Microsatellite loci in wild-type and inbred Strongylocentrotus purpuratus.

Strongylocentrotus purpuratus, a major research model in developmental molecular biology, has been inbred through six generations of sibling matings. Though viability initially decreased, as described earlier, the inbred line now consists of healthy, fertile animals. These are intended to serve as a genomic resource in which the level of polymorphism is decreased with respect to wild S. purpuratus. To genotype the inbred animals eight simple sequence genomic repeats were isolated, in context, and PCR primers were generated against the flanking single-copy sequences. Distribution and polymorphism of these regions of the genome were studied in the genomes of 27 wild individuals and in a sample of the inbred animals at F2 and F3 generations. All eight regions were polymorphic, though to different extents, and their homozygosity was increased by inbreeding as expected. The eight markers suffice to identify unambiguously the cellular DNA of any wild or F3 S. purpuratus individual.

Embryonic and post-embryonic utilization and subcellular localization of the nuclear receptor SpSHR2 in the sea urchin.

SpSHR2 (Strongylocentrotus purpuratus steroid hormone receptor 2) is a nuclear receptor, encoded by a maternal RNA in the sea urchin embryo. These maternal SpSHR2 transcripts, which are present in all cells, persist until the blastula stage and then are rapidly turned over. A small fraction of the embryonic SpSHR2 protein is maternal, but the majority of this nuclear receptor in the embryo is the product of new synthesis, presumably from the maternal RNA after fertilization. In agreement with the mRNA distribution, the SpSHR2 protein is also detected in all embryonic cells. Contrary to the RNA though, the SpSHR2 protein persists throughout embryonic development to the pluteus stage, long after the mRNA is depleted. Following fertilization and as soon as the 2-cell stage, the cytoplasmic SpSHR2 protein enters rapidly into the embryonic nuclei where it appears in the form of speckles. During subsequent stages (from fourth cleavage onward), SpSHR2 resides in speckled form in both the nucleus and the cytoplasm of the embryonic cells. The cytoplasmic localization of SpSHR2 differs between polarized and non-polarized cells, maintaining an apical position in the ectoderm and endoderm versus a uniform distribution in mesenchyme cells. Following the end of embryonic development (pluteus stage), the SpSHR2 protein is depleted from all tissues. During the ensuing four weeks of larval development, the SpSHR2 is not detected in either the larval or the rudiment cells which will give rise to the adult. Just prior to metamorphosis, at about 35 days post-fertilization, the protein is detected again but in contrast to the uniform distribution in the early embryo, the larval SpSHR2 is specifically expressed in cells of the mouth epithelium and the epaulettes. In adult ovaries and testes, SpSHR2 is specifically detected in the myoepithelial cells surrounding the ovarioles and the testicular acini. Nuclear SpSHR2 in blastula extracts binds to the C1R hormone response element in the upstream promoter region of the CyIIIb actin gene indicating that the latter may be a target of this nuclear receptor in the sea urchin embryo.

Twins raised from separated blastomeres develop into sexually mature Strongylocentrotus purpuratus.

The first two blastomeres of Strongylocentrotus purpuratus embryos were separated and the resulting twins raised in pairs through larval life and to sexual maturity. We derive two conclusions from this study: changes in the pattern of specification of embryonic cell fates following blastomere separation result in the creation of two sets of fully functional coelomic pouches and imaginal rudiments, and sex determination in sea urchins is chromosomal, since the pairs of twins were always of the same sex.

Development of sibling inbred sea urchins: normal embryogenesis, but frequent postembryonic malformation, arrest and lethality.

Inbred lines of Strongylocentrotus purpuratus descended from a single pair of wild animals were constructed by sibling mating. We describe results from a systematic series of crosses in which eggs from F2 and from F3 females were fertilized respectively with sperm from their sibling males. Observations were also made on self-fertilized cultures derived from several naturally occurring hermaphrodites. Morphological development, survival efficiency, and expression of three territorial embryonic markers were assayed in the embryos developing from these crosses. Unexpectedly, out of > 90 controlled crosses, we observed no developmental failures whatsoever, up to the end of embryogenesis (i.e., onset of feeding) that could be attributed to homozygous, zygotically acting recessive genes. However, during postembryonic larval development, lethality, morphological malformation, and arrest are observed in inbred cultures at a high frequency. The incidence of these zygotic developmental failures is such that it appears that there is at least one recessive genetic defect affecting larval development per haploid parental genome. The relative imperviousness of the basic embryonic process to defects arising from homozygosity is consistent with other evidence implying that territorial specification in sea urchin embryogenesis is controlled by maternally rather than zygotically expressed gene products.

Loss of yolk platelets and yolk glycoproteins during larval development of the sea urchin embryo.

The fate of the yolk platelets and their constituent yolk glycoproteins was studied in Strongylocentrotus purpuratus eggs and embryos cultured through the larval stage. Previous studies have shown that the yolk glycoproteins undergo limited proteolysis during early embryonic development. We present evidence that the yolk glycoproteins stored in the yolk platelets exist as large, disulfide-linked complexes that are maintained even after limited proteolysis have occurred. We provide additional evidence that acidification of the yolk platelet may activate a latent thiol protease in the yolk platelet that is capable of correctly processing the major yolk glycoprotein into the smaller yolk glycoproteins. Because we previously showed that these yolk glycoproteins are not catabolized during early embryonic development, it was of interest to study their fate during larval development. Using a specific polyclonal antibody to a yolk glycoprotein, we found that both yolk glycoproteins and the yolk platelets disappeared in feeding, Day 7, larval stage embryos, but that starvation did not significantly affect the levels of the yolk glycoproteins. We also found that the yolk glycoproteins reappeared in 30-day-old premetamorphosis larvae.

Sea urchin actin gene linkages determined by genetic segregation.

Genetic linkage between the actin genes of Strongylocentrotus purpuratus was investigated by observing the segregation of restriction fragment length polymorphisms (RFLPs). Specific RFLPs of actin gene pairs CyI/CyIIa and CyIIIa/CyIIIb always cosegregated, confirming the linkage groups CyI-CyIIa-CyIIb previously previously determined by molecular cloning. In contrast, RFLPs of actin genes CyI/CyIIa, CyIIIa/CyIIIb, and M all segregated at random with respect to one another. This demonstrates that the known actin gene clusters CyI-CyIIa-CyIIb, CyIIIa-CyIIIb, and the M actin gene are not closely linked.

A large-scale laboratory maintenance system for gravid purple sea urchins (Strongylocentrotus purpuratus).

A large-scale laboratory facility for the maintenance of several thousand gravid sea urchins (S. purpuratus) is described. Gametes of this species provide an important research resource for studies of animal development. Methods are described for culturing adult S. purpuratus for periods up to two years or more with almost no mortality after the first three to four weeks following collection. Adult females can be maintained in a fertile state in the culture system for four to six months. When spawned, gravid females living in the culture facility will routinely regenerate normal sized populations of fertile oocytes at 1 to 2-month intervals. Such females can be used repeatedly as a source of mature gametes for laboratory research. The reproductive performance of these females can be predicted approximately by the number of late vitellogenic oocytes present in their ovaries. After several months the pool of these oocytes is exhausted and no further mature oocytes can be found for a long period. We show, however, that such females are able to carry out a complete annual cycle of oogenesis if held for a long enough period of time in the culture system.