To study development genes already identified in fish, with major emphasis on the superfamily of growth factors and their receptors (in model and commercial fish). To set up, and operate large-scale research of original genes by enhancer trap, gene trap and mutagenesis (in model fish) and differential cloning of cDNAs (in commercial fish). To develop technologies required for all these approaches including uniparental reproduction, gene transfer and ES cells (in model and commercial fish).
Remarkable progress has been recorded in the study of two already known genes (the Xmrk EGF receptor-related proto-oncogene, and the TGF-beta related Radar gene), using a combination of all tools available in fish developmental genetics (whole-mount in situ hybridization, ectopic expression, use of development markers, use of existing mutants, induction of new mutant phenotype by mutagens and dominant-negative strategies). It is now very clear that Xmrk has a major role in the early proliferation of the central nervous system, and that Radar is involved in neural crest development and retina patterning, through dorsalising effects. A novel Radar-related gene (V5), normally expressed in the trunk neural tube is shown to be regulated by sonic hedgehog, a well known inducer of ventral neural tube. A number of homeobox genes involved in the development of CNS have also been cloned, and intense efforts are devoted to study the role of two of them (Prx and engrailed2) in the development of midbrain and hindbrain. Recloning of such genes in economically-important fish species is in progress. Other methodologies are under development to identify and study key developmental genes. So far, difficulties are encountered in the mass production of transgenic lines in laboratory fish, which would permit to start large insertional screens such as enhancer trapping, and new investigations should permit to enhance the rate of DNA integration in classical transgenesis. These methods are already very potent in freshwater commercial fish to understand gene regulation mechanisms, and are tentatively extended to marine fish in the project. A major achievement of the project is the development of one feeder layer-free protocol to culture embryonic cells and possibly develop totipotent ES cell lines for reimplantation into embryos. This would open the way to homologous recombination and gene trap strategies. An ES-like cell line has been maintained for more than 18 months, and meets all the criteria (morphology, gene expression, differentiation potential) suggesting in vitro totipotency including a stable diploid karyotype. Alternative protocols using feeder layer cells are also currently tested, but the major challenge is now the demonstration of in vivo totipotency, through cell and nuclear transplantation. The methodological developments in the project should permit in part to undertake large scale insertional strategies, aimed at discovering novel developmental genes and cloning them easier than after chemical mutagenesis. This genetic approach is so far replaced in the project by a major effort to isolate genes differentially expressed during salmon early development, by differential hybridization and DDRT-PCR. A number of clones issued from these methods are currently characterized.
MAJOR SCIENTIFIC BREAKTHROUGHS:
Detailed functional study of original genes related to TGFbeta and EGF receptor demonstrating their implication in neural development. Isolation and functional studies of other genes of the same and other families of genes involved in CNS development. Development of long-term cultures of embryonic cells keeping morphology, differentiation and karyotype features similar to those of mouse ES cell lines. Large-scale isolation of genes differentially expressed during salmon early development.
Funding SchemeCSC - Cost-sharing contracts