experimental and control cultures and examine them microscopically It is sible to observe differences in ciliation with an ordinary light microscope if the iris diaphragm is closed down,but you will get better results using some form of phase-contrast microscopy or the dark-field or Rheinberg tech-niques (see p 206) The Rheinberg technique is especially useful for observing cilia Do you see differences inswimming? In ciliation?

pos-3 Repeat the observations in Steps 1 and 2 every few minutes for the next 60 to 90 minutes If you haveseen almost complete settling of the experimental embryos, watch for them to resume swimming Try to corre-late resumption of swimming with reappearance of cilia that you might observe directly How much time do youestimate is required for complete cilia regeneration?

Possible Further Observations

1 You might be able to observe the process of cilia loss directly if you mix a drop of seawater containingsome embryos with several drops of hypertonic seawater on a slide Can you see cilia that have fallen off ? Try topipette away the hypertonic seawater and replace it with normal seawater If you succeed in doing so, place theslide on a piece of wet paper towel in a petri dish and cover the dish to reduce evaporation How many minutespass before you can see regenerating cilia? How long does it take for these small cilia to double in length? When

do the blastulae begin to swim?

2 If you have a culture of embryos that have successfully regenerated their cilia, you might wish to do other experiment to test the ability to regenerate cilia several times in succession

E QUIPMENT

Basic equipment and supplies for sea urchin and sand dollar experiments as listed in Laboratory 1

PVC pipe with filter-covered end—35 to 40 ␮m mesh size (two are needed for the alternative method)

Turkey baster

S OLUTIONS AND C HEMICALS

Seawater or appropriate salt mixture (artificial seawater—see Appendix A)

0.5 M KCl solution

Hypertonic seawater (seawater with 30 g NaCl added per liter)

organi-of the types organi-of experiments that have been part organi-of this long series organi-of investigations.

DEVELOPMENTAL EFFECTS OF LITHIUM CHLORIDE

The results of the experimental exposure of echinoderm embryos to lithium ions played an portant role in development of the historical double-gradient hypothesis of developmental control.Although the double-gradient hypothesis has undergone considerable reevaluation, the sometimesstriking effects of lithium ions on major morphogenetic events in echinoderm embryos remain of in-terest For example, abnormal development of structures derived from the vegetal area can result in for-mation of embryos with a proportionately large archenteron or even with an archenteron that bulgesoutward from the surface rather than invaginating properly into the blastocoel This phenomenon is

im-called exogastrulation.

In light of lithium’s known effects on cellular signaling systems (see Suggestions section), it is notsurprising that lithium has significant effects on developmental processes that depend upon cellular in-teractions Lithium has been used more recently as a tool for the investigation of major organizationalprocesses in early development

Techniques

1 Use a 60 mM solution of LiCl in seawater to prepare 30 mM and 15 mM solutions as well

2 Fertilize sea urchin or sand dollar eggs in a 250 ml or larger beaker, using the standard techniques duced in Laboratory 1 Once you are satisfied that most of the eggs have fertilization membranes, swirl the cul-ture and pour off samples into several smaller beakers Cover each culture loosely with aluminum foil and leavethe beakers undisturbed until most of the zygotes have settled to the bottom Then pour off as much of the su-pernatant water in each culture as you can, and replace it with normal seawater Set the cultures aside or returnthem to an appropriate culture chamber

intro-3 When the embryos reach the two-cell stage, pour off as much of the supernatant water in each culture

as you can and replace it with seawater containing LiCl at each of the concentrations that you have chosen totest Keep one culture as a control in which you replace the discarded seawater with normal seawater

4 Sometime between the time when hatching begins and the mesenchyme blastula stage, replace the

lithium-containing seawater in the cultures with normal seawater (It is probably best to transfer Lytechinus

species while they are hatching; transfer other species closer to the mesenchyme blastula stage.) If you haveenough separate culture containers, leave some embryos in lithium solutions To replace the lithium-containingseawater with normal seawater, concentrate each culture of embryos by drawing off almost all of the seawater

in the beaker using a filter-covered piece of PVC pipe and a turkey baster (see p 13) Be especially careful not

to use strong suction during this process because embryos can be damaged if they are drawn forcefully againstthe filter Replace the discarded lithium-containing seawater with normal seawater Repeat the process to furtherwash the embryos

