Schmidt JE et al. (1996), Regulation of dorsal-ventral patterning: the ve...

XB-ART-18148

Development 1996 Jun 01;1226:1711-21. doi: 10.1242/dev.122.6.1711.

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Regulation of dorsal-ventral patterning: the ventralizing effects of the novel Xenopus homeobox gene Vox.

Schmidt JE , von Dassow G , Kimelman D .

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The formation of the dorsal-ventral axis in Xenopus laevis is elicited by a signaling cascade on the dorsal side of the embryo initiated by cortical rotation. These early developmental events impart an initial axial polarity to the embryo. By the time gastrulation occurs, the embryo has established opposing dorsal and ventral regulatory regions. Through a dynamic process, the embryo acquires a definitive pattern that reflects the distribution of future cell fates. Here we present a novel homeobox gene, Vox, whose expression reflects this dynamic process. Vox is first expressed throughout the embryo and subsequently eliminated from the notochord and neural plate. Ectopic expression of Vox demonstrates that the normal function of this gene may be to suppress dorsal genes such as Xnot and chordin, and induce ventral and paraxial genes such as Bmp-4 and MyoD. Ectopic expression of BMP-4 ventralizes embryos and positively regulates the expression of Vox, suggesting that these genes are components of a reciprocal regulatory network.

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Species referenced: Xenopus laevis
Genes referenced: bmp4 chrd evx1 gbx2.2 gsc hmx3 myod1 not otx2 sia1 tbx2 tbxt ventx2 wnt8a

