Suri C et al. (2004), Inhibition of mesodermal fate by Xenopus HNF3be...

XB-ART-4212

Dev Biol 2004 Jan 01;2651:90-104. doi: 10.1016/j.ydbio.2003.09.017.

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Inhibition of mesodermal fate by Xenopus HNF3beta/FoxA2.

Suri C , Haremaki T , Weinstein DC .

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The winged-helix transcription factor HNF3beta/FoxA2 is expressed in embryonic organizing centers of the gastrulating mouse, frog, fish, and chick. In the mouse, HNF3beta is required for the formation of the mammalian node and notochord, and can induce ectopic floor plate formation when misexpressed in the developing neural tube; HNF3beta expression in the extraembryonic endoderm is also necessary for the proper morphogenesis of the mammalian primitive streak. In the frog Xenopus laevis, several lines of evidence suggest that the related winged-helix factor Pintallavis functions as the ortholog of mammalian HNF3beta in both axial mesoderm and neurectoderm; the role of Xenopus HNF3beta itself, however, has not been clearly defined, and is the subject of this study. HNF3beta is widely expressed in the vegetal pole but, as previously suggested, is excluded from the gastrula-stage mesoderm. We find that expression of an HNF3beta-Engrailed repressor fusion protein induces ectopic axes and inhibits head formation in Xenopus embryos, while ectopic HNF3beta inhibits mesoderm and anterior endoderm formation in explant assays and in vivo. Our studies suggest that HNF3beta target genes function to limit the extent of mesoderm formation in the Xenopus gastrula, and point to related roles for Xenopus HNF3beta and the extraembryonic component of mammalian HNF3beta during vertebrate gastrulation.

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Species referenced: Xenopus laevis
Genes referenced: chrd foxa2 foxa4 tfap2a ttr

