Worm Breeder's Gazette 14(4): 32 (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.
Dept. of Molecular Biology, Princeton University, Princeton, NJ 08544
SPARC is a calcium binding, extracellular matrix protein found in the basement membranes and bones of multicellular organisms. In nematodes, SPARC is expressed by the body wall and sex muscle cells and is also found in the germline. Over-expression of wild type SPARC in N2 animals gives an Unc phenotype with a protruding vulva, other body deformities, paralysis, and sterility (Schwarzbauer and Spencer (1993) Mol. Biol. Cell 4, 941). To study the functions of SPARC in transgenic nematodes, we decided to regulate SPARC expression by putting the gene under the control of the hsp16-1/48 heat shock promoter which reportedly shows expression predominantly in body muscle and hypodermis (Stringham et al. (1992) Mol. Biol. Cell 3, 221). A 349 bp segment containing the 1.48 intergenic region plus the transcriptional initiation site for hsp16-48 was fused to the SPARC gene (ost-1) within the 5' untranslated region. The resulting plasmid contains all introns and exons of the SPARC gene plus an additional 800 base pairs of 3' flanking sequence. This plasmid (pHS-SPC) was used to establish several transgenic lines by coinjection with the rol-6 dominant marker. One integrated line N2;Ex[hsp-1/48::ost-1;rol-6(d)] was used to assess the effects of SPARC induction at different larval stages. For most experiments, animals were incubated at 33 degrees C. for 2 hours and examined microscopically through subsequent larval stages and adulthood. Induction of SPARC expression by heat shock during late larval stages (L3 and L4) gave variable results and a number of different phenotypes including vulval protrusions, paralysis, death, deformed embryos, and intermittent rolling. Heat shock of earlier L1-L2 larvae gave a more limited repertoire of phenotypes. 42% of the animals developed a Dpy phenotype as determined by visual inspection. This could be quantified by measuring the diameter at the mid-section of adult hermaphrodites and showed a clear difference in arbitrary units of 70 +/- 3 versus 60 +/- 3 for plus and minus heat shock, respectively. The most dramatic phenotype was the complete absence of any rolling. While all of the N2;Ex[hsp-1/48::ost-1;rol-6(d)] transgenic animals developed the Rol phenotype when grown at room temperature, very few of the animals subjected to one 2 hour heat shock were rollers. Of the animals tested so far, 90% showed no evidence of rolling from the time of heating through adulthood. Heat shock treatment did not affect the development of the Rol phenotype in control N2;Ex[rol-6(d)] nematodes. These results suggest that induction of SPARC expression in early larval stages can affect the assembly or function of the cuticle. It is possible that SPARC is ectopically expressed in the hypodermis and interacts with cuticle collagens or affects the hypodermal cells as they are assembling the cuticle. In this way, it might either prevent inclusion of the Rol protein or correct the structural defects that cause rolling. It is also possible that over-expression by muscle cells at early stages can affect the structure of the hypodermis.