XB-ART-8165Differentiation September 1, 2001; 68 (2-3): 115-25.
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Xenopus Na,K-ATPase: primary sequence of the beta2 subunit and in situ localization of alpha1, beta1, and gamma expression during pronephric kidney development.
The osmoregulatory function of the pronephric kidney, the first excretory organ of the vertebrate embryo, is essential for embryonic survival. The transport systems engaged in pronephric osmotic regulation are however poorly understood. The Na,K-ATPase is the key component in renal solute transport and water homeostasis. In the present study, we characterized the alpha, beta, and gamma subunits of the Na,K-ATPase of the developing Xenopus embryo. In addition to the known alpha1, beta1, beta3 and gamma subunits, we report here the identification of a novel cDNA encoding the Xenopus beta2 subunit. We demonstrate by in situ hybridization that each Xenopus Na,K-ATPase subunit exhibits a distinct tissue-specific and developmentally regulated expression pattern. We found that the developing pronephric kidney expresses alpha1, beta1, and gamma subunits uniformly along the entire length of the nephron. Onset of pronephric Na,K-ATPase subunit expression occurred in a coordinated fashion indicating that a common regulatory mechanism may initiate pronephric transcription of these genes. The ability to engage in active Na+ reabsorption appears to be established early in pronephric development, since Na,K-ATPase expression was detected well before the completion of pronephric organogenesis. Furthermore, Na,K-ATPase expression defines at the molecular level the onset of maturation phase during pronephric kidney organogenesis. Taken together, our studies reveal a striking conservation of Na,K-ATPase subunit expression between pronephric and metanephric kidneys. The pronephric kidney may therefore represent a simplified model to dissect the regulatory mechanisms underlying renal Na,K-ATPase subunit expression.
PubMed ID: 11686233
Article link: Differentiation
Species referenced: Xenopus laevis
Genes referenced: atp1a1 atp1b1 atp1b2 atp1b3 atp4b fxyd2 igf2bp3
Article Images: [+] show captions
|Fig. 1 Relationship of the Xenopus Na,K-ATPase b2 subunit to other vertebrate b subunits. A Alignment of the deduced amino acid sequences of Xenopus, Bufo and human Na,KATPase b2 subunits. Hyphens represent gaps that were introduced for optimal alignment. Amino acids shared by all three proteins are highlighted in blue. The predicted transmembrane domain is underlined. The asterisks denote conserved cysteine residues of the extracellular domain. Conserved potential N-glycosylation sites (N-X-S/T) are indicated with black dots. The amino acids are numbered on the right side of the sequences. B Sequence comparison of Xenopus b2 and selected vertebrate b subunits. The matrix of percent amino acid identity among the b subunits was calculated using the MegAlign program. Sequences were obtained from the GenBank database. C Phylogenetic tree showing the relationship of vertebrate Na,K-ATPase b subunits. The tree is based on an alignment of amino acid sequences performed with the Clustal method. The Xenopus gastric H,K-ATPase b subunit (Chen et al., 1998) was used as an outgroup. The scale bar measures the distance between the sequences. Units indicate the number of substitution events. The evolutionary distance between any two sequences is the sum of the horizontal branch length separating them. Vertical distances are for illustration purposes only. The GenBank accession numbers are AJ293961; Bufo b2, Z25812; human b3, U51478; mouse b3, as follows: human Na,K-ATPase b1 subunit, X03747; mouse b1, U59761; chicken b3, L13208; Bufo b3, Z11799; Xenopus b3, X16646; chicken b1, J02787; Bufo b1, Z11797; Xenopus b1, M37788; and Xenopus gastric H,K-ATPase b subunit, U17061; human b2, U45945; mouse b2, X16645; Xenopus b2, AF042812.|
