Morphology and Genetics of Sea Urchin Development

Transcription

1 AMER. ZOOL., 15: (1975) Morphology and Genetics of Sea Urchin Development RALPH T. HINEGARDNER Division of Natural Sciences, University of California, Santa Cruz, California SYNOPSIS. Sea urchins can be raised from egg to egg in the laboratory. With proper food, the larvae can be grown to maturity in about 3 weeks. When mature larvae are exposed to the proper chemical cues metamorphosis occurs. Over the next 5 days the small urchins develop internal organs and then begin to feed. Sexual maturity can be reached in as little as 4.5 months. By then the urchin is about a centimeter in diameter. Several different approaches to the study of developmental genetics are covered. These include: (i) hybrids between the sand dollars Dendraster and Encope, in which both crosses produce offspring that have predominantly paternal characteristics; (ii) a preliminary description of two mutants, one which produces abnormally shaped blastula that may lead to a significant number of exogastrulae, and another that produces a large number of fourpart symmetrical urchins; (iii) urchins produced by parthenogenetic activation and from reaggregated larval cells. INTRODUCTION The first reasonably accurate description of sea urchin early development was written by M. Derbes, in He described the embryological development of Echinus esculentus. On the whole, Derbes' description was fairly complete. He deduced the presence of the jelly coat, described formation of the fertilization membrane, and recognized that the mature egg arose from an earlier germinal vesical stage. He pointed out that it was possible to identify the sexes by the appearance of the gonads, and he added that the female gonads "tasted more agreeable and more pleasurable." Some of Derbes' observations were not totally accurate. Had they been, the history of biology might have been different. Because he could not see the male nucleus in the fertilized egg, he concluded that the sperm did not contribute to the embryo but only activated the egg. His conclusions were in keeping with the beliefs of his time. Ironically, 30 years later, the main piece of evidence Hertwig (1876) used to show that the male gamete did contribute to the embryo was the fact that the male nucleus could be seen fusing with the female nucleus in sea This research was supported by a National Science Foundation Grant. I wish to thank Margaret Swanson and Kathryn Boyer for their conscientious assistance. 679 urchin eggs. Derbes was misled in a few other places. He concluded that the blastopore became the mouth. This is a mistake that is easy to make unless development is observed carefully. He attempted to follow the complete life cycle and he thought that the starved pluteus represented further development toward the urchin and that the little ciliated blob the pluteus finally degenerates into after many days without food was a particularly critical state in the urchin's life cycle. Subsequent observations corrected Derbes' mistakes, and all that he attempted to do has since been accomplished. It wasn't until the last decades of the 19th century, with the work of Hertwig, Boveri, Herbst, Driesch and others, that the sea urchin egg began to play a large role in the study of development. Since then something like 3,000 papers have been published that in one way or another are concerned with the sea urchin embryo. Around the turn of the century, a number of biologists succeeded in raising larvae to maturity. Usually this was done by frequently changing the sea water the cultures were growing in, the food source being the planktonic algae that came in with the water. Growth was slow, but judging by published illustrations, it was normal. Some of these larvae were taken through metamorphosis. Bury (1895) was one of the

