Figure 1.
alfa-1 Mutations Cause a Defect in Yolk Storage or Transport in the C. elegans Embryo (A) Genomic organization of
alfa-1. Two alleles of
alfa-1,
gk498021 and
ok3062, are both likely to be null.
gk498021 is a G-to-A transition leading to a tryptophanto-opal stop codon at amino acid 54, and
ok3062 is a partial deletion of exons 3 and 4. Black bar, sequence deleted in the
ok3062 mutant.(B) Differential interference contrast (DIC) and confocal images of wild-type,
alfa-1(
ok3062), and
alfa-1(
gk498021) comma-stage (top) and 3-fold (bottom) embryos containing the yolk protein transgene pwIs98[
vit-2::tdimer2] or nIs755[
vit-2::bfp,
vit-2::pHluorin], respectively. Arrowheads, blobs (abnormal yolk presencein extra-embryonic fluid). Scale bars, 10 um.(C) Confocal images of comma-stage embryos of the indicated genotypes containing the transgene nIs755[
vit-2::bfp,
vit-2::pHluorin]. Mutation of
rme-2, theoocyte's yolk receptor gene, suppresses yolk uptake and the blob phenotype. Scale bar, 10 um. Animals also contained the reporter qxIs257[
nuc-1::mCherry]. (D) Percentage of 3-fold or older embryos of the indicated genotypes containing blobs, showing the suppression of the
alfa-1 blob phenotype caused by loss ofthe yolk receptor gene
rme-2 function. For the quantification of the penetrances of the blob phenotype, we studied embryos at the 3-fold stage or older but beforethe initiation of pumping and derived from 1-day adults, unless otherwise stated. Nomarski differential interference contrast (DIC) optics was used.(E) Survival curve of wild-type (black),
alfa-1(
ok3062) (red), and
alfa-1(
gk498021) (blue) L1 larvae that hatched in the absence of food. The data shown arerepresentative of two independent experiments.(F) Anti-HA tag immunofluorescence of proliferative (left) and comma-stage (right) embryos expressing the reporter nIs759[Palfa-1::
alfa-1 gDNA::HA::
alfa-1 3'UTR]showing ALFA-1::HA cytoplasmic localization. Scale bar, 10 mm.(G) Percentage of 3-fold or older embryos of the indicated genotypes containing blobs. Overexpression of either wild-type
alfa-1 or codon-optimized C9orf72 forexpression in C. elegans (ceC9orf72) completely or partially rescued the
alfa-1 blob phenotype, respectively. z, animals expressed a Palfa-1::4xnls::gfp::alfa-130UTR transcriptional reporter. See also Figure S1.