Materials

E QUIPMENT

Basic equipment and supplies for sea urchin and sand dollar experiments as listed in Laboratory 1

PVC pipe with filter-covered end—mesh size 35 to 40 ␮m

S OLUTIONS AND C HEMICALS

Seawater or appropriate salt mixture (artificial seawater—see Appendix A)

0.5 M KCl solution

60 mM LiCl solution in seawater

L IVING M ATERIAL

Sea urchins or sand dollars

DEVELOPMENTAL EFFECTS OF NICKEL IONS

Development of the larval skeleton involves an easily observed series of events.After primary enchyme cells enter the blastocoel, they migrate to establish a ring of cells At two points within thering, primary mesenchyme cells aggregate in two ventrolateral clusters and form the syncytial clumpsthat produce the first two skeletal spicules.This cell activity, along with expression of specific genes in-volved in differentiation of ventral and dorsal ectoderm, is involved in converting the previously radi-ally patterned embryo into the bilaterally arranged prism-stage larva Some ions, such as nickel, are known

mes-to affect this characteristic patterning process In this experiment, you’ll be experimenting with thisnickel effect

Techniques

1 Use a 2 mM stock solution of NiCl2in seawater to prepare 1 mM and 0.1 mM solutions in seawater bydilution If your experiments are limited by beaker availability or culture space and you can test the effect of onlyone concentration of nickel ions, consult classmates who test the other two concentrations so that you have op-portunity to share observations of the effects of the several concentrations

2 Fertilize sea urchin eggs in a 250 ml or larger beaker, using the standard techniques introduced inLaboratory 1 Once you are satisfied that most of the eggs have fertilization membranes, swirl the culture and pouroff samples into several smaller beakers Leave the beakers undisturbed until most of the zygotes have settled tothe bottom Then pour off as much of the supernatant water in each culture as you can and replace it with sea-water containing NiCl2at each of the concentrations that you have chosen to test Keep one culture as a control

p 13) Be especially careful not to use strong suction during this process because embryos can be damaged if they are drawn forcefully against the filter Replace the discarded nickel-containing seawater with normal sea-water Listen carefully for instructions regarding disposal of the nickel-containing solutions.

4 Repeat this washing step at least twice and refill the beakers with normal seawater each time Listen fully for instructions regarding disposal of the nickel-containing solutions

care-5 Re-cover the beakers and maintain the cultures for further observation

3 When you observe definitely bilaterally symmetrical prism-stage embryos in your control cultures, fully examine embryos from your experimental cultures Check for differences in continuing skeletal spicule growthand patterns of archenteron growth Does the archenteron in the nickel-treated embryos bend to contact a flat-tened oral ectoderm area? Do the nickel-treated embryos appear to be bilaterally symmetrical?

care-Materials

E QUIPMENT

Basic equipment and supplies for sea urchin and sand dollar experiments as listed in Laboratory 1

PVC pipe with filter-covered end—mesh size 35 to 40 ␮m

Turkey baster

S OLUTIONS AND C HEMICALS

Seawater or appropriate salt mixture (artificial seawater—see Appendix A)

0.5 M KCl solution

2 mM solution of NiCl2in seawater; Please consult your institution’s Hazardous Material Officer for instructions regarding disposal of nickel-containing solutions

ENZYME SYSTEM DIFFERENTIATION

A number of developmental biologists are investigating patterns of gene expression in echinodermembryos and larvae They detect specific gene activation in particular areas of embryos by techniquessuch as in situ hybridization, using specific probes for gene expression An inexpensive and relativelysimple approach to studying gene activation is to detect enzyme systems characteristic of areas or struc-tures in embryos and larvae as they become functionally differentiated One such enzyme system is thealkaline phosphatase that is specifically expressed in the developing gut You will have an opportunity

to test for alkaline phosphatase activity in this experiment

Techniques

Preparing for staining:

1 Rear sea urchin or sand dollars to the gastrula, prism, and early pluteus stages using the techniques troduced in Laboratory 1

FIGURE 3.1 An early pluteus larva stained with an indicator that

is converted from colorless to colored in conjunction with reactions catalyzed by alkaline phosphatase The colored substance indicates sites where genes for the alkaline phosphatase enzymes have been expressed and the enzyme system is functioning actively.

