Zhang J and King ML (1996), Xenopus VegT RNA is localized to the vegetal co...

XB-ART-17323

Development 1996 Dec 01;12212:4119-29. doi: 10.1242/dev.122.12.4119.

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Xenopus VegT RNA is localized to the vegetal cortex during oogenesis and encodes a novel T-box transcription factor involved in mesodermal patterning.

Zhang J , King ML .

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An RNA localized to the vegetal cortex of Xenopus oocytes encodes a novel T-box protein (VegT) capable of inducing either dorsal or posterior ventral mesoderm at different times in development. VegT is a nuclear protein and its C-terminal domain can activate transcription in a yeast reporter assay, observations consistent with VegT functioning as a transcription factor. Zygotic expression is dynamic along the dorsoventral axis, with transcripts first expressed in the dorsal marginal zone. By the end of gastrulation, VegT is expressed exclusively in posterior ventral and lateral mesoderm and is excluded from the notochord. Later expression is confined to a subset of Rohon-Beard cells, a type of primary sensory neuron. In animal cap assays, VegT is capable of converting prospective ectoderm into ventral lateral mesoderm. Such ectopic expression of VegT induces its own expression as well as that of Xwnt-8 in caps, suggesting that a Wnt pathway may be involved. Mis-expression of VegT in dorsal animal blastomeres fated to contribute to brain suppresses head formation. Our results suggest that VegT is a localized transcription factor, which operates sequentially in several developmental pathways during embryogenesis, including dorsoventral and posterior patterning of mesoderm.