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	FIG. 1. RT-PCR analysis of HNF3β expression during early Xenopus development. (A) Temporal expression of Xenopus HNF3β. ornithine decarboxylase (ODC), expressed uniformly throughout early embryogenesis, is used as a loading control (Bassez et al., 1990). chordin expression, like that of HNF3β, is initiated zygotically. (B, C, D) Spatial localization of HNF3β transcripts during early gastrula stages. Schematic diagrams of stage 10+ embryos, showing regions isolated for expression analysis, are shown above gels. (B) HNF3β transcripts are excluded from gastrula-stage dorsal mesoderm. meso: epithelial and deep dorsal mesoderm; endo: dorsal endomesoderm. (C) HNF3β is expressed throughout the endoderm during early gastrula stages. AE: anterior endoderm; PE: posterior endoderm. (D)HNF3β transcripts are excluded from the ectoderm and ventral mesoderm during early gastrula stages. AnC: animal cap; PC: posterior cortex; PD: posterior deep. EF1-α is used as a loading control (Krieg et al., 1989). The –RT lane contains all reagents except reverse transcriptase, and is used as a negative control.
	FIG. 2. Effects of ectopic expression of HNF3β. (A, B) HNF3β disrupts gastrulation and inhibits mesoderm formation in intact embryos. (A) 8-cell stage embryos were injected dorsally with 2 ng HNF3β RNA and cultured until stage 32; top embryo is an uninjected control. (B) 8-cell embryos were injected with both 100 pg HNF3β and 200 pgβ-Gal RNA; top embryo is an uninjected control. Embryos were probed with the somite-specific antibody 12/101 (Kintner and Brockes, 1984). (C) Gel shift analysis of HNF3β and HNF3β-MT; in vitro translation products are shown at left. TTR, specific competitor; AP2, non-specific competitior; control, no RNA included in in vitro translation. (D) HNF3β-MT does not affect dorsal development. 8-cell stage embryos were injected dorsally with 250 pg of either HNF3β RNA (top) or HNF3β-MT RNA (bottom). (E) HNF3β is a more potent inhibitor of dorsal development than Pintallavis. 8-cell stage embryos were injected dorsally with 250 pg of either HNF3β RNA (left) or pintallavis RNA (right). All embryos are lateral views, anterior is to left in (A), (B); anterior is to right in (D), (E).
	Fig. 3. Effects of HNF3β on marginal zone explants. (A) Injection of HNF3β RNA inhibits development of dorsal mesoderm. RT-PCR analysis of dorsal marginal zone (DMZ) explants harvested at late neurula stages. (B) Ventral injection of HNF3β RNA dorsalizes mesoderm, but does not induce secondary axis formation. RT-PCR analysis of ventral marginal zone (VMZ) explants harvested at late neurula stages (top); lateral views of stage 35 embryos, anterior is to right (bottom). (C) Ventral injection of pintallavis RNA dorsalizes mesoderm. RT-PCR analysis of ventral marginal zone (VMZ) explants harvested at late neurula stages. (D) Injection of HNF3β RNA inhibits mesoderm induction and dorsoanterior development in early gastrulae. RT-PCR analysis of marginal zone explants harvested immediately after dissection at early gastrula stages. 250 pg (A, B, C) or 500 pg (D) HNF3β or pintallavis RNA was injected, as listed, into 4–8 cell stage embryos.
	FIG. 4. Chimeric HNF3β constructs used in this study. See text for details.
	FIG. 5. Inhibition of mesoderm by activated HNF3β constructs. (A) Injection of VP-HDNAB RNA disrupts gastrulation and inhibits mesoderm formation in intact embryos. Embryos were injected dorsally with VP-HDNAB RNA, cultured until stage 39, and probed with the somite-specific antibody 12/101 (Kintner and Brockes, 1984). Embryos are lateral views; anterior is to left. (B) Dorsal and ventral marginal zone expression of VP-HDNAB RNA inhibits mesoderm formation. RT-PCR analysis of marginal zone explants harvested immediately after dissection at early gastrula stages. (C) VP-HDNAB-mediated inhibition of mesoderm formation is rescued by co-expression of EnR-HNF3β RNA. RT-PCR analysis of animal caps dissected at late blastula stages and cultured until midgastrula stages. 1 ng VPHDNAB or EnR-HNF3β RNA was injected at early cleavage stages, as listed. 0.5 ng/mL Activin was added to stage 9 animal caps, as listed.
	FIG. 6. Effects of HNF3β target gene inhibition in the anterior endoderm. (A) Dorsal vegetal injection of 250 pg EnR-HNF3β RNA inhibits head formation. Left panel: lateral view of stage 40 embryos; anterior is to left. Embryo at top is an uninjected control. Right panel: EnR-HNF3β RNA induces ectopic structures, resembling secondary axes, in a subset (approximately 10%) of injected embryos; these structures do not contain somitic mesoderm (data not shown). Lateral views of stage 28 embryos; anterior is to left. Embryo at top is an uninjected control. (B) Dosal vegetal cells expressing EnR-HNF3β populate the head and anterior gut. Embryos injected in dorsal vegetal blastomeres with 200 pg β-Gal RNA (top) or both 250 pg EnR-HNF3β and 200 pg β-Gal RNA (bottom), and stained with X-gal as a substrate. Lateral views of stage 32 embryos; anterior is to left; embryo in bottom panel, viewed at higher magnification, was cleared in 2:1 benzyl benzoate/benzyl alcohol. (C) EnR-HNF3β-MT does not affect dorsal development. Top panels: gel shift analysis of EnRHNF3β and EnR-HNF3β-MT; in vitro translation products are shown at left. TTR, specific competitor; AP2, non-specific competitior; control, no RNA included in in vitro translation. Bottom panel: lateral views of stage 35 embryos; anterior is to left. 8-cell stage embryos were injected dorsovegetally with 250 pg of EnR-HNF3β-MT RNA. (D) Dorsal suppression of HNF3β target genes alters mesendodermal gene expression. RT-PCR analysis of dorsal explants dissected and immediately harvested at early gastrula stages; explants used for lane 1 were isolated from embryos injected with 250 pg EnR-HNF3β RNA in both dorsal vegetal blastomeres at the 8-cell stage. (E) Pintallavis-EnR does not affect dorsal development. Top panel: RT-PCR analysis of dorsal explants dissected and immediately harvested at early gastrula stages; explants were isolated from embryos injected with 250 pg of either EnRHNF3β RNA or pintallavis-EnR RNA in both dorsal vegetal blastomeres at the 8-cell stage. Bottom panel: Lateral view of stage 28–30 embryos injected with 250 pg pintallavis-EnR RNA in both dorsal vegetal blastomeres at the 8-cell stage; anterior is to right.
	FIG. 7. Effects of HNF3β target gene inhibition in the posterior/ventral endoderm. (A) Ventral vegetal blastomeres expressing EnR-HNF3β RNA (250 pg) contribute directly to ectopic axes. 8-cell stage embryos were co-injected with 250 pg EnR-HNF3β and 200 pgβ-Gal RNA, and stained with X-gal as a substrate; stain is found predominantly in the ectopic structures. Lateral view of stage 32 embryos; anterior is to right. (B) Secondary axes induced by ventral vegetal injection of EnR-HNF3β RNA (250 pg) contain dorsolateral mesoderm. Embryos were co-injected as in (A), stained with X-gal as a substrate, and probed with the somite-specific antibody 12/101 (Kintner and Brockes, 1984). Lateral view of stage 32 embryos; anterior is to right. Embryos were cleared in 2:1 benzyl benzoate/ benzyl alcohol. (C) Injection of EnR-HNF3β RNA dorsalizes ventral explants. RT-PCR analysis of ventral explants harvested at mid-neurula stages; explants used for lane 1 were isolated from embryos injected with 250 pg EnR-HNF3β RNA in both ventral vegetal blastomeres at the 8-cell stage. (D) Ventral suppression of HNF3β target genes induces dorsoanterior mesendoderm. RT-PCR analysis of ventral explants dissected and immediately harvested at early gastrula stages; explants used for lane 1 were isolated from embryos injected with 250 pg EnR-HNF3β RNA in both ventral vegetal blastomeres at the 8-cell stage.

References [+] :

Ang, A targeted mouse Otx2 mutation leads to severe defects in gastrulation and formation of axial mesoderm and to deletion of rostral brain. 1996, Pubmed