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Previous Article | Next Article 
Molecular and Cellular Biology, May 2005, p. 3639-3647, Vol. 25, No. 9
0270-7306/05/$08.00+0 doi:10.1128/MCB.25.9.3639-3647.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Howard Hughes Medical Institute, Department of Neurology, Beth Israel Deaconess Medical Center, and Program in Neuroscience, Division of Medical Sciences, Graduate School of Arts and Sciences, Harvard University, Boston, Massachusetts 02115
Received 8 January 2005/ Accepted 5 February 2005
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ABSTRACT |
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INTRODUCTION |
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Although Drosophila achintya and vismay are transcriptional activators (20), mammalian TGIF has been implicated as a transcriptional repressor and corepressor based on in vitro studies (32, 50). TGIF exhibits several different modes of repression (51). First, TGIF represses transcription by competing with retinoid receptors for the DNA binding sites, the retinoid X receptor (RXR) responsive element, in the promoter regions of the regulated genes (4, 18, 23). Second, TGIF represses transcription by acting as a Smad corepressor (50). Smad proteins are the cellular signaling transducers of the TGF-ß signaling pathway (22). Upon TGF-ß family ligand binding to the cell surface receptor serine/threonine kinases, the receptor multimerizes and phosphorylates Smad proteins (28). The phosphorylation-activated Smad proteins then translocate to the nucleus to activate gene expression by interacting with coactivators (29, 45). TGIF replaces the coactivators to bind to Smad2 and Smad3 and also recruits other general corepressors, including histone deacetylases (HDACs) and Sin3, to the complex, thus repressing TGF-ß-activated transcription (44, 49). In addition, TGIF may also regulate ubiquitin-dependent degradation of Smad2 (43). Third, TGIF can repress gene expression by recruiting CtBP, which may repress transcription via interactions with polycomb group proteins (32).
Mutations in the human TGIF gene have been associated with holoprosencephaly (HPE [MIM 236100]). HPE is the most common congenital malformation of the developing human forebrain, in which the two cerebral hemispheres fail to separate and the two lateral ventricles are fused into one (13, 30, 39, 41). Heterozygous nonsense and missense mutations in the genes SIX3, SHH, TGIF, ZIC2, and PTCH have been identified to be responsible for familial HPE2, HPE3, HPE4, HPE5, and HPE7, respectively (3, 7, 16, 33, 47). TGIF falls within the minimal critical region of the HPE4 locus (HPE4 [MIM 142946]) on human chromosome 18p11.3 (40). Six missense mutations and one nonsense heterozygous mutation in TGIF have been reported in HPE patients to date (1, 8, 16, 25). The nonsense mutation generates an early stop codon within the homeodomain (1). HPE has been identified in partial chromosome 18p deletion cases, which implicates that HPE4 may result from partial loss of TGIF function (10, 17, 36). On the other hand, HPE4 has also been implied in partial trisomy 18 cases, which suggests a gain-of-function mechanism (9, 26, 52). It remains unclear how TGIF mutations cause HPE. Whereas targeted disruption of Shh, Zic2, or Six3 in mice results in HPE-like malformation of the forebrain (12, 24, 37), we report here that Tgif–/– mice are, surprisingly, viable, fertile, and indistinguishable from wild-type littermates.
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MATERIALS AND METHODS |
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ISH. For in situ hybridization (ISH), time-pregnant mice were sacrificed by carbon dioxide euthanasia. The embryos were dissected in cold 0.01 M phosphate-buffered saline (PBS) (pH 7.4). For section ISH, the heads of the embryos were immediately frozen in isopentane on dry ice and stored at –80°C until processed. The embryonic heads were sectioned coronally at 16 µm in a cryostat, mounted on Superfrost plus microscope slides (Fisher Scientific, Pittsburgh, PA), and stored at –80°C until processed. For whole-mount ISH, whole embryos were fixed in 4% paraformaldehyde, dehydrated with methanol, and stored at –20°C in 100% methanol for up to 3 months.
