Delayed accumulation of maternal histone mRNA during sea urchin oogenesis

We have used in situ hybridization and RNA blotting analysis to compare the timing of accumulation of poly(A) and alpha-subtype histone mRNA during oogenesis in the sea urchin Strongylocentrotus purpuratus. In situ hybridization with 3H-poly(U) shows that the content of poly(A) in the developing oocyte increases four- to sixfold during vitellogenesis, implying a similar increase for polyadenylated maternal RNAs. In contrast, both RNA blotting and in situ hybridization demonstrate that there is little, if any, alpha-subtype histone mRNA in large oocytes. These results suggest that these maternal mRNAs accumulate in the pronucleus of the haploid egg after completion of meiotic maturation where they are stored until their release during the breakdown of the pronucleus during prophase.     

LvNotch signaling mediates secondary mesenchyme specification in the sea urchin embryo

Cell-cell interactions are thought to regulate the differential specification of secondary mesenchyme cells (SMCs) and endoderm in the sea urchin embryo. The molecular bases of these interactions, however, are unknown. We have previously shown that the sea urchin homologue of the LIN-12/Notch receptor, LvNotch, displays dynamic patterns of expression within both the presumptive SMCs and endoderm during the blastula stage, the time at which these two cell types are thought to be differentially specified (Sherwood, D. R. and McClay, D. R. (1997) Development 124, 3363-3374). The LIN-12/Notch signaling pathway has been shown to mediate the segregation of numerous cell types in both invertebrate and vertebrate embryos. To directly examine whether LvNotch signaling has a role in the differential specification of SMCs and endoderm, we have overexpressed activated and dominant negative forms of LvNotch during early sea urchin development. We show that activation of LvNotch signaling increases SMC specification, while loss or reduction of LvNotch signaling eliminates or significantly decreases SMC specification. Furthermore, results from a mosaic analysis of LvNotch function as well as endogenous LvNotch expression strongly suggest that LvNotch signaling acts autonomously within the presumptive SMCs to mediate SMC specification. Finally, we demonstrate that the expansion of SMCs seen with activation of LvNotch signaling comes at the expense of presumptive endoderm cells, while loss of SMC specification results in the endoderm expanding into territory where SMCs usually arise. Taken together, these results offer compelling evidence that LvNotch signaling directly specifies the SMC fate, and that this signaling is critical for the differential specification of SMCs and endoderm in the sea urchin embryo.     

The expression of embryonic primary mesenchyme genes of the sea urchin, Strongylocentrotus purpuratus, in the adult skeletogenic tissues of this and other species of echinoderms

Adult tissues of the sea urchin, Strongylocentrotus purpuratus, were analyzed for the products of a set of genes whose expression, in the embryo, is restricted to the skeletogenic primary mesenchyme (PM). Three embryonic PM-specific mRNAs were found to be abundant in adult skeletal tissues (test and lantern), but not in a variety of soft tissues. Homologous mRNAs were also found in skeletal tissues of the congeneric sea urchin, S. droebachiensis, as well as a more distantly related echinoid, Dendraster excentricus, and an asteroid, Evasterias troschellii. The distributions of two of these RNAs were analyzed in regenerating spines of adult S. purpuratus using in situ hybridization. These gene products were localized primarily in the calcoblasts that accumulated at the regeneration site. In nonregenerating spines SpLM 18 RNAs, the most abundant of these gene products, were localized in a small population of noncalcoblast cells scattered through the spine shaft, and were absent from calcoblasts. These observations suggest that a program of gene expression associated with the process of calcification is conserved both developmentally through the period of metamorphosis and evolutionarily among the echinoderms.     

Three sea urchin actin genes show different patterns of expression: muscle specific, embryo specific, and constitutive.

The expression of three different actin genes in the sea urchin, Strongylocentrotus purpuratus, was monitored in embryos and adult tissues by using untranslated mRNA sequences as specific hybridization probes. Three distinct patterns of expression were found: muscle specific, embryo specific, and constitutive (i.e., present in all tissues examined). The actin genes encoding the muscle-specific and constitutively expressed genes were each found to be present once in the haploid genome. The embryo-specific probe could derive from either a single gene or a small subset of actin genes. These data demonstrate that at least three members of the sea urchin actin gene family are expressed in distinct ways and thus are probably associated with different regulatory programs of gene expression necessary for development of this metazoan.     

The genomic repertoire for cell cycle control and DNA metabolism in S. purpuratus

A search of the Strongylocentrotus purpuratus genome for genes associated with cell cycle control and DNA metabolism shows that the known repertoire of these genes is conserved in the sea urchin, although with fewer family members represented than in vertebrates, and with some cases of echinoderm-specific gene diversifications. For example, while homologues of the known cyclins are mostly encoded by single genes in S. purpuratus (unlike vertebrates, which have multiple isoforms), there are additional genes encoding novel cyclins of the B and K/L types. Almost all known cyclin-dependent kinases (CDKs) or CDK-like proteins have an orthologue in S. purpuratus; CDK3 is one exception, whereas CDK4 and 6 are represented by a single homologue, referred to as CDK4. While the complexity of the two families of mitotic kinases, Polo and Aurora, is close to that found in the nematode, the diversity of the NIMA-related kinases (NEK proteins) approaches that of vertebrates. Among the nine NEK proteins found in S. purpuratus, eight could be assigned orthologues in vertebrates, whereas the ninth is unique to sea urchins. Most known DNA replication, DNA repair and mitotic checkpoint genes are also present, as are homologues of the pRB (two) and p53 (one) tumor suppressors. Interestingly, the p21/p27 family of CDK inhibitors is represented by one homologue, whereas the INK4 and ARF families of tumor suppressors appear to be absent, suggesting that these evolved only in vertebrates. Our results suggest that, while the cell cycle control mechanisms known from other animals are generally conserved in sea urchin, parts of the machinery have diversified within the echinoderm lineage. The set of genes uncovered in this analysis of the S. purpuratus genome should enhance future research on cell cycle control and developmental regulation in this model.     

