Identification of Xenopus TG-interacting Factor Homologues
Previous studies showed that maternal Xenopus Zic2 represses nodal-related gene expression (Houston and Wylie,2005). To determine whether homologues of other genes implicated in human HPE might also control nodal-related expression, we looked for expression of these homologues in Xenopus oocytes. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of oocyte RNA showed that tgif1 (tg-interacting factor 1) was expressed in Xenopus oocytes, whereas other major HPE-associated genes, shh, six3, or ptch1, were not expressed (data not shown). We used human TGIF1 to query Xenopus EST databases, and we identified clones encoding complete Tgif1 proteins from both X. laevis (accession no. BC044016; Klein et al., 2002) and X. tropicalis (accession no. BC064716). A composite protein sequence, generated from overlapping ESTs, had been reported for Xenopus Tgif1 (Hyman et al.,2003) and the X. laevis EST encoded an identical protein. The predicted X. laevis protein is 96% identical to X. tropicalis Tgif1, and shows 73% and 71% identity to the human and mouse proteins, respectively (Fig. 1A). The Xenopus proteins are 272 amino acids in length and contain conserved regions including a homeodomain, a putative binding motif for CtBP, a putative nuclear localization signal and a short Tgif1-specific motif (Hyman et al.,2003; Fig. 1A). The C-termini of Tgif1 homologues are also nearly identical across species. This C-terminal region mediates transcriptional repression through recruitment of the Sin3 corepressor complex (Wotton et al.,2001) and includes a known ERK phosphorylation site, thought to control Tgif1 stability (Lo et al.,2001). Residues mutated in human HPE (Gripp et al.,2000) are also conserved in the Xenopus proteins (Fig. 1A). We obtained the full-length X. laevis clone and used this cDNA in expression and functional studies.
Figure 1. Sequence and expression of Xenopus tgif1. A: Alignment of human (TGIF1), mouse (Tgif1), Xenopus tropicalis (xtr Tgif1), and Xenopus laevis (xla Tgif1) translated amino acid sequences. Identical residues are in black, similar residues are in grey. Boxes above the sequences mark the locations of a CtBP-binding motif, a nuclear-localization sequence, a Tgif-specific motif and an ERK phosphorylation site. The homeodomain is underlined with a dashed line. Arrowheads mark the sites of HPE mutations. Amino acid (a.a.) numbers are indicated for the X. laevis protein. B: Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of tgif1 and tgif2 expression in animal (An) and vegetal (Vg) oocyte halves, compared with whole oocytes (Oo) and stage 10.5 embryos (10.5). The −RT indicates a stage 10.5 sample processed in the absence of reverse transcriptase. C: Quantitative real-time PCR (QPCR) analysis of tgif1 and tgif2 expression during early development. Samples were normalized to odc and values displayed as a percentage relative to an uninjected oocyte sample (relative expression %). D–I: in situ hybridization of tgif1. D: Stage 10.5 (top row) and stage 13 (bottom row) embryos. Both rows show an animal pole view. In the bottom row, anterior is to the left. E: Stage 18, anterior to the left. F,G: Stage 24 (F) and stage 36 (G). H,I: Sections through stage 30 embryos, hindbrain level section (H), spinal cord level section (I). Anterior is to the left. no, notochord.
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Vertebrates have multiple Tgif paralogues, including Tgif2, whose expression is similar to Tgif1 in mouse (Jin et al.,2005; Shen and Walsh,2005). Tgif-related genes are also present on the sex chromosomes in mammals (Tgiflx and Tgifly; Blanco-Arias et al.,2002). We identified two tgif2 sequences in Xenopus laevis, likely representing allo/pseudoalleles, and one tgif2 gene in X. tropicalis (accession nos. BC081024, BC081037, CT030554, respectively, not shown). We did not identify any ESTs homologous to the sex chromosome-specific Tgif genes. Synteny analysis using Metazome indicated that the X. tropicalistgif1 and tgif2 loci correspond with the mammalian Tgif genes (data not shown).
