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To investigate the potential function of ATP4 in formation of the ciliated Xenopus embryonic epidermis, we analyzed the expression of ATP4a protein in the epidermal ectoderm ( Fig. S1). ATP4a was found throughout the animal hemisphere during gastrulation (Fig. S1A and A‵) and neurulation (Fig. S1B and C), within the outer and deep layer of the skin ectoderm (Fig. S1B). The deep layer forms multiciliated cells (MCCs), ion secreting cells (ISCs) and small secretory cells (SSCs) which later intercalate into the outer epithelium ( Deblandre et al., 1999; Dubaissi et al., 2014 ; Stubbs et al., 2006). Furthermore, ATP4a protein was enriched in the epidermal ectoderm during neurulation (Fig. S1C), and continued expression in the epidermis throughout tailbud (Fig. S1D) and early tadpole stages (Fig. S1F and G). |
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Fig. 5.
foxj1 and atp4a are co-expressed in the gastrointestinal tract which transiently harbors motile cilia in the stomach. (A–F) WMISH for atp4a (A,C,D) and foxj1 (E,F) in the GI tract of stage 43–45 tadpoles (A–C, E; stomach highlighted by arrowheads; ventral views) and isolated GI tracts (D, F). (B) atp4a sense control revealed no staining. Note the co-expression of atp4a and foxj1 at stage 45 (C–F). (G–M) GI tract ciliation as shown by immunofluorescent staining of cilia/tubulin (acetylated-α-tubulin staining, red) and staining for actin (phalloidin, green) as well as nuclei (DAPI, blue) on cryosections from stage 45 tadpoles (planes indicated in J). MCCs (M) were found in the esophagus (es; G–I,K), stomach (sto; G–I,L) and the proximal part of the small intestine (smi; H, I). (N, N`) Scanning electron microscopy analysis of gastric epithelia from adult frogs revealed the presence of short monocilia, but lack of MCCs. (O) Injection of atp4aSplMO targeted to the endoderm prevented normal development of the GI tract. Targeting was monitored by co-injection of fluorescent rhodamine dextrane (red). Embryos are shown in ventral view. a, anterior; d, dorsal; es, esophagus; l, left; p, posterior; r, right; st., stage; smi, small intestine; sto, stomach; v, ventral. |
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atp4a (ATPase, H+/K+ exchanging, alpha polypeptide) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 45, dissected gut. |