MUTATIONS AFFECTING THE FORMATION OF THE NOTOCHORD IN THE ZEBRAFISH, DANIO-RERIO

In a large scale screen for mutants with defects in the embryonic deve lopment of the zebrafish we identified mutations in four genes, floati ng head (flh), memo (mom), no tail (ntl), and dec, that are required f or early notochord formation. Mutations in flh and ntl have been descr ibed previously, while mom and doe are newly identified genes. Mutant mom embryos lack a notochord in the trunk, and trunk somites from the right and left side of the embryo fuse underneath the neural tube. In this respect morn appears similar to flh. In contrast, notochord precu rsor cells are present in both ntl and doc embryos. In order to gain a greater understanding of the phenotypes, we have analysed the express ion of several axial mesoderm markers in mutant embryos of all four ge nes. In flh and mom, Ntl expression is normal in the germ ring and tai lbud, while the expression of Nd and other notochord markers in the ax ial mesodermal region is disrupted. Nd expression is normal in doc emb ryos until early semitic stages, when there is a reduction in expressi on which is first seen in anterior regions of the embryo. This suggest s a function for doc in the maintenance of ntl expression. Other notoc hord markers such as twist, sonic hedgehog and axial are not expressed in the axial mesoderm of ntl embryos, their expression parallels the expression of ntl in the axial mesoderm of mutant doc,flh and mom embr yos, indicating that ntl is required for the expression of these marke rs. The role of doc in the expression of the notochord markers appears indirect via ntl. Floor plate formation is disrupted in most regions in flh and mom mutant embryos but is present in mutant ntl and doc emb ryos. In mutant embryos with strong ntl alleles the band of cells expr essing floor plate markers is broadened. A similar broadening is also observed in the axial mesoderm underlying the floor plate of ntl embry os, suggesting a direct involvement of the notochord precursor cells i n floor plate induction. Mutations in al of these four genes result in embryos lacking a horizontal myoseptum and muscle pioneer cells, both of which are thought to be induced by the notochord. These somite def ects can be traced back to an impairment of the specification of the a daxial cells during early stages of development. Transplantation of wi ld-type cells into mutant doc embryos reveals that wild-type notochord cells are sufficient to induce horizontal myoseptum formation in the flanking mutant tissue. Thus dec, like flh and ntl, acts cell autonomo usly in the notochord. In addition to the four mutants with defects in early notochord formation, we have isolated 84 mutants, defining at l east 15 genes, with defects in later stages of notochord development. These are listed in an appendix to this study.