Characterization of a cDNA clone coding for a sea urchin histone H2A variant related to the H2A.F/Z histone protein in vertebrates.

A cDNA clone coding for a sea urchin histone H2A variant has been isolated. The coding region of the clone has been sequenced and the sequence found to be closely related to the H2A.F sequence in chickens. The nucleotide sequence of the sea urchin H2A.F/Z is 74% conserved when compared to chicken H2A.F and 51% conserved compared to sea urchin H2A early and 60% compared to sea urchin H2A late. The nucleotide-derived amino acid comparisons show that H2A.F/Z is 97% homologous with H2A.F in chickens and 57% and 56% homologous when compared to sea urchin H2A early and late respectively. There are between 3-6 copies of the H2A.F/Z sequence in the S. purpuratus genome. The H2A.F/Z gene sequence codes for the previously identified H2A.Z protein. All embryonic stages and adult tissues tested contain mRNA for H2A.F/Z. The mRNA appears in the poly A+ RNA fraction after chromatography over oligo dT cellulose.     

Intermediary metabolism in sea urchin: the first inferences from the genome sequence

The genome sequence of the purple sea urchin Strongylocentrotus purpuratus recently became available. We report the results of functional annotation and initial analysis of more than 2300 proteins predicted to be involved in metabolite transport and enzymatic conversion in sea urchin. The comparison of various reconstructed biosynthetic and catabolic pathways in sea urchin to those known in other genomes suggests the overall similarity of the sea urchin metabolism to that of the vertebrates, with relatively small but non-trivial differences from both vertebrates and protostomes. There are several examples of two parallel, non-orthologous solutions for the same molecular function in sea urchin, in contrast with the other completely sequenced metazoans that tend to contain just one version of the same function. There are also genes that appear to be close phylogenetic neighbors of plant or bacterial homologs, as opposed to homologs in other Metazoa. The evolutionary and functional significance of these variations is discussed.     

A genome-wide survey of the evolutionarily conserved Wnt pathways in the sea urchin Strongylocentrotus purpuratus

The Wnt pathways are evolutionarily well-conserved signal transduction pathways that are known to play important roles in all Metazoans investigated to date. Here, we examine the Wnt pathway genes and target genes present in the genome of the echinoderm Strongylocentrotus purpuratus. Analysis of the Wnt genes revealed that eleven of the thirteen reported Wnt subfamilies are represented in sea urchin, with the intriguing identification of a Wnt-A ortholog thought to be absent in deuterostomes. A phylogenetic study of the Frizzled proteins, the Wnt receptors, performed throughout the animal kingdom showed that not all Frizzled subfamilies were present in the metazoan common ancestor, e.g. Fz3/6 emerged later during evolution. Using sequence analysis, orthologs of the vast majority of the cellular machinery involved in transducing the three types of Wnt pathways were found in the sea urchin genome. Furthermore, of about one hundred target genes identified in other organisms, more than half have clear echinoderm orthologs. Thus, these analyses produce new inputs in the evolutionary history of the Wnt genes in an animal occupying a position that offers great insights into the basal properties of deuterostomes.     

A regulatory sequence near the 3'' end of sea urchin histone genes.

The 3' flanking sequences of all five histone genes have been sequenced in the histone DNA clone h19 of the sea urchin Psammechinus miliaris. A large (23 bp) and a small (10 bp) conserved sequence was found by sequence comparison, some 29-40 bp downstream from the termination codon. 12 bases of the larger homology block show a dyad symmetry. The available sequences of clone h22 of the same species and those of the histone clones pSp2 and pSp17 of Strongylocentrotus purpuratus, another sea urchin species, fit well into this comparison. Two types of sequences are involved in the dyad symmetry; one is H1, H3 and H4 specific, the other is H2A and H2B specific. If these conserved sequences are transcribed, a hairpin loop could form in the RNA molecules. This secondary structure might serve as a recognition signal for a regulatory protein.     

Correct cell-type-specific expression of a fusion gene injected into sea urchin eggs

A fusion gene construct containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of CyIIIa actin gene regulatory sequences was injected into unfertilized eggs of the urchin Strongylocentrotus purpuratus, and early pluteus stage embryos that developed from these eggs were fixed and sectioned for analysis by in situ hybridization. A [3H]RNA antisense probe for CAT mRNA was hybridized to 5-micron embryo sections. Autoradiographic signal denoting the presence of CAT mRNA was detected only over aboral ectoderm cells, in which the CyIIIa gene is normally expressed, and not over any recognizable regions of gut or oral ectoderm included in the same sections.     

The evolution of repetitive DNA sequences in sea urchins.

Molecular hybridization of nuclear DNAs has been employed to study the evolution of the repetitive DNA sequences in four species of sea urchin. The data show that relative to S. purpuratus there has been approximately 0.1% sequence divergence per million years in the repetitive DNA sequences of S. droebachiensis, S. franciscanus, and L. pictus. These results confirm that repetitive DNA sequences are strongly conserved during evolution. However, comparison of the extent of base pair mismatch in the repetitive DNA heteroduplexes formed at Cot 20 with those formed at Cot 200 during the hybridization of S. purpuratus and L. pictus DNAs reveals that highly repetitive sequences of sea urchins may diverge more rapidly than do the more moderately repetitive sequences.