|Fig. 2 Expression of Na,K-ATPase b subunit genes during Xenopus embryogenesis. Whole-mount in situ hybridizations were per- formed on Xenopus embryos with antisense probes for b1, b2, and b3 subunits. Sections of stage 29 (S), stage 30 (T) and stage 37 (R) embryos stained in whole-mount were cut at 30–40 mm. Dorsal (A, C, D) and lateral (E-N) views are shown with anterior to the left. Frontal views (B, O-Q) and sections (R-T) are oriented with dorsal to the top. A Stage 19 embryo with punctate b1 expression in the epidermis. B, C b2 expression was confined to the non-neural ectoderm at stage 15 (B) and throughout the epidermis at stage 19 (C). D Stage 19 embryo showing b3 expression in the developing brain and neural tube. E-G b1 transcripts appeared at stage 26 (E) initially in the otic vesicles, pronephric anlage, somites, and proctodeum. Later, at stage 29 (F) staining was noticeable in the trigeminal (V) nerves. By stage 37 (G), b1 transcripts were also evident in the olfactory bulbs, facial (VII) and glossopharyngeal (IX) nerves. H-J Extensive epidermal expression of b2 transcripts was observed in stage 25 (H), stage 29 (I), and stage 38 (J) embryos. Arrowhead indicates high levels of b2 in the gills. Note the increasing b2 expression in the brain and eyes. K-M b3 gene expression was found at stage 26 (K) in the brain, eyes, otic vesicles, somites, and cranial neural crest (arrowheads). By stage 30 (L), strong stain ing was evident at the forebrain-midbrain junction (arrowhead), in the region of the hepatic primordium, the proctodeum, and olfac- tory bulbs. b3 transcripts were seen at stage 38 laterally in the migrating abdominal muscle anlagen (arrowheads). N High magni fication of a stage 37 embryo illustrating b1 expression in the pronephric kidney. O-Q Frontal views showing b1 (O), b2 (P), and b3 (Q) expression in the developing head. R-T Transverse sections through the midtrunk of stage 37 (R) and at the level of the pro nephros of stage 29 (S) and stage 30 (T) embryos. b1 transcripts (R) were present in the pronephric duct, b2 expression was confined to the epidermis, and b3 was observed in spinal nerves and the hepatic primordium. Abbreviations: br, brain; e, eye; ed, ectoderm; ep, epidermis; hp, hepatic primordium; m, mouth, np, neural plate; nt, neural tube; ob, olfactory bulb; ov, otic vesicle; p, proctodeum; pd, pronephric duct; pt, pronephric tubules; pn, pronephros; sc, spinal cord; sm, somites; sn, spinal neurons.|
|Fig. 3 Expression of Na,KATPase a1 and g subunit genes during Xenopus embryogenesis. Whole-mount in situ hybridizations were performed with antisense probes for a1 and g subunits. Transverse sections of stage 28 (K, L) were cut at 30–40 mm. Lateral views (A-H) are shown with anterior to the left. Frontal views (I, J) and sections (K, L) are oriented with dorsal to the top. A-D a1 subunit expression was found throughout the epidermis of tailbud embryos (A, stage 23; B, stage 26; and C, stage 28). Expression in the pronephric anlage can be anticipated from stage 26 on (white arrowheads). At stage 37, a1 transcripts were prominently detected in the olfactory bulbs, otic vesicles, the gills (arrowheads), the pronephric kidneys, and facial (VII) and glossopharyngeal (IX) nerves. E-H Stage 22 embryo (E) showed a punctate staining pattern for the g subunit. At stage 25 (F), g expression was overall low, but became at stage 28 (G) apparent in the brain, eyes, otic vesicles, and pronephric primordia. In the stage 38 embryo (H), strong expression of g transcripts was confined to the pronephric kidneys. I, J Frontal views of stage 37/38 embryos stained for a1 and g expression. Strong staining for a1 transcripts (J) was seen in the olfactory bulbs. K, L Transverse sections of stage 28 embryos revealed a1 (K) in the epidermis and g (L) expression in the pronephric and hepatic primordia. For abbreviations, see legend to Fig. 2.|
|atp1b2 (ATPase, Na+/K+ transporting, beta 2 polypeptide) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage25, lateral view, anterior left, dorsal up.|