Cholinergic regulation of the sea urchin embryonic and larval development

Choline esters of polyenoic fatty acids block cleavage divisions of sea urchins and evoke the formation of one-cell multinuclear embryos. If the fatty acids AA-Ch or DHA-Ch are added at the mid or late blastula stage, many cells are extruded, forming extra-embryonic cell clusters near the animal pole of embryos or larvae. Both effects are prevented by dimethylaminoethyl esters of polyenoic fatty acids (AA-DMAE or DHA-DMAE) or their 5-hydroxytryptamides. Nicotinic acetylcholine receptor antagonists, imechine, d-tubocurarine or QX-222 provide partial protection against AA-Ch or DHA-Ch. The organophosphate pesticide, chlorpyrifos, or a combination of (-)-nicotine + phorbol 12-myristate 13-acetate, also evoke the mass extrusion of transformed embryonic cells at the animal pole of larvae. These effects are similarly antagonized by AA-DMAE, DHA-DMAE, or fatty acids 5-hydroxytryptamides. Taking together, these results suggest that AA-Ch and DHA-Ch act on sea urchin embryos and larvae as agonists of acetylcholine receptors, whereas AA-DMAE and DHA-DMAE act as antagonists. The ability of fatty acids 5-hydroxytryptamides to prevent the effects of AA-Ch or DHA-Ch may be due to restoration of the normal dynamic balance of cholinergic and serotonergic signaling during cleavage divisions and gastrulation.     

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.     

Cell type specification during sea urchin development

Recent discoveries indicate that cell lineages and fates play a key role in the establishment of spatially restricted gene expression during sea urchin development. Unique sets of founder cells generate five territories of gene expression by means of an invariant pattern of complete cleavage. Cell lineage analysis demonstrates that the second embryonic axis, the oral-aboral axis, is specified with reference to the first cleavage plane. In the undisturbed embryo, clones that contribute to one territory or another begin to appear at the third cleavage, and founder cell segregation to all five territories is completed by the sixth cleavage. Founder cell segregation is a key feature of mechanisms that establish the spatially defined gene activity of sea urchin embryogenesis.     

PKCgamma regulates syndecan-2 inside-out signaling during xenopus left-right development

The transmembrane proteoglycan syndecan-2 cell nonautonomously regulates left-right (LR) development in migrating mesoderm by an unknown mechanism, leading to LR asymmetric gene expression and LR orientation of the heart and gut. Here, we demonstrate that protein kinase C gamma (PKCgamma) mediates phosphorylation of the cytoplasmic domain of syndecan-2 in right, but not left, animal cap ectodermal cells. Notably, both phosphorylation states of syndecan-2 are obligatory for normal LR development, with PKCgamma-dependent phosphorylated syndecan-2 in right ectodermal cells and nonphosphorylated syndecan-2 in left cells. The ectodermal cells contact migrating mesodermal cells during early gastrulation, concurrent with the transmission of LR information. This precedes the appearance of monocilia and is one of the earliest steps of LR development. These results demonstrate that PKCgamma regulates the cytoplasmic phosphorylation of syndecan-2 and, consequently, syndecan-2-mediated inside-out signaling to adjacent cells.     

Acetylcholine synthesis and possible functions during sea urchin development

Cholinergic neurotransmitter system molecules were found to play a role during fertilisation and early cell cycles of a large number of invertebrate and vertebrate organisms. In this study, we investigated the presence and possible function of choline acetyltransferase (ChAT, the biosynthetic enzyme of acetylcholine) in gametes of the sea urchin, Paracentrotus lividus, through localisation and functional studies. ChAT-like molecules were detected in oocytes, mature eggs and zygotes with indirect immunofluorescence methods. Positive immunoreactivity was found in the ovarian egg cytoplasm and surface as well as at the zygote surface. This suggests the eggs' capacity to autonomously synthesise acetylcholine (ACh), the signal molecule of the cholinergic system. Acetylcholinesterase (AChE, the lytic enzyme of acetylcholine) was also found in ovarian eggs, with a similar distribution; however, it disappeared after fertilisation. Ultrastructural ChAT localisation in sperms, which was carried out with the immuno-gold method, showed immunoreactivity in the acrosome of unreacted sperms and at the head surface of reacted sperms. In order to verify a functional role of ACh during fertilization and sea urchin development, in vivo experiments were performed. Exposure of the eggs before fertilisation to 1 mM ACh + 1 microM eserine caused an incomplete membrane depolarisation and consequently enhanced polyspermy, while lower concentrations of ACh caused developmental anomalies. The exposure of zygotes to 0,045 AChE Units/mL of sea water caused developmental anomalies as well, in 50% of the embryos. Altogether, these findings and other previously obtained results, suggest that the cholinergic system may subserve two different tasks during development, according to which particular type of ACh receptor is active during each temporal window. The first function, taking place in the course of fertilisation is a result of autonomously synthesised ACh in sperms, while the second function, taking place after fertilisation, is due to maternal ChAT molecules, assembled on the oolemma along with egg maturation and fertilisation processes.     