Expression of tgif1 in Normal Development
We detected expression of tgif1 by RT-PCR at all stages analyzed, from egg through the swimming tadpole stage (not shown). Notably, tgif1 transcripts are present in stage VI oocytes, and are evenly distributed between animal and vegetal hemispheres (Fig. 1B, and in situ data not shown), suggesting that the RNA is not localized. Expression of tgif2 was also detected maternally, in a similar distribution as that found for tgif1 (Fig. 1B). Comparison of tgif1 and tgif2 message levels over a series of developmental stages showed that tgif1 transcripts decline in abundance after fertilization, whereas tgif2 levels showed a marked increase during the gastrula stages (Fig. 1C).
Spatial analysis of tgif1 expression identified expression in animal cap ectoderm (Fig. 1D, top row) and surrounding the blastopore. Expression in two stripes flanking the notochord plate in late gastrula embryos was also evident (Fig. 1D, bottom row). By the neurula stages, tgif1 is expressed in the nervous system, particularly in the anterior and posterior regions of the neural plate, with some lateral expression along the periphery of the neural folds (Fig. 1E). Tail bud embryos express high levels of tgif1 in the nervous system, branchial arches, pronephros, ear vesicle, and in the eye (Fig. 1F,G). Sectioning of these embryos revealed that in the nervous system, tgif1 is expressed in the dorsal neural tube, predominantly in the mid- and hindbrain (Fig. 1H,I). These data show that tgif1 is maternally expressed in Xenopus, and that its embryonic expression pattern is consistent with roles in early development, as well as in neural development and organogenesis.
Antisense Inhibition of Maternal tgif1 Results in Excess Anterior Development
To elucidate the function of tgif1 in early embryonic development, we used antisense oligos to deplete the maternal stores of tgif1 mRNA. DNA-based antisense oligonucleotides (oligos) cause the degradation of complementary mRNAs in Xenopus oocytes through an endogenous RNase-H activity (Dash et al.,1987). We screened several antisense oligos specifically targeting tgif1, but not tgif2, and identified two that were effective in degrading tgif1 mRNA. Figure 2A shows a dose-dependent reduction of tgif1 mRNA levels in oocytes injected with 3.0 or 6.0 ng of oligo tgif1-as2mp. Quantitative real-time PCR (QPCR) analysis of the same cDNA samples found that tgif1 was reduced to 23% and 6% of control levels, at the 3.0 ng and 6.0 ng doses respectively. The oligos used are not complementary to tgif2 mRNA, and levels of tgif2 were unaffected in these experiments (data not shown). A 3.0-ng dose of tgif1-as2mp was used in the following experiments because higher doses resulted in fewer surviving embryos. Another DNA oligo targeting a different sequence in the mRNA gave similar results (not shown). We also designed a morpholino oligo complementary to the start codon to block tgif1 translation.
Figure 2. Depletion of maternal tgif1 anteriorizes embryos. A: Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of tgif1 mRNA levels in uninjected (Un) and tgif1-depleted oocytes, injected with 3.0 ng or 6.0 ng of oligo tgif1-as2mp. Oocytes were analyzed after overnight progesterone (Pg) treatment. Values from separate Quantitative real-time PCR (QPCR) runs on the same cDNA samples are shown below each marker as a percentage of the uninjected sample, normalized to odc. B: QPCR analysis of tgif1 mRNA levels in uninjected (Un) and tgif1-depleted oocytes and embryos (tgif1-; 3 ng tgif1-as2mp). Samples were normalized to odc and values displayed as a percentage relative to the uninjected oocyte sample (relative expression %). C–H: Phenotypes of representative control (C, E, G, Uninjected), 3 ng of tgif1-as2mp-injected (D, F, tgif1-) and 30 ng of tgif1-MO-injected embryos (H) tail bud stage embryos. Embryos in C,D and E,F are from separate host-transfer experiments. Arrows indicate enlarged head and anterior endoderm (D), and a secondary head (F). I: RT-PCR of late patterning markers in uninjected (Un) and tgif1-depleted (tgif1-) stage 30 embryos, obtained by fertilization of tgif1-as2mp-injected oocytes (3.0 ng) by the host-transfer method.
