Davies CS et al. (1996), Primary sequence and developmental expression p...

XB-ART-18422

Dev Biol 1996 Mar 15;1742:431-47. doi: 10.1006/dbio.1996.0086.

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Primary sequence and developmental expression pattern of mRNAs and protein for an alpha1 subunit of the sodium pump cloned from the neural plate of Xenopus laevis.

Davies CS , Messenger NJ , Craig R , Warner AE .

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Expression of a catalytic alpha subunit of the sodium pump was followed in early Xenopus embryos for correlation with physiological experiments showing that the sodium pump controls cavity expansion and the differentiation of neurones from the neural plate. Two cDNAs (one full length, one partial) for alpha1 subunit isoforms were cloned from a neural plate stage Xenopus library and sequenced. Other isoforms were not detected. Temporal and spatial expression patterns for alpha1 subunit transcripts and protein revealed extensive developmental regulation. At all stages, cells involved in cavity generation (outer ectoderm and cells lining the archenteron) expressed alpha1, transcripts with protein confined to the lateral and basal membranes. Before gastrulation, transcript levels were low and predominantly in animal cells. During gastrulation, alpha1 mRNAs rose significantly. Transcripts and protein were down-regulated in future outer neural plate cells as the mesoderm invaginated. Protein appeared at the blastopore on apical surfaces of lip cells and apposing surfaces of invaginating cells, suggesting that the Na pump opposes entry of fluid. In early neurulae, alpha1 mRNAs rose sharply. Transcript expression remained low in outer neural plate cells and increased in the endoderm, and protein appeared in the notochord. In midneurulae, transcripts returned in outer neural plate cells. Protein expression appeared on basal surfaces of deep neural plate cells and the floor plate, matching physiological observations. After neural tube closure, transcripts were detected in all dorsal structures. Protein was retained in the notochord and floor plate, was eliminated from the outer layer of the neural tube, and appeared on ependymal cells. The results are discussed in relation to previous physiological observations.

