Human Molecular Genetics, 2002, Vol. 11, No. 9 1129-1135
© 2002 Oxford University Press
Pallister–Hall syndrome phenotype in mice mutant for Gli3
Institute for Animal Developmental and Molecular Biology, Heinrich-Heine University, Düsseldorf, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations in the GLI3 gene have been identified in several human malformation syndromes. One of these autosomal dominant developmental disorders is Pallister–Hall syndrome (PHS; MIM146510), which is associated with central polydactyly and other malformations. Interestingly, the mutations in the GLI3 transcription factor gene identified in patients with PHS are restricted to the region 3' of the zinc finger-encoding domain, leaving this DNA-binding domain intact. We have investigated the consequences of this mutation on the development of multiple organ systems by introducing a targeted mutation in mice. We found that mice homozygous for the mutation showed a central polydactyly, thus modeling one of the major abnormalities of the human syndrome. Moreover, Gli3-mutant mice displayed a wide range of developmental abnormalities encompassing almost all of the common PHS features, including imperforate anus, gastrointestinal, epiglottis and larynx defects, abnormal kidney development, and absence of adrenal glands. Thus, our Gli3-mutant mice provide an excellent model for studies of both the pathogenesis of PHS and Gli3 functions in the development of the affected organ systems.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Pallister–Hall syndrome (PHS; MIM146510) was first described in 1980 and represents a pleiotropic disorder of human development, usually associated with central polydactyly, imperforate anus, hypothalamic hamartoma and other malformations (1,2). This disorder is inherited as an autosomal dominant trait and has been mapped to chromosome 7p13 (3). Mutations in the transcription regulator gene GLI3 has been identified in patients with PHS (4). Interestingly, mutations in GLI3 are also associated with four other different autosomal dominant phenotypes: the Greig cephalopolysyndactyly syndrome (GCPS; MIM175700), preaxial polydactyly type IV (PPD-IV; MIM 174700), postaxial polydactyly type A (PAP-A; MIM 1472000) and postaxial polydactyly type A/B. Most of the clinical characteristics of these syndromes are distinct, but all share digit abnormalities. These limb malformations show a wide range of phenotypes, including preaxial (GCPS, PPD-IV), central (PHS) and postaxial (PAP-A/B) polydactyly. It has been suggested that the site of the GLI3 mutation might be correlated to the resulting phenotypes (4–6). PHS and PAP-A are caused by mutations that are located exclusively 3' of the zinc finger domain. However, other publications showed that GCPS and PPD-IV are associated with mutations that map throughout the coding region (7,8).
Several mouse mutants without a functional Gli3 zinc finger domain serve as an animal model for GCPS (9–11). A deletion 3' of the first zinc finger in the XtJ mouse mutant results in preaxial polydactyly, neural tube closure defects and embryonic lethality in hetero- and homozygous animals, respectively (9,10). The XtJ allele is thought to be a null allele. The different Gli3 mouse mutants are caused by mutations before or within the zinc finger domain and display phenotypic variability. Importantly, they all exhibit a preaxial polydactyly (9–11). To better understand the etiology of human PHS, we were interested in the development of a mouse model. Therefore, we generated mice carrying a targeted mutation 3' of the zinc fingers at the Gli3 locus. Indeed, homozygous mutant mice exhibited a wide range of developmental anomalies encompassing almost all of the common PHS features.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation of Gli3
699-mutant miceTo evaluate the importance of specific GLI3 mutations in the etiology of PHS in a model system, a selection marker cassette was inserted by gene targeting in mouse embryonic stem (ES) cells into the Gli3 locus. This mutation (Gli3
) was supposed to result in a premature termination of translation C-terminally of the zinc finger region, generating a shortened protein of 744 amino acids (Fig. 1). Homologous recombinant ES cell clones were identified by Southern blot analysis and two independent clones were injected into C57BL/6 blastocysts. The resulting chimeric mice were crossed with C57BL/6 and 129P2 mice, respectively, to produce (Gli3/+) F1 progeny.
