December 1, 1996;
Xenopus VegT RNA is localized to the vegetal cortex during oogenesis and encodes a novel T-box transcription factor involved in mesodermal patterning.
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
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
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
[+] show captions
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.
Xenopus and mammals
and is expressed both
(A) Alignment of VegT
with the T-box DNAbinding
domain in other
proteins. mTbx2, mouse
Tbox2; omb, Drosophila
protein from the
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
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
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.