Uochi T et al. (1997), The Na+,K+-ATPase alpha subunit requires gastru...

XB-ART-15903

Dev Growth Differ 1997 Oct 01;395:571-80. doi: 10.1046/j.1440-169x.1997.t01-4-00004.x.

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The Na+,K+-ATPase alpha subunit requires gastrulation in the Xenopus embryo.

Uochi T , Takahashi S , Ninomiya H , Fukui A , Asashima M .

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Na+,K+-ATPase participates in reabsorption of ions and water and produces an electrochemical gradient between the intra- and extracellular spaces across the cell membrane. It also plays an important role in many developmental phenomena such as a blastocoele formation and neural formation. To elucidate the expression pattern of Na+,K+-ATPase in the Xenopus embryo, the spatial expression patterns of the Na+,K+-ATPase alpha subunit were studied in a normal embryo by whole-mount in situ hybridization. These transcripts were localized around the dorsal blastopore at the gastrula stage, in the neural tube at the neurula stage, and then in the pronephros and cloaca at the tail-bud stage. To study the function of Na+,K+-ATPase in embryogenesis after mid-blastula transition, the expression of the Na+,K+-ATPase alpha subunit was inhibited by the injection of specific antisense RNA. Embryos injected with Na+,K+-ATPase antisense RNA showed inhibition of gastrulation. When antisense RNA was injected into the dorsal blastomeres, head differentiation was markedly inhibited. These results suggest that this transcript plays an important role during gastrulation and head differentiation.

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Species referenced: Xenopus laevis
Genes referenced: inhba tbx2

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	Fig. 1. Whole-mount in situ hybridization analysis of Na+, K+ATPase ex subunit transcripts in Xenopus embryos. (A) Lateral view of St. 10 embryo showing intensive staining in the blastopore lip. (B) Vegetal view of St. 12 embryo showing staining in the dorsal side around the yolk plug. (C) Lateral view of St. 13 embryo showing staining in the rostral region of the invaginating mesoderm and the blastopore. (D) Dorsal view of of St. 21 embryo showing extensive staining in the neural tube. The expression in the neural tube disappeared until the late tail-bud stage. (E) Ventral view of St. 21 embryo showing extensive staining in the presumptive cloaca. The expression at this position was maintained throughout embryogenesis. Lateral view of St. 27 embryo (F) and St. 35 embryo (G), showing expression in the ear vesicle and in the pronephros. The expression in the pronephros was maintained until the late tail-bud stage. (H) Crosssection of the embryo shown in G. Left is dorsal side. bl, blastopore lip; clo, cloaca; dor, dorsal side; ev, ear vesicle; nt, neural tube; p, presumptive pronephros; pd, pronephric duct; pt, pronephric tubules; ven, ventral side, yp, yolk plug.
	Fig. 2. Whole-mount in situ hybridization analysis of Na+, K+ -ATPase a subunit transcripts in explants treated with activin A and retinoic acid (RA). Explants were cultured for (A, B) 1 day or (C , D) 3 days after treatment for 3 h with 10 ng/ml activin A and 10-4 moi/L RA. (A) Na+, K" -ATPase a subunit transcripts were not expressed in the explant. (B) Cross-section of the explant shown in A. (C) Extensive staining was seen in the explant. (D) Cross-section of the explant shown in C. Na+, K+-ATPase a subunit transcripts were expressed and localized at pronephric tubules. pi, pronephric tubules. Arrow indicates the staining point.
	Fig. 3. Expression of NaT, K+ -ATPase a subunit transcripts in dissected mesoderms. (A) Shows a scheme of St. 10 embryo. Samples were dissected from embryos. The dissected mesoderm was divided into three parts, the DMZ, LMZ, and VMZ as shown in A. (B) The expression pattern of Na+, K+ -ATPase a subunit transcripts was shown by RT-PCR analysis. Lane 1, DMZ (dorsal marginal zone); lane 2, LMZ (lateral marginal zone); lane 3, VMZ (ventral marginal zone); lane 4, St. 10 embryo (negative control); lane 5, St. 10 embryo (positive control) The negative control had no reverse transcriptase added when first strand eDNA was synthesized.
	Fig. 4. Microinjection of Na+, K+-ATPase a subunit antisense RNA in Xenopus embryos. The embryo was injected with Na+, K+ATPase a subunit antisense RNA into (B, D) dorsal or (C, E) ventral blastomeres at the 4-cell stage. (A) The embryo was injected with Na-, K+-ATPase a subunit sense RNA into dorsal blastomeres at the same stage. This embryo showed no effect. The same results were shown in embryos injected with Gurdon's buffer or sense RNA into dorsal or ventral blastomeres at the same stage. (B) The embryo injected with antisense RNA showed inhibition of gastrulation and head differentiation. (C) The embryo showed inhibition of gastrulation. These embryos did not close the blastopore (B, C). (D, E) shows various phenotypes of embryos. (D). The upper embryo is the same embryo as A. Head differentiation was limited in these embryos. (E) All embryos showed head differentiation but failed to undergo gastrulation, as seen with the embryos in D. Arrow head indicates a head structure.
	Fig. 5. Cross-sections of embryos injected with Na+, K+ -ATPase et subunit sense or antisense RNA into dorsal or ventral blastomeres at the 4-cell stage. (A) shows a lateral view of an embryo injected with sense RNA into the dorsal blastomeres at the 4-cell stage; b and c show the positions of cross-sections of B and C, respectively. (B) Cross-section of A. Head specific tissues such as forebrain and eyes are differentiated in this embryo. (C) Second cross-section shown in A. Trunk-tail structures such as neural tube, somitic muscle, and notochord differentiated in this embryo. (D) shows a lateral view of the embryo injected with antisense RNA into the dorsal blastomeres at the 4-cell stage; e and f show the positions of cross-sections of E and F, respectively. (E) Cross-section of D. Head specific tissues such as forebrain and eyes did not differentiate in this embryo, although differentiation of neural tissue was observed. (F) Cross-section of D. Trunk-tail structures such as neural tube, somitic muscle, and notochord differentiated in this embryo as seen in the embryo in A. (G) shows a lateral view of the embryo injected with antisense RNA into the ventral blastomeres at the 4-cell stage; hand i show positions of cross-sections of Hand I, respectively. (H) Cross-section of G. Head specific tissues such as forebrain and eyes differentiated in this embryo. (I) Cross-section of G. Trunk-tail structures such as neural tube, somitic muscle, and notochord differentiated in this embryo. The tissue differentiation that was observed in embryos injected with antisense RNA into dorsal or ventral blastomeres was more immature than that in embryos injected with sense RNA. Arrow head indicates a head structure. eye, eye; lb. forebrain; nt, neural tube; not, notochord; sm, somitic muscle.