Yamada A et al. (2010), Highly conserved functions of the Brachyury gen...

XB-ART-40798

Dev Biol 2010 Mar 01;3391:212-22. doi: 10.1016/j.ydbio.2009.12.019.

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Highly conserved functions of the Brachyury gene on morphogenetic movements: insight from the early-diverging phylum Ctenophora.

Yamada A , Martindale MQ , Fukui A , Tochinai S .

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Brachyury, a member of the T-box transcription family identified in a diverse array of metazoans, was initially recognized for its function in mesoderm formation and notochord differentiation in vertebrates; however, its ancestral role has been suggested to be in control of morphogenetic movements. Here, we show that morpholino oligonucleotide knockdown of Brachyury (MlBra) in embryos of a ctenophore, one of the most ancient groups of animals, prevents the invagination of MlBra expressing stomodeal cells and is rescued with corresponding RNA injections. Injection of RNA encoding a dominant-interfering construct of MlBra causes identical phenotypes to that of RNA encoding a dominant-interfering form of Xenopus Brachyury (Xbra) in Xenopus embryos. Both injected embryos down-regulate Xbra downstream genes, Xbra itself and Xwnt11 but not axial mesodermal markers, resulting in failure to complete gastrulation due to loss of convergent extension movements. Moreover, animal cap assay reveals that MlBra induces Xwnt11 like Xbra. Overall results using Xenopus embryos show that these two genes are functionally interchangeable. These functional experiments demonstrate for the first time in a basal metazoan that the primitive role of Brachyury is to regulate morphogenetic movements, rather than to specify endomesodermal fates, and the role is conserved between non-bilaterian metazoans and vertebrates.

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Species referenced: Xenopus
Genes referenced: chrd foxa4 tbxt wnt11b

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Fig. 3. Injection of RNA encoding MlBra-EnR into Xenopus embryos leads to the similar phenotype to those injected with RNA encoding Xbra-EnR. Embryos injected with RNA encoding MlBra-EnR (A, E, and I–I′), Xbra-EnR (B, F, and J–J′), and EnR (C, G, and K–K′) were compared to one another and to uninjected control embryos (D, H and data not shown). (A–D) Vegetal views of the early gastrulae (stage 10.5) and (E–H) those of the neural fold stage (stage 15) embryos were shown with dorsal to the up. Injection of MlBra-EnR and Xbra-EnR had no influence on the development of normal early gastrulae (compared A and B with C and D) but caused gastrulation defects (E and F, asterisks) while EnR injection was unaffected (G), compared to controls (H). (I–K, I′–K′, and I′–K′) Tailbud embryos derived from the injection were shown laterally. Anterior is to the left. Injections of MlBra-EnR and Xbra-EnR RNA led to posterior truncation (I, J) differently to controls (K) although muscle and notochord were present in such embryos shown by immunostaining with the muscle-specific antibody 12/101 (I′–K′) and the notochord-specific antibody MZ15 (I′–K′), respectively. bp, blastopore; cg, cement grand; yp, yolk plug. Scale bar, 500 μm.

Fig. 5. Xenopus embryos injected with RNA encoding MlBra-EnR and Xbra-EnR show the similar responses at a molecular level. Vegetal views of the injected embryos hybridized using probes for Xbra (A–D and A'–D'), Xwnt11 (E–H and E'–H'), chordin (I–L), and pintallavis (M–P) at the middle gastrula (stage 11) with dorsal to the top. Hybridization signals were detected in purple. LacZ RNA was co-injected into all embryos and its activity was stained in pink. Embryos were injected with RNAs encoding MlBra-EnR (A, A′, E, E′, I and M), Xbra-EnR (B, B′, F, F′, J and N), and EnR (C, C′, G, G′, K and O), together with lacZ RNA. As injection controls, lacZ alone were injected (D, D′, H, H′, L and P). The regions inheriting the injected RNA were enlarged from photographs A–D and E–H to A′–D′ and E′–H′, respectively. The expressions of Xbra and Xwnt11 were down-regulated in cells expressing lacZ activity, that is, including RNA encoding MlBra-EnR (A, A′ and E, E′, respectively) and Xbra-EnR (B, B′ and F, F′, respectively), but not in embryos introduced RNA encoding EnR alone (C, C′ and G, G′, respectively) and in controls (D, D′ and H, H′, respectively). Frequency of the abnormal phenotype was shown in each panel (A–D and E–H). By contrast, the expressions of chordin and pintallavis were little affected in any embryos, irrespectively of the kinds of injected RNA (I–L and M–P, respectively). Frequency of the similar expression pattern to controls was shown in each panel (I–L and M–P). Scale bar, 500 μm.