Horb ME and Thomsen GH (1997), A vegetally localized T-box transcription facto...

XB-ART-16557

Development 1997 May 01;1249:1689-98. doi: 10.1242/dev.124.9.1689.

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A vegetally localized T-box transcription factor in Xenopus eggs specifies mesoderm and endoderm and is essential for embryonic mesoderm formation.

Horb ME , Thomsen GH .

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Pattern formation in early embryogenesis is guided by maternal, localized determinants and by inductive interactions between cells. In Xenopus eggs, localized molecules have been identified and some, such as Vg1 and Xwnt-11, can specify cell fates by functioning as inducers or patterning agents. We have used differential screening to identify new Xenopus genes that regulate mesodermal patterning, and we have isolated a new member of the T-box family of transcription factors. This gene, named Brat, is expressed maternally and its transcripts are localized to the vegetal hemisphere of the egg. During early embryonic cleavage, Brat mRNA becomes partitioned primarily within vegetal cells that are fated to form the endoderm. Zygotic expression of Brat begins at the onset of gastrulation within the presumptive mesoderm of the marginal zone. Consistent with its zygotic expression pattern, Brat induces, in a dose-dependent manner, a full spectrum of mesodermal genes that mark tissues across the dorsal-ventral axis, from the blood through the Spemann organizer. Brat also induces endoderm, consistent with its vegetal localization, making Brat a good candidate for a maternal determinant of the endoderm. We tested whether endogenous Brat is required for mesoderm formation by expressing a dominant-negative, transcriptional repressor form of Brat in embryos. This treatment inhibited mesoderm formation and severely disrupted normal development, thereby establishing that Brat plays a critical role in embryonic mesoderm formation and body patterning.

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Species referenced: Xenopus laevis
Genes referenced: acta4 actc1 actl6a bmp4 chrd clstn2 evx1 fabp2 gdf1 gsc hoxa9 hoxb9 hoxc9 myod1 pdx1 tbx2 tbxt trg vegt wnt8a