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	Fig. 1. The Xenopus Vox sequence. (A) The nucleotide and predicted amino acid sequence of Vox. The homeodomain is underlined. (B) Comparison of the Vox homeodomain sequence to other homeodomain sequences. The dashes indicate identical amino acids. The percentage amino acid identity within the homeodomain is shown. Xgbx-2 (von Bubnoff et al., 1996), eve (MacDonald et al., 1986; Frasch et al., 1987), Xhox3 (Ruiz i Altaba and Melton, 1989a), Hox1.11 (Tan et al., 1992), and Nkx-5.1 (Bober et al., 1994).
	Fig. 2. Temporal expression of Vox. (A) Northern analysis of the developmental expression of Vox. Poly(A) RNA was isolated from 50 unfertilized eggs or embryos at various stages, separated on a denaturing gel, blotted and hybridized with a 32P-labeled full-length probe made from the Vox cDNA. (U) unfertilized egg; (8) stage 8, midblastula; (9) stage 9, late blastula; (10) stage 10, early gastrula; (12) stage 12, late gastrula; (15) stage 15, midneurula; (18) stage 18, late neurula; (24) stage 24, tailbud. The blot was rehybridized with an EF-1a probe (Krieg et al. 1989) to verify the loading of RNA in each lane (not shown). (B) RNase protection assay showing the temporal expression of Vox. RNA was isolated from five unfertilized eggs (U) or embryos at the indicated hours postfertilization. RNA was analyzed by RNase protection using a mixture of Vox and EF-1a probes. EF-1a is a ubiquitously expressed gene in Xenopus embryos and the levels of expression increase from the MBT. MBT occurs at approximately 6 hours postfertilization.
	Fig. 3. Spatial expression of Vox transcripts during early development. Vox expression was analyzed by whole-mount in situ hybridization using albinos, except for the embryo in D. (A) Vegetal view of a stage 10 embryo, dorsal is upper left. Vox expression is cleared from the region above the blastopore lip. (B) Dorsal view of the same embryo as in A, animal pole is up. Vox is expressed throughout the embryo except for a small region above the dorsal lip. (C) Dorsal view of a stage 11 embryo, animal pole is up. The clearing of Vox expression from the embryo has expanded anteriorly and delineates the regions of the future anterior neural plate and notochord. (D) Dorsovegetal view of a stage 12 embryo, anterior is up. Vox expression flanking the presumptive notochord has become sharpened and the anterior dorsal clearing has broadened and extended anteriorly. (E) Dorsal view of a stage 13-14 embryo, anterior is up. Vox is expressed in the posterior paraxial region of the embryo. Two new spots of anterior expression are emerging (arrowhead) which mark the region dorsal to the developing eye. (F) Lateral view of a stage 19 embryo, anterior is to the left. Vox is expressed diffusely ventrally and with sharp borders along the posterior notochord and floorplate in the posterior paraxial region of the embryo. The staining above the future eye is more distinct at this stage. (G) Lateral view of a stage 24 embryo, anterior is to the left. Diffuse staining is present but diminishing on the ventral side of the embryo. The paraxial expression is now confined to a small region of the developing tailbud. (H) Lateral view of a stage 26 embryo, anterior is to the left. Ventral expression has diminished. Vox is now expressed solely in the developing tailbud flanking the notochord and floorplate and above the developing eye. The dark spot in the dorsal anterior half is a defect in this particular embryo.
	Fig. 4. Histological analysis of Vox expression in sections of albino embryos processed for whole-mount in situ hybridization. (A) Sagittal section of a stage 11 embryo, dorsal is to the left. Vox expression is absent from the dorsal side. (B) Transverse section of a stage 11 embryo, dorsal is up. Vox is cleared from a broad region on the dorsal side of the embryo. (C) Sagittal section of a stage 12 embryo, the blastopore is to the lower right-hand corner of the panel. Ventral is to the right. Vox expression is absent from the involuted and noninvoluted cells on the dorsal side of the embryo. On the ventral side, Vox expression is found in both the involuted mesoderm and the noninvoluted overlying ectoderm, however the expression in the noninvoluted cells appears much stronger. (C¢) Higher magnification of the ventral side of the embryo shown in C, the blastopore is at the bottom of the panel. Vox expression is much stronger in the noninvoluted cells. (C¢¢) Higher magnification of the dorsal side of the embryo shown in C, the blastopore at the lower right-hand corner. Vox expression is absent from the involuted mesoderm as well as the noninvoluted ectoderm on the dorsal side. (D) Parasagittal section of a stage 12 embryo, the blastopore is to the lower right-hand corner of the panel. Ventral is to the right. Vox expression is strongest in noninvoluted cells of the animal ectoderm and the ventral ectoderm, and weaker expression is found in the involuted ventral mesoderm. On the dorsal side, Vox expression is weak in the anterior region of the presumptive neural plate or the anterior noninvoluted cells. However, posteriorly, Vox is expressed in both the involuted and noninvoluted cells. (D¢) Higher magnification of the dorsal side of the embryo shown in D, the blastopore is to the right. Vox expression is weaker in the anterior dorsal noninvoluted cells of the neuroectoderm and is stronger in the noninvoluted and just involuted cells in the posterior region of the embryo.
