Expression of Hox Genes in Early Development of Irregular Sea Urchin Scaphechinus mirabilis (Echinodermata, Echinoidea)

The quantitative assessment of the expression of 11 genes of the Hox cluster of the sea urchin Strongylocentrotus intermedius (SiHox1, 2, 3, 5, 6, 7, 8, 9/10, 11/ 13a, 11/13b, 11/13c) in the larvae at early developmental stages (blastula – 13 hours post fertilization (h.p.f.), gastrula – 35 h.p.f., prism stage – 46 h.p.f., echinoplutei – 4 and 9 days p.f.) was carried out. At the blastula stage, the expression of the SiHox-7, 11/13b, and 11/13c is observed; as for the latter the early activation was noted for the first time for regular sea urchins. At the gastrula and prism stages, a very low level of expression is observed. At the pluteus stage, there is significant increase of the expression of all Hox cluster genes. Breakages of temporal colinearity during the expression of Hox genes in echinoderms are discussed.

We have characterized the temporal and spatial expression of Spec3 mRNA in embryos of the sea urchin, Strongylocentrotus purpuratus. This mRNA, 2.0 kb in length, is present at low levels in unfertilized eggs but accumulates rapidly during cleavage, increasing 50-fold by hatching blastula stage. Message levels then decline abruptly, remain constant during mesenchyme blastula and gastrula stages, and increase again during prism and pluteus stages. This accumulation pattern is quite similar to that of the ectodermally expressed beta-tubulin mRNAs described recently by Harlow and Nemer (1987a). In situ hybridization shows that although Spec3 message accumulates in all blastomeres at early blastula stages, it later becomes restricted to ectoderm. By late blastula stage, hybridization is strongest in the animal hemisphere. At gastrula, signals are variable over ectoderm, and by pluteus, grains are concentrated in the ciliary band, though present in other ectodermal cells as well. Deciliation and regeneration of cilia in gastrula-stage embryos results in a four- to fivefold increase in Spec3 mRNA levels, implying that the Spec3 gene product is associated with ciliogenesis. Spec3 mRNA is encoded by a single gene in the haploid genome, and characterization of the gene shows that it contains three exons that encode an open reading frame for a hydrophobic protein of 21.6 kD. The reading frame reveals that the carboxy-terminal part of the protein contains two long hydrophobic stretches, 31 and 37 residues long, separated by short hydrophilic regions of six to eight residues. The presence of these two distinct hydrophobic stretches suggests that the Spec3 protein contains two alpha-helical domains that either span the lipid bilayer or are associated with some other hydrophobic environment.

Cellular competence is defined as a cell's ability to respond to signaling cues as a function of time. In Xenopus laevis, cellular responsiveness to fibroblast growth factor (FGF) changes during development. At blastula stages, FGF induces mesoderm, but at gastrula stages FGF regulates neuroectoderm formation. A Xenopus Oct3/4 homologue gene, XLPOU91, regulates mesoderm to neuroectoderm transitions. Ectopic XLPOU91 expression in Xenopus embryos inhibits FGF induction of Brachyury (Xbra), eliminating mesoderm, whereas neural induction is unaffected. XLPOU91 knockdown induces high levels of Xbra expression, with blastopore closure being delayed to later neurula stages. In morphant ectoderm explants, mesoderm responsiveness to FGF is extended from blastula to gastrula stages. The initial expression of mesoderm and endoderm markers is normal, but neural induction is abolished. Churchill (chch) and Sip1, two genes regulating neural competence, are not expressed in XLPOU91 morphant embryos. Ectopic Sip1 or chch expression rescues the morphant phenotype. Thus, XLPOU91 epistatically lies upstream of chch/Sip1 gene expression, regulating the competence transition that is critical for neural induction. In the absence of XLPOU91 activity, the cues driving proper embryonic cell fates are lost.