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We used the host-transfer method (Heasman et al.,1991) to assess the developmental effects of depleting maternal tgif1 in oocytes. This procedure is necessary because cultured oocytes cannot be fertilized in vitro directly, but rather must acquire jelly coats and other surface modifications to become competent for fertilization and normal development (Heasman et al.,1991). Control and antisense oligo-injected oocytes were matured in vitro using progesterone, vital dye stained, and transferred into the body cavity of egg-laying females. The eggs were recovered, fertilized normally in vitro, and sorted. Analysis of tgif1 mRNA levels in depleted embryos over a series of stages in early development showed that tgif1 accumulates to normal zygotic levels by mid-gastrulation (stage 11). This result suggests that effects of tgif1 loss-of-function are due primarily to loss of maternal tgif1.
We examined tail bud stage tgif1-depleted embryos for phenotypic abnormalities. These embryos were shorter than controls and had swollen anterior endoderm, truncated tails and expanded cement glands (Fig. 2C–F; Table 1). We saw small secondary heads with cement glands in approximately 25% of surviving, affected tgif1-depleted embryos (Table 1, series 1; Fig. 2E,F). Similar phenotypes were seen in embryos derived from oocytes injected with 15 and 30 ng of an MO against tgif1 (Table 1, series 2; Fig. 2G,H). Injection of the same morpholino doses into fertilized eggs had no effect on development (data not shown), further suggesting that it is the maternal store of tgif1 that is relevant for anteroposterior patterning. To provide molecular evidence for anteriorization, we analyzed tailbud stage tgif1-depleted embryos by RT-PCR (Fig. 2I). We found elevated levels of anterior markers: wnt1, a hindbrain marker, and hex, a marker of liver diverticulum at this stage, along with concomitant decreased levels of ventroposterior markers: hoxb9, a spinal cord marker and globin (〈 T4 globin), a blood island marker (Fig. 2I).
Table 1. Tgif1 Regulates Anteroposterior Patterning in a Nodal-Dependent Fashiona
| ||n||Exogastrula||Expanded Anterior 2° heads||Normal||% Anteriorized % 2° heads|
|Series 1|| || || || || |
| || || ||N/A|| ||N/A|
|3 ng tgif1-as2mp||34||5/34||24/34||5/34||71|
| || || ||6/24|| ||25|
|3 ng as2mp + 50 pg tgif1 RNA||22||2/22||7/22||13/22||32|
| || || ||1/7|| ||14|
|Series 2|| || || || || |
| || || ||N/A|| ||N/A|
|15 ng tgif1-MO||10||0/10||6/10||4/10||60|
| || || ||0/6|| ||0|
|30 ng tgif1-MO||18||0/18||12/18||6/18||67|
| || || ||1/12|| ||8|
|Series 3|| || || || || |
| || || ||N/A|| ||N/A|
|3 ng tgif1-as2mp||16||2/16||10/16||4/16||63|
| || || ||3/10|| ||30|
|3 ng as2mp + 50 pg cerS RNA||17||3/17||6/17||8/17||41|
| || || ||0/6|| ||0|
We controlled for the specificity of the antisense oligos through rescue experiments, in which tgif1 mRNA was injected back into depleted oocytes. Oligo-injected oocytes were incubated for 24 hr to allow for oligo turnover, and were then injected with tgif1 mRNA (50 pg). Oocytes were transferred into host females and fertilized as above. Phenotypic examination at the tail bud stage showed that reintroduction of tgif1 mRNA could rescue the anteriorization phenotype (Table 1, series 1, also Fig. 4). In summary, our phenotypic analyses show that depletion of tgif1 results in an expansion of anterior development, which is reversed by reintroduction of tgif1 mRNA.
Figure 4. Rescue of maternal tgif1 depletion by injected tgif1 mRNA. A–E: Phenotypes of representative control (C, Uninjected), tgif1-depleted (D, tgif1-), rescued (E, tgif1-;+RNA), and 50 pg tgif1 RNA-injected (F) embryos, at stage 32. E: Quantitative real-time polymerase chain reaction analysis of nr5, gsc, xbra, and xnr3 expression at stage 10. Relative expression values are shown on the y-axis; samples were normalized to odc and values expressed as a percentage of uninjected control stage 10 embryos (100%).
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Because tgif1-depleted embryos showed abnormal embryonic body axis patterning, we analyzed their early development to identify the more proximal defects in these embryos. Depleted embryos developed normally up to the gastrula stage, but were slower than control embryos in progressing through gastrulation. Embryos deficient in tgif1 displayed open blastopores with deeply recessed yolk plugs (Fig. 3A,B), a phenotype which oftentimes indicates hyper-dorsalization. The majority (> 75%) of tgif1-depleted embryos eventually completed gastrulation 3–4 hr later than controls; the remainder exogastrulated and did not survive past the neurula stages (Table 1, series 1).