Nonradioactive ISH was performed using digoxigenin-labeled cRNA probes as previously described (15, 35). The Shh probes were derived from a cDNA clone (courtesy of C. Tabin) as described previously (14). For the Tgif probes, a SalI-digested expressed sequence tag clone (IMAGE:3595273; Invitrogen, Carlsbad, CA) of Tgif was used as the linearized template. Tgif antisense cRNA probes were in vitro transcribed with T7 RNA polymerase. For the Tgif2 probes, a 333-bp fragment of Tgif2 cDNA (nucleotide positions 381 to 724 downstream of the ATG start site) was amplified by PCR (forward primer, 5'-CTCCTTGTCTGTGTGCTCCA-3'; reverse primer, 5'-GCGTTTTCTGAGACGAAAGG-3') and cloned in the pCRII vector (Invitrogen, Carlsbad, CA). The primers were designed using Primer3 (42). The Tgif2 partial cDNA clone was linearized with EcoRV and transcribed with Sp6 RNA polymerase to generate the Tgif2 antisense probes. All probe templates were verified by direct sequencing. Sections or whole embryos were hybridized to each probe (
100 ng/ml) at 68°C overnight. Hybridized probes were visualized using alkaline phosphatase-conjugated anti-digoxigenin Fab fragments and BM Purple (Roche, Indianapolis, IN) or 5-bromo-4-chloro-3-indolyl-phosphate/Nitro Blue Tetrazolium substrate (Kierkegard and Perry Laboratories, Gaithersburg, MD).
Construction of the Tgif targeting vector, embryonic stem (ES) cell screening using Southern blotting, and generation of Tgif mutant mice. The genomic clones corresponding to the mouse Tgif locus were isolated from a genomic library prepared from the 129/SvEV mouse strain (courtesy of M. Thompson) by using a mouse Tgif probe of the 899-bp intron between the last two coding exons. The 11-kb Tgif genomic fragment from ClaI to BcgI including all exons was subcloned into a modified pSP72 vector. For the targeting vector, a 2.2-kb region of genomic DNA containing exons 2 and 3 of the Tgif gene was flanked by two loxP sites. A selectable cassette containing the genes for neomycin resistance (Neo cassette) was inserted downstream of the genomic fragment flanked by two FRT sites (InGenious Targeting, Stony Brook, NY). The Tgif targeting construct was electroporated into ES cells that were subsequently grown in medium containing G418 to select for cells with homologous recombination. Correctly targeted ES cell clones were identified by Southern analysis. Genomic DNA was digested with PstI and identified by a 3' probe downstream of the targeted Tgif locus or digested with PacI and KpnI and identified by a 5' probe upstream of the targeted Tgif locus. Two independent ES cell clones with the targeted Tgif locus were injected into B6 blastocysts to generate two lines of chimeras (MGH Gene Targeting Core, Charlestown, MA). The chimeras were crossed into B6 mice for germ line transmission (TgifloxPneo). The Neo cassette was deleted by crossing the mice into human ß-actin-driven FLPe deleter mice (courtesy of S. Dymecki) to generate the TgifloxP conditional line. The null allele (Tgif–) was generated by crossing the TgifloxP line into the human ß-actin-driven Cre deleter line. Germ line transmission of the targeted allele (TgifloxPneo), the conditional allele (TgifloxP), and the null allele (Tgif–) in mice was confirmed by Southern blotting. Genomic DNA was digested with SpeI and identified by a 3' probe downstream of the targeted Tgif locus. The accession number for this mouse line is MGI:3513135.
Genotyping of Tgif wild-type, conditional, and null alleles by genomic PCR. Mouse biopsy samples were collected and digested with 10 µg proteinase K in 100 µl buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl2, 0.1 mg/ml gelatin, 0.45% [vol/vol] Nonidet P-40, and 0.45% [vol/vol] Tween 20) at 55°C overnight. Genomic DNA (0.5 µl) containing solution was used in 10 µl PCR mixture including 0.5 U of Taq (QIAGEN, Valencia, CA), 1x PCR buffer, 0.125 mM deoxynucleotide triphosphates (Invitrogen, Carlsbad, CA), 1.5 mM MgCl2, and primers (1 µM 1f, 5'-TAACAGCAAGCCTTTCACGA-3'; 0.35 µM 1r, 5'-GCCCTCACAACCTTGTCTTT-3'; and 0.65 µM 2r, 5'-TGGGCCCTTCTGAAATACAC-3'; Sigma-Genosys, The Woodlands, TX). The PCR cycle profile was as follows: 1 cycle at 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 60°C for 45 s, and 72°C for 1 min, and finally 1 cycle at 72°C for 10 min. Primers 1f and 1r surrounding the first loxP insertion site would amplify a 112-bp fragment of the wild-type allele and a 174-bp fragment of the conditional allele. Primers 1f and 2r (downstream of the Neo cassette insertion site) would amplify a 313-bp fragment of null allele.
