UNC-6 is a laminin related protein required for circumferential migrations of pioneering axons and mesodermal cells in C. elegans. Vertebrate homologs, the netrins, have been proposed to act as chemoattractants and chemorepellants for migrating axons in the embryonic nervous system. UNC-6 is predicted to be a secreted protein with five structural domains. The N-terminal domains VI and V (V-1,V-2,V-3) are homologous to laminin-g except small region of domain VI and domain V-2 that are homologous to laminin-b. The C-terminal, C domain, is unique. The homology of domain VI to laminin suggests that UNC-6 interacts with the basement membrane through this domain. Molecular analysis of
unc-6 alleles suggests that specific functions are coded by different domains. For example, alleles that delete specifically domain V-2 affect only dorsal migrations whereas an allele carrying a mutation in domain VI selectively disrupts ventral migrations. Another allele that has a different mutation in domain VI disrupts mesodermal but not axonal migrations. We have suggested that the V-2 domain mediates binding to the UNC-5 receptor for dorsal migrations and that mutations in domain VI affects the display of UNC-6 on the face of the basement membrane. Our goal is to study structure-function relationships of UNC-6. Since several EGF-like repeats of laminin have been proposed to form binding sites for cellular receptors, the
unc-6 domains V-1, V-2, V-3 might be the ligands for receptors on growth cones and migrating cells. Therefore, we have constructed synthetic alleles of
unc-6 containing deletions of EGF-like repeats. We have also constructed an allele that has the V-2 domain replaced by domain V-3 to test whether domain V-3 can functionally substitute for domainV-2. If so, it may indicate that the spacing between EGF-like repeats is critical and not the sequence. We have constructed an allele with a deletion of C domain to examine its effect on axonal and mesodermal migrations. The C domain is rich in basic amino acids and may tether netrins to other molecules for display on the cell surfaces or it may bind to unique matrix sites. We will also test whether a distinct function is associated with the unique and highly conserved laminin-b like region of domain VI. For these experiments, we have designed an assay system that provides a strong selection for loss of function alleles. Our screen first measures the ability to rescue a select phenotype, either movement (dorsal guidance function) or egg laying (ventral guidance function). Further analysis details if other functions of
unc-6 are affected. This study may discover new functions associated with specific domains of
unc-6. Since netrins are highly conserved from nematodes to chordates and contain structures found in other extracellular matrix proteins, this analysis will provide insights into how such protein domains interact with their cellular receptors and the extracellular matrix.