Lustig KD et al. (1996), Expression cloning of a Xenopus T-related gene ...

XB-ART-17325

Development 1996 Dec 01;12212:4001-12. doi: 10.1242/dev.122.12.4001.

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Expression cloning of a Xenopus T-related gene (Xombi) involved in mesodermal patterning and blastopore lip formation.

Lustig KD , Kroll KL , Sun EE , Kirschner MW .

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We have used a functional assay to identify a putative T-box transcription factor (Xombi) that has the ability to induce sites of invagination in the ectoderm of Xenopus embryos that resemble the blastopore lip. Maternal Xombi transcript is first localized to the oocyte's vegetal cortex and cytoplasm, early sources of mesoderm and endoderm-inducing signals. Soon after zygotic transcription begins, there is a wave of Xombi expression, beginning in dorsal mesoderm and then extending to lateral and ventral mesoderm, that precedes and parallels blastopore lip formation at the border between the mesoderm and endoderm. Transcripts encoding brachyury, Xwnt8 and goosecoid colocalize with Xombi transcripts within the marginal zone; ectopic expression of Xombi induces expression of all three mesodermal genes. In ectodermal explants, Xombi expression is induced by the secreted mesoderm inducers activinA, activinB, Xnrl and eFGF, and by brachyury, another Xenopus T-box containing gene. The time course and location of Xombi expression, its biological activities and the partial dependence of Xombi expression and blastopore lip formation on fibroblast growth factor (FGF) signaling suggest that Xombi contributes to a traveling wave of morphogenesis and differentiation during Xenopus gastrulation.

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Species referenced: Xenopus
Genes referenced: dnai1 fgf4 gsc inhba nodal1 tbx2 tbxt vegt wnt8a

