Two-stage dependence for 1-methyladenine induced reinitiation of meiotic maturation in starfish oocytes.

The maturation hormone 1-methyladenine (1-MA) causes meiotic resumption of prophase arrested immature starfish oocytes. Continuous exposure to > or = 0.5 microM 1-MA causes germinal vesicle breakdown (GVBD) in approximately 20 min, but oocytes pretreated for > 30 min with a subthreshold dose of 1-MA undergo GVBD much faster (approximately 10 min) when they are exposed to 1 microM 1-MA. Furthermore, a very low subthreshold 1-MA suffices to start the maturation process: oocytes exposed to 0.005 microM 1-MA for up to 10 min followed by 1 microM 1-MA is equivalent to continuous exposure to 1 microM 1-MA. These dose and timing relationships indicate that there is a two-stage dependence on 1-MA. A possible explanation for this dependence is that there are two processes involved: an initial process that is triggered by a low dose of 1-MA, and a second process that cannot start until the first process is completed and is stimulated by a higher dose of 1-MA. These subthreshold 1-MA effects on GVBD timing are not directly coupled to changes in calcium physiology that also occur during maturation. Subthreshold 1-MA was found to cause a transient accumulation of Cdc2/cyclin B into the nucleus. The two-stage dependence indicates that there are unsuspected features in this well-studied pathway leading to GVBD. In the animal, this hormone dependence may help to synchronize maturation throughout all parts of the ovary.

Dorsoventral patterning in hemichordates: insights into early chordate evolution.

We have compared the dorsoventral development of hemichordates and chordates to deduce the organization of their common ancestor, and hence to identify the evolutionary modifications of the chordate body axis after the lineages split. In the hemichordate embryo, genes encoding bone morphogenetic proteins (Bmp) 2/4 and 5/8, as well as several genes for modulators of Bmp activity, are expressed in a thin stripe of ectoderm on one midline, historically called "dorsal." On the opposite midline, the genes encoding Chordin and Anti-dorsalizing morphogenetic protein (Admp) are expressed. Thus, we find a Bmp-Chordin developmental axis preceding and underlying the anatomical dorsoventral axis of hemichordates, adding to the evidence from Drosophila and chordates that this axis may be at least as ancient as the first bilateral animals. Numerous genes encoding transcription factors and signaling ligands are expressed in the three germ layers of hemichordate embryos in distinct dorsoventral domains, such as pox neuro, pituitary homeobox, distalless, and tbx2/3 on the Bmp side and netrin, mnx, mox, and single-minded on the Chordin-Admp side. When we expose the embryo to excess Bmp protein, or when we deplete endogenous Bmp by small interfering RNA injections, these expression domains expand or contract, reflecting their activation or repression by Bmp, and the embryos develop as dorsalized or ventralized limit forms. Dorsoventral patterning is independent of anterior/posterior patterning, as in Drosophila but not chordates. Unlike both chordates and Drosophila, neural gene expression in hemichordates is not repressed by high Bmp levels, consistent with their development of a diffuse rather than centralized nervous system. We suggest that the common ancestor of hemichordates and chordates did not use its Bmp-Chordin axis to segregate epidermal and neural ectoderm but to pattern many other dorsoventral aspects of the germ layers, including neural cell fates within a diffuse nervous system. Accordingly, centralization was added in the chordate line by neural-epidermal segregation, mediated by the pre-existing Bmp-Chordin axis. Finally, since hemichordates develop the mouth on the non-Bmp side, like arthropods but opposite to chordates, the mouth and Bmp-Chordin axis may have rearranged in the chordate line, one relative to the other.

Echinoderm eggs and embryos: procurement and culture.

The protocols outlined here hopefully will provide researchers with healthy, beautiful echinoderm oocytes, eggs, and embryos for experimental use. The large size of echinoderm oocytes and eggs, the ease with which they can be manipulated, and (in many species) their optical clarity, make them an ideal model system for studying not only the events specific to oocyte maturation and fertilization, but also for investigating more general questions regarding cell cycle regulation in an in vivo system. The quick rate at which development proceeds after fertilization to produce transparent embryos and larva makes the echinoderm an advantageous organism for studying deuterostome embryogenesis. Continued use of the echinoderms as model systems will undoubtedly uncover exciting answers to questions regarding fertilization, cell cycle regulation, morphogenesis, and how developmental events are controlled.

Identification of a starfish egg PLC-gamma that regulates Ca2+ release at fertilization.

At fertilization, eggs undergo a cytoplasmic free Ca2+ rise, which is necessary for stimulating embryogenesis. In starfish eggs, studies using inhibitors designed against vertebrate proteins have shown that this Ca2+ rise requires an egg Src family kinase (SFK) that directly or indirectly activates phospholipase C-gamma (PLC-gamma) to produce IP3, which triggers Ca2+ release from the egg's endoplasmic reticulum (ER) [reviewed in Semin. Cell Dev. Biol. 12 (2001) 45]. To examine in more detail the endogenous factors in starfish eggs that are required for Ca2+ release at fertilization, an oocyte cDNA encoding PLC-gamma was isolated from the starfish Asterina miniata. This cDNA, designated AmPLC-gamma, encodes a protein with 49% identity to mammalian PLC-gamma1. A 58-kDa Src family kinase interacted with recombinant AmPLC-gamma Src homology 2 (SH2) domains in a specific, fertilization-responsive manner. Immunoprecipitations of sea urchin egg PLC-gamma using an affinity-purified antibody directed against AmPLC-gamma revealed fertilization-dependent phosphorylation of PLC-gamma. Injecting starfish eggs with the tandem SH2 domains of AmPLC-gamma (which inhibits PLC-gamma activation) specifically inhibited Ca2+ release at fertilization. These results indicate that an endogenous starfish egg PLC-gamma interacts with an egg SFK and mediates Ca2+ release at fertilization via a PLC-gamma SH2 domain-mediated mechanism.

Egg activation at fertilization: where it all begins.

A centrally important factor in initiating egg activation at fertilization is a rise in free Ca(2+) in the egg cytosol. In echinoderm, ascidian, and vertebrate eggs, the Ca(2+) rise occurs as a result of inositol trisphosphate-mediated release of Ca(2+) from the endoplasmic reticulum. The release of Ca(2+) at fertilization in echinoderm and ascidian eggs requires SH2 domain-mediated activation of a Src family kinase (SFK) and phospholipase C (PLC)gamma. Though some evidence indicates that a SFK and PLC may also function at fertilization in vertebrate eggs, SH2 domain-mediated activation of PLC gamma appears not to be required. Much work has focused on identifying factors from sperm that initiate egg activation at fertilization, either as a result of sperm-egg contact or sperm-egg fusion. Current evidence from studies of ascidian and mammalian fertilization favors a fusion-mediated mechanism; this is supported by experiments indicating that injection of sperm extracts into eggs causes Ca(2+) release by the same pathway as fertilization.