2 Concentrate an embryo or larva culture to a very small volume by drawing off almost all of the seawater

in the beaker using a filter-covered piece of PVC pipe and a turkey baster (see p 13) Be careful not to use strongsuction as you near the end of this process because embryos can be damaged if they are drawn forcefully againstthe filter

3 Position several coverslips coated with Poly-L-Lysine near the edge of a petri dish cover or inverted tom set on a dark background Shine a bright light through the culture that you have concentrated so that youcan see the swimming individuals clearly Draw as many of them as you can into a Pasteur pipette

bot-4 Transfer a couple of drops from the pipette to each coated coverslip Be careful to avoid adding so muchseawater that it flows off the edge of the coverslip Wait several minutes until the embryos have begun to settleand adhere to the coated coverslip Shine a bright light on the coverslip so that you can see the swimming in-dividuals clearly.Then carefully draw off seawater from the coverslip, using a clean Pasteur pipette.As the volume

of seawater on the coverslip decreases, more individuals will tend to stick to the surface

5 If there are ten or twelve individuals stuck to the coverslip, proceed to the staining procedure that lows If there are only five or six or fewer individuals on the coverslip, repeat Steps 3 and 4 before beginning stain-ing It is important to have a number of individuals on the coverslip because some will be lost during transfers inthe staining process

fol-Staining procedure:

1 At each step in the enzyme reaction staining procedure, make transfers carefully Hold coverslips by theiredges and slide them very slowly into each staining jar Remove coverslips from staining jars with similar cautionbecause individuals are most likely to be washed off the coverslips as they pass through the surface films of thevarious solutions It is wise to mark a front on each staining jar and transfer the coverslips consistently so that theembryo side is always directed forward

2 Place the coverslips in ice-cold methanol (4° C or colder) in a staining jar and put them in the ator for 5 minutes

refriger-3 Carefully remove each coverslip from the jar, hold it vertically, allowing the methanol to drain by ing the bottom edge to absorbent paper (paper towel or blotting paper)

touch-4 Transfer the coverslips to ice-cold seawater,* leave for 30 seconds, withdraw them, drain them by ting the edges as in Step 3

blot-5 Repeat Step 4 by transferring the coverslips to a second staining jar containing ice-cold seawater Drainand blot

6 Transfer the coverslips to pH 9.1 buffer (room temperature) for 1 minute Drain and blot

7 Transfer to alkaline phosphatase substrate solution (room temperature) for 25 minutes.** Drain and blot

8 Transfer to phosphate buffered saline (PBS) for 3 minutes.*** Then mount on a microscope slide (makecertain that the embryo/larva side of the coverslip is toward the slide) and seal the edges

Analysis of Results

1 Examine each slide carefully, looking for the bluish purple color that indicates alkaline phosphatase tivity (fig 3.1)

ac-*Replace the seawater in the first seawater-containing staining jar after each use.

**You may need to vary the time of exposure to alkaline phosphatase substrate depending on your results.

***Replace the PBS after each use It is possible to skip the PBS wash and mount the coverslips immediately after removal from the alkaline phosphatase substrate You might consider deleting this step if you are having problems with embryos/ larvae washing off coverslips However, if you delete the PBS wash, reactions will continue, and stain eventually will diffuse out of the gut into other parts of the embryo larva.