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	Fig. 1. The Brat Protein. (A) The predicted amino acid sequence of Brat encodes a protein of 456 amino acids. The DNA-binding domain, T domain, is located between the arrows and is highlighted in bold. (B) A comparison of the T domain of Brat with six other T domain containing proteins. Identical amino acids are indicated by dots, and spaces (dash) were introduced to maximize homology. The degree of identity between these domains and the T domain of Brat is: Xenopus Brachyury (Xbra, 47%), mouse Brachyury (T, 45%), zebrafish T (Zf-T, 46%), Drosophila T-related gene (Trg, 45%), mouse Tbx2 (54%), and Drosophila optomotor blind (Omb, 51%). The accession number for Brat is U89707.
	Fig. 2. Expression pattern of the Brat gene in Xenopus development. (A) A Developmental northern blot on total embryonic RNA revealed a single Brat transcript of approximately 3 kb that is expressed maternally and zygotically into mid-neurulation (Stage 15). Stages analyzed were egg, blastula (stages 7,9), gastrula (stage 11), neurula (stages 15,18) tailbud tadpole (stage 26) and swimming tadpole (stage 38). Five embryo equivalents were loaded per lane and RNA recovery and integrity was equal across the blot (not shown). (B) Localization of Brat mRNA in oocytes by whole-mount in situ hybridization. Brat mRNA is expressed throughout oogenesis and is localized to the vegetal pole by stage II. Pigmented oocytes (P) are shown in the upper panel, animal pole up, to orient the stain relative to the animal and vegetal pole. Albino oocytes (A) are shown in the lower panel to clearly display the domain occupied by Brat transcripts. During the in situ procedure, the cortex was partially removed from several stage VI oocytes to demonstrate that the bulk of Brat transcripts are sequestered in the cortex. Arrows indicate the edge where the cortex was torn off, revealing the underlying, unstained cytoplasm. (C) Vegetal localization of Brat mRNA in early cleavage stage embryos. A northern blot of RNA from animal and vegetal halves of stage 6 embryos. Seven explants or three embryos were analyzed and the blot was reprobed with Histone H4 to contrast the localization of Brat mRNA with that of a cytoplasmic mRNA.
	Fig. 3. Comparison of the spatial distribution of Brat and Xbra transcripts during gastrulation by whole-mount in situ hybridization. Brat expression is shown in the upper panel, Xbra expression is shown in the lower panel. (A,D) Zygotic expression of Brat and Xbra is first detected just prior to the appearance of the dorsal blastopore lip (arrow), stage 10-. (B,E) By stage 10+ both Brat and Xbra are expressed throughout the marginal zone. (C,F) During late gastrulation, stage 12, Brat is expressed in the ventrolateral mesoderm surrounding the yolk plug, but not in the axial mesoderm of the future notochord (arrow). Xbra is also expressed in the ventrolateral mesoderm, but, in contrast to Brat, it is expressed in the notochord (arrow).
	Fig. 4. Analysis of mesoderm induction by Brat and Xbra in animal caps. (A) Comparison of Xbra and Brat activities reveals that both induce the ventrolateral markers Xwnt-8 (Christian et al., 1991) and XMyoD (Frank and Harland, 1991); only Brat however, induces the dorsal mesoderm markers XFKH-1 (Dirkson and Jamrich, 1992) and chordin (Sasai et al., 1994). (B) A more detailed examination of mesoderm induction by Brat. At low doses of injected mRNA (14- 42 pg), Brat induced ventralposterior mesoderm, as scored by the expression of Xwnt-8, Xhox3 (Ruiz i Altaba and Melton, 1989), Xlhbox6 (Wright et al., 1990), Xbra, and XMyoD. At intermediate doses (42-140 pg) XFKH-1 is induced, followed by organizer-specific genes goosecoid (gsc) (Cho et al., 1991) and chordin at higher doses (350-700 pg). (C) In tadpole stage 28 animal caps Brat induces globin, a marker for blood (the most ventral mesoderm derivative), and muscle actin, which marks dorsal mesoderm. Animal caps were injected with synthetic mRNA at the 2-cell stage and mesoderm induction was analyzed by RT-PCR at either stage 11 (A,B) or stage 28 (C).
	Fig. 5. Brat induces mesodermal tissues. (A) Control-injected animal caps form solid balls of atypical epidermis. (B) At low doses, 50 pg, Brat-injected animal caps form vesicles, characteristic of ventral mesoderm. Caps in (A) and (B) were scored at stage 28. (C-F) Whole-mount staining for muscle with the 12/101 antibody. (C) Sibling stage 24 whole embryo. Muscle tissue is stained in the segmented somites. (D) CS2-injected animal caps, 250 pg mRNA. No muscle tissue formed, n=11. (E) Brat-injected animal caps, 500 pg mRNA. Muscle formed in 33% of the injected caps, n=27. (F) BVg1-injected animal caps, 5 pg mRNA. Muscle staining is seen in all caps, n=22.
	Fig. 6. RT-PCR analysis of endoderm induction by Brat in animal caps at stage 28. Brat induces the endodermal markers, intestinal fatty acid binding protein (IFABP) and Xlhbox8. IFABP is a general marker of endoderm while Xlhbox8 is an anterior endoderm marker of pancreas and liver.
	Fig. 7. Brat is induced by peptide growth factors in the FGF and TGF-b families. (A) Brat displays dose-dependent induction by FGF or activin protein. Animal caps were excised at stage 8-9 and exposed to the indicated concentrations of FGF or activin protein. Caps were harvested at stage 11, and Brat induction was analyzed on a northern blot. RNA recovery was equal across the blot (not shown). (B) Brat is induced by BVg1 and BMP-4. Caps were injected with 30 pg of BVg1 mRNA or 1.8 ng BMP-4 mRNA, excised at stage 9, harvested at stage 11, and scored for Brat expression by RT-PCR. EF1-alpha expression is a control for RNA recovery and cDNA synthesis. Maternal Brat transcripts in the animal pole (see Fig 2c) account for the background signal in vector-injected animal caps.
	Fig. 8. A repressor form of Brat, Brat-EnR, inhibits expression of dorsal and ventral mesodermal markers. (A) Schematic diagram of wild-type Brat (upper) and a chimera of the N terminus of Brat and the Drosophila engrailed repressor domain, Brat-EnR (lower). The Tbox is red, while the engrailed repressor domain is black. (B) Diagram illustrating the experimental design. 2 ng Brat-EnR and 0.5 ng LacZ mRNA was injected into the marginal zone of both blastomeres along the first cleavage plane at the 2-cell stage. Albino embryos were used in which dorsal-ventral differences are not apparent and therefore the site of injection is randomized with respect to the dorsal or ventral side. (C,F) Wild-type whole-mount expression patterns of Xbra and gsc. (D,G) Vegetal view of marker expression in embryos injected with Brat-EnR. (E,H) Animal view of the same embryos in D,G to show b-gal staining in the region coinjected with Brat-EnR and Lac Z mRNA. Note that the expression of gsc and Xbra is absent on the side expressing Brat-EnR. Xlim expression was also absent in these embryos (data not shown). Since the site of injection along the dorsal-ventral axis was randomized, gsc expression was present opposite the b-gal stain in 50% of the embryos (n=30), as expected. Xbra expression, on the contrary, was always eliminated in the injected cells, whether injected dorsally or ventrally.
	Fig. 9. Brat-EnR disrupts mesodermal patterning. (A) Vegetal view stage 11 wild-type embryo, positioned with the dorsal blastopore lip at the bottom. (B) Dorsal injection of Brat-EnR inhibits dorsal blastopore lip formation. (C) Ventrally injected embryo shows normal dorsal lip formation, but ventral lip formation is blocked. (D) Stage 35 wild-type embryo. (E) Embryos injected in the dorsal marginal zone with Brat- EnR mRNA do not develop anterior head structures (note the lack of eyes) and the neural plate does not close (arrow). (F) Ventral injection of Brat-EnR disrupts posterior mesoderm development, but anterior development is not affected. A total of 2 ng of Brat-EnR was injected into two dorsal or two ventral blastomeres at the 4-cell stage.
	Fig. 10. Rescue of Brat-EnR phenotypes by Brat but not by Xbra. (A) Wild-type stage 34 embryo. (B) Embryos injected with 250 pg Brat-EnR into the lateral marginal zone. At this dose, the embryos have kinked backs and forked tails, and sometimes incomplete closure of the neural plate. (C) A 2:1 ratio of wild-type Brat: Brat- EnR rescues the embryonic defects caused by Brat-EnR. (D) A 2:1 ratio of Xbra: Brat-EnR does not rescue the Brat-EnR defects. In fact, no dose of Xbra tested (0.25:1 to 5:1) rescued the Brat-EnR phenotype.