Figure 3. Depletion of maternal tgif1 causes gastrulation defects and abnormal nodal-related gene expression. A,B: Phenotypes of control uninjected (A) and tgif1-depleted (B) stage 13 embryos. C: Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of early mesendoderm markers in uninjected (Un) and tgif1-depleted (tgif1-) stage 11 embryos, obtained by fertilization of as2mp-injected oocytes (3.0 ng) by the host-transfer method. Values from separate quantitative real-time PCR (QPCR) runs on the same cDNA samples are shown below each marker as a percentage of the uninjected sample, normalized to odc. D: QPCR analysis of nodal-related genes in uninjected (Un) and tgif1-depleted (tgif1-) stage 9.5 and 10.5 embryos, obtained by fertilization of as2mp-injected oocytes (3.0 ng) by the host-transfer method. Samples were normalized to odc and values displayed as a percentage relative to the uninjected stage 9.5 sample (relative expression %).
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We next assayed molecular markers of dorsoventral and germ layer patterning at the gastrula stage (stage 11) by RT-PCR. Compared with sibling embryos, tgif1-depleted embryos had elevated levels of dorsal markers such as goosecoid (gsc), and siamois (sia), as well as increased levels of the early endodermal nodal-related genes, nr5 (Fig. 3C) and nr6 (not shown). Representative mesendodermal (xbra, wnt8, mix.2, sox17α) markers were also increased to a lesser degree (Fig. 3C) in tgif1-depleted embryos. Markers of ectoderm (lhx5, zic1) were unchanged compared with controls (Fig. 3C), as were a subset of genes known to suppress TGFβ signaling in the animal hemisphere, foxi1 and sox3 (Suri et al.,2005; Zhang et al.,2003; data not shown). Thus, the increases in mesendodermal gene expression suggest misregulation of these genes in situ, rather than alterations in overall germ layer patterning.
Because nr5 and nr6 were prominently increased in tgfi1-depleted embryos, we examined the expression of the other nodal-related genes in pre- and early gastrula stage embryos (stage 9 and 10.5) to identify any dynamic changes in their expression (Fig. 3D). Interestingly, although nr5/6 were up-regulated at both stages, the other nrs showed differing patterns of expression. The genes nr1 and nr3 were both little affected in pregastrula embryos but up-regulated by early-to-mid-gastrulation (Fig. 3D). Of interest, nr2 and nr4 were down-regulated before gastrulation, but were normally expressed or slightly down by mid-gastrulation, suggesting that Tgif1 may positively influence their expression. These changes in gene expression are specific to deficiency in tgif1, because normal development and near-normal levels of representative markers, nr5, nr3, gsc, and xbra, were restored in tgif1-depleted embryos injected with tgif1 mRNA (Fig. 4).
The elevated levels of nr5 in tgif1-depleted embryos suggested that Nodal signaling might be involved in causing anteriorization in these embryos. Expression of nr5 is initiated coincident with, or immediately after MBT (Takahashi et al.,2000), and Nodal signaling is known to contribute to the expression of genes elevated in tgif1-depleted embryos, such as gsc and sia (Nishita et al.,2000). Therefore, we determined if blocking Nodal activity could reduce the severity of tgif1-deficiency. We injected a low dose of cerberus-short (cerS) mRNA (50 pg), a Nodal antagonist (Piccolo et al.,1999), into tgif1-depleted embryos. In controls, this dose did not significantly alter anteroposterior patterning on its own. When low-dose cerS was injected into tgif1-depleted embryos, the overall proportion of normal embryos increased (Table 1, series 3). It has been shown that overexpression of cerS in frog embryos increases nr5 expression (Kofron et al.,2004; Houston and Wylie,2005; Takahashi et al.,2006), even though overall Nodal activity is inhibited (Piccolo et al.,1999; Agius et al.,2000). These results support the idea that the phenotypic consequences of tgif1 depletion are due to mis-regulated nodal expression and signaling.