2 680 RALPH T. HINEGARDNER first to accomplish this. Development was seldom carried further. The most complete description of larval development that has been published is still that of MacBride (1903). By the 1920's the basic features and many of the details of the sea urchin life cycle were well understood. NORMAL DEVELOPMENT At various times, attempts have been made to raise urchins as laboratory animals. In retrospect, it isn't clear why these were not successful. As it turns out, the technique is not difficult once the proper procedures are worked out. One problem may have been that the larvae and young urchins were being given food organisms they do not readily eat. For example, Harvey (1949) tried to raise Arbacia larvae on diatoms. These are a poor food source for all species I have tried to raise, including Arbacia. When the right food organism is used along with the proper technique, it is not difficult to raise urchins from egg to egg in the laboratory (Hinegardner, 1969). The basic features of normal Lytechinus pictus development have been described (Hinegardner, 1969). At 18 C larvae grow in the laboratory from 0.3 mm long plutei to 1.5 mm mature larvae in approximately 3 weeks. Arbacia and several other species grow at about the same rate. Earlier we kept our animals at 24 C; however 18 is somewhat better. Growth rate is dependent upon temperature and several factors. It is decreased if the larvae are crowded. We routinely provide about 10 ml of sea water per larva, and completely change the water after 1.5 weeks. If too little food is provided, growth can be extended over several months. We use an unidentified species of Rhodomonas for food and give our cultures about as much as they will consume in 24 hr. epaulets form. Three pedicellariae appear, two on the right side and one at the posterior end of the larvae. These will later be incorporated into the anatomy of the urchin after metamorphosis. Figure 1 illustrates the anatomy of a mature larva. Larvae stop growing when they are mature, and once they reach this size they are competent to metamorphose. If metamorphosis does not occur the larvae will continue to feed, though at a much lower rate, and can be kept for several more months. However, they are able to metamorphose for a period of only a few weeks. After that, they slowly begin to degenerate, and finally end up as ciliated spheres. During larval growth, the embryonic urchin is also developing. On about the 7th day after fertilization, it begins to form out of the union of a small portion of the ectoderm on the left surface of the larva and the middle left hydrocoel, which arose from the coelmic pouches formed after gastrulation. The developing urchin, while it is in the larva, is called the rudiment. The larva Developmental morphology The external features of the developing larvae gradually become more complex as the larvae grow. New spicules and their associated arms arise, and in some species, such as Lytechinus, dense ciliary bands called FIG. 1. A mature larva of the sea urchin Lytechinus pictus. p, Pedicillaria; r, urchin rudiment; s, larval stomach.

3 serves as a source of nutrient and protection for the growing rudiment. The rudiment is not a little urchin, but only a portion of the developing ventral half of the urchin, and consists primarily of ventral skeleton and water vascular system. The rest of the ventral half, as well as almost all the dorsal and internal structures, develop subsequent to metamorphosis. Though most of the larval biomass ends up in the urchin, it does so after passing through a period during early metamorphosis when many of the cells are broken down and the larval material is little more than a lump of protoplasm on the top of the newly metamorphosed urchin. The urchin develops its own mouth, anus, and most of its internal organs. The nemertines and insects have somewhat similar development. In both these there are imaginal discs which give rise to portions of the adult. Some authors have also called the urchin rudiment an imaginal disc. However, it is not really the same thing. Unlike imaginal discs, the rudiment shows the beginnings of differentiation from its first appearance, and as it grows, the tube feet and spines are clearly visible. By metamorphosis it is well differentiated. Metamorphosis Once the larva is mature, it has to pass successfully through metamorphosis and then grow to a sexually mature adult. Metamorphosis is not obligatory, and if the mature larva is not exposed to the right cues, it never becomes an urchin, and instead, as I have already mentioned, it eventually degenerates into a ciliated sphere that finally dies. This is not necessarily true for all echinoderms. The larva of the sea star Mediaster aequalis, if it is not exposed to the tubes of the polychaete worm on which it normally settles, can live for up to 14 months and still remain capable of metamorphosis (Birkeland et al., 1971). If the larvae of Lytechinus, Arbacia, or many other species of sea urchins are exposed to the appropriate cues, they begin metamorphosis within a few minutes. The important cue for all of these species turns out to be an unidentified low molecular SEA URCHIN DEVELOPMENT 681 weight organic compound(s) that is formed by bacteria. A solid surface greatly facilitates metamorphosis, though larva will sometimes metamorphose while they are held on a suction pipette or by forcepts. The nature of the chemical cue and a more detailed description of the metamorphic process are described in Cameron and Hinegardner (1974). In an hour or less after the initial stimulus, the major external changes from larva to urchin have taken place and by 24 hr the individual looks like a little urchin. There are still major internal rearrangements that take 5 or 6 more days before the urchin begins to feed. These include formation of a complete gut along with mouth, anus and teeth, and the dorsal skeleton. When these internal changes are complete, the urchin begins to feed. Urchin growth In the laboratory, we feed our young animals a surface-adhering diatom belonging to the genusnitzschia, which is grown on plastic dishes. The urchins are transferred to fresh dishes about every 5 days, or when they have consumed most of the algae. When the urchin reaches a diameter of 9 to 10 mm it can be induced to spawn. In our laboratory the males of Lytechinus mature earlier than the females and can spawn at 9 mm. The females usually do not spawn until they are 10 mm or larger. The entire life cycle from egg to egg can take as little as 4.5 months in the laboratory if the urchins are well cared for. With more ordinary care, 6 months is the more usual maturing age. Table 1 outlines the time course of the developmental process. On the whole, sea urchins are not appreciably more difficult to raise than other TABLE 1. Development of Lytechinus pictus at 18 C. Fertilization Begins to feed Larva matures Urchin begins to feed Sexual maturity Life span 0 2 days 3 weeks 5 days after metamorphosis 4.5 to 6 months At least 7 years in the laboratory; 3-year average in the wild (Ebert, 1975).