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Species referenced: Xenopus
Genes referenced: lgals4.2 myc tbxt vegt
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	Xenopus laevis VegT expression in an ovulated egg. Image from Zhang and King (1996).
	Fig. 1. VegT contains a T-domain found in proteins from C. elegans, Drosophila, Xenopus and mammals and is expressed both maternally and zygotically. (A) Alignment of VegT with the T-box DNAbinding domain in other proteins. mTbx2, mouse Tbox2; omb, Drosophila optomotor blind; F21H11.3, presumptive protein from the Caenorhabditis genome project; VegT, Xenopus VegT; Xbra, Xenopus Brachyury. Identical residues are shown on a black background. (B) Vegetally localized expression of VegT. The total RNA equivalent of five stage VI oocyte halves was loaded on each lane and hybridized with VegT and FGFR probes. V, vegetal; A, animal; T, whole embryo. (C) Developmental expression of VegT. The RNA equivalent of 3 embryos was loaded per lane. OV, ovulated egg; 4, 4-cell embryos; other staging according to Nieuwkoop and Faber (1976). In B and C, FGFR expression was used as a control for RNA loading.
	Fig. 2. Spatial expression of VegT during oogenesis and embryogenesis. Whole-mount in situ hybridization with digoxigenin-labeled VegT RNA probe. (A) Stage I oocytes. VegT is uniformly distributed. (B) Stage IV oocyte. VegT is localized to the vegetal hemisphere. (C) Ovulated egg. VegT remains in the vegetalhemisphere. (D) Stage 9.5 embryo, side view. Zygotic VegT is expressed on the dorsal side with significantly lower expression laterally and ventrally within the marginal zone. Animal pole is at the top. Blastocoel is somewhat collapsed. (E) Stage 9.5 embryo, vegetal pole view of the embryo in D. Staining mostly restricted to the dorsal side of the embryo. (F) Stage 10.25 embryo, vegetal pole view. Staining still mostly on the dorsal side, but an increase along the lateral and ventral sides is now apparent. (G) Stage 10.5 embryo, vegetal view. Staining in entire marginal zone. (H) Stage 12.5 embryo, posterior view. Ventral and lateral staining around the blastopore. Note the dorsalmost region is not stained. (I) Dorsal view of a cleared mid neural fold embryo (stage 16). VegT RNA is restricted to the posterior end. (J) Side view of a tail bud embryo (stage 23). Newly transcribed VegT RNA within a posterior subset of primary sensory neuron cells (arrowhead). (K) Side view of a tadpole (stage 31). Staining only within the same neurons (arrowhead) as shown in J. Staining observed at the anterior end of the embryo is non-specific.(K') Transverse section of stage shown in K. Stainingis in the large primary sensory neurons found within the dorsal lateral spinal cord (arrowhead). nt, neural tube; nc, notochord. (L) High magnification of the same embryo in K.
	Fig. 3. VegT can function as a transcription factor. (A) Nuclear localization of myc-tagged VegT protein. Single blastomere of a 2- cell embryo was injected at the animal pole with MT-VegT RNA. Shown is a stage 9 embryo stained with myc antibody 9E10. Progeny of the uninjected blastomere are on the right and served as a negative control. (B) Yeast transcription reporter assay. Three colonies from each transformation group were transferred to a filter and stained with X-gal. Each construct contains the Gal4 DNA-binding domain fused with either vector alone (pGBT), VegT C-terminal 176 amino acids (pGBT/VT-AD), or the Gal4 activation domain (pGBT/G-AD).
	Fig. 4. Secondary axis formation after VegT RNA injection into vegetal/ventral blastomeres. VegT RNA was injected into one of the vegetal ventral blastomeres of the 8-cell embryo. (A) Diagrammatic representation of the polypeptides synthesized from the injected RNAs. On the top is the deduced full-length VegT peptide with the T-box in black, the nuclear localization signal (NLS) in dark-grey and the activation domain in light-grey. The bottom diagram is the truncated form missing the C-terminal region, the putative activation domain. (B) Stage 10.5 VegT-injected embryo showing the primary (I) and secondary (II) dorsal blastopore lips. (C) Control embryos (stage 10.5) injected with the truncated form VegTDAD or buffer showing only one lip. (D) VegT-injected embryos (stage 33) showing primary axes (I) and partial secondary axes (II). (E) Control embryos (stage 33) showing only primary axes. (F) Section of a VegT-injected embryo showing large blocks of muscle (in green) and a neural tube in the secondary axis. nt, neural tube; nc, notochord. (G) VegTinjected embryos were stained with a notochord marker (MZ-15) antibody at stage 33 and cleared. Primary (I) and secondary(II) notochords are indicated. Extra auditory vesicles (arrowhead) are present.
	Fig. 5. Lineage tracing of VegT-injected blastomeres. (A) VegT and nbgal RNAs were co-injected into a single ventral-vegetal blastomere at the 8-cell stage. At stage 33, the tadpoles were stained with X-gal. Specific staining is nuclear and is present almost exclusively in the endoderm underlying the secondary axes (II). (B) Transverse section of embryo in (A). Virtually all staining is in the gut region.
	Fig. 6. Ectopic effect of VegT injection on animal cap explants and whole embryos. VegT RNA was injected into the animal pole of both cells at the 2- cell stage. (A-E) Animal caps were explanted at blastula-stage (stage 8) and cultured to the equivalent of stage 41. (A,B) show cap morphology. (C-E) are histological sections. (A) Control animal caps form ciliated epidermis. (B) VegT-injected animal caps form swelled vesicles, a phenotype of ventral mesoderm. (C) Control animal caps showing atypical epidermis. (D) VegT RNA-injected cap showing block of muscle cells. (E) Another example of VegT RNA-injected cap where extensive ventral mesoderm including mesenchyme and blood were formed. (F-H) Alternatively, RNAs were injected into the animal pole at the 2-cell stage and the embryos were cultured to stage 11. (F) Stage 11.5 embryo. VegT-DAD-injected control together with VegT-injected sibling. Note the failure of the blastopore to close. (G) Section of stage 11.5 embryo injected with 0.7 ng VegT RNA showing collapse of the blastocoel. (H) Section of stage 11.5 embryo injected with 2.0 ng VegT RNA showing the complete loss of the blastocoel. Note the protrusive activity of the ectoderm in G and H.
	Fig. 7. VegT can activate expression of Xwnt-8 and itself in animal caps. 2-cell-stage embryos were injected with 2 ng VegTD 3'UTR RNAs at the animal pole. At stage 8, the animal caps were explanted and cultured until control embryos reached stage 11. Total RNA was isolated from twenty animal caps (An caps) and assayed by northern blot hybridization with VegT (left panel) or Xwnt-8 (right panel) as probes. VegT, endogenous VegT RNAs; VegTD3¢UTR, injected VegT RNA; un-inj., uninjected; inj., injected; WE, whole embryos. EF1a was used as an RNA loading control.
	Fig. 8. Ectopic expression of VegT in dorsal/animal blastomeres suppresses head formation. At the 8-cell stage, 1 ng of RNA was injected into each dorsal/animal blastomere. The embryos were allowed to develop to stage 34 for morphological assay (A,B) or to stage 28 for whole-mount in situ hybridization with digoxigenin-labeled En-2 RNA probe (C,D). (A) Control embryos injected with VegTDAD or buffer. (B) Embryos injected with VegT. (C) Control embryos showing dark-blue staining of En-2 transcripts (arrowheads). Darkly pigmented cement glands are also indicated (open arrows). (D) VegT-injected embryos showing variation of En-2 expression. The embryo on the top shows no En-2 expression, while the bottom one has normal expression of En-2, although it lacks cement gland and eyes.