Ihha is required for cell proliferation in the gastrointestinal tract. A–F, Proliferation assay using PCNA antibody. Cell nuclei are visualized with DAPI staining. A–F, Cross-section of gastrointestinal tract at the level of anterior part of swimbladder in A–C, controls and D–F, ihha-/- reveals decreased cells proliferation in both epithelial and mesenchymal layers of intestinal bulb in ihha-/- mutant. Dotted white lines demarcate epithelium and mesenchyme of intestinal bulb in control and ihha-/- mutant. G, Evaluation of epithelial and mesenchymal cell proliferation in the esophagus/intestinal bulb, mid-gut, and caudal gastrointestinal segments of control and ihha-/-. Epithelial cell proliferation in the absence of Ihha signaling is reduced significantly in the esophagus/intestinal bulb segment of ihha-/-. Most significant decrease in cell proliferation was observed in the mesenchyme of esophagus/intestinal bulb and mid-gut segments in ihha-/- comparing to controls. Solid red bars represent epithelium of esophagus/intestinal bulb, mid-gut and caudal gastrointestinal segments of control, while empty red boxes correspond to ihha-/-. Solid blue bars represent mesenchyme of esophagus/intestinal bulb, mid-gut and caudal gut of control, while others correspond to ihha-/-. There are no significant differences in cell proliferation in epithelium and mesenchyme of posterior gut in the control and ihha-/-. Abbreviations: e, epithelium; g, gut; sb, swimbladder. Scale bars: 250 μm.