Repression of nodal-related Genes 5 and 6 by Tgif1 Depends on VegT and Is Regionally Specific
Expression of nr5 is activated directly by VegT and β-catenin (Hilton et al.,2003), and is repressed by maternal transcription factors, including Zic2, Tcf3, Sox3, and FoxH1 (Houston et al.,2002; Zhang et al.,2003; Kofron et al.,2004; Houston and Wylie,2005), in addition to Tgif1 (this study). To distinguish whether Tgif1 antagonizes the VegT or β-catenin aspect of nr5 regulation, we generated embryos deficient in both Tgif1 and VegT. We depleted tgif1 mRNA as above, and we used a previously characterized morpholino oligo to inhibit VegT function (Heasman et al.,2001). We analyzed expression of the VegT target nr5, and of the Wnt/ β-catenin target gene, sia, at the gastrula stage by QPCR. Depletion of maternal VegT alone reduced nr5 expression to approximately 25% of control levels (Fig. 4A, left panel) but did not greatly affect sia levels (Fig. 4A, right panel). Both genes were elevated upon depletion of tgif1 (Fig. 4A). In embryos depleted of both tgif1 and VegT, nr5 levels were similar to that seen with VegT depletion alone, suggesting that Tgif1 represses nr5 in a VegT-dependent context. In contrast, in tgif1/VegT double depleted embryos, sia expression remained at control levels (Fig. 5A, right panel), suggesting that factors downstream of VegT are responsible for boosting sia expression in tgif1-depleted embryos. Thus, Tgif1 is likely to function as an antagonist of VegT activity, rather than of Wnt/β-catenin activity in Xenopus embryos.
Figure 5. Tgif represses nr5/6 expression in vegetal cells. A: Quantitative real-time polymerase chain reaction (QPCR) analysis of nr5 expression in embryos depleted of tgif1, VegT, or both. Relative expression values; samples were normalized to odc and values expressed as a percentage of uninjected control stage 10.5 embryos. tgif1-, embryos injected with 3.0 ng of tgif1-as2mp as oocytes, VegT-, embryos injected with 18.0 ng of vegt-MO as oocytes. B: Reverse transcriptase-polymerase chain reaction (RT-PCR) of mesendodermal markers in uninjected and tgif1-depleted explants. Cap, animal caps; Eq, equatorial/marginal zone region; Vg, vegetal mass/endodermal mass. Explants were cut at stage 9, and cultured to stage 11. Each sample contained five explants or two intact embryos. C–D′: In situ hybridization of nr5/6 expression in control uninjected (B, Un) and tgif1-depleted embryos (C,D) at stage 10, obtained by fertilization of tgif1-as2mp-injected oocytes (3.0 ng) by the host-transfer method. Dorsal is toward the top.
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In line with these results, spatial analysis of gene expression in tgif1-depleted embryos showed that nr5 was only up-regulated in vegetal cells. We analyzed explants of prospective ectoderm (animal caps), mesoderm (equatorial regions), and endoderm (vegetal masses) from either control or tgif1-depleted embryos by real-time RT-PCR (Fig. 5B). The expression of nr5 was increased vegetally, reflecting the appropriate spatial distributions of this gene. gsc and sia were increased both vegetally and equatorially, likely due to their potential to be up-regulated by TGFβ signaling (Nishita et al.,2000). These results were confirmed by in situ hybridization analysis of nr5/6 expression (Fig. 5C,D), which showed that these genes were increased only in vegetal cells of tgif1-deficient embryos. Thus, Tgif1 is only required to repress nr5/6 in cells that normally express those genes, rather than being required as a global repressor of nodal expression.
