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Sea Urchin Embryology
A Genetic Approach to Development

Mary A. Petti and Susan Terry
1994 Woodrow Wilson Collection


Teacher Sheet

Introduction

This laboratory activity involves merging a classical sea urchin embryology laboratory with a unit on gene regulation. There are many good sea urchin embryology labs that already exist in various lab manuals (see references) and it is not our intent to replicate these here. However, that portion of the lab can be done easily and you are encouraged to try it. It is recommended that you coordinate this lab with other biology and marine biology teachers at your site. The kits available from Wards or Carolina are relatively expensive but a lot of students can use the materials in different ways (see teacher laboratory preparations).

The approach taken in this laboratory is one of student exploration of developmental processes after studying gene regulation. The question students are attempting to answer is: How does an organism, which begins as the merging of two very different gametes into a single cell, become over time a complex multicellular organism composed of many specialized cells? The students will observe and record developmental events and their sequence using the sea urchin. An extended activity involves the detection of alkaline phosphatase in the various embryonic stages. Alkaline phosphatase is an enzyme which is expressed in a few cells during early stages and later is expressed in large quantities primarily in the sea urchin gut. As such it is a good indicator of an endodermal cell line.

At the end of this lab, the teacher will work with students to elucidate the known aspects of sea urchin embryology and students will then attempt to explain these developmental aspects in terms of gene regulatory mechanisms. This area is one which is actively being pursued by current researchers and represents a blend of two biological disciplines: embryology and molecular biology. A good discussion of current knowledge of the sea urchin is found in Coffman and Davidson, 1994.

Target Students: Advanced Biology, A.P. Biology, Honors Biology, Research (First year Biology and Marine Biology students will also benefit from this lab experience if the more complex aspects are omitted.)

Background Information

Evolution

Evolution is the fundamental unifying process of life on earth. Evolution involves the change in species (gene pools) over time. Elucidating homologies between living organisms has been a major approach to clarifying the process of evolution and the relationship between current taxa. This approach can now be applied at the molecular level of development. A homology at this level expresses a set of developmental decisions made by an embryonic part. If the sequence of decisions was the same for a particular part in two species, then these parts are homologous.

Development

Development involves the change in an organism's cell population (cell pool) over a period of time. We are beginning to understand these developmental changes at the molecular level. How does an individual develop from a single cell (the fertilized egg) into a multicellular organism with a large number of specialized cells each with a specific function but working harmoniously together?

There are three processes that must be elucidated to answer this question.

Regional Specification
(spatial organization or pattern formation): This is the process by which cells in different regions of the embryo become switched onto different pathways during development.

Cell Differentiation:
This is the process that leads to the synthesis by a population of cells of types of proteins different from those made by their ancestors and different from those made by other cells in the same developmental stage.

Morphogenesis:
(the creation of form) This involves the processes of cell and tissue movement that shape the embryo. Gene products are necessary for any type of cellular behaviors, but shape changes have a substantial mechanical and physical component.

By the early tailbud stage, most embryos are mosaics of regions already determined to form principal organs and structures of the body. The embryo is built up by a hierarchy of decisions. At the tailbud stage, many decisions have already been made but more decisions will be taken, in most cases, before each cell differentiates into its mature cell type. Some cell types arise from more than one lineage (see fig. 1).

Very early developmental stages have been largely covert except for the 3 germ cell layers which are named according to their physical position in the embryo. Recent in situ hybridizations of RNA made from cDNA have shown the definition of regions by early gene activity.

Two processes are known which can account for events leading to regional subdivision. The first is called cytoplasmic localization which has been studied primarily in invertebrates. Cytoplasmic localization occurs when regulatory molecules become differentially distributed in the cytoplasm of a cell. In the course of cell division, two daughter cells inherit different amounts of materials and thus enter different states of commitment. Cytoplasmic determinants are often deduced. Proof comes from the transfer of cytoplasm from one place in an embryo to another by microinjection. In this type of experiment cells inheriting a particular cytoplasm become structures normally formed by the egg region from which the original cytoplasm came. The anterior/posterior system in Drosophila is an example.