Materials

E QUIPMENT

Basic equipment and supplies for sea urchin and sand dollar experiments as listed in Laboratory 1

PVC pipe with filter-covered end—mesh size 35 to 40 ␮m

S OLUTIONS AND C HEMICALS

Seawater or appropriate salt mixture (artificial seawater—see Appendix A)

Water-miscible mounting medium or nail hardener or New Skin Liquid Bandage

Poly-L-Lysine solution

Useful Information Source

http://worms.zoology.wisc.edu/urchins/SUpattern_Ni.html

This website, developed by Jeff Hardin of the University of Wisconsin, contains information about development

of embryonic patterns, especially the roles of primary and secondary mesenchyme cells, and the effects ofnickel ions

L A B O R A T O R Y

4

Observations and Experiments on the

Living Frog Embryo

The continuous change of form in developing embryos has always provoked interest and ity, and amphibian eggs, which are large and fairly easy to collect and observe, long ago became popu-lar subjects for those interested in studying animal development Early investigators focused their at-tention on describing the orderly, normal course of developmental events, and a wealth of informationwas gathered by direct observation of the development of frog, toad, and salamander embryos.Later, this observational and descriptive work began to be supplemented by experimental studies.The distinguished German embryologist, Wilhelm Roux, who studied amphibian development in the1880s, was one of the first to take an experimental approach to investigation of animal development.From the time that Roux pricked frog cleavage cells with a hot needle, the amphibian embryo has beensubjected to a variety of experimental manipulations and to numerous alterations of its environment

curios-In the United States, embryos of the common leopard frog Rana pipiens have been employed in many experimental studies of vertebrate animal development Rana pipiens embryos are also widely used as subjects in biology courses dealing with development.This laboratory introduces the Rana pip- iens embryo and some of the basic techniques used in obtaining and studying frog embryos

We will begin our study of the frog with a descriptive and experimental examination of tion and the early stages of development In addition to uniting the haploid nuclei of the gametes toproduce a diploid zygote, fertilization also initiates a complex set of activation responses in the egg.However, in some cases, an egg can develop in the absence of sperm; such development is known asparthenogenesis The drone-producing egg of the honey bee is an excellent example of naturally oc-curring parthenogenic development Other eggs that do not ordinarily develop parthenogenetically can

fertiliza-be induced to do so by a variety of experimental manipulations For example, the eggs of frogs, manders, and toads may be stimulated to complete early stages of development in the absence of thenuclear events of normal fertilization if they are inseminated with pretreated sperm that have been ir-radiated or chemically modified Such treatment disrupts the genetic material of the sperm but does notinterfere with its mobility or the ability of the sperm to penetrate and activate the egg

testis

FIGURE 4.1 The position of the testes in relation to the other internal

organs of the frog.

Your initial experience with the embryo of the leopard frog, Rana pipiens, will be a study of

nor-mal diploid embryos accompanied by a parallel examination of parthenogenetically activated embryos

An embryo deprived by artificial means of one set of parental chromosomes exhibits markedly impaireddevelopment

The African clawed frog, Xenopus laevis, is now widely used in developmental studies Xenopus

zygotes can be obtained readily, and the embryos can be experimentally manipulated as easily as those

of the leopard frog Although the leopard frog is the organism suggested for this laboratory, Xenopus

can be substituted However, there are some differences between the methods used for obtaining and

handling Xenopus gametes and embryos and those used with Rana pipiens Thus, you should consult information about Xenopus development in Laboratory 9 and Appendix C if you are using Xenopus for

this laboratory

Techniques

Preparation of Sperm Suspension

Mature sperm are present in the testes of male leopard frogs throughout the year, although there

is a period of relatively lower spermatogenic activity from late June to mid-September

1 Pith (or anesthetize) a male frog and use a scissors and forceps to dissect out the pair of testes ( If youare not adept at pithing, that is, destroying the central nervous system of the frog with a needle, then anesthetizethe frog in a sealed container with a wad of cotton saturated with ether or chloroform.) The testes are yellowish,ovoid, paired bodies held to the kidneys by means of mesentery folds (fig 4.1)

2 After removing the testes, roll each testis gently on paper toweling to free the organ of adhering bloodand mesentery Using blunt forceps, mince the pair of testes thoroughly in 10 ml of spring water or in a diluted(10%) Amphibian Ringer’s solution in a finger bowl Tilt the finger bowl so that the testes can be macerated eas-ily in the pool of fluid at one side of the bowl

3 Allow the suspension to stand for 15 minutes, during which time the sperm will become active Place adrop of the suspension on a glass slide and examine under the high-power objective of a compound microscope.Observe the shape of the sperm and check for motility