Tgif1 Inhibits VegT-Mediated Activation of nr5
To further test the hypothesis that Tgif1 inhibits VegT activity, we performed coexpression experiments in animal caps. Injection of caps with VegT alone caused induction of nr5, whereas this expression was inhibited by coinjection of tgif1 (Fig. 6A). This was also true for nr6 (data not shown), but we focused on nr5 for simplicity. Injection of tgif1 partially inhibited the activation other markers by VegT, such as xbra and wnt8, but did not block dorsal mesoderm or endoderm markers, gsc and sox17α (Fig. 6A). These genes are also targets of VegT, (Xanthos et al.,2002) suggesting that Tgif1 does not equally repress all VegT targets. Because Tgif1 is known to repress transcriptional activation by recruiting several different corepressor complexes, we used deletion and site-directed mutagenesis to identify which potential mechanisms are required for the inhibition of VegT activity by Tgif1. The C-terminus of Tgif1 is highly similar across vertebrate species (Fig. 1A; Hyman et al.,2003), and is thought to mediate transcriptional repression through recruitment of the Sin3/HDAC complex (Wotton et al.,2001). We, therefore, made a C-terminal truncation mutant of Tgif1 (Tgif1ΔC) and tested this construct in animal cap assays. A similar deletion in human TGIF1 nearly eliminated Sin3 binding in cell line assays (Wotton et al.,2001). In caps coexpressing tgif1ΔC and vegt, induction of nr5 was unaffected (Fig. 6B) compared with caps expressing vegt alone, suggesting that the Tgif1 C-terminus is required for the regulation of nr5 expression by VegT.
Figure 6. The tgif1 C-terminus is required to inhibit VegT activation of nr5. A: Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of gene expression in uninjected (Un), vegt-injected (1.0 ng) and vegt+tgif1-injected (1.0 ng each) stage 10.5 animal caps. We, whole stage 10.5 embryo. B: RT-PCR analysis of nr5 expression in uninjected (Un) caps and in caps overexpressing vegt, vegt+tgif1, vegt+tgif1ΔC or tgif1ΔC alone (1.0 ng each). We, whole stage 10.5 embryo. C: RT-PCR analysis of nr5 expression in uninjected (Un), vegt-injected and vegt+ wild-type (tgif1) or mutant tgif1-injected stage 10.5 animal caps (1.0 ng each). We, whole stage 10.5 embryo. C′: Immunoblotting of embryos injected with tgif1, tgif1ΔC, tgif1-S28C, and tgif1-T151A. The left panel shows a representative blot using anti-TGIF1 N-terminal mAb; the right panel shows a representative blot using anti-TGIF1 C-terminal mAb. Tubulin was used as a loading control in the right panel. D: RT-PCR analysis of nr5 expression in uninfected (Un), vegt-injected and vegt+tgif1, and vegt+tgif1+cerS-injected stage 10.5 animal caps (1.0 ng each). We, whole stage 10.5 embryo. D′: RT-PCR analysis of nr5 and nr1 expression in uninjected (Un) and cerS-injected stage 10.5 embryos.
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We next tested point mutations in Tgif1 that have been identified in cases of human HPE, S28C and T151A (Gripp et al.,2000). The S28C mutation is defective in CtBP recruitment (Melhuish et al.,2000; El-Jaick et al., 2007) as well as Smad2- and retinoid-dependent transcriptional repression (Gripp et al.,2000; El-Jaick et al.,2007). The T151A mutation reduced the ability to bind activated Smad2 (Gripp et al.,2000) but a later study showed normal binding to Smad3, as well as normal transcriptional repression function in cell line assays (El-Jaick et al.,2007). Xenopus Tgif1 has a serine at position 151 rather than a threonine, but we kept the mammalian nomenclature in our constructs for consistency. In contrast to tgif1ΔC construct, tgif1-S28C and tgif1-T151A both retained the wild-type ability to inhibit nr5 induction by VegT in animal cap assays (Fig. 6C). Immunoblotting of sibling embryos using cross-reacting antibodies against human TGIF1 confirmed that wild-type and mutant Tgif1 proteins were equivalently produced in embryos (Fig. 6C′).
Because the S28C and T151A constructs were defective in Smad-dependent corepression and putatively defective in Smad2 binding respectively, we concluded that the ability to antagonize VegT was independent of Smad-associated corepression. To confirm this result, we coexpressed vegt and tgif1 in the presence or absence of the Nodal antagonist, cerS, and then analyzed nr5 expression in animal caps. Tgif1 was able to repress nr5 regardless of the presence or absence of injected cerS (Fig. 6D), showing that ongoing Nodal signals are not required for Tgif1 to inhibit VegT function. Embryos injected with cerS were analyzed separately to confirm that nr5 was not blocked under these conditions. Indeed, consistent with our observations and with previous reports (Kofron et al.,2004; Houston and Wylie,2005; Takahashi et al.,2006), cerS did not inhibit, but rather up-regulated, nr5 (Fig. 6D′). Overall, these results indicate that corepressor proteins recruited by the Tgif1 C-terminus, likely including the Sin3 complex, are involved in attenuating VegT function.