Localization of mitochondrial Hsp56 chaperonin during sea urchin development

We have previously demonstrated that Paracentrotus lividus nuclear genome encodes for the heat shock inducible chaperonin homolog Hsp 56 (1) and that the mature protein is localized in the mitochondrial matrix (2). In this paper we report that constitutive Hsp56 is maternally inherited, in fact it is present in the in unfertilized eggs, and that it has a perinuclear specific localization during cleavage. In the later stages both the constitutive and the heat shock inducible chaperonin has a specific territorial distribution. Moreover following heat shock, the Hsp56 appears in the cytoplasm and in the postmitochondrial supernatant beside the mitochondrial fraction.     

Properties of adenylate cyclase activity during early sea urchin development

The total adenylate cyclase activity in homogenates of eggs of the sea urchins Strongylocentrotus purpuratus and Lytechinus pictus was assayed in vitro and found to remain constant in eggs before and at intervals after fertilization. In S. purpuratus egg homogenates virtually all of the enzyme activity was sedimented by centrifugation at 20 000 g. The enzyme specific activity in the 20 000 g pellet remained unchanged at each point through first cleavage, though it was several-fold higher than in the whole homogenate. The adenylate cyclase from both fertilized and unfertilized eggs was maximally active in vitro when assayed with 10 mM MgSO₄ and 10 mM NaF at pH 8 using 0.2 mM AMP-PNP (an ATP analog) as the substrate. Sucrose density gradient centrifugation of egg homogenates showed that adenylate cyclase activity was present in fractions which sedimented at a variety of densities. The adenylate cyclase specific activity in cortices isolated by the method of Sakai [10] from eggs at first cleavage was 4- to 6-fold higher than in unfertilized egg cortices. The increased enzyme activity in egg cortices at first cleavage suggests that adenylate cyclase-containing membranes may become localized within the egg cortex after fertilization.     

Cell polarity emerges at first cleavage in sea urchin embryos

In protostomes, cell polarity is present after fertilization whereas most deuterostome embryos show minimal polarity during the early cleavages. We now show establishment of cell polarity as early as the first cleavage division in sea urchin embryos. We find, using the apical markers G_M1, integrins, and the aPKC-PAR6 complex, that cells are polarized upon insertion of distinct basolateral membrane at the first division. This early apical-basolateral polarity, similar to that found in much larger cleaving amphibian zygotes, reflects precocious functional epithelial cell polarity. Isolated cleavage blastomeres exhibit polarized actin-dependent fluid phase endocytosis only on the G_M1, integrin, microvillus-containing apical surface. A role for a functional PAR complex in cleavage plane determination was shown with experiments interfering with aPKC activity, which results in several spindle defects and compromised blastula development. These studies suggest that cell and embryonic polarity is established at the first cleavage, mediated in part by the Par complex of proteins, and is achieved by directed insertion of basolateral membrane in the cleavage furrow.     

The effect of salinity on larval survival and development in the sea urchin Echinometra lucunter

Summary

Reductions in salinity can have adverse effects on larval development and larval survival in some invertebrate taxa but not others. Salinity tolerance of larvae may be particularly important in echinoderms because they are both poor ion regulators and stenohaline. I examined the effect of six levels of salinity (15, 18, 21, 24, 27 and 33 PSU) on survival and rate of development of larvae in the subtropical sea urchin Echinometra lucunter. In the short-term, mortality rate was significantly lower in 33 PSU than in all other salinities except 27 PSU, and it was significantly greater in 15 and 18 PSU than in all higher salinities. In the long-term, daily and cumulative mortality were significantly greater in 15 PSU than in most other salinities over 11 days of development (except for cumulative mortality in 18 PSU). They were significantly greater in 18 PSU than in 21 PSU or 33 PSU over a period of 13 days. Furthermore, daily mortality was significantly greater in 18 PSU than in 24 PSU or 27 PSU at 13 d after fertilization. Daily and cumulative mortality were significantly lower in 33 PSU than in 21, 24 or 27 PSU over a period of 17 days. Although in the control (33 PSU) 75% of larvae completed development to the 8-arm stage at 35 d, no larvae developed further than the 4-arm stage in 18, 21, 24 or 27 PSU; in 15 PSU, ~60% of larvae did not develop further than swimming blastulae. Since prolonged exposure to salinities as high as 27 PSU (frequently recorded in the adult habitat) can result in great larval losses, adaptive behaviours that prevent larvae from entering water layers of low salinity will enhance their chance for survival.     

Methylation profile of P. lividus sea urchin genes during development

Methylation pattern has been studied in two genes of sea urchin Paracentrotus lividus using sodium bisulfite method to understand the possible role of DNA methylation during invertebrate development. Three regions of the gene for the hatching enzyme have been analyzed and all of them resulted unmethylated in embryos at different stages of development. Four CpG rich regions have been studied in the gene for DNA methyltransferase: upstream, upstream-exon1, intron 1 and exon 20. The upstream-exon 1 region is always unmethylated, while intron 1 and exon 20 are heavy methylated. Only the upstream fragment changed its pattern of methylation during development. For none of the studied regions the reported data show a general direct correlation between gene expression and methylation process during development.