The hermaphrodite distal tip cells (DTCs) lead through their migration the extension of the hermaphrodite gonad arms. The DTCs undergo a 3-phase pattern of migration sequentially moving along the AP, DV and AP axes. The second migration phase, from the ventral to the dorsal side, requires the UNC-6/Netrin guidance cue and its receptors UNC-5 and UNC-40 (1). UNC-5, which is transcriptionally induced at the L3/L4 transition, is both necessary and sufficient to cause ventral to dorsal migration.
emb-9 promoter-driven UNC-5 in the DTCs causes a precocious ventral to dorsal migration (2). Analysis of the
emb-9::
unc-5 strain led to two interesting observations. First, the
emb-9::
unc-5 induced precocious migration was partially dependent upon the endogenous
unc-5 suggesting that
unc-5, albeit undetected, is probably expressed and tightly regulated during the L3 stage. In addition, it was also suggested that some putative unknown negative regulation is relieved at the time of the turn (2). This prompted us to conduct modifier screens for both suppression and enhancement of this UNC-5 induced precocious migration phenotype. We believe that those screens will identify molecules that work upstream of
unc-5 to regulate its function or molecules that act downstream of
unc-5 to mediate its action. In addition, since the precocious migration is occurring prematurely under unfavorable conditions, it is reasonable to assume that it would be very sensitive to any changes with respect to the ability of the DTC to detach from the ventral side and reorient dorsally. This sensitivity, we believe, will allow us to identify molecules that participate in this process, but may act redundantly or have no marked phenotype when mutated on their own. As a starting strain for the screen we generated a strain that carries
emb-9::
unc-5 along with
emb-9::gfp (evIs129). This strain has higher levels of the transgene compared to the original
emb-9::
unc-5 and results in a more complex phenotype. The DTCs undergo precocious migrations at a higher penetrance (~90%) and in addition, once the DTCs reach the dorsal side, instead of reorienting towards the midbody, they migrate towards either the head or the tail. This sometimes involves extra turns of the DTC. This third phase migratory defect suggests that similar to the situation on the ventral side, in which
unc-5 must be tightly regulated for the normal process to occur, here the reduction in
unc-5 levels which occur as the DTC approach the dorsal muscle band (2) is necessary for the normal execution of the 3rd phase of migration. Apart from being informative, this phenotype allows the classification of the different suppressors with respect to their ability to suppress the 2nd or the 3rd or both migratory defects. We have conducted a clonal screen looking for mutations that would suppress
emb-9::
unc-5 induced precocious migration. Out ~1000 genomes 5 suppressors were identified. All 5 are recessive and suppress the precocious migration from 80-90% to about 15-20%. (
ev768)- has no phenotype on its own and suppresses the precocious migration as well as the 3rd phase migration defect. (
ev767)- suppresses the precocious migration but, once the DTC approach the midbody, they turn and migrate away from it. (
ev764)- suppress the precocious migration, but after the initiation of the 3rd migration phase the distal arm is frequently detached from the dorsal muscle band. This mutant might shed light on the cross talk between the migratory cell and the substratum it migrates upon. (
ev765)- has a short gonad in 90% of the population on its own, as well as in the presence of evIs129. The precocious migration is probably not suppressed in this mutant but the DTC do migrate to the midbody. We are in the process of mapping these mutants by using both genetic and snip-snip mapping. We are also generating a balanced evIs129 heterozygote strain that would serve as the starting strain for an enhancer screen.