Worm Breeder's Gazette 12(2): 67 (January 1, 1992)
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.
We are trying to understand how the her-1 gene specifies male sexual fates. This gene has two XO-specific transcripts; a rare 1.2 kb mRNA appears to encode a small secreted polypeptide with an N-terminal signal sequence that should allow its secretion, while a relatively abundant 0.8 kb mRNA could encode the C-terminal half of the same polypeptide. To determine if either of these mRNAs can supply her-1 function we inserted their cDNAs into plasmids ( pPD30 .38)that carry an enhancer and promoter from the major body wall muscle myosin gene, unc-54 (Fire, pers. comm.). Expression from the plasmid should be restricted to non-pharyngeal muscle cells and should start about 5 hrs. post-fertilization, prior to embryonic twitching (i.e., during the her-1 TSP).
Plasmid DNA was mixed with pRF4 [ rol-6 ( su1006 )](Mello, et al., 1991. EMBO J.10:3959.), injected into the distal gonad of N2 XX hermaphrodites, and F1 Rol progeny were scored for sexual phenotype. The construct carrying the larger cDNA with the putative secretion signal sequence dramatically masculinized these animals. Phenotypes ranged from Egl hermaphrodites-some with Tra tails, to intersexual animals (more male tail structures and abnormal gonads), to pseudo-males (unilobed gonads producing sperm, no pseudocoelomic yolk protein), showing that expression in muscle can non-cell-autonomously transform sexual fates of dimorphic lineages derived from AB, E, MS, and P4 .We could not establish heritable lines with this construct due to sterility and poor transmittance. The small abundant her-1 cDNA did not overtly affect sexual phenotype in this assay. However, since body wall muscle is not dimorphic, any cell-autonomous sexual transformations would be difficult to score ( unc-54 is expressed in sex muscles during differentiation, but we assume that her-1 made in those cells may be too late to affect their fate). Therefore, we also fused the her-1 cDNAs to an HSP16 promoter (on pPD49 .78and pPD49 .83).These lines were subjected to several heat shock regimens (Stringham and Candido, pers. comm.), confirming our initial results: the 1.2 kb cDNA overtly transforms XX animals while the 0.8 kb cDNA has no effect (yet).
To assess the tissue specificity of the muscle constructs, and test if the her-1 signal sequence could direct secretion of a heterologous protein, we used the unc-54 enhancer+promoter to drive expression of a her-1 -lacZfusion gene containing a second, internal, trans-membrane domain (derived from pPD34 .110(Fire et al., 1990. Gene 93:189)). This should anchor ß-galactosidase to the cytoplasmic faces of the ER, golgi, and plasma membrane. Indeed, lines bearing this construct show strong lacZ staining of perinuclear regions in body wall muscle cells only (the muscle actin bundles were stained with rhodamine-conjugated phalloidin).
Our experiments provide molecular evidence supporting genetic models for cell signaling interactions during sex determination in C. elegans (WBG v11 , n4 , p86 ;and, VIIIth International mtg. on C. elegans, 1991, Hunter; Kuwabara; Perry), and show that a her-1 product from the larger transcript must be secreted.