Fig 5.
anoh-1 is required for the efficient removal of necrotic touch cells but not apoptotic cells. (A) Gene structure of two isoforms of
anoh-1. The blue open boxes indicate the region deleted in the
anoh-1(
tm4762) allele. (B) Domain structure of ANOH-1b protein. Dark grey bars indicate 8 predicted transmembrane domains. Red color labels amino acids 1-18, which exists in ANOH-1b but missing from ANOH-1a. (C) The
anoh-1(
tm4762) mutant embryos are normal in the removal of somatic apoptotic cells. (D) The
anoh-1(
tm4762) mutant adults are normal in the removal of apoptotic germ cells. (E) The engulfment and degradation processes of embryonic apoptotic cells C1, C2, and C3 are normal in
anoh-1(
tm4762) mutant embryos, measured by timelapse microscopy. (F) The
anoh-1(
tm4762) mutation perturbs the removal of necrotic cells, and the
anoh-1b but not the
anoh-1a form rescues the necrotic cell-removal defect when expressed in touch cells under the control of Pmec-7. For each sample, 6 groups of 10 worms at indicated stages were scored anddisplayed as mean. Error bars indicate standard deviations. *, 0.01<p<0.05 (student t-test). ***, p<0.001 (student t-test). ns, no significant difference,p>0.05 (student t-test). (G) The expression of
anoh-1b in the necrotic cells, not engulfing cells, rescues
anoh-1(
tm4762) mutant phenotype. In
anoh-1(
tm4762);
mec-4(
e1611dm) mutant animals expressing indicated transgenes, the number of necrotic cells in the tail of each of the 6 groups of 10 newlyhatched L1 larvae were scored. Data are presented as mean ± sd. (H) The GFP fusion forms of ANOH-1b but not ANOH-1a are localized to the plasmamembrane. GFP (a, c, e, g) and DIC (b, d, f, h) images of the tails of
mec-4(
e1611dm) L1-stage larvae. Arrowheads in (a, b, e, f) label necrotic cells on which ANOH-1b is observed on the cell surface. Arrows in (c, d, g, h) label necrotic cells in which ANOH-1a is observed on the nuclear surface. All GFP reportersare expressed under the Pmec-7 promoter control. (I) The
anoh-1b promoter is expressed in touch neurons. Shown here are epifluorescence (b-c) and thecorresponding DIC (a) images of the tail region of a wild-type L2 larva co-expressing Pmec-7 mCherry (b), which is a touch neuron-specific reporter, and Panoh1b NLS::GFP (c). White arrowheads mark a touch neuron. Dorsal is up. Scale bars are 5μm.