Western analysis. Total protein was extracted from embryonic mouse tissue. Mouse embryos were immediately frozen in liquid nitrogen. Tissues were homogenized in protein extraction buffer containing 100 mM Tris, pH 6.8, 2% sodium dodecyl sulfate (SDS), 25 mM dithiothreitol, and protease inhibitors. Proteins were separated on SDS-20% polyacrylamide electrophoresis gels and transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore, Billerica, MA). Western blots were blocked in 3% dry milk for 2 h at room temperature, incubated with a rabbit polyclonal antiserum against TGIF (TGIF H-172; Santa Cruz Biotechnology, Santa Cruz, CA) or against tubulin as a control at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature, and detected by the ECL detection kit (Amersham, Piscataway, NJ).
Histological analysis. Four-week-old wild-type, heterozygous, and Tgif–/– mice were weighed and sacrificed by carbon dioxide euthanasia. Sixty mice (10 per sex per genotype) were examined. The body length and tail length were measured with a ruler. The major organs were dissected, examined, rinsed with PBS, blotted dry, and weighed on a digital scale. For histology, 4- and 7-week-old age- and sex-matched wild-type, heterozygous, and Tgif–/– mice were anesthetized with 20 µl avertin per gram body weight via intraperitoneal injection. Avertin was prepared by dissolving 1.25 mg 2,2,2-tribromoethanol (Sigma-Aldrich, St. Louis, MO) in 2.5 ml 2.5% 2-methyl-2-butanol (Fisher Scientific, Pittsburgh, PA) water solution. The anesthetized mice were perfused transcardially with PBS, followed by Bouin's solution (Sigma-Aldrich, St. Louis, MO). Tissues from various organs were dissected and fixed in Bouin's fixative. Tissue samples were processed routinely and embedded in paraffin, sectioned at 5 µm, stained with hematoxylin and eosin (Harvard Medical School Rodent Histopathology Core and BIDMC Histology Core, Boston, MA), and analyzed by light microscopy.
Retinoic acid treatment. Tgif–/– female mice were mated with Tgif+/– male mice overnight. The time-pregnant mice were weighed and fed with 7.5 mg/kg body weight all-trans-retinoic acid (ATRA) (Sigma-Aldrich, St. Louis, MO) suspended in 100 µl canola oil at 9 a.m. on GD 7. The ATRA-treated pregnant mice were sacrificed by carbon dioxide euthanasia in the late afternoon of GD11. The embryos were dissected and genotyped by PCR. The morphology of each embryo was examined and recorded.
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RESULTS |
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We performed whole-mount ISH on E9.5 and E10.5 mouse embryos. Tgif mRNA was widely expressed in the developing mouse embryo at E9.5 (Fig. 1A), with the highest expression in the forebrain, the branchial arches, the otic pit, and the limb buds, but not in the heart. The front view at a higher magnification revealed the highest expression in the heart-shaped ventricular lining of the bulging telencephalic hemispheres (Fig. 1B). The expression pattern of Tgif was very different from that of Shh, the human HPE3 gene (Fig. 1C and 1D). In E10.5 embryos, Tgif expression was high in the forebrain and in the nasal pits (Fig. 1E).
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Generation of Tgif–/– mice.
The interesting expression patterns of Tgif prompted us to investigate the in vivo function of Tgif. We decided to create a mouse model lacking Tgif. Because most human HPE fetuses are spontaneously aborted and known mouse models lacking other HPE genes are embryonically lethal, we hypothesized that mice with a simple deletion of Tgif would probably die prenatally. Therefore, we employed a conditional knockout strategy using the Cre/loxP system to overcome the potential lethality.
We targeted the mouse Tgif locus in ES cells by homologous recombination. A detailed scheme for the targeting construct is shown in Fig. 2. Using this strategy, we obtained four correctly targeted ES clones from 191 G418-resistant clones (Fig. 3A). Two independent ES cell clones were injected, and germ line transmission of both TgifloxPneo/+ lines was achieved. The TgifloxP/+ conditional lines and the Tgif–/– knockout lines were produced by subsequent breeding, as genotyped by PCR (Fig. 3C) and Southern blotting (Fig. 3B). Tgif protein, which migrates as a doublet around 30 kDa on SDS-polyacrylamide gels (27), was not detectable in Tgif–/– embryos by Western blotting (Fig. 3D).