The second process is induction. This has been widely studied in vertebrates, particularly amphibians. During induction, tissue becomes differentially determined in response to the concentration of a chemical signal from another region of the embryo. Induction involves intercellular interactions. Usually regulation will only occur if certain regions are present. These are the signaling centers or organizers. The ability to respond to the inductive signal is called competence. The range of choices open to competent tissue is a property of its state of determination. For example, once mesoderm is formed it is competent to become somite, kidney, mesenchyme, or blood cells. It is no longer competent to become one of the ectodermal derivatives. (see fig. 1)

Recent work on Drosophila has revealed the presence of homeotic genes. These genes when mutated cause conversion of one body part into another. These are believed to be the initial elements which respond to cytoplasmic determinants or to inductive signals. Homeotic genes have also been called selector genes. It is their activity which selects a particular developmental pathway. They work by regulating the activity of other genes.

Example of how a homeotic gene might work.

A morphogen is an inducing factor which can evoke more than one positive response from responding tissue. Assume that a gene has two states: on=1 and off=0. If a morphogen gradient turns on 3 homeotic genes at different concentrations, the embryonic field can be divided into four territories: 111, 011, 001, 000. If there is a mutation in the 2nd homeotic gene and it can not be turned on, then the embryonic field will be divided into only 3 fields one of which is novel: 101, 001, 001, 000. The second and third territories will be identical and the first territory will be novel since it now has a new combination of the three homeotic genes turned on.

Genetic Regulation During Sea Urchin Development

Example of cytoplasmic localization in the sea urchin.
During oogenesis in the vegetal position the vegetal center is specified. Structures later determined by this region are the micromeres.

Example of Induction in the Sea Urchin
In the blastula stage the vegetal region is the signaling region and the responding region is the animal hemisphere. The outcome of this induction is the formation of the oral arms.

An Overview of Genetic Regulation in the Sea Urchin

  1. There is no extensive switching on of new genes during early development.

  2. Most of the mRNA sequences found in the gastrula stage are stored maternal mRNA.

  3. The structural complexity of the early embryo progresses during development while the molecular complexity of mRNA is highest at the beginning of development. Most mRNA's are replaced by new transcripts of the same set of genes that were active in oogenesis.

  4. James Lee of Cal Tech and Robert Angerer at the U. of Rochester have elucidated the CyIIIa gene that codes for the protein actin. It is active 15-20 hours after fertilization and only in cells destined to be the outer covering of the larva.

  5. Transcription and translation of most macromolecules needed for cell division and morphogenesis occur during oogenesis.

  6. Fertilization triggers biosynthetic activities in the oocyte as well as spatial reorganization of the cytoplasm.

  7. During cleavage, blastulation and gastrulation diverse domains are established.

  8. Nuclei of cells in these different domains are induced to function differentially in a micromere, a macromere, and a mesomere.

  9. Maternal mRNA and proteins are replaced by embryonic mRNA and proteins and other products from genes which are differentially activated in cells of a given lineage.

  10. Termination of embryogenesis occurs when specific patterns of gene expression which are characteristic of the major mature cell lineages are established.

Fig 1. Hierarchy of Decisions Made by Cells throughout Development


Teacher Sheet

Sea Urchin Normal Development Sea Urchins belong to Class Echinoida in phylum Echinodermata. True sea urchins are radially symmetrical as adults although they are bilaterally symmetrical in the larval stage.

Eggs are about 75 to 150 microns in diameter and mature in the ovary before being shed. Eggs are surrounded by a vitelline membrane. After fertilization, a new fertilization membrane is formed by a combination of the vitelline membrane and material released from cortical granules. A surface coat, the hyaline layer is secreted shortly afterwards.

  1. The first two cleavages are vertical and the third is equatorial.

  2. During the fourth cleavage the four cells at the animal pole cleave equally and form 8 mesomeres while the four cells at the vegetal pole cleave unequally and form 4 large cells (macromeres) and four small cells (micromeres).

  3. Subsequently each micromere divides to form 2 small micromeres and 8 primary mesenchyme cells.

  4. After the sixth cleavage, divisions are asynchronous. A blastocoel develops in the interior with a basement membrane on its surface.

  5. During the eighth division one cilium appears on each cell and shortly afterward the blastula hatches from its fertilization membrane.

  6. The blastula wall thickens and forms a plate at the vegetal pole. 32 primary mesenchyme cells invaginate into the blastocoel and arrange themselves in a ring.

  7. During gastrulation these cells begin the formation of calcareous spicules which later become the larval skeleton.

  8. The future oral side flattens and the mouth is formed.

  9. At the other end of what is now the alimentary canal the blastopore becomes the anus and the oral/aboral polarity becomes clearly visible.

  10. The embryo now elongates to form two long oral arms and two short anterolateral arms. These are reinforced by skeletal rods laid down by the primary mesenchyme. In the coelomic sacs are 8 small micromeres which are to contribute to the adult sea urchin. This is the pluteus larva.

