Modifier mutations induce cell invasion in mlt heterozygotes. (A-D) Lateral views of live 6-dpf larvae. Arrowhead points to the mid-intestine. (A) A wild-type (WT) larva. (B) A homozygous mlt larva in which there is invasive expansion of the mid- and posterior intestine. (C) A mlt/S237Y compound heterozygote. Arrowhead points to a region of altered intestinal morphology. Inset shows a more severely affected mlt/S237Y compound heterozygote. (D) mlt/L1287M compound heterozygote. Mild and moderate (inset) phenotypes are shown as in C. (E,F) Histological section through the posterior intestine of 6-dpf mlt/S237Y and mlt/L1287M compound heterozygotes following their immunostaining with anti-Keratin (red) and anti-Laminin-1 antibodies (green). Dashed line indicates predicted position of basement membrane disrupted by the invasive epithelial cells; e, intestinal epithelium; sm, smooth muscle. (G,H) Histological section through the posterior intestine of 3-dpf Tg(miR194-Lifeact-GFP) wild-type and sibling mlt/S237Y larvae following immunostaining with anti-GFP (green) and anti-Laminin-1 (red) antibodies, showing GFP-labeled invadopodia (arrowheads) of epithelial cells extending through gaps in the basement membrane. Inset shows a higher-magnification view. (I-K) Confocal projections through the mid- and posterior intestine of 4-dpf Tg(miR194-Lifeact-GFP) wild-type and sibling mlt/S237Y (myh11^mlt/S237Y) larvae following anti-GFP immunostaining (green). Arrowheads point to invadopodia protruding from the basal epithelial cell plasma membrane in the mlt/S237Y mutants. Blue, DAPI-stained nuclei.

mlt modifier mutants are sensitized to oxidative stress. (A-D) Lateral views of 82-hpf wild-type (WT), homozygous mlt, heterozygous mlt and L1287 homozygous larvae. Wild-type (A), mlt heterozygote (C) and L1287M homozygote (D) larvae treated with menadione (1.5µM; mena). Morphological changes of the mid- and posterior intestine that are characteristic of epithelial invasion (arrowheads) are seen in the mlt larva (B) and the menadione-treated mlt heterozygote and L1287M homozygote (C,D). In all three mutant larvae, the ventral intestine appears to extend into the yolk. Intestinal morphology is normal in the wild-type larva (A). (E-H) Lateral views of 6-dpf KRAS-axin larvae that either have homozygous wild-type myh11 (E,F), S237Y (G) or L1287M (H) alleles. The intestine is expanded in KRAS-axin larvae as a result of epithelial hypertrophy (brackets, E,F). Further expansion as a result of epithelial invasion is seen in menadione-treated KRAS-axin S237Y and L1287M homozygotes (brackets, G,H), as previously reported for menadione-treated KRAS-axin mlt heterozygotes (Seiler et al., 2012). (I) Quantification of intestinal width in KRAS-axin compound-mutant larvae treated with menadione compared with untreated siblings. ****P<0.001; n.s., not significant. Unpaired Student′s t-test performed; mean±s.e.m. (J,K) Histologic analyses of immunostained larvae showing epithelial cell invasion in the intestine of a menadione-treated homozygous S237Y KRAS-axin larva (K) vs a wild-type KRAS-axin larva (J). Green, anti-Laminin-1; red, anti-Keratin; blue, DAPI-stained nuclei. Arrowhead (K) points to invasive cells. Scale bars: 100µm.

Zebrafish myh11 mutations alter intestinal transit. (A,B) Intestinal transit as measured by the expulsion of fluorescent microspheres in wild-type and sibling homozygous mlt larvae injected with a myh11 morpholino (MO) (A), and wild-type and sibling S237Y homozygous larvae (B). Anterior: beads remain in anterior intestinal bulb; posterior: beads remain in mid- and posterior intestine; expelled: no beads remaining in the intestine. ****P<0.001. Chi-squared test performed with 2 degrees of freedom. (C) Time lapse of a wild-type (WT) larva beginning when the fluorescent beads are present in the anterior intestine and followed until bead expulsion. A, M, P, location of anterior intestine, mid-intestine and posterior intestine, respectively.

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