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In the present study, we have isolated a novel gene that is specifically expressed in the ventral region of Xenopus neurula and tailbud embryos. This gene, referred to as ventrally associated leucine-zipper (val), encodes for a novel class of protein consisting of a leucin-zipper motif, a glutamic acid-rich sequence and 5 repeats of proline-rich sequence. Expression of val started at the mid-gastrula stage, peaked at the early tailbud stage, and disappeared by the end of tailbud stage, and the endogenous expression of val was strictly dependent on BMP signaling. Myc-tagged val protein injected at early stage was accumulated in the nucleus at the gastrula stage and later, suggesting involvement of val in the process of ventraltissue formation during the neurula and tailbud stages.
Fig. 3 (Right). Whole-mount in situ hybridization analysis of val in developing embryos. A very faint expression of val message was first detected in the yolk plug (presumptive endoderm) at the mid-gastrula stage (st. 11) (A,B). Expression was gradually accumulated in the posteriorventral part of the neurulaembryo (st. 13-20) (C-F) and peaked at the early tailbud stage (st. 24) (G,H). Dissection of the stained embryo (st. 20) showed that val was expressed in all three germ layers (K). Kshows a control dissected embryo hybridized with the sense probe. During the tailbud stages, the expression of val extended toward anterior and posterior directions (st. 26-28) (I,J,L,M) and two domains of expression areas were separated each other (arrowheads in M). Positive areas of val (N,O), α-globin (P), hex (Q,R) and myosin heavy chain (MHC) (S) at the late tailbud stage (st. 32) were compared each other, suggesting that val-positive area overlapped with hex-positive area (a region of the liver rudiment as indicated by arrowheads in N, O, Q and R). Positive messages were visible in the two spots of anterior and posterior regions (arrowheads in U) in the swimming tadpoles (st. 37/38) (T,U). Scale bars in A and Kshow 1 mm and 500 μm, respectively.
Fig. 4. Regulation of val expression by the BMP signal. (A-C) Embryos were injected with 2ng BMP-4 (B) or 1.5 ng tBR (C) RNA and cultured until the early tailbud stage (st. 23). Uninjected control embryos were also cultured (A). These embryos were fixed and subjected to the whole- mount in situ hybridization analysis for detection of val mRNA. (D) Dorsal marginal zone (DMZ) or ventral marginal zone (VMZ) explants were prepared from the embryos that had been injected with BMP-4 (0.6 or 3 ng) or tBR (1.5 ng) RNA at the 4-cell stage and cultured until the early tailbud stage (st. 20). Explants were subjected for the RT-PCR analysis to detect the expression of val, vent-1, gata-2, nrp-1 and ef1α. Note that val expression is essentially depending on the BMP signal.
Fig. 5. Localization of val protein in nucleus at the gastrula stage. (A)
Biochemical detection of myc-tagged val and mif (Suzuki et al., 2004) by Western blot analysis. Embryos were injected with myc-val and myc-mif RNA (4ng/embryo) into two blastomeres at the 2-cell stage, and they were allowed to develop until st. 24. Soluble proteins extracted from these embryos were loaded in 12.5 % SDS-PAGE. Anti-myc antibody- positive bands were visualized with a peroxidase-based chemi-lumines- cence substrate. (B-G) Whole-mount immunostaining shows subcellular localization of myc-val (B,C) and myc-mif (D,E) proteins at st. 8 (B, D) or st. 12 (C, E). (F,G) Section through the immunostained embryo shows the localization of myc-val protein (F) and nuclei by Hoechst staining (G). Note that myc-val protein is localized in the nucleus (arrowheads in F and G) at the gastrula stage, but not at the early blastula stage. Scale bars indicate 500 μm (B) and 50 μm (F), respectively.
Supplementary Fig. S1. Regulation of val expression by wnt signal. (A-C) Embryos were injected with 50 pg (B) or 200 pg (C) dkk1 RNA and cultured until the tailbud stage (st. 32). Uninjected control embryos were also cultured (A). These embryos were fixed and subjected to the wholemount in situ hybridization analysis for detection of val mRNA. (D) Ventral marginal zone (VMZ) or dorsal marginal zone (DMZ) explants were prepared from the embryos that had been injected with dkk1 RNA (250 pg) or wnt8 DNA (100 pg) at the 4-cell stage and cultured until the tailbud stage (st. 32). Explants were subjected for the RT-PCR analysis to detect the expression of val, pox2 and ef1α. Injection of dkk1 causes anteriorization of the embryos in in situ hybridization analysis (A-C), but the RT-PCR assay indicates that val message is not significantly enhanced by the injection of dkk1 (D).
val (val protein) gene expression in Xenopus laevis embryos, NF stage 11, as assayed by in situ hybridization, vegetal view, anterior up.
val ( val protein ) gene expression in Xenopus laevis embryos, NF stage 28, as assayed by in situ hybridization, lateral view, anteriorleft, dorsal up.
val ( val protein ) gene expression in Xenopus laevis embryos, NF stage 32, as assayed by in situ hybridization lateral view, anteriorleft, dorsal up.
val ( val protein ) gene expression in Xenopus laevis embryos, NF stage 37 and 38, as assayed by in situ hybridization, ventral view, anteriorleft.