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Since Tgif was expected to play a role in forebrain patterning and proliferation, we performed morphological analysis of the brains of Tgif–/– mice. The brains were normal in size and weight (Fig. 4B; also see Fig. S1A and S2A in the supplemental material). Major brain structures appeared normal (see Fig. S1A in the supplemental material). The midline structures, including the thalamus, the hypothalamus, and the pituitary gland, were maintained. The two cerebral hemispheres were well formed, and the two lateral ventricles were well separated (Fig. 4B; also see Fig. S1B in the supplemental material). Cerebral cortical lamination was preserved (Fig. 4C; also see Fig. S1D in the supplemental material). In addition, the lamination of the retina also appeared normal (Fig. 4D; also see Fig. S1E in the supplemental material). Histological analysis of other major organ systems, including the heart, the lungs, the mandibular glands, the thyroid glands, the esophagus, the stomach, the small intestines, the colon, the liver, the pancreases, the kidneys, the adrenal glands, the thymus, the spleen, the bones and bone marrow, the bladder, the ovaries, the uterus, and the testes, showed no discernible pathological derangements (see Fig. S2 in the supplemental material).
No direct genetic interaction between Shh and Tgif.
Mutations in both SHH and TGIF have been found in a human HPE patient, whose mother, carrying only the SHH mutation, appeared phenotypically normal (16). This suggested that SHH and TGIF might interact genetically. We mated Tgif+/– mice with Shh+/– mice. Such a cross resulted in approximately Mendelian ratios of progeny (Table 1). Tgif–/–; Shh+/– mice were viable and fertile (Fig. 4A). The progeny of the intercross of Tgif–/–; Shh+/– mice also fit Mendelian ratios (Table 1). Furthermore, whole-mount ISH showed that Tgif mRNA was expressed in E10.5 Shh–/– embryos, with the highest expression in the primitive forebrain, the branchial arches, the otic pit, and the limb buds comparable to that of wild-type embryos (see Fig. S3 in the supplemental material). Tgif protein was detected in E11.5 and E12.5 Shh–/– embryos (see Fig. S3 in the supplemental material). We also observed Tgif–/–; Shh–/– double-mutant embryos from E10.5 to E13.5. These double-deficient embryos were indistinguishable from Shh–/– embryos. These results did not provide evidence to support the hypothesis that Shh and Tgif had direct genetic interaction in mouse.
Unaltered susceptibility to retinoic acid induced teratogenic effects.
Mice that ingest ATRA during early pregnancy are more likely to produce HPE offspring (46). ATRA could activate RXR. Since TGIF represses RXR function, TGIF mutations in humans may cause HPE by deregulating the retinoid signaling pathway. If this were the case, ATRA would have a more potent teratogenic effect on Tgif–/– embryos than on Tgif+/– embryos. To test this hypothesis, we fed ATRA to time-pregnant Tgif–/– mice on GD 7 and examined the phenotypes of the embryos in the late afternoon of GD 11. We collected 10 litters of E11.75 embryos but did not observe any embryo with an HPE-like phenotype. We did observe some deformed dead embryos. We genotyped all live and dead embryos. The percentages of dead embryos were not significantly different between Tgif–/– and Tgif+/– embryos (Fig. 5). These results suggest that Tgif–/– embryos were not more susceptible to ATRA-induced teratogenic effects.
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Like TGIF, TGIF2 binds Smad and HDAC, playing the role of a corepressor in the TGF-ß signaling pathway (31). The homeodomains of TGIF and TGIF2 demonstrate 77% identity, but the similarity is only 49% outside of the homeodomain. TGIF2 is slightly smaller, with 237 amino acids, an expected molecular mass of 25.9 kDa, and a pI (isoelectric point) of 7.96, in comparison to the 272-amino-acid TGIF, with a molecular mass of 29.7 kDa and a pI of 8.06, calculated by Lasergene Protean v5.07 (DNASTAR, Madison, WI).