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To confirm that the desired mutation resulted in an altered Gli3 transcript, reverse-transcription polymerase chain reaction (RT–PCR) analysis of Gli3 was performed using different primers flanking the insertion site (Fig. 1A). In contrast to wild-type embryos, in both heterozygous and homozygous mutant embryos, Gli3 transcripts were altered and an abnormal band was amplified from the mutant allele (Fig. 1E). Sequencing of this PCR amplification product identified an unexpected transcript. Detailed analysis revealed an abnormal splicing into the 3' part of the tk cassette, which caused a loss of Gli3 coding sequences, an apparent frameshift and a predicted protein termination codon (Fig. 1F). Use of different primer combinations such as forward 4 and backward 2 (Fig. 1A) did not reveal any other transcript than the one shown in Figure 1F. The mutant allele terminates just C-terminally of the zinc finger domain (amino acid position 699), in close proximity to the mutations identified in PHS patients (4), with an additional 21 residues of abnormal protein sequence between the splice site and the stop codon (Fig. 1F). As a consequence, the mutation in these mice predicts a truncated Gli3 protein that consists of 720 amino acids, compared with the predicted length of 1588 amino acids of the wild-type Gli3 (12), and encodes a similar aberrant transcription factor as was identified in PHS patients (4). In relation to the end of the Gli3 protein sequence at position 699, we called this allele Gli3
699.
Gli3699/Gli3699 mutants die shortly after birth
Adult mice heterozygous for the mutant allele, Gli3699, were viable and fertile on an inbred 129 or in a (129xC57) background. On the inbred 129 background, we observed a postaxial extra digit at very low frequencies (6%) on forelimbs of Gli3699/+ mice (data not shown). This digit was not well formed and frequently occurred in the form of a unilateral skin tag (postminimus). On a mixed 129xC57/BL6 background, the appearance was further reduced (3%). Independently of the background, we observed no evidence of any other phenotype in heterozygous animals. Intercrossing heterozygotes yielded no homozygous mutant mice at weaning. However, homozygous animals were recovered at the expected Mendelian frequency at birth and during embryonic stages. Homozygous mutants died within 12–18 hours after birth (Fig. 2A). Both male and female Gli3699/Gli3699 mice were obtained.
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The Gli3
699 mutation causes imperforate anus and visceral abnormalitiesTo determine the etiology of the early lethality, we examined Gli3
699/Gli3699 embryos from timed pregnancies throughout development. By gross morphology, homozygous mutant embryos were retarded between E10.5 and E12.5; however, at later time points in development, no differences in the size of the embryos were obvious (data not shown). Importantly, they had an imperforate anus (Fig. 2B), and neonatal Gli3699/Gli3699 mice accumulated air in their stomachs and intestines (data not shown). Comparison of transverse sections of E15.5 wild-type and Gli3699/Gli3699 gastrointestinal tracts revealed that the colon terminated in a blind dilation that was not fused to the surface ectoderm (insets in Fig. 2C). In addition, we observed a dilation of parts of the intestine, a reduction in size of the villi and an abnormally thin wall of the colon (Fig. 2C), which is reminiscent of Hirschsprung's disease (13).
Additionally, Gli3699/Gli3699 embryos showed malformations of the respiratory system. Histological examination at E15.5 revealed an absence of the epiglottis (data not shown) and a laryngeal cleft resulting in an abnormal connection between the esophagus and the larynx (Fig. 2D). Another finding among the homozygous mutants analyzed between E15.5 and birth was the complete absence of the adrenal gland and the absence of one kidney combined with phenotypic abnormalities in the remaining kindney (Fig. 3). By E11.5, the bilateral anlagen of the metanephric blastema were visible in normal embryos and in homozygous mutants (data not shown). Thus, full-length Gli3 is not essential for the initiation but is for the subsequent development of the kidney.
