Walentek P et al. (2015), ATP4 and ciliation in the neuroectoderm and end...

XB-ART-51004

Data Brief 2015 Apr 20;4:22-31. doi: 10.1016/j.dib.2015.04.003.

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ATP4 and ciliation in the neuroectoderm and endoderm of Xenopus embryos and tadpoles.

Walentek P , Hagenlocher C , Beyer T , Müller C , Feistel K , Schweickert A , Harland RM , Blum M .

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During gastrulation and neurulation, foxj1 expression requires ATP4a-dependent Wnt/β-catenin signaling for ciliation of the gastrocoel roof plate (Walentek et al. Cell Rep. 1 (2012) 516-527.) and the mucociliary epidermis (Walentek et al. Dev. Biol. (2015)) of Xenopus laevis embryos. These data suggested that ATP4a and Wnt/β-catenin signaling regulate foxj1 throughout Xenopus development. Here we analyzed whether foxj1 expression was also ATP4a-dependent in other ciliated tissues of the developing Xenopus embryo and tadpole. We found that in the floor plate of the neural tube ATP4a-dependent canonical Wnt signaling was required for foxj1 expression, downstream of or in parallel to Hedgehog signaling. In the developing tadpole brain, ATP4-function was a prerequisite for the establishment of cerebrospinal fluid flow. Furthermore, we describe foxj1 expression and the presence of multiciliated cells in the developing tadpole gastrointestinal tract. Our work argues for a general requirement of ATP4-dependent Wnt/β-catenin signaling for foxj1 expression and motile ciliogenesis throughout Xenopus development.

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Species referenced: Xenopus laevis
Genes referenced: atp4a ctnnb1 foxj1 foxj1.2 nsd1 ptch1 shh tbx2 tuba4b
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	Fig. 1. Floor plate foxj1 expression requires ATP4a and β-catenin downstream or in parallel of Hedgehog signaling. (A–F) WMISH for foxj1 expression in control and manipulated embryos at stage 16. (A, B) Normal foxj1 expression in the floor plate of control uninjected (uninj.; A) and ethanol (1%; EtOH; B) treated specimens. (C) Strong reduction of foxj1 signals in atp4a morphants was partially rescued by co-injection of β-catenin DNA (ß-cat.; E). (D) Inhibition of Hedgehog signaling by cyclopamine treatment decreased foxj1 expression in the floor plate and was partially rescued by injection of β-catenin DNA (ß-cat.; F). (G) Quantification of results. a, anterior; l, left; n, number of embryos; p, posterior; r, right; st., stage.
	Fig. 2. Normal floor plate induction and Hedgehog signaling in ATP4a-deficient embryos. (A–E) Normal floor plate formation in atp4a morphants. WMISH for foxj1 (A–C) and shh (D, E) revealed attenuated foxj1 expression but unaffected floor plate formation, as judged by apically constricted cells (histological vibratome sections in A–C`; planes indicated in A–C) and shh expression in atp4a morphants. (F–K) Unaffected Hedgehog signaling in atp4a morphants with (H) or without (G) co-injection of ß-catenin (β-cat.) DNA, as judged by WMISH for ptch1, a direct Hedgehog signaling target, to monitor activity of Hedgehog signaling, as compared to uninjected controls (uninj.). ptch1 expression was decreased in cyclopamine-treated embryos (J, K), as compared to ethanol (1%) controls (I), but independent of β-catenin DNA injection (K, K`). a, anterior; l, left; p, posterior, right; st., stage.
	Fig. 3. ATP4a is required for foxj1-dependent cerebrospinal fluid in the tadpole brain. To investigate whether ATP4 was required for brain cilia, we analyzed cerebrospinal fluid (CSF) flow as a proxy. Injection of fluorescent beads into the brain ventricles at stage 45 revealed a significant reduction of CSF velocity in atp4a morphants. In contrast, velocity of CSF flow in atp4a morphants was partially rescued by co-injection of either atp4a or foxj1 DNA constructs. cf. Movie 1.
	Fig. 4. The atp4a splice-site MO causes atp4a intron 2 retention. To facilitate late analysis of atp4a morphants, a splice-site MO (atp4aSplMO) was used, which targeted the second exon/intron boundary of zygotically expressed mRNA and caused atp4a intron 2 retention. atp4aSplMO targeted to gastrointestinal tract caused intron2 retention, as shown by RT-PCR using primers (blue arrows) which bind to exon 2 (yellow) and intron 2 (black). Genomic DNA served as positive control. Total RNA extracts without reverse transcription (-RT) and water (H2O) served as negative controls. RT-PCR of ef1α served as loading control. λ-phage DNA digested with Pst1 (λ PST) served as size marker.
	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.
	atp4a (ATPase, H+/K+ exchanging, alpha polypeptide) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 45, dissected gut.

References [+] :

Aamar, Isolation and expression analysis of foxj1 and foxj1.2 in zebrafish embryos. 2008, Pubmed