XB-ART-54803Dev Dyn January 1, 2018; 247 (7): 903-913.
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 and VegT, are essential for the proper formation of mesoderm and endoderm, 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 and gastrula stages. Ectopic expression of Tbx2 represses mesoderm and endoderm, 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.
PubMed ID: 29633424
PMC ID: PMC6105467
Article link: Dev Dyn
Genes referenced: bix1.3 bmp4 chrd.1 dand5 dll1 eomes flrt3 foxi1 gdf3 gsc krt12.4 mab21l3 nodal sox17b.1 sox2 sox3 szl tbx2 tbx4 tbx5 tbxt vegt ventx2.1 ventx2.2 wnt11 wnt11b wnt8a
GO keywords: dorsal/ventral neural tube patterning
Morpholinos: foxi1 MO3 foxi1 MO6 tbx2 MO5
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|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 differentiation.|