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Skeletal malformations in Gli3
699/Gli3699 miceExamination of skeletal preparations of newborn mice revealed additional defects in Gli3
699/Gli3699. The newborn mutants had an overall length that was similar to that of their wild-type littermates, but a curvature of the spine and a slightly enlarged skull were observed (Fig. 4A). The analysis of the chondrocranium in Gli3699/Gli3699 neonates showed a malformation at the base of the skull. The posterior basisphenoid bone, which is the hypophyseal cartilage surrounding the pituitary, was misshapen and reduced in size, and a hole in its center was evident (Fig. 4B). The secondary palate was also affected in homozygous mutants – the two palate shelves were present but showed a delayed mineralization (Fig. 4B). In addition, we analyzed the mineralization of appendicular and axial skeletal elements in Gli3699/Gli3699 neonates. Advanced mineralization was found in the sclerotome-derived vertebrae. The premature mineralization did, however, not occur throughout the vertebral column. In the lumbar and sacral region, ossification in the dorsal-arch unit already extended to fuse with the ossification centers of the vertebral bodies. A fusion of neighboring vertebrae in homozygous neonates was apparent (Fig. 4C). In contrast, we observed a delayed mineralization in the lateral mesoderm-derived skeletal elements of the autopod (Fig. 5D). Detailed examination of skeletal preparations and histological transverse sections of wild-type and Gli3699/Gli3699 mice revealed additional limb malformations. All Gli3699/Gli3699 mice showed different degrees of bilateral and anterior bending of long bones, including ulna, radius and tibia, with the most severe bending always being observed in the tibia (Figs 4A and 5D). The severity of bending varied among the long bones from slight bowing (radius) to obvious angulation (tibia). In addition, a proximo-distal shortening of the fore- and hindlimbs and a central/insertional polydactyly in the autopod of homozygous mutant mice was obvious (Fig. 5D). The position of the additional digit varied among the Gli3699/Gli3699 animals. In half of them, the extra digit was located in the same plane as the remaining digits, whereas it was localized ventral of digit 3 in the other 50% of Gli3699/Gli3699 mice (Fig. 5D and C). To further understand the defects of the appendicular skeleton of Gli3699/Gli3699 mutants, we examined the developmental course of the limb malformations. At E14.5, the limbs of Gli3699/Gli3699 fetuses already showed the described central polydactyly and the proximo-distal shortening of the fore and hindlimbs, as well as soft tissue syndactyly and dysplastic nails (Fig. 5A,C and D, and data not shown). By E13.5, whole-mount in situ hybridizations revealed a difference in Sox9 expression between wild-type and mutant embryos (Fig. 5B). The alterations of Sox9, an early chondrogenic marker, were identical to the morphological defects that were observed one day later in the limbs of homozygous mutant embryos (compare Fig. 5A and 5B). These data suggest that the Gli3699 mutation has an effect not only on bone development but already on patterning of the limb bud. However, at E11.5, we could not detect any differences in the localization of various marker gene transcripts for anterior–posterior (Shh, dHAND), proximo-distal (Fgf8) and dorso-ventral (Lmx1b) patterning of the developing limb or for Bmp4 or Hoxd13 in the limb buds of homozygous Gli3699/Gli3699 animals (data not shown).
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Programmed cell death is impaired in the limbs of Gli3
699/Gli3699 miceWhen we examined the limbs of Gli3
699/Gli3699 animals by TUNEL staining, we observed a reduced number of apoptotic cells in the interdigital web (Fig. 5E), consistent with the presence of syndactyly in fore and hindlimbs (Fig. 5A). In contrast, in situ hybridization indicated that the pattern of Msx2, a gene known to be involved in the realization of interdigital cell death (14), was not obviously altered in E12.0 limbs of Gli3699/Gli3699 mice (Fig. 5F). This finding suggested that either Msx2 signalling is interrupted or the alterations in programmed cell death in Gli3699/Gli3699 animals are caused by an Msx2-independent mechanism. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have investigated the consequences of a Gli3 mutation that maps 3' of the zinc finger region on mouse development and have shown that Gli3
699/Gli3699 mice display multiple abnormalities. Our study confirms and extends previous evidence suggestive of an involvement of C-terminally truncated GLI3 proteins in PHS. Detailed analyses of the Gli3699/Gli3699 mice indicated that a full-length Gli3 is essential for several aspects of normal development of vertebrates.
