MUTATIONS AFFECTING MIDLINE SIGNALING IN THE ZEBRAFISH

Signals from the vertebrate midline have for a long time been implicated in patterning the paraxial mesoderm and the ventral neural tube. In the zebrafish, a number of mutations affecting the midline have been described. Among these are mutations affecting formation of the notochord, mutations affecting somite patterning and mutations affecting the ventral neural tube.

Mutations in six genes affect the formation of the horizontal myoseptum, which in the wildtype separates the somites into a ventral and a dorsal part. This class of mutants, termed the you-type mutants, forms U-shaped instead of V-shaped somites. Earlier in development,
these mutants are affected in  the adaxial cells and the muscle pioneers. The similarity of the somite phenotype in mutants lacking the notochord and in the you-type mutants suggests that the you-type genes are involved in a signaling pathway from the notochord involved in patterning the somites.

Sonic hedgehog (Shh) was identified as an important signaling molecule at the midline involved in patterning of the paraxial mesoderm and the CNS. Three hedgehog homologues, including shh, tiggy-winkle hedgehog (twhh) and echidna hedgehog (ehh), are known to be expressed in the zebrafish midline. We are currently investigating the possibility that some of the you-type genes encode members of the hedgehog gene family, using fine structure mapping as well as sequencing approaches. We are also testing zebrafish homologues of other genes involved in hedgehog signaling as candidates for mutations affecting midline signaling.

Shh has also been implicated in the induction of left-right asymmetry of the heart. By screening the zebrafish stock collection, we identified several mutations with defects in left-right asymmetry of the heart. In addition, some of the genes affected by these mutations show phenotypes affecting the midline. We are currently analyzing the midline of these mutants using whole mount in situ hybridization and antibody labeling techniques. We are also identifying molecular markers that are closely linked to these mutations with the aim of
identifying the affected genes by positional cloning.

GENETIC MAPPING OF MUTATIONS IN THE ZEBRAFISH

A large number of developmental mutants of the zebrafish have been isolated in a recent mutagenesis screen (Haffter et al., 1996a; Haffter et al., 1996b). Our aim is to place 665 of these mutations on a simple sequence length polymorphism map as a first step towards molecular analysis. For our project we have selected 182 SSLP markers (Knapik, E. W. et al. (1996) Development 123, 451-60; Goff, D. J. et al. (1992) Genomics 14, 200-2) that give strong and easily distinguishable PCR products when amplified from the Tü line, in which the mutagenesis was done, and from the newly established reference line WIK (available from the Tübingen stockcenter).

We cross Tü fish carrying each of the mutations with WIK fish and collect up to 2000 mutant F2 embryos and their wildtype siblings. SSLPs are amplified from pools of 48 mutants and siblings, and separated on agarose gels. A difference in band intensity between the mutant and sibling pools indicates a potential linkage to the SSLP in question, which is confirmed and refined by genotyping single embryos. We find a significant linkage to at least one marker in approximately 90 % of the mutations tested. We are planning to make these data available to the community on the Internet.

Our lab pursues different strategies to further analyze some of the mapped mutations, with a particular focus on those affecting the midline:

(a) Matching with previously cloned candidate genes. We have created a set of Southern blots of reference fish that allow us to place selected candidate genes on the SSLP map through restriction fragment length polymorphisms (RFLPs). We are very interested in collaborating
with researchers who would like to share candidate gene data with us. Additional map positions are obtained through a collaboration with the laboratory of J. Postlethwait.

(b) Positional cloning, which is currently feasible only for a few mutations due to the limited density of the map. We were able to identify markers closely linked to iguana and detour, two mutations that affect midline signalling, and are performing genomic walks of these regions.

(c) Generation of additional closely linked markers by the AFLP technique, which is greatly facilitated by preexisting map information. We are currently following this approach for several
mutations.