A new subfamily of high molecular mass CDC2-related kinases with PITAI/VRE motifs.

Kinases of the CDC2 family play a key role in cell cycle regulation and gene expression. In the present work, we identified sea urchin and human cDNAs encoding homologues of a high molecular mass CDC2-like kinase (designated CDC2L5) sharing respectively a PITAVRE and PITAIRE motif. The human cDNA encodes the full-length amino acid sequence of the cholinesterase-related cell division controller (CHED) kinase, a previously published partial coding sequence. CDC2L5 overexpressed in mammalian cells is an approximately 170-kDa nuclear protein. The mRNA is present during the sea urchin early embryogenesis and is ubiquitously expressed in human tissues.

Cdk2 activity is dispensable for the onset of DNA replication during the first mitotic cycles of the sea urchin early embryo.

Earlier work reported the important role of Cdk2 as a regulator of DNA replication in somatic cells and in Xenopus extracts. In the present report we analyze in vivo the involvement of Cdk2 in DNA replication during early embryogenesis using the first mitotic cycles of sea urchin embryos. Unfertilized Sphaerechinus granularis eggs are arrested after the second meiotic cytokinesis. Fertilization resumes the block and induces DNA replication after a short lag period, making sea urchin early embryo a good model for studying in vivo the onset of DNA replication. We show that Cdk2 as well as its potential partner cyclin A are present in the nucleus in G1 and S phase and therefore available for DNA replication. In accordance with data obtained in Xenopus egg extracts we observed that Cdk2 kinase activity is low and stable during the entire cycle. However, in contrast with this in vitro system in which Cdk2 activity is required for the onset of DNA replication, the specific inhibition of Cdk2 kinase by microinjection of the catalytically inactive Cdk2-K33R or the inhibitor p21(Cip1) does not prevent DNA replication. Because olomoucine, DMAP, and emetine treatments did not preclude DNA synthesis, neither cyclin A/Cdk1 nor cyclin B/Cdk1 kinase activities are necessary to replace the absence of Cdk2 kinase in promoting DNA replication. These data suggest that during early embryogenesis Cdks activities, in particular Cdk2, are dispensable in vivo for the initiation step of DNA replication. However, the specific localization of Cdk2 in the nucleus from the beginning of M phase to the end of S phase suggests its involvement in other mechanisms regulating DNA replication such as inhibition of DNA re-replication and/or that its regulating role is achieved through a pathway independent of the kinase activity. We further demonstrate that even after inhibition of Cdk activities, the permeabilization of the nuclear membrane is required to allow a second round of DNA replication. However, in contrast to Xenopus egg extracts, re-replication can take place in the absence of DMAP-sensitive kinase.

A presumptive developmental role for a sea urchin cyclin B splice variant.

We show that a splice variant-derived cyclin B is produced in sea urchin oocytes and embryos. This splice variant protein lacks highly conserved sequences in the COOH terminus of the protein. It is found strikingly abundant in growing oocytes and cells committed to differentiation during embryogenesis. Cyclin B splice variant (CBsv) protein associates weakly in the cell with Xenopus cdc2 and with budding yeast CDC28p. In contrast to classical cyclin B, CBsv very poorly complements a triple CLN deletion in budding yeast, and its microinjection prevents an initial step in MPF activation, leading to an important delay in oocyte meiosis reinitiation. CBsv microinjection in fertilized eggs induces cell cycle delay and abnormal development. We assume that CBsv is produced in growing oocytes to keep them in prophase, and during embryogenesis to slow down cell cycle in cells that will be committed to differentiation.

Skeletogenesis in sea urchin larvae under modified gravity conditions.