Irradiation of Sperm

Nuclear damage is one of the demonstrable effects of ultraviolet ( UV ) irradiation of the sperm cell

In particular, the energy of U V radiation induces abnormal bonding of the pyrimidine bases of nucleicacids Chromatin material of the sperm cell can be disrupted by U V radiation without diminishing thesperm cell’s capacity to enter the egg

A phenomenon observed in cases of U V exposure is that of photoreactivation, in which the effect

of the U V irradiation is perceptibly lessened by the presence of intense visible light (for example, head illumination).Accordingly, it is advisable to irradiate in a dimly lit room.An inexpensive U V source

over-is a 15-watt germicidal lamp mounted in a fluorescent fixture The inverse square law operates for U Vradiation—the greater the distance, the longer the exposure time required to accomplish the desiredeffect

1 Transfer a small quantity of the sperm suspension to each of two petri dishes The sperm suspensionshould be spread thinly over the bottom of each dish Label one petri dish “control” and the other “experimental”.Set aside the control dish

2 Position the ultraviolet lamp 38 to 40 cm (15 to 16 in) above the table top Place the uncovered mental petri dish beneath the lamp and expose the sperm suspension to the rays of the lamp for 15 minutes.Occasionally swirl the sperm suspension gently to ensure equal exposure of all sperm to the rays Do not exposeyour skin to direct UV radiation and do not look directly at the lamp

experi-You are now ready to inseminate eggs with the control and experimental sperm

FIGURE 4.2 Two different views showing how to hold the female frog while “stripping” her eggs.

FIGURE 4.3 Formation of the gray crescent in the frog’s egg A and B represent unfertilized eggs; A⬘ and B⬘ represent eggs shortly after fertilization.

Ovulation and Fertilization

Ovulation can be induced by injecting frog pituitary extracts or fragmented pituitaries into ture, healthy female frogs The pituitary hormones cause ovulation within 24–48 hours at room tem-perature (20–24° C) or within 4–5 days at 10° C Injected female frogs will be available in the labora-tory (Pre-injected females can be purchased from commercial suppliers, or female frogs can be injected

ma-to provide eggs when needed in the laborama-tory.The procedures for inducing ovulation in Rana pipiens

are described in Appendix B.)

The female frog need not be sacrificed to release her eggs Eggs are removed from the oviducts bythe technique of “stripping.”

1 Hold the female frog with her back against the palm of one hand Grasp and extend the frog’s hindlimbs with your other hand Position the frog’s back in your hand so that your fingers partially encircle thebody just posterior to the forelimbs.The tips of your fingers will come to rest on the ventral surface of the frog(fig 4.2)

2 Eggs can be forced from the cloaca by initially applying gentle pressure to the anterior part of the bodyand then progressively closing the hand toward the cloaca region First, squeeze the female over paper towelinguntil she releases several eggs Usually, they will be accompanied by cloacal fluid Discard the first few eggs issuedfrom the oviduct and wipe the cloacal region dry.Then proceed to strip 100 or more eggs into each of the petridishes containing the unirradiated or irradiated sperm suspensions When stripping the eggs, squeeze gently andmove the female around over dishes to produce several chains of eggs rather than a single, heaped mound.To as-sure complete exposure to sperm, repeatedly draw sperm suspension into a clean pipette and squirt the spermover the eggs Observe the orientation of the black-pigmented area (animal pole) and the creamy white vegetalpole of the eggs

3 Allow the eggs to remain in the sperm suspension for 15 minutes After the 15-minute period, pour thesperm suspensions out of the petri dishes and flood the eggs with spring water (or 10% Ringer’s solution).Note: A mature female frog can release approximately 2000 eggs A “stripped” female can be stored at 4° C,and she will subsequently yield viable eggs each day for about 4 days When removed from the 4° C storage eachday, she should be allowed to sit at room temperature for 30 minutes to effect temperature equilibration

