Migrations of cells and growth cones along the dorsoventral and anteroposterior body axes establish the pattern of the C. elegans nervous system. We are undertaking several screens for axon guidance mutants to identify genes needed specifically for longitudinal axon growth as well as genes required generally for all axon extension and pathfinding. PVQ is a pair of bilaterally symmetric neurons located in the tail and each extends a single axon that enters the ventral nerve cord and continues to the nerve ring in the head. PVQ can be visualized in living animals using a
sra-6::gfp transgenic strain and fluorescence microscopy. To identify mutants defective in the growth of the PVQ axons, we treated
sra-6::gfp animals with either the mutagen EMS or ENU, isolated behavioral and morphological mutants in the F2 generation, and then examined their progeny for axon growth defects. To date, we have recovered over ninety mutants with axon outgrowth and pathfinding defects. While some mutations cause the PVQ axons to terminate their growth prior to reaching the nerve ring, most cause both axon outgrowth and pathfinding defects, such as the inappropriate extension along the dorsal nerve cord and lateral midline or across the ventral midline. We found over thirty mutants with defects in either cell fate, cell migration, cell position or programmed cell death. We also performed a screen to identify genes needed for the growth of the AVA, AVB and AVD axons. The roughly 150 mutations found in these two screens define over forty genes, including around twenty-five previously known genes. We have begun the molecular analyses of several new genes discovered in our screens (see below and abstract by Yam & Clark).
zag-1 mutations cause axons to defasciculate and branch inappropriately as well as neurons to extend ectopic axons. Both
zag-1 alleles are nonsense mutations, suggesting that they greatly reduce or eliminate
zag-1 function. We cloned
zag-1 and found that it encodes a protein containing multiple zinc finger domains and a homeodomain (F28F9.1). ZAG-1 is similar in sequence and structure to the products of the Drosophila
zfh-1 gene, which is expressed in mesoderm and the CNS and functions in mesoderm differentiation, and the vertebrate ZEB genes. Like these proteins, ZAG-1 likely acts as a transcriptional repressor, as it possesses a conserved sequence motif needed for association with the CtBP corepressor protein. A transcriptional
zag-1::gfp transgene is expressed in both neuronal and nonneuronal cells.
zag-1::gfp expression is observed in ventral cord neurons in a
zag-1 mutant yet not in wild type, indicating that
zag-1 regulates, either directly or indirectly, its own expression in some neurons. Our results suggest that
zag-1 regulates the expression of genes involved in axon formation and guidance. We are currently investigating how
zag-1 activity is regulated as well as the genes that act downstream of
zag-1 .