Gli3699/+ mutant mice are characterized by a postaxial polydactyly with variable penetrance. On the inbred 129 back-ground, we observed the postaxial extra digit on both forelimbs at low frequencies (6%). On the mixed 129xC57/BL6 background, the appearance was reduced (3%) and this additional digit occurred in the form of a unilateral skin tag. A similar influence of the genetic background was also observed on the XtJ allele. Backcrossing these animals (C3HxC57) with 129P2 mice resulted in an extra postaxial digit in heterozygous offspring at a low frequency (data not shown). These observations support the strong influence of the genetic background on the appearance of a postaxial polydactyly in different Gli3 mouse mutants and the implication of genetic modifiers (15).
At birth, the Gli3699/Gli3699 mice were viable and showed no obvious behavioral abnormalities. However, the neonates became lethargic and died between 12 and 18 hours postpartum. The cause of death is thought to be a combination of the severe structural malformations found in the Gli3699/Gli3699 mice and probably acute hypoglycemia due to insufficient suckling of the newborns and lack of energy to compete for nourishment (16).
Gli3699/Gli3699 mice showed several malformations, such as central polydactyly, imperforate anus, gastrointestinal, epiglottis and larynx defects, abnormal kidney development, and absence of adrenal glands. In contrast, mice carrying a Gli3 null allele (Xt) do not show these phenotypic alterations (9,17). Instead, these mice display a preaxial polydactyly at a high frequency, an absence of olfactory bulbs and the cerebral cortex, and other pleiotropic malformations (9,17,18). These differences suggest that the different alleles generate Gli3 proteins with very different functions in development. This interpretation is in line with a current model about the function of Gli3 that considers two forms: either a full-length protein that acts as a transactivator or a physiologically generated C-terminally truncated form with characteristics of a transcriptional repressor (19,20). A repressor function is also indicated by the findings in our Gli3-mutant mice. The observed phenotypic alterations in Gli3699/Gli3699 mice revealed striking overlap with the gastrointestinal tract phenotype of Shh and Ihh mutant mice (13). These similarities indicate that Gli3699 efficiently interferes with normal development in the same way as inactivation of Shh and Ihh. However, in other developmental processes such as limb patterning, Gli3699 does not seem to block Shh signaling. Here, a block a Shh signaling should result in a loss of antero-posterior identity and a loss of digits, as has been shown for Shh-negative mice (21). During limb development, one function of Gli3 is to suppress a second Shh domain anteriorly (22). This function is still realized by the C-terminally truncated Gli3 protein. Instead, signaling appears to be affected at a central position in the antero-posterior axis, with a tendency towards ventral, as can be invoked from an ectopic digit or a branching at the second phalange of the third digit. Our analysis has not revealed any obvious change in the expression of several important genes for limb development. It could be that the expression levels are only marginally altered and therefore not detectable by whole-mount in situ hybridization. Further investigations will have to clarify this problem.
The observed phenotypic abnormalities bear striking similarities to those observed in PHS patients (Table 1). There is, however, an important issue that has not been resolved with respect to the applicability of this mouse model to the human PHS. Patients with this syndrome has been shown to be heterozygous for mutations in GLI3 3' of the zinc finger region (4), whereas heterozygosity of the Gli3699 mutation in mice did not result in a phenotype reminescent to PHS. Nonetheless, the abnormalities are of the same type and affect mostly the same organs in both cases. One possible explanation is a species difference in dosage sensitivity resulting in different penetrance of the phenotypes, as has been reported for several other genes, such as GATA3, LMX1b, MSX2 and TBX1. In all these cases, heterozygous effects are evident in humans but not mice, and the homozygous effects in mice are similar in nature to the human heterozygous effects (23–26). Similarly, in the case of Gli3699, we postulate that humans are more sensitive than mice to lower levels of the gene product, and thus display increased penetrance. In that context, one could consider that the mRNA of the targeted allele might have a reduced half-life compared with the mRNA of PHS patients. Alternatively, it might be possible that human–mouse differences in timing and spatial expression are responsible for the observed discrepancies, as has been described, for example, for Wnt genes (27). Further studies will be required to address these possibilities.