4 After fertilization, the egg becomes free to rotate slowly within the space (the perivitelline space) locatedbetween the egg itself and the vitelline membrane Gravity causes the vegetal hemisphere containing the relativelyheavier yolk to rotate to the underside Soon after fertilization, and before the first cleavage begins, a lightcrescent-shaped area, the “gray crescent,” appears on one side of the egg in the boundary between the animal andvegetal regions (fig 4.3), although it is often difficult to locate The gray crescent is formed by the shifting of cy-toplasmic components in the egg, and the position of the crescent is related to the penetration path of the spermcell; the gray crescent appears on the side of the egg opposite the sperm entry point One of the earliest obser-vations made by embryologists is that the gray crescent establishes the bilateral symmetry of the frog egg Theplane of the first cleavage of the frog egg, in most cases, passes through the gray crescent.Thus, the first cleavageeffectively divides the egg into bilaterally equal halves

5 The jelly envelope, which initially is dense and viscous, absorbs water and swells to several times its inal thickness.This thick envelope of jelly, comprised of two or three concentric layers, protects the egg from me-chanical injury The jelly layers swell maximally about 1 hour after eggs enter water

orig-The jelly mass generally sticks to the glass bottom of the petri dishes, so use a clean scalpel or section lifter

to free the jelly from the glass, and then gently lift the cluster of eggs from the bottom of the dish With sharpscissors, cut the mass of eggs into small clusters of 5 to 10 eggs You need not be hesitant or overly cautious incutting the mass as it is almost impossible to shear an egg since the jelly-coated eggs are very resistant to me-chanical injury

6 Lift the small egg clusters with forceps and place the clusters in several finger bowls (4 inches in ameter) containing spring water (or 10% Ringer’s solution) Development will proceed well with 15 to 20 eggs

di-in 250 ml of solution, but more crowded conditions generally should be avoided if it is practical to do so Toreduce evaporation, cover the finger bowls with loose aluminum-foil covers No change of solution is required

FIGURE 4.4 Scanning electron micrographs of frog embryo cleavage stages (a) First cleavage division The cleavage furrow deepens

more quickly in the animal pole (AP) than in the yolky vegetal pole (VP) (b) The 8-cell stage The size difference between the smaller animal pole blastomeres and the larger, yolky vegetal pole blastomeres is apparent (c) Early blastula stage viewed from above and to

one side.

SEM photographs of Xenopus laevis embryos courtesy of Dr Robert E Waterman.

throughout embryonic development At room temperature (20–24°C), first cleavage of the egg occurs within

2 or 3 hours after fertilization The first cleavage furrow is evident first at the animal pole and later at the etal pole, and it divides the egg into two equal blastomeres (fig 4.4) Subsequent cleavages partition the egginto an increasing number of smaller blastomeres

veg-External Features of Development

Various stages of development can be made available at different times by distributing both thecontrol (diploid) and experimental (parthenogenetically activated) eggs to various temperature-controlcabinets set at different temperatures, if they are available.The eggs of the leopard frog can tolerate tem-peratures as low as 6° C and as high as 28° C However, for optimum development, restrict the range

to temperatures between 12° C and 26° C

1 Repeated reference to figure 4.5 and table 4.1 will aid in identifying the stages of development.The stagenumbers are those originally assigned by Waldo Shumway in 1940 Plan to make frequent observations during thecourse of the week to witness the continuous change in the form of the embryo

TABLE 4.1 Embryonic Development of the Frog

1 Unfertilized 5 8-cell 9 Late cleavage 13 Neural plate

4 4-cell

17 Tail bud

21 Mouth open

20 Gill circulation

25 Operculum complete

24 Operculum closed on right

FIGURE 4.5 Normal stages in the development of the frog embryo (Rana pipiens), based on Waldo Shumway’s

The process of development takes place in a series of stages that occur in a regular sequence Cleavage of

the fertilized egg leads to the blastula stage (stages 8–9), which is followed by the process of gastrulation (stages

10–12) Gastrulation is a complex set of cell movements that results in the establishment and positioning of the

three germ layers (ectoderm, endoderm, and mesoderm) Subsequently, the gastrula elongates, and there is velopment of neural folds and the neural tube (the neurula stages of development, stages 13–16) Various out-

de-pocketings, inde-pocketings, thickenings, and other changes produce the sundry organs of the body