4 682 RALPH T. HINEGARDNER laboratory animals. The inconveniences that do exist almost all stem from the fact that urchins live in water and we do not. Consequently, there is a lot of water handling. Aside from this the animals are almost as hardy as mice or Drosophila and present no particular problems. LABORATORY MAINTENANCE The previous sections of this paper cover the essential features of laboratory culture. In order to utilize individuals in genetic studies mortality has to be kept low once the animals have matured. This section will describe the methods we use for maintaining animals over long periods and in good health. " At present we have about 400 adult urchins averaging about 2 cm in diameter. Most of these are laboratory raised and are distributed among eight 20-gallon tanks. The tanks are kept at a temperature of about 15 C either in a cold room or by refrigeration. Each tank is aerated and has its own external water filter which contains a layer each of Dacron wool and crushed dolomite. About 4 liters of sea water are removed and replaced with fresh sea water once a week for each tank. Food is almost solely the giant kelp Macrocystis. This species is used primarily because it is convenient. Several other species of algae, such as Egregia, Laminaria or Viva can also be used. The urchins are fed as much algae as they will consume. With this feeding they can be induced to spawn about once a month. Spawning Many of our urchins have been made to spawn repeatedly, sometimes as often as once every 2 months for more than a year. Those carrying unique developmental characteristics have been particularly well used. For these, as well as others, we take particular care not to kill the source of our eggs. After trying various spawning procedures, we have settled on injection of 0.5 M KC1 as our routine method. Acethylcholine (Hinegardner, 1967) can also be used but is less convenient. The animals are never allowed to remain out of the water for more than a minute. KC1 itself is not harmful if used in moderate amounts; however Lytechinus is very sensitive to drying and though an individual may appear normal for several days after it has been left out of the water, it soon begins to lose spines and dies within a week or so. To spawn our animals we inject them only with enough KC1 to induce spawning, which is seldom more than 0.5 ml for a large urchin, and usually closer to 0.25 ml. We immediately place the urchin in a 500-ml beaker containing sea water from the tank the urchin was living in, and allow the urchin to crawl around freely. The animal almost invariably crawls up the side to the water surface and eggs or sperm collect undisturbed on the bottom. Broken spines and debris are later removed by filtering the eggs through HO-jitm nylon mesh. If sperm are to be saved, they are drawn off the bottom with a pipette and centrifuged at 800 g for 10 min. The sea.water is removed and the concentrated sperm pellet stored on ice or in the refrigerator. Before fertilization, the eggs are washed several times in fresh sea water to remove any fertilization inhibitors that come from the adult animals. From this point on, the eggs and sperm are handled under standard procedures such as those described by Harvey (1956), Costello et al. (1957), Tyler and Tyler (1966), and Hinegardner (1967). DISEASES It is surprising that in the more than 5 years we have been raising animals in closed laboratory systems, we have had no serious outbreak of any disease. This is in spite of the fact that all the food for the adults comes from the ocean and that we take no particular precaution to keep out potential disease organisms. The only outbreak of any kind that we have had in some of our tanks was an infection caused by an amoeboid flagellated protozoa that grew in dense patches on the urchins. This caused massive congregation of echinochromecarrying cells in the infected area which made the patches bright red. Though these patches became necrotic and spines fell off,