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Species referenced: Xenopus laevis
Genes referenced: atp1a1 nucb1 odc1 sst.1 tbx2
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	FIG. 1. Western blot of protein extracts from Xenopus embryos at stage 9 (lane 1), stage 20 (lane 2), stage 35 (lane 3), adult Xenopus kidney (lane 4), and rat brain (lane 5) probed with NPSE antibodies. Sixty micrograms of protein was loaded per lane. Note that in all cases the antibodies recognize a band at a molecular mass of about 100 kDa, which is appropriate for the predicted size of pXen9 protein. Molecular weight markers are shown to the left with arrowheads.
	FIG. 2. (A) The complete primary sequence of pXen9 cDNA and derived protein. The deduced amino acid sequence is given below. (B) Comparison between the 5* end sequence of pXen9 cDNA (first 120 bases) and the A6 Xenopus kidney cell line cDNA obtained by Verrey et al. (1989). (C) Sequence of 20us6 partial cDNA derived from stage 20 Xenopus embryos compared with that of pXen9. Amino acid differences between pXen9 and the 20us6 clone are indicated above and below the cDNA sequence, in boldface.
	FIG. 3. The time course of expression of pXen9 RNAs. (A) Northern blot of poly(A)/ RNAs from neurula stage Xenopus embryos probed with full-length pXen9. A sample at stage 10.5 run in the same experiment did not bind pXen9. Note clear signal at stage 12, which increases in intensity during neurulation. (B) Expression of pXen9 RNAs during development followed by RNase protection assay with the 20us6 probe. (Top band) ODC signal; (bottom band) 200-nt protected pXen9 fragment. Note that in this experiment the RNA loading at stage 12 was very low. (C) Time course of expression of pXen9 RNAs during development summarized from several experiments of the kind illustrated in B. The a subunit RNAs were numdetected with the 20us6 probe and in each experiment were normal-ized relative to the intensity of an ODC probe. Ordinate: a1 subunit mRNAs (relative units). Abscissa: upper, Nieuwkoop and Faber developmental stage; lower, time after fertilization (hr). The number of determinations at each stage is given above each column. Bars, standard error of the mean, calculated when n > 3.
	FIG. 4. The spatial distribution of pXen9 mRNAs prior to gastrulation. Methacrylate sections through embryos that had been hybridized with a whole mount in situ schedule (see Materials and Methods). (A) 2 cell. (B) 4 cell. nuc, nuclei which have been lost during sectioning because of their very large size. (C) 64-cell, animal pole (AP). (D) 64-cell, vegetal pole (VP). (E) Sense control, 2 cell. (F) Outermost animal pole cells at stage 7. (G) Animal pole cells lining the blastocoel (blast) at stage 7 (early blastula). (H) Equatorial region of stage 9 (late blastula) AN, animal pole; VEG, vegetal pole. blast, blastocoel. (I) Sense control: stage 7. Bars, 100 mm.
	FIG. 5. The spatial distribution of pXen9 RNAs during gastrulation and neurulation. (A) Longitudinal section through the dorsal lip of an early gastrula (stage 10.5). Note absence of signal in outer layer of dorsal ectoderm behind the dorsal lip, with a sharp boundary between nonexpressing and expressing cells (arrow). The nonexpressing cells are destined to form the outer layer of the neural plate. DL, dorsal lip. (B) Embryo at stage 11. The region of nonexpressing dorsal ectoderm cells now extends further back (arrow), paralleling invagination of the mesoderm. (C, D) Longitudinal sections through dorsal (C) and ventral (D) regions of an embryo at the end of gastrulation (stage 12). Note the lack of expression in the outer layer of the dorsal ectoderm, the putative neural plate. DL, dorsal lip; VL, ventral lip; YP, yolk plug. (E) Stage 14, transverse section through the neural plate. Note transcript expression is lower in superficial cells than deep cells of the neural plate (NP). The notochord (no), somites (som), and endoderm cells lining the archenteron show transcript expression. (F) Stage 17. Expression is most intense in the neural plate (NP) and lateral ectoderm (LE). The notochord (no) and somites (som) now show more intense expression than endoderm cells lining the expanding archenteron. (G) Stage 22. NT, neural tube; no, notochord; som, somites. (H) Stage 36. All dorsal structures now show intense expression of Na pump a1 subunit mRNAs. (I) Sense control, stage 17. (J) Stage 36; expression of collagen mRNAs. Note expression restricted to the notochord and floor plate (FP). (K) Stage 36. Expression of actin mRNAs is restricted to the somites. Bars, 100 mm for all except H, J where bars are 25 mm.
	FIG. 6. The expression of Na pump a1 subunit protein prior to gastrulation detected with NPSE or NASE antibodies. The relatively high background apparent in some images stems from inherent autofluorescence of the early Xenopus embryo. (A) Oblique section through embryo at the 4-cell stage. The cleavage furrow (indicated by arrows) stains intensely for a1 subunit protein. (B) 64-cell stage; outer animal pole cells. Note stain restricted to lateral and basal regions of the cell. (C) 64-cell stage; animal pole cells lining the blastocoel (blast). (D) Stage 7, outer animal pole cells. ext, the outside of the embryo. a1 subunit protein is limited to the lateral and basal membranes. (E) Stage 7, animal pole cells facing the blastocoel (blast). (F) Stage 7 deep vegetal pole cells. Note protein stain on all cell surfaces. (G) Stage 9, part of section through the embryo showing outer animal pole cells and cells facing the blastocoel. Protein stain is absent from the outer surface, but stain of varying intensity is present on all other cell membranes. ext, external surface of embryo. b, blastocoel facing surface. (H) Stage 7, antibody stain is abolished in the presence of competing peptide. ext, extraembryonic surface; AP, animal pole cells. Bars in A, B, D, 25 mm; in remainder, 100 mm.
	FIG. 7. The expression of a1 subunit protein during gastrulation. (A, B) Sections through the dorsal lip of two embryos at the beginning of gastrulation. DL, dorsal lip. Note protein stain is limited to cells lining the developing blastopore. (C, D) Sections through two regions of the yolk plug of a single embryo at stage 12. DL, dorsal lip; YP, yolk plug. Note intense protein stain on the apposing surfaces of ectoderm and endoderm as cells move through the blastopore. This is particularly clear in C, which shows endoderm cells as they move into the embryo. Bars, 100 mm.
	FIG. 8. The expression of a1 subunit protein during neurulation. (A) Stage 13. The rostral notochord (no) begins to stain for a1 subunit protein. The neural plate (NP) and somites (som) show little stain. (B) Stage 14, rostral. The notochord begins to show prominent staining for a1 subunit protein. (C) Stage 14, caudal. Notochord staining (no) is absent. There is some patchy staining elsewhere. som, somites. (D) Stage 17. The deep surface of neural plate cells (NP) stain for a1 subunit protein. The notochord stains intensely and the somites (som) show only weak and patchy stain. (E) Stage 17. The boundary (arrow) of a1 subunit protein between deep neural plate (NP) and lateral ectoderm (LE) cells. Part of the intensely stained notochord is visible and the somite (som) shows little stain. (F) Stage 17: an example of a1 subunit protein expression in floor plate cells lying immediately above the intensely staining notochord. (G) Stage 17. Negative control. NP, neural plate; no, notochord; som, somite. Bars, 25 mm for F, G; otherwise, 100 mm.
	FIG. 9. The expression of a1 subunit protein after closure of the neural tube. (A) Stage 22/23. Note intense stain of notochord, floor plate (FP), and apical surfaces of neural tube cells (NT). som, somites. (B) Enlargement of A to show the region just above the notochord to further illustrate floor plate (FP) and neurocoel lining stain. NT, neural tube. (C) Ventral region of same section to show pattern of a1 subunit protein expression in the expanding archenteron (arch). The ventral side of the notochord is just visible. The somites (som) continue to show little expression. (D) Stage 36. The dorsal notochord and ventral region of the neural tube (NT). Note marked expression along the cell surfaces lining the neurocoel, particularly in the floor plate. (A, C) Bar, 100 mm; (B, D) Bar, 25 mm.