Ouatas T et al. (1998), Differential expression of nucleoside diphospha...

XB-ART-15337

Int J Dev Biol 1998 Jan 01;421:43-52.

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Differential expression of nucleoside diphosphate kinases (NDPK/NM23) during Xenopus early development.

Ouatas T, Sélo M, Sadji Z, Hourdry J, Denis H, Mazabraud A.

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In Xenopus laevis, three nucleoside diphosphate kinase (NDPK) monomers have been described (NDPK X1, X2 and X3) (Ouatas et al., 1997). In eucaryotes, this kinase is known as a hetero- or homohexamer. Here, we examine the distribution of the enzyme and its different subunit mRNAs during oogenesis and early embryogenesis of Xenopus laevis, respectively by immunohistofluorescence and whole-mount in situ hybridization. These analyses show that NDPKs and their mRNAs are differentially distributed throughout the oocyte and early embryos with a high level of transcription in somites and brain. We emphasize two points. First, each mRNA displays a distinct subcellular localization in somites, suggesting a complex regulation of NDPK genes both at the transcriptional and translational level and a possible involvement of NDPK X2 homohexamers in the dorsal muscle differentiation. Second, in oocytes and early embryos, the proteins are mainly localized in the nucleus, suggesting a new mechanism for their nuclear import, since they do not possess any known nuclear import sequences.

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Species referenced: Xenopus laevis
Genes referenced: nme2 rel tbx2 tnni3 tubb2b

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	Fig. 1. Schematic representation of the different anti-sense probes asX specifically recognizing each of the NDPK mRNAs. The open reading frame is hatched. The initiation codon (A TG) and the stop codon (TAA) are indicated. The differently shaded rectangles correspond to the 5' or 3' UTR regions. Construction of asXl. asX2, and asX3 which recognize NDPK X1, NDPK X2, and NDPK X3 mRNAs, respectively, and of the antl~sense probe asNDPK Xt recognizing every NDPK mRNA, is descnbed in methods.
	Fig. 2. NDPK X1 mRNA stability in acellular extracts. 1ng mRNA was incubated alone (0, H), with a yeast extract (A. E, I), or with a S 100 Xenopus oocyte extract (8, C, F, G, J, K) at Qac for 1h (A. a, C), at 20aC for 5 min (0, E. F, G) or 30 min (I, J, K).
	Fig. 3. Specific detection of NDPKs transcripts by whole-mount in situ hybridization on gastrula IA-CI and neurula embryos ID-FI. IA and DI NDPK Xl mANA. IB and E) NDPK X2 mRNA. IC and FI NDPK X3 mANA. a. anterior; an, animal,- d. dorsal; dl, dorsal/lp of the blastopore: nf, neural fold: np. neural plate. op, optIC vesicle; P. posref/or; v, vegetative; vn, veneral: ym. yolk mass; yp, yolk plug. Bars. 200 pm.
	Fig. 4. Specific detection of NDPK transcripts by whole-mount in situ hybridization on late neurula (A-C) and tail bud embryos IDFl. IAandDI NDPKXl mRNA. (BandEl NDPK X2 mRNA;IC and FI NDPKX3mRNA Nore rhe staining 10the bralO, optiC vesicle and branChial arches for all mRNAs at the tail bud stage. a, anteflor; p, posterior.
	Fig. 5. Tissue section visualization of the in toto hybridized tail budembryos.IAI NDPKX1 mRNA locahzatlon.IB.EI NDPK X2 mRNA localtzatlon.IF and GI NDPK X3 mRNA. Secllons show the presence of NDPK X, mRNA In the optiC vesicle, telencephalon and brachial arches_ NDPK X2 mRNA is present in the oric vesicle. neural tube, bram and branchial arches. NDPK X3 mRNA IS mainly derecred In the brain, spinal cord and branchial arches. Section 0 shows the Hoechst stamlng of secrlon E 00, branchial arches: en, endoderm: Fb. forebram: Mb. midbrain: Hb, hindbram g, gut; nc, notOChord: ne, neurocoele: nf. neural tube: orv, OtiCvesicle: ov, optic vesicle: p, pharynx: sc, spinal cord: te, telencephalon.
	Fig. 6. NDPK X1 and NDPK X2 mRNA localization in the somites of tail bud embryos. (A-B) NDPK Xl mRNA localization. (D-F) NOPK X2 mRNA localization, IB, F) Hoechst staining of sections In A and In E. respectively. ICI Scheme of the rail bud samires (so) showing rhe alrgnement of nuclei in rhe myotome (n and n+ 1 represent adjacent somites). Arrows pomt on the nuclear localization of X2 mRNA and cytoplasmic localization of Xl mRNA. NDPK XI mRNA;s present in the two anterior and posterior cyroplasmic parts of the samiric cells (A). NDPK X2 mRNA co-localize wirh the nuclei in the center of the sam/tiC cells (D. E, F). NDPK X3 mRNA has the same localization as of NDPK Xl mRNA (data not shown). Section in 0 was revealed by the NBT-BCIP procedure, as opposed to sections in A and E, revealed with the 8M purple procedure (cf methods).
	Fig_ 7. Indirect immunohistofluorescence detection of NDPKs in Xenopus oocytes and early stage embryos. lA-D) Oocytes. A prevlre/logenic oocyte: S, mature oocyre; C. II/rel/agenic oocyte: D. morula macromeres: note the weak staining (faint green' of nuclei compared to cytoplasm. IF' Gastrula endodermaf cells: nore the strong stdlning(yellowJofnuclei.IG-HI stage 44 larvae. G, optic vesIcle cells; note the weak staining of nuclei (arrows). H, Hoechsr sraming of section G. b. Balbiam body: gll, germmalvesic!e of the oocyre, en, endoderm; m, mesoderm; n, nucleoli. Arrows point to nucleI. Bars, 50 pm.