5 in time most of the urchins cured themselves and we have since had no trouble. All this suggests that Lytechinus, at least, is remarkably resistant to disease. This makes their culture relatively easy. The whole area of disease resistance is an aspect of echinoderm biology that deserves more attention than it has been given. The only observation we have made so far that relates to sea urchin defenses against disease is the frequent appearance of the echinochrome-carrying cells in areas of infection, or all over animals that are obviously not healthy. What role they actually play is not clear. GENETICS Almost all of sea urchin genetics has been limited either to studies of inter-specific and inter-generic hybrids or to the area of molecular biology. To some extent, hybrid studies have been forced on the sea urchin embryologist because genetics at a more refined level has not been possible. Hybrid studies have been useful and they played a particularly important part in the early investigation of the role of the nucleus vs. that of the cytoplasm. Horstadius (1973) presents an extensive discussion of these early investigations. In many ways, research at the molecular level is just beginning, in spite of the fact that the literature is already very extensive. The general features of echinoderm development that have emerged so far from the use of both hybrids and molecular biology are these: During oogenesis, messenger RN A is synthesized and transported to the cytoplasm in an inactive form. Upon fertilization, or shortly afterward, this RNA begins to participate in protein synthesis. In general, most of the proteins synthesized up to mesenchyme blastula are translated from this RNA. The early embryo, therefore, bears primarily maternal characteristics. Shortly before gastrulation, the proteins synthesized on RNA from the embryonic genome, which consists of both maternal and paternal chromosomes, begins to play a role, and from then on the embryo bears characteristics of the combined genomes. There is a vast literature on SEA URCHIN DEVELOPMENT 683 this subject which has been reviewed many times. The following are a few of the books covering this subject: Davidson (1968), Giudice (1973), and Czihak (1975). Hybrids In contrast to this extensive literature on the timing and overall pattern of gene action, the literature on single gene effects and their timing is much sparser. Most of the work comes from studies of hybrids, though the techniques of molecular biology are also beginning to yield conclusive results. Only the use of hybrids will be considered here. Barrett and Angelo (1969) used hybrids between Strongylocentrotus purpuratus and S. franciscanus and demonstrated that hatching enzyme has characteristics derived from the maternal genome. At the prism stage, the enzyme alkaline phosphatase produced by hybrids between S. purpuratus (female) and the sand dollar Dendraster excentricus (male) has activity intermediate between the two species (Flickinger, 1957). The same holds for the enzyme aryl sulfatase from a cross between Allocentrotus fragilis and S. purpuratus. Electrophoretic mobility of the enzyme was also intermediate (Fedecka-Bruner et al., 1971). In general, hybrids show characteristics of the maternal parent prior to gastrulation. After gastrulation their characteristics are intermediate between the parents. This is in agreement with the evidence from molecular biology. Another form of hybridization is the use of eggs that have had their nucleus removed. These are called merogones. Merogones can be formed either by centrifugation into halves, using the anucleate half, or a portion of the cytoplasm and the nucleus can be cut off prior to fertilization. Some eggs are particularly well suited to the latter procedure since the nucleus lies close to the cell membrane prior to fertilization. ' The subsequent larvae, if they are viable, usually have characteristics of the male species. This technique played a key role in early research demonstrating that the nucleus carried the hereditary characteristics of the

