January 1, 2018;
Tbx2 is required for the suppression of mesendoderm during early Xenopus development.
BACKGROUND: T-box family proteins are DNA-binding transcriptional regulators that play crucial roles during germ layer formation in the early vertebrate embryo
. Well-characterized members of this family, including the transcriptional activators Brachyury
, are essential for the proper formation of mesoderm
, respectively. To date, T-box proteins have not been shown to play a role in the promotion of the third primary germ layer, ectoderm
RESULTS: Here, we report that the T-box factor Tbx2
is both sufficient and necessary for ectodermal differentiation in the frog Xenopus laevis. Tbx2
is expressed zygotically in the presumptive ectoderm
, during blastula
stages. Ectopic expression of Tbx2
, while loss of Tbx2
leads to inappropriate expression of mesoderm
- and endoderm
-specific genes in the region fated to give rise to ectoderm
. Misexpression of Tbx2
also promotes neural tissue
in animal cap explants, suggesting that Tbx2
plays a role in both the establishment of ectodermal fate and its dorsoventral patterning.
CONCLUSIONS: Our studies demonstrate that Tbx2
functions as a transcriptional repressor during germ layer formation, and suggest that this activity is mediated in part through repression of target genes that are stimulated, in the mesendoderm
, by transactivating T-box proteins. Taken together, our results point to a critical role for Tbx2
in limiting the potency of blastula
-stage progenitor cells during vertebrate germ layer differentiation. Developmental Dynamics 247:903-913, 2018. © 2018 Wiley Periodicals, Inc.
dorsal/ventral neural tube patterning
[+] show captions
References [+] :
Fig. 1. tbx2 expression in the blastula and gastrula stage ectoderm is
dependent on Xema/Foxi1e activity. A: Xema morpholino-mediated
knockdown (Xema MO) inhibits expression of tbx2. RT-PCR analysis of
early gastrula stage explants. Ornithine decarboxylase (ODC) was used
as a loading control (Bassez et al., 1990). B: Whole-mount in situ
hybridization of early gastrula stage embryos (stage 10.5). Tbx2 expression
is seen as a blue stain throughout the animal pole of albino
embryos; tbx2 expression is excluded from the vegetal pole. Expression
of the panmesodermal marker Xbra is only detected in the marginal
zone of gastrula stage embryos, as expected (Smith et al., 1991).
C: RT-PCR analysis of tbx2 in late blastula stage explants. tbx2 is
expressed in the animal cap and excluded from vegetal pole explants;
vegt is only expressed in vegetal explants, as expected (Lustig et al.,
1996; Stennard et al., 1996; Zhang and King, 1996; Horb and Thomsen,
1997). D: Inner and outer layers of early gastrula stage ectoderm
were separated and analyzed by RT-PCR for expression of tbx2. tbx2
is expressed in the inner layer of the ectoderm; delta is only expressed
in the inner layer, and epidermal keratin is only expressed in the outer
layer (Jonas et al., 1985; Chalmers et al., 2002). All experiments shown
or described in this figure, and throughout the manuscript, were performed
at least three times.
Fig. 2. Ectopic Tbx2 suppresses mesendoderm induction. A: Tbx2
inhibits Activin-induced mesendoderm. RT-PCR analysis of animal cap
explants dissected at late blastula stages and cultured until midgastrula
stages. Activin (0.25 ng/ml) was added to stage 8 animal caps excised
from uninjected embryos or from embryos injected with tbx2 RNA into
the animal pole of both blastomeres at the two-cell stage. B: Inhibition
of FGF-mediated mesoderm induction by Tbx2. RT-PCR analysis of
animal cap explants dissected at late blastula stages and cultured until
midgastrula stages. Basic FGF (10 ng/ml) was added to stage 8 animal
caps excised from uninjected embryos or from embryos injected with
tbx2 RNA, as listed. Unless otherwise noted, 1 ng of tbx2 RNA was
used in this and in subsequent experiments.
Fig. 3. Tbx2 promotes neural fate. A: Ectopic tbx2 induces the preneural markers sox2 and sox3. RT-PCR analysis was performed on animal cap
explants from uninjected embryos and from embryos injected with tbx2 RNA; explants were harvested at gastrula stages. B: Tbx2 represses the
BMP targets bmp4, sizzled, and vent-2 (Lee et al., 2002; Collavin and Kirschner, 2003). RT-PCR analysis of mid-gastrula stage animal caps. C:
Tbx2 misexpression inhibits expression of a Vent-2 luciferase reporter fusion protein (TCFm-Vent2-LUC); this construct includes a mutation in a
TCF binding site that renders it insensitive to Wnt activation (Hikasa et al., 2010). Truncated BMP receptor (tBR) was used a positive control (Graff
et al., 1994). Embryos at the two-cell stage were injected with 5 pg of pRLTK, 50 pg of TCF and 1 ng tbx2 RNA or 1 ng tBR RNA in the animal
pole. D: Tbx2 misexpression inhibits expression of a Vent-2 luciferase reporter fusion protein with a mutation in the putative T-box binding element
(TBEm-TCFm-Vent2-LUC). TCFm-Vent2-LUC and TBEm-TCFm-Vent2-LUC were injected in the absence or presence of tbx2 RNA at the two-cell
stage and collected for analysis of Luciferase expression. Samples of five whole embryo lysates were assayed in triplicate for Firefly and Renilla
luciferase activity at mid-gastrula stages. Fifteen embryos were used for each trial, and each experiment was repeated at least 3 times to confirm
the observed trends. Error bars indicate standard error.
