The Zebrafish Science Monitor Vol 3(4)
ECTODERMAL PATTERNING AND NEUROGENESIS IN THE ZEBRAFISHC. Sagerstrom, Y. Grinblat and H. Sive. Whitehead Institute, Cambridge, MA.
We are exploring the mechanisms directing ectodermal patterning and neurogenesis in the zebrafish. To gain access to these events we have devised an assay that permits the culture of presumptive ectoderm from blastula stage embryos in vitro. The explants consist of 30-50 cells and, when cultured in aggregates of ten to fifteen explants, they survive for at least 48 hrs in a balanced salt solution. The explants undergo extensive cell division during the culture period, as evidenced by a ten-fold increase in total cell number. This system is now being used to study cell interactions and signals required for induction of the ectoderm. Initially, wholemount in situ hybridization and quantitative PCR with a battery of molecular markers was used to assay the final fate of ectoderm explanted from blastula stage embryos. Neural marker expression was undetectable by in situ hybridization, but the PCR assay detects low levels of two markers (pax6 and NCAM), while four others (Zash A, Zash B, eng3 and wnt1) are essentially undetectable by PCR. Taken together, this suggests that the ectoderm has not yet received inductive signals to differentiate as neural tissue. The explants do differentiate however, as they go on to express high levels of a non-neural cytokeratin, cyt1, that we recently cloned. The explants also express only low levels of five different mesodermal markers, arguing that they are essentially free of contaminating mesoderm. Current studies are aimed at studying neural induction in these explants by culturing them in contact with putative inducing tissues (e.g. gastrula stage mesoderm) as well as by co-culture with defined factors.
Acknowledgement: We are grateful to our colleagues Greg Conway, Anders Fjose, David Grunwald, Christiane Nüsslein-Volhardt, Robert Riggleman and Eric Weinberg, for generously providing cDNA clones and sequences.
Zebrafish Science Monitor Vol 3(4)
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