Teacher Laboratory Preparations

It is best to unpack sea urchins as soon as they arrive. Discard shipping water and place them in an established or temporary saltwater aquarium that has been properly prepared and aerated. Use the sea urchins as soon as possible. We have left them for up to 2 weeks in the aquarium before injection for gametes and still obtained a large number of gametes.

Directions for obtaining gametes may vary depending on the laboratory directions you are following. You cannot distinguish a male sea urchin from a female sea urchin so you may have to inject several before you get both egg and sperm. Collect the gametes from individual sea urchins separately. If the sperm are collected and not diluted in sea water they may remain viable for a number of days in the refrigerator. Eggs should be diluted in sea water after collection. Aspirate the sea water and replace with new sea water. Eggs may be kept in the refrigerator for several days in sea water.

Fertilizations on microscope slides require only small numbers of egg and sperm and can be accomplished by students in each class; however, probably only one or two classes can participate in the initial injections to obtain gametes. A video camera will be useful to record this process for other classes to observe. A video camera attached to a microscope is useful in recording subsequent development.

For the accumulation of different embryonic stages to be used for alkaline phosphatase detection, the teacher can initiate separate fertilizations over a two or three day period. Embryos can be stored in small beakers, cups, or Petri dishes. The student will then have a variety of stages to test for alkaline phosphatase. (You may try refrigerating some to retard development.) Alternatively the teacher can preserve stages periodically in cold 100% methanol and maintain these in covered containers in the refrigerator.

Lab Materials

  • Basic Sea Urchin Embryology Kit.......Carolina or Wards
  • Natural Sea Water or Instant Ocean...... Local Ocean or Any Pet Store
  • Salt Water Aquarium (20 gal or larger)...Pet Store or Biological Supply Co.
  • Microscope Slides (depression slides are best)
  • Cover slips
  • Plastic or glass pipettes
  • 0.5M KCL (comes with kit)

For Alkaline Phosphatase Detection: (All prices are as of Summer 1994)

  • methanol (Carolina cat # 87-4950): 500 ml: $8.15
  • Nitroblue Tetrazolium Chloride (ICN Biochemicals cat. # 100416) 50 mg.: $10.35
  • 5-Bromo-4-chloro-3-indolyl phosphate (ICN Biochemicals cat #150042) 50 mg.: $15.75

Tris pH 9.5 Tris Buffer:

  • 50 mM Tris
  • 5 mM MgCl
  • 100 mM NaCl

To make 1 liter:

  • 6.06 g Tris
  • 1.02 g MgCl
  • 5.44 g NaOH

Make up to 1 liter with distilled water and adjust pH

Access to a centrifuge and/or analytical balance will be helpful but is not essential. Embryos will settle to the bottom if tubes are allowed to stand for 10-15 minutes.

Please contact either Mary Petti or Susan Terry if you are interested in doing this lab. We are trying to get Carolina to package the chemicals in an "easy-to-use" kit form. We are also interested in developing other markers for differential gene expression during development of the sea urchin that can be used in the high school laboratory.

References

Coffman, J.A. and E. H. Davidson. 1994. "Regulation in Gene Expression in the Sea Urchin Embryo." Journal of the Marine Biological Assoc. of the United Kingdom. 74:1:17-26.

Davidson, E.H. et al. 1982. "The Molecular Biology of the Sea Urchin Embryo." Science 217:17-26.

Gehring, Walter J. 1985. "The Molecular Basis of Development." Scientific American 253(4):152-163.

Gilbert, Scott F. 1991. Developmental Biology. 3rd ed. Sinauer Assoc. Inc.

Glover, D.M. and B.D. Hanes (ed.) 1989. Genes and Embryos. IRL Press.

Horstadius, V. 1973 . Experimental Embryology of Echinoderms. Oxford: Clarendon Press.

Merriam, Robert W. 1986. Experiments in Animal Development. Sinauer Assoc. Inc.

Micklos, D.A. and G. A. Freyer. 1990. DNA Science. Cold Spring Harbor Press.

Oppenheimer, S.B. Sept. 1989. "The Sea Urchin Embryo: A Remarkable Classroom Tool." The American Biology Teacher. vol. 5 (6).

Oppenheimer, S.B. and G. LeFevre, Jr. 1992. Introduction to Embryonic Development from Egg to Adult. A Report from the Howard Hughes Medical Institute.