Fig. 4. Tbx2 knockdown leads to ectopic expression of mesendodermal
marker genes. A: Tbx2 morpholino (Tbx2MO) blocks translation of
tbx2 RNA in vitro. MObs-Tbx2 is a tbx2 construct that contains the
Tbx2MO-binding site. B: Mesendodermal markers are up-regulated in
ectodermal explants derived from Tbx2 morpholino-injected embryos.
RT-PCR analysis of animal cap explants dissected at late blastula
stages and harvested at mid-gastrula stages; embryos were injected
with Tbx2 morpholino (80 ng) at early cleavage stages. For the rescue
experiment, 80 ng of Tbx2 morpholino and 1 ng of tbx2 RNA were coinjected
at early cleavage stages. C: Injection of a control (80 ng),
“scrambled” morpholino does not lead to extra-ectodermal gene
expression, while injection of Tbx2 morpholino (80 ng) results in ectopic
expression of both mesodermal and endodermal markers.
Fig. 5. The Tbx2 C-terminus is required for mesendoderm suppression.
A: Expression of Tbx2DC does not suppress mesoderm induction
by Activin. RT-PCR analysis of animal caps dissected at late blastula
stages and cultured until mid-gastrula stages. tbx2DC RNA (1 ng) was
injected at early cleavage stages, as indicated. Activin (0.5 ng/ml) was
added to stage 8 animal caps, as indicated. B: Expression of Tbx2DC
stimulates expression of mesodermal marker genes in the absence of
exogenous growth factors. RT-PCR analysis of animal caps dissected
at late blastula stages and cultured until mid-gastrula stages. tbx2DC
(1 ng) was injected at early cleavage stages, as indicated.
Fig. 6. Tbx2 functions as a repressor during germ layer differentiation.
A: Expression of Tbx2-DBD-EnR inhibits mesendoderm induction by
Activin (left panel); expression of Tbx2-DBD-Vp16 does not inhibit
Activin-mediated mesendoderm induction (right panel). RT-PCR analysis
of animal caps dissected at late blastula stages and cultured until midgastrula
stages. tbx2-DBD-EnR or tbx2 DBD-VP16 RNA (1 ng) was
injected at early cleavage stages, as indicated. Activin (0.5 ng/ml) was
added to stage 8 animal caps, as indicated. B: Expression of Tbx2 DBDVP16
fails to inhibit BMP target gene expression (left panel), and stimulates
expression of mesodermal and endodermal marker genes in the
absence of exogenous growth factors (left and right panels). RT-PCR
analysis of animal caps dissected at late blastula stages and cultured
until mid-gastrula stages. tbx2-DBD-VP16 RNA (1 ng) was injected at
early cleavage stages, as indicated. The “-RT” lane contains all reagents
except reverse transcriptase and was used as a negative control.
Fig. 7. Tbx2 represses downstream targets of VegT and Brachyury. A: Tbx2 represses expression of derriere, xwnt11, and bix4 (Zhang and King,
1996; Casey et al., 1999). RT-PCR analysis of animal caps dissected at late blastula stages and cultured until mid-gastrula stages, as indicated.
Activin (0.5 ng/ml) was added to stage 8 animal caps, as indicated. B: tbx2 represses expression of a Bix4 reporter construct. Embryos at the
two-cell stage were injected with bix4-LUC (20 pg) and pRLTK (7 pg), in the absence and presence of tbx2 RNA (250 pg). Whole embryo lysates
from gastrula stage embryos were assayed in triplicate for Firefly and Renilla luciferase activity. C: Mutation of three T-box binding sites renders
bix4 insensitive to repression by Tbx2. Embryos were injected at the two-cell stage with pRLTK (7 pg) and bix4-LUC (20 pg), Td-LUC (20 pg), or
Tdmp-LUC (20 pg) in the absence and presence of tbx2 RNA. Tbx2 blocks the expression of Td-LUC less efficiently than it does bix4-LUC, while
mutation of the three T-box sites renders Tdmp-LUC insensitive to repression by Tbx2. Samples of five whole embryo lysates from gastrula stage
embryos were assayed in triplicate for Firefly and Renilla luciferase activity. Error bars indicate standard error.
Fig. 8. A model of Tbx2-mediated ectodermal specification. Tbx2
binds to and represses target genes in animal pole progenitor cells,
thereby inhibiting mesoderm and endoderm formation. In the marginal
zone and vegetal pole, other T-box proteins bind to and activate an
overlapping set of target genes, stimulating mesodermal and endodermal
The T-box transcription factor Tbx2: its role in development and possible implication in cancer.