The developmental history of internal structures will be considered in the next laboratory; only the nally visible changes during development are considered here

exter-2 Cleavage of the frog egg is total (holoblastic); cytokinesis divides the entire egg, notwithstanding the

rel-atively large amount of yolk Notice that the first cleavage is meridional (vertical); the second is also meridional,but at right angles to the first.The third is latitudinal, separating four smaller, upper animal hemisphere cells fromfour larger vegetal hemisphere cells

After the first few divisions, cleavage of the cells in the vegetal hemisphere lags behind that of the animalhemisphere cells that contain relatively little yolk and continue to divide quite synchronously Throughout earlydevelopment, yolky vegetal cells are larger than the animal pole cells

3 Notice the externally visible changes that occur during gastrulation.An essential feature is epiboly, a

spread-ing overgrowth by animal hemisphere cells that eventually envelop and enclose the vegetal hemisphere cells.Gastrulation movements are first indicated by the appearance of a crescent-shaped groove, or depression (stage

10), on one side of the blastula below the equator The depression itself is known as the blastopore, and the rim above the depression is referred to as the dorsal lip of the blastopore (fig 4.6) The tips of the blastopore pro-

gressively extend around until they meet—passing through a succession of shapes: quarter circle (stage 10), halfcircle (stage 11), and full circle (stage 12) At stage 12, only a small area of vegetal pole cells is visible This re-maining visible yolk area is called the yolk plug The ring-shaped blastopore will soon close, that is, narrow to abarely visible slit (stage 13)

4 The body elongates during neurulation, and the neural folds are conspicuously elevated at stage 14 (fig.4.6).At the tail-bud stage (stage 17 ), the embryo can easily be removed from its surrounding jelly coats and vitellinemembrane Use two pairs of fine forceps to grasp the vitelline membrane and rip it apart without harming or dis-torting the embryo Leave some of the embryos in their membranes so that you can determine when normal hatch-ing occurs

Become familiar with some of the important landmarks of the embryo at the tail-bud stage (fig 4.7) At the

anterior end is the prominent oral sucker (cement gland), a V-shaped groove with prominent lips Between the lips of the sucker may be seen a depression called the stomodaeum, or mouth invagination An olfactory pit and the optic bulge are evident at each side of the head.The gill plate has become subdivided by transverse furrows into three bars: the first, second, and third branchial (visceral ) arches Behind the gill plate, a lateral swelling marks the position of the pronephros, or early larval kidney The tail bud appears as an outgrowth of the poste-

rior end of the body Note that the embryo rotates continuously within its jelly coat, propelled by the cilia thatcover its body

5 Try to detect the beating of the heart at stage 19 by using a bright light source and focusing carefully onthe ventral surface immediately posterior to the V-shaped sucker Look for circulation of blood through the ex-ternal gills developed as branched filaments on the branchial arches of a stage 20 embryo In late embryonic de-

velopment, the tail is differentiated into a dorsal and ventral fin, and myotomes (muscle segments) become clearly visible On the ventral side, at the base of the tail, the proctodaeum is evident in the area where the blastopore

closed

In the final stages of embryonic development (stages 21–25), a mouth forms, and rows of horny teeth

de-velop Observe the development of an operculum on each side as the external gills are resorbed and replaced by

internal gills.These membranous operculum folds grow back from the visceral arches and become fused with the

trunk, leaving only one aperture, the spiracle, on the left side The spiracle provides the means by which water

taken through the mouth passes through the gill region to the exterior The completion of operculum ment on both sides (stage 25) marks the end of embryonic development

develop-Early gastrula Late gastrula

YP DL

pronephros

tail bud

FIGURE 4.6 Selected stages during gastrulation and neurulation of the frog embryo (DL: dorsal lip of

blastopore, and YP: yolk plug).

Photographs from R G Kessel and C Y Shin, Scanning Electron Microscopy in Biology, 1976, Springer-Verlag.

FIGURE 4.7 A tail-bud stage (stage 17) frog embryo.

FIGURE 4.8 Mitotic figures as seen in aceto-orcein squash preparations of tail-region cells of (a) haploid (n ⫽ 13) and (b) diploid (2n⫽ 26) embryos.