6 684 RALPH T. HINEGARDNER organism. Horstadius (1936) used it in a particularly sophisticated way to demonstrate the role of the micromeres. He formed a heterospecific merogone using Paracentrotus cytoplasm and Psammechinus sperm. At the 16-cell stage, the micromeres were removed and fused to a normal Paracentrotus embryo that had previously had its micromeres removed. The skeleton and general shape of the pluteus had the appearance of a Psammechinus pluteus, even though the nuclei of the primary mesenchyme cells were all that had a Psammechinus genome. The reciprocal cross gave reciprocal results. Many hybrids fail to develop much beyond blastula. An example is the cross between Paracentrotus lividus (female) and Arbacia lixula (male) which was extensively studied by Whiteley and Baltzer (1958). There are many other papers on this subject dating from the last century to the present. In fact the first review of sea urchin hybridization is by Tennent in More recent reviews are in Harvey (1956), Giudice (1973), and Horstadius (1973). Almost no hybrids have been raised beyond plutei. With the development of procedures for laboratory culture, this is now possible, and one cross between two different genera can grow at least to young adults. This cross is between the sand dollars Dentraster excentricus and Encope californicus (Hinegardner and Vacquier, unpublished). Figure 2 illustrates the appearance of the mature larvae and the young urchins produced by the various possible crosses. The most striking feature is the dominance of paternal characteristics in both hybrids. The shape of the mature larva is close to that of the paternal species. Pigmentation is also paternal. The young Dendraster sand dollars have elongate black pigment cells, particularly in their ventral epithelium, and very little red pigment. In contrast, young Encope at the same stage have bright red spherical pigment cells and little or no black pigment. The hybrids, Dendraster (female) x Encope (male) had only red pigment. Pigment cells in the reciprocal cross were black, though the cells were not as elongate as those in pure Dendraster. There were only about 1% red spherical cells. This same paternal dominance also extends to the amount of the enzyme /3-1, 3-glucanase that is synthesized by the plutei (Vacquier, personal communication). Plutei carrying the Encope sperm genome produce more. However, the time at which synthesis begins is determined by the egg. The same general features of paternal dominance that have been described here also apply to crosses between Dendraster and Encope grandis. Again, the male genome is dominant. At first glance, at least, these results are hard to explain. They suggests a more complex control of gene activity than current theories of gene control provide for. One of the odd features of these crosses is that the Encope x Dendraster cross is not able to hatch from its jelly coat. They have to be hatched artificially with the aid of proteolytic enzymes. Unfortunately, we were never able to follow our crosses through to maturity. The incubation system failed on a hot day and the young sand dollars died. At the time, both crosses were feeding and growing, although not as well as the parental types. Even this accident yielded some information. Encope comes from the warm waters of Baja California; Dendraster from the California coast. The inside temperature in our incubator reached about 35 C for a period of several hours. Only the Encope x Encope adults survived. The Dendraster (female) x Encope (male) individuals did not, indicating that they were not totally Encope in all their characteristics. Classical genetics All the foregoing examples of sea urchin genetics used hybrids. These can tell a lot, FIG. 2. Mature larvae and one-day-old urchins from for each cross. The Dendraster x Encope larva pictured normal and hybrid crosses between Dendraster excentricus (D) and Encope californicus (E). A, D x D; B, Ex eny of that cross. here has somewhat longer arms than the typical prog- E; C, D x E; D, E x D. The female parent is given first

7 SEA URCHIN DEVELOPMENT 685

8 686 RALPH T. HINEGARDNER but there is little room for manipulation or experimentation, and it is not possible to examine the effects of selected genes in homozygotic conditions. Hybrids are the ultimate in hererozygosity. The literature on sea urchin genetics using classical procedures such as back crossing and inbreeding is non-existent. This situation has begun to change. In our laboratory we now have a number of inbred lines of the sea urchin Lytechinus pictus. Many of our animals are descendants from larvae of crosses that yielded high instances of developmental abnormalities. Two of these inherited abnormalities will be described here. We have not had enough time to do the crosses necessary to define their exact genetic basis. Square. This is the term we have used to designate urchins that are four-part, rather than the usual five-part, symmetrical. Figure 3 is a photograph of one of these urchins. This is an abnormality that shows up infrequently in out crosses, but in one of our inbred lines between 1 and 10% (depending on the particular parents used) of the progeny will be square. Actually square is only one consequence of this inheritance. The urchins can be two-, three-, four-, fiveand sometimes six-part symmetrical. In other words, there seems to be a loss of symmetry control. However, only the four-, five- and six-part urchins have ever been able to develop. Four is the common abnormality, thus the designation square. Square is apparently controlled by more than one gene, since in only one out of six crosses between unrelated square urchins were a significant number of square offspring produced. In that cross about onethird of the Fi urchins were less than 5-part symmetrical. Further analysis has to wait until our inbred urchins reach maturity. Exogastrula. This is a maternal effect and the number of abnormal embryos that are produced is unaffected by the male used to fertilize the eggs. Like square, we have not yet had sufficient time to determine the genetic basis of this characteristic. We do know that it is not temperature sensitive. At present we have one female from an inbred line that consistently produces embryos with some degree of this characteristic. Usually less than 1% of the embryos exogastrulate. The more prevalent effect is a condition earlier in development which leads to the formation of embryos that are distinctly oval at the start of gastrulation. Most of the embryos have that appearance; those that exogastrulate may be the ones that do not recover from their abnormal shape. Figure AA illustrates a normal early gastrula and 4B, a normally gastrulating, but oval, embryo produced by this female urchin. Figure 4C is a slightly older embryo that was beginning to exogastrulate, and 4D, a still older embryo. Figure 4 shows a pluteus with a completely everted gut. The latter is the extreme condition. Others may only have partially protruding guts and in a few the guts barely protruded, with the mouth end therefore not quite reaching the stomadeal opening. As Horstadius (1949) has shown, the stomadeal opening forms whether or not the gut is there. As with LiCl treatment, which also induces exogastrulation, the gut is able to differentiate into a tripartite structure even when it is wrong side out and in an abnormal position. EXPERIMENTAL TECHNIQUES FIG. 3. Four-part symmetrical Lytechinus pictus. Animal is about 2 cm in diameter. Sea urchin eggs can be manipulated by a