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In conclusion, our Gli3
699/Gli3699 mice provide a valuable model for studying the molecular mechanisms underlying proper development of the affected organ systems (kidney, limbs, adrenal gland and gastrointestinal tract). Furthermore, our in vivo data support previous conclusions (4–6) of a genotype–phenotype correlation for GLI3 mutations. Finally, our Gli3699/Gli3699 mice will facilitate investigations of the function of GLI3 in the fields of congenital birth defects seen in PHS. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Gene targeting
We used homologous recombination to target Gli3 to produce the Gli3
699 allele. The targeting construct was designed to introduce a PGKneoNTRtkpA cassette (28) into the first exon 3' of the zinc finger region of the mouse Gli3. The vector included the BglII–ClaI 7.5 kb and ClaI–SacII 3.5 kb genomic DNA fragments isolated from a mouse 129/SvJ genomic BAC library (IncyteGenomics). The linearized vector was electroporated into E14 embryonic stem (ES) cells (29). After G418 selection (350 µg/ml), cells were screened by Southern blot analysis using the 3' external probe (3' probe) with a diagnostic BamHI digest to distinguish a 18 kb endogenous allele from a 4 kb targeted allele. Additional Southern analyses with SpeI, SacI and KpnI confirmed the targeting event. The targeting efficiency was 12%. Targeted ES cells were used to create chimeras that passed the Gli3699 mutation on to their progeny. Chimeric mice were mated with C57BL/6 mice to produce (129P2xC57BL/6)F1 mice. Gli3699/+ F1 animals were intercrossed to derive homozygous mutant F2 newborns and embryos by timed mating. Genotyping was carried out by either Southern blot analysis or PCR of DNA extracted from the organs or tail tips of neonates or the yolk sacs of embryos. The 18 and 4 kb BamHI-digested fragments corresponding to the wild-type (+) and targeted () allele were identified by hybridization with the 3' external probe (3' probe). PCR analysis of embryos derived from matings between heterozygous mice using primer sequences 1 and 2 showed the wild-type and targeted fragment. Total RNA was isolated using TRIZOL (GibcoBRL) followed by DNaseI digestion. First-strand cDNA was synthesized with the Expand RT system (Roche) according to the manufacturer's instructions. PCR amplification was carried out using the prime sequences 3, 4 and 5, respectively. Primer sequences for amplification for genotyping and RT–PCR are available upon request.
Analysis of Gli3699/Gli3699 mutants
Embryos were collected for analysis between E10.5 and term. Standard methods were used for Alcian blue/Alizarin red skeletal preparations (30). In brief, embryos were fixed in 80% ethanol, skinned and eviscerated, and stained in Alcian blue/Alizarin Red for 6 h at 37°C. Embryos were then rinsed with ethanol, and finally were run through an 1% potassium hydroxide : glycerol series and stored in pure glycerol. Embryos for histology were fixed in 4% paraformaldehyde in PBS, and paraffin sections were prepared according to a standard protocol and processed for H&E staining of serial sections. Whole-mount in situ hybridizations were performed essentially as described previously (31). For the detection of apoptotic cells in the limb by the TUNEL procedure, we followed exactly the protocol described previously (32).
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ACKNOWLEDGEMENTS |
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We are indepted to K. Götz, A. Eggert and K. Rajewsky for their help in the initial phase of the project. We thank B. Robert and R. Lovell-Badge for probes and Thomas Theil and Karl-Heinz Grzeschik for comments and critical reading of the manuscript. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Ru 376/7).
