Zebrafish intestinal microvilli establishment requires Fip5b. (A) Domain schematic of zebrafish Fip5b protein containing a C2 domain at the N-terminus and a Rab-binding domain (RBD) at the C-terminus. The red arrowhead with STOP denotes premature termination codon in fip5b mutant alleles. (B) qRT-PCR for fip5b in wild-type and fip5bCO40 mutant larvae at 6 dpf. n=50 larvae for each condition. (C) 6 dpf larvae expressing Tg(hsp:GFP:Rab11a) labeling the intestine. The intestinal bulb is denoted by a bracket and the midgut by a dashed box. All following images are representative cross sections through the midgut region. Wild-type siblings are used as controls. For all experiments, three separate animals for each condition were analyzed. (D) Electron micrographs showing 3 dpf wild-type and fip5bCO40 mutant larvae. Luminal space is lighter gray region. (E) High magnification electron micrographs showing 3 dpf wild-type and fip5bCO40 mutant larvae. Yellow box shows zoomed in view on a region with subapical microvillus inclusion-like structures. (F) Quantitation of the mean number of inclusion-like structures per cell in 3 dpf wild-type and fip5bCO40 mutant larvae. (G) Electron micrographs showing 6 dpf wild-type and fip5bCO40 mutant larvae. Arrows point to larger than 500 nm organelles and boxes outline magnified region shown in (G′). (G′) Magnified view of apical cell surface showing terminal web and microvilli. Brackets denote electron dense line and organelle-free zone comprising the terminal web or lack thereof in mutants. (H) Quantitation of less than 500 nm apical vesicles in 6 dpf larvae. (I) Quantitation of greater than 500 nm organelles in 6 dpf larvae. (J) Quantitation of microvilli length in the intestinal bulb and midgut in 6 dpf larvae. Each dot represents a single microvillus length combined across three animals. All plots show mean±s.e.m. A t-test was used for Gaussian data and a Mann–Whitney test for all other statistics. ***P<0.0005, **P<0.005.
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