Nuclear basic protein acetylation during early sea urchin development

First molecular evidence of the toxicogenetic effects of copper on sea urchin Paracentrotus lividus embryo development

Expression patterns of three Par-related genes in sea urchin embryos

Estradiol and endocrine disrupting compounds adversely affect development of sea urchin embryos at environmentally relevant concentrations

Five developmentally regulated sea urchin mRNA sequences which increase in abundance between the blastula and pluteus stages of development were isolated by molecular cloning of cDNA. The regulated sequences all appeared in moderately abundant mRNA molecules of pluteus cells and represented 4% of the clones tested. There were no regulated sequences detected in the 40% of the clones which hybridized to the most abundant mRNA, and the screening procedures were inadequate to detect possible regulation in the 20 to 30% of the clones presumably derived from rare-class mRNA. The reaction of 32P[cDNA] from blastula and pluteus mRNA to dots of the cloned DNAs on nitrocellulose filters indicated that the mRNAs complementary to the different cloned pluteus-specific sequences were between 3- and 47-fold more prevalent at the pluteus stage than at the blastula stage. Polyadenylated RNA from different developmental stages was transferred from electrophoretic gels to nitrocellulose filters and reacted to the different cloned sequences. The regulated mRNAs were undetectable in the RNA of 3-h embryos, became evident at the hatching blastula stage, and reached a maximum in abundance by the gastrula or pluteus stage. Certain of the clones reacted to two sizes of mRNA which did not vary coordinately with development. Transfers of RNA isolated from each of the three cell layers of pluteus embryos that were reacted to the cloned sequences revealed that two of the sequences were found in the mRNA of all three layers, two were ectoderm specific, and one was endoderm specific. Four of the regulated sequences were complementary to one or two major bands and one to at least 50 bands on Southern transfers of restriction endonuclease-digested total sea urchin DNA.

Five developmentally regulated sea urchin mRNA sequences which increase in abundance between the blastula and pluteus stages of development were isolated by molecular cloning of cDNA. The regulated sequences all appeared in moderately abundant mRNA molecules of pluteus cells and represented 4% of the clones tested. There were no regulated sequences detected in the 40% of the clones which hybridized to the most abundant mRNA, and the screening procedures were inadequate to detect possible regulation in the 20 to 30% of the clones presumably derived from rare-class mRNA. The reaction of 32P[cDNA] from blastula and pluteus mRNA to dots of the cloned DNAs on nitrocellulose filters indicated that the mRNAs complementary to the different cloned pluteus-specific sequences were between 3- and 47-fold more prevalent at the pluteus stage than at the blastula stage. Polyadenylated RNA from different developmental stages was transferred from electrophoretic gels to nitrocellulose filters and reacted to the different cloned sequences. The regulated mRNAs were undetectable in the RNA of 3-h embryos, became evident at the hatching blastula stage, and reached a maximum in abundance by the gastrula or pluteus stage. Certain of the clones reacted to two sizes of mRNA which did not vary coordinately with development. Transfers of RNA isolated from each of the three cell layers of pluteus embryos that were reacted to the cloned sequences revealed that two of the sequences were found in the mRNA of all three layers, two were ectoderm specific, and one was endoderm specific. Four of the regulated sequences were complementary to one or two major bands and one to at least 50 bands on Southern transfers of restriction endonuclease-digested total sea urchin DNA.

An Orthodenticle-Related Protein from Strongylocentrotus purpuratus

Developmental abnormalities and DNA-protein crosslinks in sea urchin embryos exposed to three metals