Slack, J.W. 1991. From Egg to Embryo. 2nd ed. Cambridge University Press.


STUDENT SHEET

Sea Urchin Development

PURPOSE:

The purpose of this lab is to observe and record the differences in the sea urchin gametes (sperm and egg); the changes that occur during and shortly after fertilization; and the changes that occur in the developing embryo over a period of several hours and days. After you have made careful observations you may use a direct method of assaying for tissue- specific gene expression.

Procedure:

  1. Observe, draw, and label an adult sea urchin.

  2. Write a brief paragraph recording your observations.

  3. Mature sea urchins are either female or male. Unfortunately you can not determine the gender of a sea urchin just by looking at it. When the gametes are ripe, they are released through pores on the aboral (top) side of the sea urchin. Fertilization is external and takes place in sea water. In this laboratory you will induce the urchins to release their gametes so that they can be collected separately and so you can observe fertilization on a microscope slide.

  4. You may be asked to inoculate several sea urchins with 0.5M potassium chloride. (Your teacher will give you more detailed instructions)

  5. Eggs and sperm are collected separately. Do not dilute the sperm until you are ready to fertilize the eggs. Eggs will be diluted in sea water immediately and allowed to settle to the bottom of a cup or beaker. Sea water should be removed with a pipette and replaced with new sea water.

  6. Place a small drop of sea water with eggs on a depression slide. Observe and draw the unfertilized egg. Include the cell diameter, color and texture of the cytoplasm, appearance of the cell membrane, and any internal organization that is visible. Under low power add a drop of sperm diluted in sea water. You may add the sperm to the edge of the drop containing the eggs or you may mix both with a toothpick while observing. Observe and draw the changes in an egg 2 minutes after being mixed with the sperm. Switch to the high power objective and observe and draw a sperm.

  7. Your teacher will set up additional fertilizations in Petri dishes or beakers over the next few days. You should draw and observe as many stages as possible. In addition, you may be asked to video tape particularly good specimens.

  8. You should submit a series of drawings of several stages from the first cleavage to the pluteus larva. Refer to the various references provided by your teacher, but only after you have made your own observations and drawings.

  9. Write a summary of the major changes that you observed as the embryo developed into a larva. Be sure they are in the proper sequence.


Direct Detection of
Alkaline Phosphatase in Sea Urchin Embryos

Alkaline phosphatase is an enzyme found in the gut and other tissues of most organisms. In the sea urchin it is expressed primarily in the gut lining. In the sea urchin it is a good marker for endoderm. The technique used in visualizing the presence of alkaline phosphatase is called whole-mount histochemistry.

  1. Pipette 1 ml of embryo suspension from each developmental stage into a labeled Eppendorf tube.

  2. Spin in a centrifuge briefly or allow embryos to settle by gravity and remove the sea water.

  3. Resuspend embryos in ice cold 100% methanol.

  4. Let the embryos remain in the cold methanol for at least 20 minutes. Your teacher may have already preserved embryos in the refrigerator. If this is so you can move directly to step #5.

  5. Bring the embryos to room temperature and remove the supernatant from above the settled embryos.

  6. Resuspend the embryos in 1 ml Tris ph 9.5 buffer.

  7. Repeat step 5.

  8. Add 3.3 microliters of Alkaline Phosphatase Substrate (BCIP...bromochloroindolyl phosphate) and 6.6 microliters of NBT (nitrolbluetetrazolium). In the presence of alkaline phosphatase, phosphate is removed from the BCIP, causing a blue precipitate to settle at the site of enzymatic activity. The NBT enhances the signal.

  9. Rock the tube gently for 5-15 min.

  10. Pulse spin the tube or allow embryos to settle by gravity.

  11. Resuspend the fixed, stained embryos in cold normal sea water.

  12. Put the embryo suspension from each tube into a well in a depression slide.

  13. Observe each sample for blue-stained cells which indicate the presence of alkaline phosphatase.

  14. Draw the stained areas in an embryo at each of 4 stages.

  15. Write a brief description of the expression pattern of the gene for alkaline phosphatase in the development of sea urchin embryos.

IV. Gene Regulation During Development

Summary Question:

You have learned that the eukaryotic chromosome is far from a simple linear sequence of genes. Discuss the types of transcriptional and post-transcriptional regulation of this genome that might determine the development of the sea urchin embryo. Include determination and differentiation as they relate to the development of this deuterostomic, coelomate which is initially bilaterally symmetrical and radial symmetrical as an adult.


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