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	Fig. 1. Ectopic lip formation by Xombi in normal and ventralized embryos. Xombi RNA (100 pg or 300 pg) was injected at the fourcell stage of development into the animal pole region of a ventral blastomere. (A) Lateral view of a normal early gastrula-stage embryo with a slightly curved ectopic lip. (B) Lateral view of a normal late gastrula-stage embryo with an almost circular ectopic lip. At this stage, the endogenous blastopore is almost closed. (C) Sagittal section through a ventralized embryo showing both endogenous and ectopic lips. (D) A higher magnification view of the endogenous lip shown in C. (E) A higher magnification view of the ectopic lip shown in C. In A, B and C, the endogenous blastopore is indicated by a white arrowhead and the ectopic lip is indicated by a black arrowhead.
	Fig. 2. Xombi encodes a T-box protein related to Xenopus brachyury, mouse Tbx2 and Drosophila optomotor-blind. (A) DNA and putative amino acid sequence of the Xombi cDNA. The putative DNA-binding domain (the T-box) is underlined. (B) Alignment of the putative DNA-binding domains of several Trelated proteins. Amino acid residues identical to those in the putative Xombi T-box are boxed in black.
	Fig. 3. Xombi is expressed maternally and zygotically. Embryos were harvested at the indicated stage and assayed by RT-PCR for expression of Xombi. Elongation factor 1-a (EF-1a) expression was also assayed as a loading control (after blastula stages) and to illustrate the timing of the onset of general zygotic transcription. Each PCR reaction (23 cycles) contained the cDNA equivalent of one-tenth of an embryo.
	Fig. 4. Spatial expression of Xombi during normal development. Oocytes or embryos were hybridized with a digoxigenin-labeled Xombi antisense RNA probe. (A) Animal-vegetal section of a stage III oocyte. (B) Higher magnification view of a different section from the same oocyte. (C) Vegetal view of a stage V oocyte. (D) Animal-vegetal (side) view of a stage VI oocyte. (E) Vegetal view of a stage 9.6 embryo. (F) Vegetal view of a stage 10+ embryo. The arrow denotes the blastopore groove. (G) Vegetal view of a stage 10.5 embryo. (H) Vegetal view of a stage 11 to 11.5 embryo. (I) Parasagittal section through a mid-gastrula embryo. (J) Parasagittal view of a mid-gastrula embryo that has been split open. In I and J, the black arrows denote the forming archenteron and the white arrows denote the boundary of Xombi expression. (K) Dorsal view of a mid-gastrula stage stained for Xombi (light blue stain) and brachyury (magenta stain). Where both co-localize in the marginal zone, a dark blue stain is detected. In dorsal notochord-forming cells, where only brachyury is expressed, only a magenta stain is detected (arrow at anterior end). (L) Dorsal view of a neurula stage embryo. Note that Xombi is still specifically excluded from the notochord. (M) Lateral view of an early tailbud stage embryo. (N) Differential interference contrast (DIC) image of a posterior transverse section of an early tailbud stage embryo showing Xombi staining only within cells in the neural tube. In A, B and D, the animal pole is at the top. In E, F, G, H, I and J, dorsal is to the right. In K, L and M, anterior is to the right. In M and N, dorsal is at the top. All oocytes and embryos except for those in G, H, J and K were cleared in a 2:1 mixture of benzyl benzoate and benzyl alcohol.
	Fig. 5. Xombi expression in ventralized and dorsalized embryos. Embryos were hybridized with a digoxigenin-labeled Xombi antisense RNA probe. (A-C) Uninjected embryos. (D-F) Dorsalized embryos. (G-I) Ventralized embryos. (A,D,G) Vegetal view of stage 9.6 embryos (about 90 minutes prior to the onset of gastrulation). (B,E,H) Vegetal view of stage 10.5 embryos. (C, F,I) Posterior view of stage 13 embryos. Except for A, D and G, which show cleared sibling albino embryos, sibling pigmented embryos were used. The arrows in B and E indicate the location of the blastopore pigment line.
	Fig. 6. Xombi is a general mesoderm inducer. (A) Presumptive ectoderm was explanted from embryos injected with the indicated dose of Xombi RNA and assayed by RT-PCR for the expression of brachyury (Xbra), Xwnt8 or goosecoid (Gsc). EF-1a expression was assayed as a loading control. (B) Lateral view of a Xombi RNAinjected gastrula embryo that has been stained by in situ hybridization for Xwnt8. Xwnt8 expression (light blue stain) is detected in the endogenous marginal zone above the forming blastopore lip as well as in a region of cells above the forming ectopic lip. Dorsal is to the left.
	Fig. 7. Induction of Xombi expression by mesoderm-inducing proteins. Ectodermal explants were harvested from uninjected blastula-stage embryos and then treated with the indicated concentration of activin A protein. In parallel, presumptive ectoderm was also explanted from embryos that had been injected in the animal pole at the two-cell stage with the indicated amount of activinbB, Xnr1, eFGF or brachyury (Xbra) RNA. Explants were harvested when sibling embryos reached the mid-gastrula stage, and then assayed by RT-PCR for Xombi expression. EF-1a expression was assayed as a loading control.
	Fig. 8 Xombi expression and blastopore lip formation are FGFindependent early in gastrulation but FGF-dependent late in gastrulation. Embryos were co-stained by double in situ hybridization for Xombi (magenta stain) and brachyury (light blue stain). A dark blue stain is detected wherever the genes are co-expressed. (A-C) Uninjected embryos. (D-F) Embryos injected dorsally with XFD RNA (1 or 2 ng). (G-I) Embryos injected ventrally with XFD RNA (1 or 2 ng). (A,D,G) Stage 10 embryos in which apically pigmented superficial cells are just becoming apparent. (B,E,H) Stage 10.25 embryos in which the dorsal lip has formed but not extended very far laterally. (C,F,I) Stage 11.5 embryos. All embryos are shown in vegetal view with dorsal at the top.
	Fig. 9. Kinetic model of Xombi expression and blastopore lip formation. A vegetal view of a schematized late blastula to early gastrula Xenopus embryo is shown. Xombi expression (blue) begins dorsally during late blastula stages and has moved circumferentially towards the ventral side of the embryo by early gastrula stages. Blastopore lip formation (red) follows this traveling wave although the site of lip formation is always several cell diameters from the border of Xombi expression in the mesoderm. As mesoderm involution proceeds through the nascent lip (green), Xombi is expressed in a standing wave that forms orthogonal to the traveling wave of lip formation.