1. Lipid metabolism during the early embryonic development of the sea urchin, Paracentrotus lividus, was studied, using acetate-1-C14 and glycerol-1-C14 as precursors.2. The content of total lipids per embryo showed no significant change up to the mesenchyme blastula stage but began to decrease considerably with the onset of gastrulation. Phospholipids, which account for about one-third of the total lipids, showed the same change as the total lipids. Total nitrogen, on the other hand, continued to decrease slightly until the gastrula stage and then began to increase.3. The total lipids were fractionated through a silicic acid column. The predominant components were found to be triglycerides and phospholipids. For example, the figures obtained with lipids extracted from early gastrulae were 41.5% of triglycerides, 27.4% of phospholipids and 8.8% of free cholesterol. Only small amounts of cholesterol esters and free fatty acids were found.4. Acetate-1-C14 was only slightly incorporated into the total lipids of unfertilized eggs. The incorporation, however, increased very rapidly in an S-shaped curve with progressive development, reaching a maximum at the prism stage. The incorporation rate was somewhat reduced thereafter. Most of the C14 was found to be incorporated into fatty acid moieties.5. After the administration of acetate-1-C14, much of the C14 was incorporated into the phospholipid and triglyceride fractions throughout the early embryonic development. The highest specific activity was always obtained with the cephalin fraction, the lecithin fraction having much lower specific activity. The specific activity of the triglyceride fraction, on the other hand, was fairly low as compared with those of other fractions.6. Similar results were obtained with glycerol-1-C14, except that the incorporation of this precursor occurred to a much lesser extent than that of acetate-1-C14. In this case, however, the incorporation into the total lipids decreased somewhat after fertilization and reached the maximum at the pluteus stage, with a shoulder during the blastula stage.7. The highest specific activity was also found in the cephalin fraction after the administration of glycerol-1-C14. As a considerable amount of C14 was found in the saponifiable fraction as well as in the unsaponifiable fraction, some glycerol seems to be metabolized before being incorporated into lipids.8. The above results indicate that lipid metabolism is much intensified during the embryonic development of the sea urchin, especially in its later phase. As C14, once taken up into phospholipids at the blastula stage, was not lost in any significant amount during the subsequent development to the prism stage, it seems likely that new synthesis occurs in some lipid components, especially in phospholipids, which are essential for subsequent development.

The sensitivity to some chemical agents was examined comparatively at sperm, fertilization, cleavage, blastula, gastrula, pluteus and metamorphosis stages of a sand dollar from Japanese waters (Peronella japonica) and a sea urchin from the Pacific coast of Australia (Heliocidaris erythrogramma). These agents included Cu sulphate, ABS and NH3 chloride. Responses observed included departures from control rates of fertilization and developmental reduction at the attainment of first cleavage, gastrula, pluteus or metamorphosis. Developmental anomalies were noted at the fertilization, 2-cell, gastrula, pluteus and metamorphosis stages. Using minimum effective concentrations of the 3 chemicals at various developmental stages of P. japonica, it was found that sensitivity to chemicals varies from fertilization to metamorphosis. It seems that sperm activity is the most sensitive, and that fertilization and gastrulation are more sensitive than first cleavage, blastulation and pluteus formation. H. erythrogramma seems to show nearly the same responses to Cu, but is more sensitive at metamorphosis.

The mitogen activated protein (MAP) kinase signaling cascade has been implicated in a wide variety of events during early embryonic development. We investigated the profile of MAP kinase activity during early development in the sea urchin, Strongylocentrotus purpuratus, and tested if disruption of the MAP kinase signaling cascade has any effect on developmental events. MAP kinase undergoes a rapid, transient activation at the early blastula stage. After returning to basal levels, the activity again peaks at early gastrula stage and remains high through the pluteus stage. Immunostaining of early blastula stage embryos using antibodies revealed that a small subset of cells forming a ring at the vegetal plate exhibited active MAP kinase. In gastrula stage embryos, no specific subset of cells expressed enhanced levels of active enzyme. If the signaling cascade was inhibited at any time between the one cell and early blastula stage, gastrulation was delayed, and a significant percentage of embryos underwent exogastrulation. In embryos treated with MAP kinase signaling inhibitors after the blastula stage, gastrulation was normal but spiculogenesis was affected. The data suggest that MAP kinase signaling plays a role in gastrulation and spiculogenesis in sea urchin embryos.

Expression of 11 genes of the Hox cluster (SiHox1, 2, 3, 5, 6, 7, 8, 9/10, 11/13a, 11/13b, and 11/13c) was assessed in the sea urchin Strongylocentrotus intermedius at early developmental stages, including the blastula (13 h post fertilization (hpf)), gastrula (35 hpf), prism (46 hpf), and pluteus (4 and 9 days post fertilization (dpf)) stages. Expression of SiHox7, 11/13b, and 11/13c was observed at the blastula stage; early activation of 11/13c was detected for the first time in regular sea urchins. The expression level was very low at the gastrula and prism stages. The pluteus stage showed a significant increase in expression of all Hox cluster genes. Deviations from temporal collinearity of Hox gene expression in echinoderms are discussed.