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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.
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.