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.

Ectopic Expression of the Extracellular Matrix Protein SPARC during Early Larval Stages can Suppress the Rol Phenotype

Jean E. Schwarzbauer

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.