The
unc-52 gene encodes components of the body-wall muscle extracellular matrix that are essential and required for proper myofilament lattice assembly during embryogenesis (Rogalski et al., 1993, Genes and Development 7:1471). Studies using antibodies that recognize different regions of UNC-52 have indicated that the UNC-52 proteins found in the basement membrane between muscle and hypodermis in early embryos are produced by muscle (most recently Moerman et al., 1996, Dev. Biol. 173:228 and Mullen et al., 1999, Mol. Biol. Cell 10:3205). These studies demonstrated that faint intracellular staining of UNC-52 could be seen in the body-wall muscle cells (but not hypodermal cells) of early morphogenesis stage embryos. We have been focusing on
mec-8 , a gene that encodes a probable RNA binding protein that may interact directly with
unc-52 pre-mRNA. Our studies of MEC-8 have led us to hypothesize that a significant amount of UNC-52 protein is produced by the hypodermis. Loss-of-function mutations in
mec-8 enhance the phenotypes of hypomorphic mutations in
unc-52 found in alternatively spliced exons. It was shown that
mec-8 function is required to generate
unc-52 mRNA isoforms that skip these exons (Lundquist et al., 1996, Development 122:1601). We recently found that expression of MEC-8 by the main body hypodermis, but not by body-wall muscles or their precursors, is sufficient to rescue the genetic interaction between
mec-8 and
unc-52 , and that high levels of MEC-8 in the hypodermis can delay or completely suppress the onset of paralysis conferred by the hypomorphic
unc-52(
e669) allele. These experiments were performed by expressing a
mec-8 cDNA under the control of either
lin-26 (main body hypodermis) or
hlh-1 (body-wall muscle) regulatory elements (generous gifts from M. Labouesse and A. Fire, respectively); antibody staining of embryos indicated that MEC-8 was expressed appropriately in each case. We previously reported making a minigene containing the alternatively spliced region of
unc-52(
e444) translationally fused to gfp and driven by a muscle-specific promoter,
myo-3 (Davies et al., 1997, IWM abstract 116).
unc-52(
e444) is a nonsense mutation in an alternatively spliced exon that must be removed by MEC-8-promoted alternative splicing for expression of GFP. The production of GFP in morphogenesis stage embryos containing this minigene was diminished by
mec-8 mutation. We have found that expression of MEC-8 by the body-wall muscles and their precursors, but not by the main body hypodermis, results in extremely strong activation of this minigene in morphogenesis stage embryos. This result strongly suggests that overexpression of MEC-8 protein results in enhanced
unc-52 exon-skipping, but only when these two gene products are present in the same cell. Since MEC-8 is able to rescue
unc-52(
e669) when it is made by the hypodermis but not when it is made by muscle we infer that a significant amount of UNC-52 must be produced by the hypodermis. We are currently performing mosaic analysis on
unc-52 to test this hypothesis.