2 Remove the solution around the tail tip and add several drops of distilled water Leave the tail tip in tilled water for 2 to 10 minutes Distilled water causes osmotic swelling of the nucleus that helps to untangle thechromosomes from each other.

dis-3 Draw off the distilled water and replace it with a large drop of a 2% aceto-orcein solution Quickly add aclean coverslip over the tissue to prevent crystal formation in the stain drop

4 After 5 minutes, place a paper towel above the coverslip and exert strong pressure on the coverslip withyour thumb Seal the edges of the coverslip with melted glycerine jelly applied with a fine brush (A comparablesemipermanent preparation can also be prepared by using a nonresinous mounting medium to ring the edges of thecoverslip.) Examine the preparation microscopically for mitotic figures, especially cells in metaphase of mitosis

5 The slides may be stored in a refrigerator at 2–4° C.The preparation will last for several days, and indeed,the quality of staining might actually improve with time

Materials

E QUIPMENT

Wooden-handled probe (dissecting needle) for pithing

Scissors and forceps for dissection

Clean microscope slides and coverslips

4-inch finger bowls or other containers for developing eggs

UV light source (see Section B)

Compound microscope

Dissecting microscope

Illuminator

S OLUTIONS AND C HEMICALS

Spring water or 10% Amphibian Ringer’s solution (see Appendix B)

Ethyl m-aminobenzoate methane sulfonate solution (1⬊3000 in spring water or 10% Amphibian Ringer’s solution) Distilled water

P RESERVED M ATERIAL O PTION

Some biological supply companies provide sets of preserved Rana pipiens embryos at a number of stages of

development Embryos at each stage are supplied in a small separate container that can be conveniently openedfor direct observation

Some Useful Information Sources

W EBSITES

http://sdb.bio.purdue.edu/

This is the very useful website of the Society for Developmental Biology It includes several sources of

information about amphibian development, which can be found by following links to “Virtual

Library-Developmental Biology” and “Education.”

V IDEO —A D OZEN E GGS

This video includes a video sequence of frog gastrulation photographed by Ray Keller and John Shih, University

of California, Berkeley The video was produced under the auspices of the Society for Developmental Biologyand is available from Sinauer Associates, Inc., P O Box 407, 23 Plumtree Road, Sunderland, MA 01375-0407

L A B O R A T O R Y

5

Patterns of Frog Development

The embryonic development of amphibians, like that of other animal embryos, begins with a

se-ries of mitotic cell divisions called cleavage During cleavage, chromosomal replication, mitosis, and

cy-tokinesis occur repeatedly without any intervening cell growth This pattern of repeated division, but

no growth, establishes a large population of cells, each of which contains a small part of the cytoplasm

of the original, very large zygote.At the end of the cleavage phase of development, these many cells areorganized as the blastula, a sphere of cells enclosing an internal fluid-filled cavity

Subsequent development involves the extensive cell migrations of gastrulation that reposition thecells of the embryo so that they are prepared to proceed with further development and differentiation

Gastrulation establishes the three basic body layers ( germ layers): ectoderm, mesoderm, and endoderm.

In some developing animals (for example, sea urchins) that produce eggs containing very littleyolk, the cells of the blastula are arranged in the form of a relatively thin-walled, hollow ball.The process

of gastrulation consists of the pushing inward of the cells of the vegetal hemisphere (prospective doderm and mesoderm) in a manner comparable to pushing in one side of a hollow rubber ball In am-phibians, however, the presence of a large mass of yolk-filled cells precludes such a simple inpushingprocess.The eventual interior positioning of the endoderm and mesoderm of the amphibian embryo iseffected by more complex processes

en-Gastrulation establishes the three germ layers in their final positions relative to one another Each

of the three germ layers is defined by its developmental fate.The ectoderm contains cells that will duce the epidermal covering of the body as well as the nervous system and sense organs; the endo-derm produces the lining of the digestive tract and its various derivatives (for example, the lungs, di-gestive glands, and bladder); and the mesoderm, which lies between the other two, gives rise to a variety

pro-of tissues (for example, muscular, skeletal, and circulatory systems)