	Fig. 5. Comparison of Vox, Xnot, chordin and Bmp-4 expression in Xenopus embryos. (A) Dorsovegetal view of uncleared, stage 11 embryos stained for chordin, left, Vox, middle, and Xnot, right, dorsal is up. The expression of Vox is most visible in uncleared embryos since clearing will cause the ventral expression to be visible through the dorsal side. Vox is cleared from the regions in which chordin and Xnot are expressed. (B) Lateral view of stage 24 embryos stained for Xnot, top; Vox, second; chordin, third, and Bmp- 4, bottom, anterior is to the left. At this stage, Xnot and Vox expression is limited to the posterior region of the embryo, with Xnot expressed in the posterior notochord and floorplate and Vox expression cleared from these two developing tissues. Chordin, in contrast, is expressed throughout the notochord at this stage. Bmp-4 expression at this stage is found in ventral anterior and posterior regions of the embryo and is in the developing tail fin. (C) Lateral view of the posterior region of stage 26 embryos stained for Xnot, top and Vox, bottom. Posterior is to the right. Xnot is expressed in two clear regions of expression in the developing tailbud, the developing floorplate and notochord. Vox expression is absent from these two regions in the posterior of the embryo. (D) Stage 31 embryos stained for Xnot, top; Vox, second; chordin, third and Bmp- 4, bottom, anterior is to the left. At this stage, only the most posterior tip of the body axis (the tailbud) contains Xnot and chordin expression. Vox expression surrounds the domains of chordin and Xnot expression. Bmp-4 is expressed ventral to the developing tailbud region and in the surrounding tail fin.
	Fig, 6. Phenotypic effects of Vox RNA injections. 4-5 ng of Vox RNA were injected into the dorsal (A) or ventral (B) marginal zone of 4-cell embryos. (A) The top four embryos were injected with Vox RNA, the closed blastopore is to the right. The bottom embryo is an uninjected, stage 35 control, anterior is to the left. (B) The top two embryos were injected with Vox RNA, the bottom embryo is an uninjected control embryo, anterior is to the left. Embryos are at stage 35.
	Fig. 7. Gene expression in embryos injected with Vox RNA. In all experiments, a percentage of injected and uninjected control embryos were allowed to develop until tadpole stages to score the late-stage phenotype in a each experiment. Injected embryos received 4-5 ng of Vox RNA dorsally, except in the experiment shown in L where embryos were injected ventrally. Vegetal views with dorsal up, except where noted. (A) gsc expression in an uninjected, stage 10.5 embryo. (B) An embryo injected dorsally with Vox RNA; gsc expression is eliminated from the dorsal side of the embryo (100% had no detectable gsc expression, n=29). (C) Xnot expression in an uninjected, stage 12.5 embryo. (D) An embryo injected dorsally with Vox RNA; Xnot expression is eliminated from the dorsal side of the embryo (85% had no detectable notochordal Xnot expression, the remainder had reduced expression, n=39). (E) Dorsovegetal view of Xbra expression in an uninjected, stage 12.5 embryo. Arrowhead indicates notochordal Xbra expression. (F) Xbra expression in a stage 12 embryo injected dorsally with Vox RNA; no notochordal Xbra expression is detectable (100% had no notochordal Xbra expression, n=10). (G) MyoD expression in an uninjected, stage 12 embryo. (H) MyoD expression in a stage 12 embryo injected dorsally with Vox RNA; MyoD is expressed along the dorsal midline (100% had radial MyoD expression, n=23). (I) Xwnt-8 expression in an uninjected, stage 10 embryo. (J) Xwnt-8 expression in a stage 10 embryo injected dorsally with Vox RNA; Xwnt-8 expression appears unaffected (100% had normal Xwnt-8 expression, n=17). (K) Xwnt-8 expression in an uninjected, stage 10 embryo. (L) Xwnt-8 expression in a stage 10 embryo injected ventrally with Vox RNA; Xwnt-8 is eliminated from the ventral region of the embryo (100% lacked ventral expression of Xwnt-8, n=20). (M) Dorsovegetal view of Bmp-4 expression in an uninjected, stage 12 embryo. (N) Bmp-4 expression in a stage 12 embryo injected dorsally with Vox RNA; Bmp-4 is expressed both dorsally and ventrally (95% had radial Bmp-4 expression, n=21). (O) Dorsovegetal view of chordin expression in an uninjected, stage 12.5 embryo, dorsoanterior is up. (P) An embryo injected dorsally with Vox RNA; chordin expression is eliminated from the dorsal side of the embryo (96% had little or no detectable chordin expression, n=23). (Q) Dorsovegetal view of Xotx2 expression in an uninjected, stage 12.5 embryo, dorsoanterior is up. (R) An embryo injected dorsally with Vox RNA; Xotx2 expression is eliminated from the dorsal side of the embryo (100% had no detectable Xotx2 expression, n=12). (S) Dorsovegetal view of Hairy II expression in an uninjected, stage 12.5 embryo, dorsoanterior is up. (T) An embryo injected dorsally with Vox RNA; Hairy II is weakly expressed on the dorsal side and no longer delineates the neural plate or floorplate (100% had faint, diffuse, dorsal Hairy II expression, n=27).
	Fig. 8. RNase protection analysis of the levels of Bmp-4 expression in dorsal and ventral quarters of uninjected stage 12.5 embryos and stage 12.5 embryos injected dorsally with 4 ng Vox RNA. An EF-1a probe was included to control for RNA loading. Except for the whole embryo control, each sample was derived from 10 dorsal or ventral quarters. The bar graph at the bottom represents levels of Bmp-4 expression in each lane normalized to EF-1a. Bmp-4 is expressed in nearly equal amounts on the dorsal and ventral sides of Vox RNAinjected embryos. This experiment was repeated and produced the same results (data not shown).
	Fig. 9. Ectopic BMP-4 expression prevents the dorsal clearing of Vox expression in stage 11 embryos. Vegetal views, dorsal is up. A. Vox expression in an uninjected, stage 11 embryo. B. Vox expression in a stage 11 embryo injected dorsally with 4 ng Bmp-4 RNA; Vox is radially expressed (69% had radial Vox expression, 31% had reduced dorsal clearing, n=16).
	Fig. 10. A hypothetical model of the gene regulatory network active during gastrulation in Xenopus. The set of interactions shown in this diagram reflect the minimum necessary to account for the observations discussed here, and is not intended to be the only possible hypothesis. Arrows indicate positive regulation, boxes indicate negative regulation; the interactions between the molecules shown may be either direct or indirect, transcriptional or posttranscriptional. As shown in this work, BMP-4 and Vox promote each other’s expression. Chordin, meanwhile, inhibits BMP-4 function, but is itself repressed by Vox. The positive loop between Vox and BMP-4 is self-perpetuating. Vox may either directly repress chordin transcription, or function indirectly by regulating Xnot and gsc. Siamois may provide an early bias that distinguishes the dorsal from the ventral side of the embryo.