In screens for epidermal morphogenesis mutants, we identified a mutation,
ju71, that affects one of the C. elegans intermediate filament (IF) genes,
ifb-1 (Woo and Chisholm. 2001 Intl. worm meeting).
ifb-1(
ju71) mutants display abnormal head epidermal morphology and 50% embryonic and larval lethality. The mutant embryos arrest at the three-fold stage of embryonic elongation and eventually rupture at the head epidermis.
ifb-1 encodes two isoforms, differing at the N-termini due to alternative first exons. The lesion in
ju71is a 617 bp deletion whose 3 breakpoint lies 250 bp upstream from the predicted IFB-1S (short isoform) initiator ATG. A genomic clone encoding only IFB-1S fully rescues
ifb-1(
ju71) mutants, suggesting that the
ju71 mutation specifically affects the short isoform. Consistent with these results, RNAi of both
ifb-1 isoforms resulted in 100% lethality (also reported by Karabinos, et. al. Proc. Natl. Acad. Sci. USA 98: 7863-8). The
ifb-1(RNAi) animals either arrested at the two-fold stage during embryonic elongation with lumpy epidermal morphology, or as paralyzed larvae. We are currently examining the individual functions of the two
ifb-1 isoforms using isoform-specific RNAi and transgene analysis. We are also performing Northern and Western blot analysis to determine the effect of the
ju71mutation on transcript and protein levels. A functional IFB-1S::GFP localizes to trans-epidermal attachments (TEAs, a.k.a. fibrous organelles) in embryos and larvae. To characterize the role of IFB-1 in TEAs, we are examining the expression patterns of other components of TEAs in
ifb-1(
ju71) mutants, and also the localization of IFB-1S::GFP in other embryonic elongation mutants. Since
mup-4 mutants display defects in embryonic morphogenesis overlapping with those of
ifb-1,and the intracellular domain of MUP-4 is homologous to an IF-associated protein (Hong et. al. J. Cell. Biol. 154: 403-414), we are testing whether
ifb-1 and
mup-4function in the same pathway in embryonic morphogenesis. The lumpy two-fold arrested phenotype of
ifb-1 RNAi embryos also resembles that of other mutants with defects in embryonic trans-epidermal attachments such
vab10, which encodes the conserved hemidesmosome component Plectin (Bosher et al., 2001 Intl. Worm Meeting) and
vab19, which encodes a novel conserved protein (Ding and Chisholm, 2001 Intl. worm meeting). To identify new genes functioning in IF-mediated epidermal morphogenesis we performed a semi-clonal screen for new elongation-arrested lumpy mutants (see abstract of Ding et. al., this meeting). We are currently characterizing these mutants.