Knock-down foxi3a Expression Severely Reduces Epidermal Ionocyte Progenitor Number and Abolishes the Later Differentiation Program. (A–G) Interfering with foxi3a and foxi3b functions of epidermal ionocyte differentiation by a morpholino (0.5 mM/embryo) injection. Morphants were fixed at 24 hours post fertilization (hpf) and stained with atp1b1b (green), ca2a (red), and P63 (blue) to detect Na+,K+-ATPase rich cells (NaRCs), H+-ATPase rich cells (HRCs), and epidermal stem cells, respectively. (H–I) A rescue experiment to show the specificity of foxi3a morpholinos. The foxi3a mRNA used for the rescue experiment does not contain a binding site for MO2. (J–L) Comparison of the vital dye uptake ability between the wild type (wt), foxi3a morphants, and foxi3b morphants. NaRCs and HRCs in either wild-types or foxi3b morphants can absorb MitoTracker (red) and Con-A (green) through to their apical openings. For foxi3a morphants, no MitoTracker or Con-A staining was detected due to blockage of the entire differentiation program. (M–O) Detection of the apical opening of epidermal ionocytes in wild-types, foxi3a morphants, and foxi3b morphants by scanning electron microscopy. The apical openings of NaRCs and HRCs in wild-types are shaped as deep holes (green box) or a mesh (red box), respectively. The apical openings were totally undetected in foxi3a morphants due to blockage of the entire differentiation program. For foxi3b morphants, the apical openings for both NaRCs and HRCs were reduced. Embryos in (A–I) were scored at 24 hpf, while in (J–O), they were scored at 72 hpf. In I, embryos are orientated in a dorsal-up and anterior-top position.
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