By ISH, we observed that Tgif2 mRNA was expressed in a similar pattern in the proliferating neuroepithelium to that of Tgif at E11.5 and E12.5 (Fig. 6). Interestingly, Tgif2 expression also displayed a medial to lateral gradient at E12.5. While Tgif expression was absent in Tgif–/– embryos, Tgif2 expression was unaltered. Thus, Tgif2 is coexpressed with Tgif and hence could compensate functionally for loss of Tgif.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several TGIF paralogues are present in the mammalian genome. Human TGIF2 is located on chromosome 20, and mouse Tgif2 is located on chromosome 2 (UCSC Genome Browser). TGIFLX and TGIFLY are two TGIF-like genes on the sex chromosomes (6). Our results showed that there was no bias in the sex ratio of Tgif–/– mice, suggesting that Tgiflx and Tgifly are not the compensating products. On the other hand, Tgif2 shares high homology with Tgif in the TALE homeodomain, which is the DNA binding domain of homeoproteins, suggesting that Tgif and Tgif2 may bind to similar DNA sequences (21). TGIF2 has also been shown to interact with Smad2 and Smad3 as a corepressor (31). Our results showed that the expression pattern of Tgif2 was strikingly similar to that of Tgif, and Tgif2 expression was maintained in Tgif–/– mice. Therefore, Tgif2 may compensate for the loss of Tgif function.
It remains unclear whether the seven heterozygous mutations in human TGIF cause haplo-insufficiency due to partial loss of TGIF function, have a dominant-negative effect, or produce novel abnormal functions. Although TGIF is partially lost in chromosome 18 deletion cases, the patients also lack other genes in the adjacent genomic region. Therefore, the molecular mechanism may be more complicated in these cases than partial loss of TGIF function. Since none of the TGIF mutations found to date leads to complete loss of TGIF, the mutated TGIF may have a dominant-negative effect, which will not only eliminate normal TGIF function but also interfere with the function of other TGIF-like proteins, such as TGIF2 (1, 8, 16). Alternatively, a gain-of-function mechanism may underlie the etiology of HPE4. TGIF is a Smad corepressor that potentially antagonizes the functions of Nodal, a TGF-ß family member, and Smad2. HPE-like phenotypes occur in Smad2–/– and Smad2+/–; Nodal+/– mouse embryos (19, 38). Therefore, TGIF mutations in human HPE patients may overrepress the downstream target genes in the Nodal signaling pathway.
It is conceivable that having mutated forms of the TGIF protein may be more detrimental than complete loss of the protein. TGIF has been shown to autoregulate its own expression level (11). The human TGIF promoter region contains TGF-ß-responsive elements. TGIF represses the transcriptional activation of its own promoter by TGF-ß signaling, thus maintaining balanced expression levels of TGF-ß target genes. As a result, partial loss of wild-type TGIF may lead to overproduction of mutated TGIF, which interferes with normal developmental processes. In the event of total loss of TGIF, there is no mutated TGIF. Therefore, normal forebrain patterning may still take place in Tgif null mice with a little help from other compensating genes.
HPE is an extremely heterogeneous disease. It has been proposed that it is a multigenic disorder (34). Since currently identified HPE genes only account for a small portion of all sporadic HPE cases, there are still unidentified disease genes. The difficulties in identifying these genes may relate to the multigenic nature of HPE. Loss of function in a single HPE gene may not lead to the disease. In human HPE4 patients, there may be other modifier genes in addition to TGIF mutations.
It is well established that environmental and epigenetic factors contribute to the etiology of HPE. The normal-appearing Tgif–/– mice may be predisposed to certain environmental conditions that increase the frequency of HPE, although our data with retinoic acid did not reveal such an effect. Therefore, Tgif–/– mice can still be a good mouse model to screen for modifier genes with a "second hit" mutation to cause HPE, which is not possible with other early-lethal HPE mouse models.
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ACKNOWLEDGMENTS |
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We thank members of the Walsh laboratory for their input throughout the course of this project, Clifford Tabin for the mouse Shh cDNA plasmid, Susan Dymecki for the FLP deleter mice, Margaret Thompson for the 129 genomic library, Urs Berger for the help with the ISH, Roderick Bronson for consultation on histology, Susanne White for help with histology, and Massachusetts General Hospital Gene Targeting Core for ES cell work and microinjections.
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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
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