Worm Breeder's Gazette 14(2): 41 (February 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.
Department of MCDB, University of Colorado, Boulder
We have reported previously on the cloning, molecular characterization, expression, and ectopic overexpression of a C. elegans TGF-beta homologue (previously called ceg-1: WBG 13(4):65, 1994; 1995 Meeting Abstracts: 555), which we have now named dbl-1 (dpp-BMP2,4-like). We have continued analysis of dbl-1 expression, using a transgenic him-8 line isolated after coinjection of two dbl-1::GFP constructs containing 4.4 kb of upstream sequence and differing only in the presence or absence of a nuclear localization signal. The expression of both reporters allows us to identify cells both by nuclear position and by cell body and axonal morphology. Fluorescing cells in adults include the anal depressor muscle, neurons of the retro- vesicular ganglion, and VNC motor neurons including those of the pre-anal ganglion and the PDA motor neuron. Male-specific expression is seen in the CP neurons of the VNC, in cells of the dorsal-rectal ganglion, and in two spicule associated neurons of adults, as well as transiently in several still unidentified tail cells of L3-L4 larvae. We have also continued to analyze effects of ectopic dbl-1 overexpression in transgenic lines carrying a dbl-1 cDNA construct (hsp::dbl-1) made with the heat-shock promoter plasmid pPD49.78, which induces well in neuronal and hypodermal cells (A. Fire, personal communication). Since both the dbl-1 expression pattern and existing evidence (Savage et al., 1995 Meeting Abstracts: 56) suggest a role for signaling mediated by a TGF- beta homologue during ray and spicule formation, we examined the effects of dbl-1 overexpression on ray morphogenesis. Heat shocks of hsp::dbl-1 transgenic him-8 L3 males result in a variety of ray transformations, the most common of which (50% penetrance) is transformation of ray 4 to ray 3 with 4-3 fusion, a phenotype also associated with decreased mab-5 activity (Chow and Emmons, Development 120:2579, 1994). Other phenotypes seen at lower frequencies include extra V-rays and multiple-ray fusions. These results suggest a role for dbl-1 in the specification of ray identities. We have refined the genetic map position of dbl-1 on LGV by using PCR to assay for dbl-1 sequences in DNA from inviable embryos homozygous for each of several mapped deficiencies in the region. The results locate dbl-1 in a small region of <.5 map unit (uncovered by nDf32 but not by nDf18) which includes unc-70 and four other genes defined by lethal mutations (Johnsen and Baillie, Genetics 129:735, 1991). To determine whether any of these genes could be dbl-1, we have done DNA coinjection rescue experiments with a rol-6 marker plasmid and cosmid ZC421, which contains the dbl-1 coding region and approximately 20kb of upstream sequence. We have so far injected homozygous unc-70 hermaphrodites as well as heterozygous hermaphrodites from balanced strains carrying lethal mutations representing three of the four essential genes. None of these mutants was rescued. We have also undertaken PCR screening of a high-copy-number Tc1 strain (Andachi and Kohara, 1995 Meeting Abstracts: 96) in attempts to identify nearby Tc1 insertions that would allow generation of a dbl-1 allele.