Kitaguchi T et al. (2002), Xenopus Brachyury regulates mesodermal expressi...

XB-ART-7562

Dev Growth Differ 2002 Feb 01;441:55-61. doi: 10.1046/j.1440-169x.2002.00624.x.

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Xenopus Brachyury regulates mesodermal expression of Zic3, a gene controlling left-right asymmetry.

Kitaguchi T , Mizugishi K , Hatayama M , Aruga J , Mikoshiba K .

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The Brachyury gene has a critical role in the formation of posterior mesoderm and notochord in vertebrate development. A recent study showed that Brachyury is also responsible for the formation of the left-right (L-R) axis in mouse and zebrafish. However, the role of Brachyury in L-R axis specification is still elusive. Here, it is demonstrated that Brachyury is involved in L-R specification of the Xenopus laevis embryo and regulates expression of Zic3, which controls the L-R specification process. Overexpression of Xenopus Brachyury (Xbra) and dominant-negative type Xbra (Xbra-EnR) altered the orientation of heart and gut looping, concomitant with disturbed laterality of nodal-related 1 (Xnr1) and Pitx2 expression, both of which are normally expressed in the left lateral plate mesoderm. Furthermore, activation of inducible type Xbra (Xbra-GR) induces Zic3 expression within 20 min. These results suggest that a role of Brachyury in L-R specification may be the direct regulation of Zic3 expression.

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Species referenced: Xenopus laevis
Genes referenced: bix1.1 bix1.3 nodal nodal1 pitx2 tbxt wnt11 zic3

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	Fig. 1. Reversal of heart and gut looping in Xbra-injected embryos. (A) Xbra constructs used in this study. EnR and GR indicate the repressor derived from the Drosophila engrailed protein and the hormone-binding domain of human glucocorticoid receptor protein, respectively. (B) Wild-type Xenopus embryo (stage 47; control) with a rightward-looping heart and a counterclockwise-coiled gut, and an embryo that was injected with Xbra mRNA showing a leftward-looping heart and clockwise coiled-gut (Xbra). (C) The frequency of the reversed organs in left-sided (L) or right-sided (R) injection of Xbra (2000 pg), Xbra-EnR (1000 pg), activinB (0.5 pg) mRNA, and uninjected (control) embryos (see also Table 1).
	Fig. 2. Asymmetric expression of Xnr1 and Pitx2 is disturbed by Xbra or Xbra-EnR. (A) In situ hybridization of Xnr1 and Pitx2. Xnr1 and Pitx2 expression patterns were observed in the left lateral plate mesoderm (LPM) of the uninjected control embryos (control). Xbra-injected embryos show bilateral or right side expression (Xbra). (B,C) The frequency of Xnr1 and Pitx2 expression disturbed by Xbra (2000 pg), Xbra-EnR (1000 pg), activinB (0.5 pg), and uninjected (control) embryos (see also Table 2).
	Fig. 3. Xbra specifies left–right (L–R) laterality at the early gastrula stage. Disturbed expression of Pitx2 in Xbra-GR (400 pg)-injected embryos was observed when dexamethasone was added at several stages. The frequency of Pitx2 expression disturbed by Xbra-GR injection (see also Table 3). DEX, dexamethasone.
	Fig. 4. Xbra can induce Zic3 in the mesoderm. (A) Zic3 expression (purple) is shown in the cross-section of the gastrula. Animal pole is top. The embryos were cut along the broken line shown. Zic3 is expressed symmetrically in wild-type embryos (control). Zic3 expression was enhanced in the mesoderm at stage 10.5 when Xbra (2000 pg) was injected into the lateral side of one blastomere at the 2-cell stage (Xbra). Xbra-EnR suppressed the Zic3 expression at the injected side (Xbra-EnR). Coinjected LacZ expression is indicated in blue. inj, injected side; D, dorsal side; V, ventral side; bp, blastopore. (B) Reverse transcription (RT)–polymerase chain reaction (PCR) analysis of animal cap explants from embryos injected with Xbra (2000 pg) and/or Xbra-EnR (1000 pg). Zic3 was induced by Xbra whereas the expression was reduced in the presence of Xbra-EnR. (C) RT-PCR analysis of Zic3 (100 pg) overexpressed animal cap explants. Xnr1 and Pitx2 were induced by Zic3, but Xbra was not. (D) Zic3 was induced by Xbra-GR (400 pg) as early as 20 min after adding dexamethasone. This temporal profile is comparable to that of Bix1 and Wnt11 which have been shown to be regulated directly by Xbra. RT-PCR analyses were also carried out with RNA that had not been reverse transcribed (RT–), RNA from sibling embryos (Embryo) and uninjected animal cap explants (uninj).