9 FIG. 4. A, Normal early gastrula of Lytechinus pictus. note the elongate shape. C and D, Two stages in B, Gastrulating embryo from exogastrula mutant; exogastrulation. E, Exogastrulated pluteus.

10 688 RALPH T. HINEGARDNER large number of different experimental techniques. Many of these are simple enough to be used as routine procedures. Twinning Lytechinus embryos can be twinned by separating the blastomeres at the two-cell stage. With the methods we now have, about 50% of the twin pairs will develop to mature larvae. We have raised some of these to mature, sexually productive adults. The mature larvae as well as the young urchins are the same size as normal animals and behave normally in all respects. Parthenogenesis Unlike the eggs of most other animals that are used in experimental embryology, sea urchin eggs can be artificially activated, and therefore, a homozygous population of animals can, in principle, be produced. Harvey (1956) has reviewed the earlier literature on urchin parthenogenesis. Until recently, Lytechinus pictus was an exception, and as far as I know no one had been able to induce parthenogenetic development. We have been able to obtain parthenogenesis in this species and the general procedure and some of the early results will be published elsewhere (Brandriff et al., 1975). Of approximately 30 females we have tried, only three consistently produce eggs that can both be activated and raised to maturity. Even with these females, only about one out of 10 million eggs can be raised to a feeding urchin, and all of these are at leasta little odd. The tube feet may be shorter than normal, the test malformed, or the genital openings present in the wrong places. All are stupid, even by sea urchin standards, and require special care. The male is supposed to be the digametic sex in urchins, and older parthenogenetic urchins that have died have all been females, as would be expected. Reaggregation In 1962, Giudice discovered that the cells of early sea urchin embryos could be disaggregated, then reaggregated, and that reasonably normal plutei would reform. FIG. 5. Three Arbaciapunctulata urchins grown from reaggregated cells from 16-cell embryos. He used Paracentrotus lividus, but a number of other species can also be used. This discovery is of particular importance, since at the 16-cell stage the sea urchin embryo is composed of three different cell types, the micromeres, mesomeres, and macromeres. Each cell type gives rise to a particular embryonic structure (Horstadius, 1949). For example, the micromeres are involved in spicule formation. Methods are available for dissociating and isolating each of the three cell types as pure cell suspensions (Spiegel and Rubenstein, 1972; Whiteley et al., 1975). These can be reaggregated and some of the resulting embryos will grow to maturity. Figure 5 shows three Arbacia formed from reaggregates. Lytechinus also reaggregates and some of the plutei will also grow to normal mature larvae that metamorphose into feeding urchins. With the use of different genetic strains, it will now be possible to produce allophenic larvae and sea urchins. REFERENCES Barrett, D., and G. M. Angelo Maternal characteristics of hatching enzymes in hybrid sea urchin embryos. Exp. Cell Res. 57: Birkeland.C, F. S. Chia.and R. R. Strathmann Development, substrate selection, delay of

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