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FOOTNOTES |
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* To whom correspondence should be addressed at: Heinrich-Heine University, Universitätsstrasse 1, 40225 Düsseldorf, Germany. Tel:+49 (0)211 81 11391; Fax: +49 (0)211 81 15113; Email: ruether{at}uni-duesseldorf.de
Present address: Jens Böse, Division of Microbiology, Research Group Infection Genetics, German Research Centre for Biotechnology (GBF), Mascheroder Weg 1, 38124 Braunschweig, Germany
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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 I. Matera, D. E. Watkins-Chow, S. K. Loftus, L. Hou, A. Incao, D. L. Silver, C. Rivas, E. C. Elliott, L. L. Baxter, and W. J. Pavan A sensitized mutagenesis screen identifies Gli3 as a modifier of Sox10 neurocristopathy Hum. Mol. Genet., July 15, 2008; 17(14): 2118 - 2131. [Abstract] [Full Text] [PDF]
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 R. H. Wallace, J. L. Freeman, M. R. Shouri, P. A. Izzillo, J. V. Rosenfeld, J. C. Mulley, A. S. Harvey, and S. F. Berkovic SOMATIC MUTATIONS IN GLI3 CAN CAUSE HYPOTHALAMIC HAMARTOMA AND GELASTIC SEIZURES Neurology, February 19, 2008; 70(8): 653 - 655. [Full Text] [PDF]
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P. Hill, B. Wang, and U. Ruther The molecular basis of Pallister Hall associated polydactyly Hum. Mol. Genet., September 1, 2007; 16(17): 2089 - 2096. [Abstract] [Full Text] [PDF]
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 J. Bok, D. K. Dolson, P. Hill, U. Ruther, D. J. Epstein, and D. K. Wu Opposing gradients of Gli repressor and activators mediate Shh signaling along the dorsoventral axis of the inner ear Development, May 1, 2007; 134(9): 1713 - 1722. [Abstract] [Full Text] [PDF]
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M. C. Hu, R. Mo, S. Bhella, C. W. Wilson, P.-T. Chuang, C.-c. Hui, and N. D. Rosenblum GLI3-dependent transcriptional repression of Gli1, Gli2 and kidney patterning genes disrupts renal morphogenesis Development, February 1, 2006; 133(3): 569 - 578. [Abstract] [Full Text] [PDF]
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 M. Merchant, M. Evangelista, S.-M. Luoh, G. D. Frantz, S. Chalasani, R. A. D. Carano, M. van Hoy, J. Ramirez, A. K. Ogasawara, L. M. McFarland, et al. Loss of the Serine/Threonine Kinase Fused Results in Postnatal Growth Defects and Lethality Due to Progressive Hydrocephalus Mol. Cell. Biol., August 15, 2005; 25(16): 7054 - 7068. [Abstract] [Full Text] [PDF]
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M. Parrish, T. Ott, C. Lance-Jones, G. Schuetz, A. Schwaeger-Nickolenko, and A. P. Monaghan Loss of the Sall3 Gene Leads to Palate Deficiency, Abnormalities in Cranial Nerves, and Perinatal Lethality Mol. Cell. Biol., August 15, 2004; 24(16): 7102 - 7112. [Abstract] [Full Text] [PDF]
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Y. Chen, V. Knezevic, V. Ervin, R. Hutson, Y. Ward, and S. Mackem Direct interaction with Hoxd proteins reverses Gli3-repressor function to promote digit formation downstream of Shh Development, May 15, 2004; 131(10): 2339 - 2347. [Abstract] [Full Text] [PDF]
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O. Krebs, C. M. Schreiner, W. J. Scott Jr, S. M. Bell, D. J. Robbins, J. A. Goetz, H. Alt, N. Hawes, E. Wolf, and J. Favor Replicated anterior zeugopod (raz): a polydactylous mouse mutant with lowered Shh signaling in the limb bud Development, December 15, 2003; 130(24): 6037 - 6047. [Abstract] [Full Text] [PDF]
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S. M. Kiefer, K. K. Ohlemiller, J. Yang, B. W. McDill, J. Kohlhase, and M. Rauchman Expression of a truncated Sall1 transcriptional repressor is responsible for Townes-Brocks syndrome birth defects Hum. Mol. Genet., September 1, 2003; 12(17): 2221 - 2227. [Abstract] [Full Text] [PDF]
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 M. Persson, D. Stamataki, P. te Welscher, E. Andersson, J. Bose, U. Ruther, J. Ericson, and J. Briscoe Dorsal-ventral patterning of the spinal cord requires Gli3 transcriptional repressor activity Genes & Dev., November 15, 2002; 16(22): 2865 - 2878. [Abstract] [Full Text] [PDF]
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