Tgif1 Does Not Inhibit Retinoid Signaling or TGFβ Signaling in Animal Cap Cells
Our functional analysis of Tgif1 function in early Xenopus development indicated a role in the inhibition of VegT, either directly or indirectly. In cultured cells and in adult mouse tissues, Tgif1 can repress both Smad2 and retinoid signaling, although whether Tgif1 regulates these pathways during embryonic development is not known. We used animal cap assays to determine whether either of these pathways could account for the regulation of VegT activity and nodal expression by Tgif1. We first determined the extent to which Tgif1 interacts with retinoic acid (RA) signaling by testing the ability of Tgif1 to block RA-mediated posteriorization of experimentally induced neural tissue. Animal caps expressing tgif1, with or without the bone morphogenetic protein (BMP) antagonist noggin to induce neural tissue, were cultured in the presence or absence of all-trans retinoic acid (RA). We then assayed markers of anteroposterior neural patterning at the neural groove stage (Fig. 7A). As expected, noggin expression alone induced anterior markers (NCAM, six3), and the addition of RA caused the expression of posterior markers, such as the spinal cord marker, hoxb9. Tgif1 did not alter either neural induction by noggin, or neural posteriorization by RA (Fig. 7A). Additionally, Tgif1 alone did not induce neural markers, suggesting that Tgif1 also does not block BMP/Smad1 signaling in this context, an activity that causes neural induction in animal cap assays.
Figure 7. Tgif1 does not regulate retinoid or Smad2 signaling in animal caps. A: Reverse transcriptase-polymerase chain reaction (RT-PCR) of neural markers in stage 22 caps injected with 200 pg of noggin mRNA (nog), 500 pg of tgif1, or both (nog+tgif1), cultured alone or in the presence of 1 μM all-trans retinoic acid (+ATRA). B: RT-PCR analysis of xbra expression in uninjected (Un) and tgif1-injected stage 11 animal caps treated with 10 ng/ml activin (act). Note weak xbra expression in caps injected with tgif1. We, whole stage 11 embryo. C: RT-PCR analysis of xbra and mix.2 expression in uninjected (Un), tgif1-injected (tgif1), smad2-HA-injected and tgif1+smad2-HA-injected animal caps. We, whole stage 11 embryo. D: Immunoblotting of overexpressed Smad2-HA and Tgif1 levels in sibling whole embryos from the experiment in C. E–F′; Examples of Tgif1 overexpression phenotype (F–F′) compared with an uninjected control embryo (E).
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We next determined the ability of Tgif1 to inhibit Smad2-dependent signaling, using mesoderm induction assays in animal caps. Control or tgif1-injected caps were cultured in the presence or absence of recombinant activin protein, to stimulate Smad2 activation, and mesoderm was assayed by detection of xbra in RT-PCR assays. In these experiments, Tgif1 was unable to block induction of xbra by activin (Fig. 7B). To test the role of Tgif1 in Smad2 signaling more directly, we coexpressed Tgif1 with Smad2 (HA-smad2) in animal caps. In line with our results using activin, Tgif1 failed to block Smad2-mediated activation of xbra or mix.2, another well-characterized direct transcriptional target of Smad2 (Fig. 7C). Because the injected tgif1 had no effect in these experiments, we confirmed the presence of the exogenous proteins by extracting total protein from sibling whole embryos and immunoblotting for Smad2-HA and Tgif1 (Fig. 7D). In other experiments, we inferred that tgif1 was active because sibling embryos injected with a high dose of tgif1 mRNA (500 pg to 1 ng) developed a characteristic phenotype (Fig. 7E–F′), typified by truncated posterior tissues and open blastopores. This gain-of-function phenotype is likely the result of either a cell movement defect or a deficiency in posterior mesoderm development, and is currently under investigation. Overall, these data indicate that antagonism of RA and Smad signaling are not the major activities of Tgif1 in early Xenopus development. Taken together with the results from the VegT experiments, these results suggest the interesting possibility that Tgif1 may be a more general regulator of gene expression in early development, and not simply a modulator of other pathways.