Worm Breeder's Gazette 14(4): 54 (October 1, 1996)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
|1||Department of Cell Biology, Yale University, New Haven, CT 06520|
|2||Department of Genetics, Yale University, New Haven, CT 06520|
Mutations in egl-15 and egl-17 can confer severely posteriorly displaced sex myoblasts (SMs) and an egg-laying defective phenotype (Egl). egl-15 encodes a fibroblast growth factor receptor (FGFR) homologue (1) and egl-17 encodes a fibroblast growth factor (FGF) that we propose acts as a specific ligand for the EGL-15 FGFR during SM migration (2). To understand the role of this signalling event in controlling SM migration guidance, we wished to determine the relevant sites of egl-15 and egl-17 expression. We began investigating where egl-17 is expressed by creating reporter constructs in which the egl-17 promoter drives the expression of GFP or b-galactosidase products that contain nuclear localization signals (NLS). The Fire Lab Vector Kit was used as a source for the GFP and lacZ sequences. Constructs were introduced by germ-line transformation and expression patterns were observed in stable lines. During larval stages, transformed progeny show GFP or lacZ staining in two places: a head neuron tentatively identified as M4, and in P6.p and its descendants. Expression in the neuron can be seen soon after hatching. Expression in P6.p can be seen at the single cell stage in some animals and becomes brightest after the first division of P.6p. Expression persists in P6.p descendants through L4 and then fades in the young adult. The staining pattern observed is independent of the co-transformation marker used. An additional construct was made consisting of the full length coding region of egl-17 fused to the coding region of GFP. This construct is capable of rescuing the Egl and SM defects associated with egl-17. The GFP expression seen in these transgenic worms is consistent with that observed with the constructs above but appears to be at the cell periphery of the neuron and P6.p descendants suggesting the resulting protein is secreted but some portion is retained at the cell membrane. We used a spontaneously integrated egl17::NLS-GFP array (ayIs4) to determine if the expression observed in P6.p and its descendants was intrinsic to P6.p or characteristic of primary vulval cell fates. Mutations in lin-39(n1490) and let-23(sy1) result in too few primary cell fates and a vulvaless phenotype. In ayIs4; lin-39 and ayIs4; let-23 doubles, no GFP expression in P6.p was observed although expression could still be observed in the head neuron. By contrast, ayIs4 doubles with lin-15(n309) or lin-31(n301) in which extra primary cell fates occur, GFP expression was observed in multiple vulval precursor cells (VPCs) in the L3 stage and in ectopic pseudovulvae. These results suggest that the expression of GFP from the egl-17 promoter in P6.p and its descendants is dependent upon P6.p induction to a primary vulval cell fate. The SMs normally migrate anteriorly to final positions that flank the anchor cell in the precise center of the developing gonad. A gonad-dependent signal is responsible for this precise positioning. The anchor cell is responsible for inducing P6.p to its primary cell fate allowing the expression of egl-17 reporter constructs in P6.p. These results are consistent with the idea that expression of EGL-17 in P6.p may be providing positional cues to the SMs that are gonad-dependent. However, additional data show that expression of egl-17 in primary cells is not necessary for proper SM migration. In worms in which all six VPCs have been ablated and no primary cell can be induced, SMs migrate to flank the anchor cell at the center of the gonad. Also, Z1.pp-/Z4.aa- (ac-) animals have precise SM positioning but no primary cell fates (3). In lin-39 Vul mutants where no primary cell is generated and no GFP expression from ayIs4 is observed in the VPCs, SMs again migrate to their precise final positions. SM positions in these animals are dramatically different from those in egl-17 mutants which have posteriorly displaced SMs. Since the loss of the primary cell does not mimic the effects of loss of egl-17, we conclude that expression of egl-17 in P6.p and its descendants is not necessary for proper SM migration and that egl-17 must be expressed elsewhere. We are currently conducting mosaic analysis for egl-15 and egl-17 to determine where expression of these genes is necessary for proper SM migration. 1. DeVore, D.L., Horvitz, H.R., and Stern, M.J. (1995) Cell 83, 611-620. 2. Burdine, R.D. and Stern, M.J. (1996) WBG 14(3):28. 3. Thomas, J.H., Stern, M.J., and Horvitz, H.R. (1990) Cell 62, 1041-1052.