From many points of view, skeletogenesis in sea urchins has been well described. Based on this scientific background and considering practical aspects of sea urchin development (i.e. availability of material, size of larvae, etc.), we wanted to know whether orderly skeletogenesis requires the presence of gravity. The objective has been approached by three experiments successfully performed under genuine microgravity conditions (in the STS-65 IML-2 mission of 1994; in the Photon-10 IBIS mission of 1995 and in the STS-76 S/MM-03 mission of 1996). Larvae of the sea urchin Sphaerechinus granularis were allowed to develop in microgravity conditions for several days from blastula stage onwards (onset of skeletogenesis). At the end of the missions, the recovered skeletal structures were studied with respect to their mineral composition, architecture and size. Live larvae were also recovered for post-flight culture. The results obtained clearly show that the process of mineralisation is independent of gravity: that is, the skeletogenic cells differentiate correctly in microgravity. However, abnormal skeleton architectures were encountered, particularly in the IML-2 mission, indicating that the process of positioning of the skeletogenic cells may be affected, directly or indirectly, by environmental factors, including gravity. Larvae exposed to microgravity from blastula to prism/early pluteus stage for about 2 weeks (IBIS mission), developed on the ground over the next 2 months into normal metamorphosing individuals.

The sea urchin larva, a suitable model for biomineralisation studies in space (IML-2 ESA Biorack experiment '24-F urchin').

By the ESA Biorack 'F-24 urchin' experiment of the IML-2 mission, for the first time the biomineralisation process in developing sea urchin larvae could be studied under real microgravity conditions. The main objectives were to determine whether in microgravity the process of skeleton formation does occur correctly compared to normal gravity conditions and whether larvae with differentiated skeletons do 'de-mineralise'. These objectives have been essentially achieved. Postflight studies on the recovered 'sub-normal' skeletons focused on qualitative, statistical and quantitative aspects. Clear evidence is obtained that the basic biomineralisation process does actually occur normally in microgravity. No significant differences are observed between flight and ground samples. The sub-normal skeleton architectures indicate, however, that the process of positioning of the skeletogenic cells (determining primarily shape and size of the skeleton) is particularly sensitive to modifications of environmental factors, potentially including gravity. The anatomical heterogeneity of the recovered skeletons, interpreted as long term effect of an accidental thermal shock during artificial egg fertilisation (break of climatisation at LSSF), masks possible effects of microgravity. No pronounced demineralisation appears to occur in microgravity; the magnesium component of the skeleton seems yet less stable than the calcium. On the basis of these results, a continuation of biomineralisation studies in space, with the sea urchin larva as model system, appears well justified and desirable.

Completely Direct Development of Abatus cordatus, a Brooding Schizasterid (Echinodermata: Echinoidea) from Kerguelen, With Description of Perigastrulation, a Hypothetical New Mode of Gastrulation.

Development of the brooding schizasterid Abatus cordatus, a subantarctic echinoid endemic to Kerguelen, is described. Females spawn nonbuoyant eggs 1300 µm in diameter, which are fertilized by elongated sperm (head 1 µm wide and 15 µm long). The main characteristics of this development are (1) incomplete cleavage beginning at the animal pole that becomes holoblastic, giving a filled wrinkled blastula 26 days after fertilization; (2) apparent (fate-mapping studies have not been done) external migration of mesenchyme cells, in the perivitelline space, from the animal to the vegetal pole during gastrulation while the archenteron invaginates; (3) hatching occurring at the end of the gastrulation (65 days after fertilization); (4) differentiation of the vestibule from a thickening of the oral epidermis as soon as the end of gastrulation is attained; and (5) production of a juvenile directly from the gastrula without any larval stage. The juvenile that leaves the brood chamber is 2 mm in diameter and about 250 days old. A. cordatus is a true completely direct developer (no larva and no metamorphosis). We propose to use (1) the term perigastrulation, as a tentative one until more definitive studies are available, to describe the hypothetical peculiar movement of cells during gastrulation and (2) the terms of direct development only for completely direct developing species and abbreviated development for species that have more or less transformed plutei.

Fertilization of sea urchin eggs in space and subsequent development under normal conditions.

Sea urchin eggs are generally considered as most suitable animal models for studying fertilization processes and embryonic development. In the present study, they are used for determining a possible role of gravity in fertilization and the establishment of egg polarity and the embryonic axis. For this purpose, eggs of the particularly well known and suitable species Paracentrotus lividus have been automatically fertilized under microgravity conditions during the Swedish sounding rocket flights MASER IV and MASER V. It turns out, that fertilization "in Space" occurs normally and that subsequent embryonic and larval development of such eggs, continued on the ground, is normal, leading to advanced pluteus stages.