Correct wiring of the nervous system requires that in the developing embryo each axon finds its proper position and interaction partners. To identify new genes involved in axonal guidance and fasciculation a genetic screen was performed. Mutagenized worms expressing GFP in interneurons under the control of the
glr-1 promoter were scored for fasciculation defects in the ventral cord. One of the mutations recovered was
rh315. It mapped to chromosome IV between
unc-17 and an SNP-marker on cosmid W03D2. Rescue of the mutant phenotype was obtained with both of the overlapping cosmids T08C8 and F28F9 and with a 10.4 kB PCR fragment containing the predicted open reading frame F28F9.1. This gene encodes a transcription factor with a central homeodomain and two clusters of C2H2-type zincfingers (ZF) at either end (zag = zinc finger involved in axon guidance). Sequencing of the mutant revealed a stop codon in exon five which deletes the C-terminal ZFs. Sequencing of 6 cDNA clones from Y. Koharas lab revealed no alternative splice forms. Zag-1 has homologs in D. melanogaster (
zfh-1), and vertebrates (eg. EF1 in mouse). Common to all are N- and C- terminal ZF clusters, a homeobox and a corepressor binding site, while the number of ZFs within the clusters is not fully conserved. In Drosophila
zfh-1 is expressed in muscle precursors and neurons during embryogenesis, mutants have subtle muscle defects. The mouse homolog has a similar expression pattern, with additional expression in tissues not present in invertebrates. Knock-out mutants have defects in thymus development and a variety of skeletal defects. Zag-1 expression in C. elegans was analyzed using two different GFP reporter constucts: a putative
zag-1 promoter (4.5kb upstream sequence) fusion and a translational fusion containing the same promoter plus the entire coding region. Transgenic lines carrying the integrated promoter construct express GFP in a number of head neurons, and in intestinal and anal depressor muscles from late embryo to adult stages. Expression of the ZAG-1::GFP fusion protein however, is very dynamic, starting at the comma stage in many neurons of head and tail. It is shut down in most neurons at hatching, but in late L1 it appears in the differentiating motorneurons of the ventral cord. Worms carrying the mutation
zag-1(
rh315) are phenotypically Unc. They are fairly active but tend to curl and change direction of movement quite often. Axon guidance defects can be detected with a variety of neuronal GFP markers. Defasciculation, i.e. crossing of ventral cord axons from right to left side is observed in some mutant worms. Motorneuron commissures sometimes fail to reach the dorsal cord and run laterally. In addition, one to several interneuron axons can be seen running dorsally and/or laterally. Another aspect of the zag- 1
(rh315) mutant phenotype is misexpression of neuronal markers. Some motorneuron markers are not expressed in all the cells they should be. Others are expressed in more cells than in the wildtype, eg.
glr-1::GFP expression in PDB or in some cell(s) of the PDE cluster. Using various muscle GFP markers muscle defects in mutant animals could not be detected. The dynamic expression of
zag-1 at times when neurons differentiate and its effects on the expression of other neuronal markers indicate that it regulates late steps in neuronal differentiation.