Birth Defects Caused by Mutations in Human GLI3 and Mouse Gli3 Genescga

doi:10.1111/j.1741-4520.2009.00266.x Congenital Anomalies 2010; 50, 1–7 1

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Birth defects caused by mutations in human GLI3 and mouse Gli3 genescga_

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Ichiro Naruse1, Etsuko Ueta1, Yoshiki Sumino1, Masaya Ogawa1, and Satoshi Ishikiriyama2 1School of Health Science, Faculty of Medicine, Tottori University, Yonago, and 2Division of Clinical Genetics and Cytogenetics, Shizuoka Children’s Hospital, Shizuoka, Japan

ABSTRACT GLI3 is the gene responsible for Greig cepha- type-A (PAP-A) and preaxial polydactyly type-IV (PPD-IV). A lopolysyndactyly syndrome (GCPS), Pallister–Hall syndrome mimic phenomenon is observed in mice. Investigation of human (PHS) and Postaxial polydactyly type-A (PAP-A). Genetic poly- GLI3 and Gli3 genes has progressed using the knowledge of dactyly mice such as Pdn/Pdn (Polydactyly Nagoya), XtH/XtH Cubitus interruptus (Ci) in Drosophila, a homologous gene of GLI3 (Extra toes) and XtJ/XtJ (Extra toes Jackson) are the mouse and Gli3 genes. These genes have been highly conserved in the homolog of GCPS, and Gli3tmlUrtt/Gli3tmlUrt is produced as the animal kingdom throughout evolution. Now, a lot of mutant mice of mouse homolog of PHS. In the present review, relationships Gli3 gene have been known and knockout mice have been pro- between mutation points of GLI3 and Gli3, and resulting phe- duced. It is expected that the knowledge obtained from mutant and notypes in humans and mice are described. It has been con- knockout mice will be extrapolated to the manifestation mecha- firmed that mutation in the upstream or within the zinc finger nisms in human diseases to understand the diseases caused by the domain of the GLI3 gene induces GCPS; that in the post-zinc mutations in GLI3 gene. finger region including the protease cleavage site induces PHS; and that in the downstream of the GLI3 gene induces PAP-A. A Phenotype of GCPS mimicking phenomenon was observed in the mouse homolog. Greig cephalopolysyndactyly syndrome is a disorder that affects Therefore, human GLI3 and mouse Gli3 genes have a common the development of the limbs, head and face. The features of this structure, and it is suggested here that mutations in the same syndrome are highly variable, ranging from very mild to severe. functional regions produce similar phenotypes in human and GCPS might characterized by a set of craniofacial defects (e.g. mice. The most important issue might be that GCPS and PHS macrocephaly, broad nasal root, ocular hypertelorism and promi- exhibit an autosomal dominant trait, but mouse homologs, such nent forehead) (Fig. 1A,B), and one or more extra fingers or toes as Pdn/Pdn, XtH/XtH, XtJ/XtJ and Gli3tmlUrt/Gli3tmlUrt, are autoso- (polydactyly) (Fig. 1C,D) or having an abnormally wide thumb or mal recessive traits in the manifestation of similar phenotypes hallux. The skin between the fingers and toes might exhibit cuta- to human diseases. It is discussed here how the reduced neous syndactyly (Fig. 1C,D) (Greig 1926; Gollop and Fontes amounts of the GLI3 protein, or truncated mutant GLI3 1985; Biesecker, 2009). Rarely, affected individuals have more protein, disrupt development of the limbs, head and face. serious medical problems, including seizures, developmental delay, hydrocephalus and intellectual disability (Biesecker 2009). It is Key Words: GCPS, Gli3, PAP-A, Pdn,PHS,Xt impossible to determine the incidence of GCPS, because reliable clinical criteria and molecular diagnostics are not yet readily available (Biesecker 2008). INTRODUCTION Phenotype of Pdn mouse A responsible gene can be identified using blood and/or fibroblast The Pdn (Polydactyly Nagoya) mouse (Gli3Pdn) was derived from cells of the human birth defects, but it is impossible to know how Jcl : ICR (Hayasaka et al. 1980) and has been inbred in Naruse’s the phenotypes appear. We can investigate the mechanisms of how laboratory. It is now of 146th generation (Mouse Genome Informat- the phenotype manifests in the animal homologous disease by ics, ID: MGI:1856282, http://www.informatics.jax.org) (RIKEN observing the phenomena during embryogenesis. In this review, we BioResource Center BRC no. 00387, http://www.brc.riken.jp). would like to describe the birth defects caused by the mutation of Pdn/+ mice exhibited broad thumbs of the first digit in the fore- the GLI3 gene in humans and the Gli3 gene in mice. GLI3 and Gli3 limb (Fig. 2A), preaxial polydactyly of distal phalangeal type in the genes have zinc finger domains (transcriptional regulation domains) hindlimb (Fig. 2B,F) (Hayasaka et al. 1980; Naruse and Kameyama and are peculiar genes; the full length of the GLI3 protein functions 1982) a normal brain with the olfactory bulb (Fig. 2I), normal as a transcriptional activator, and the N terminal part functions as a spaced eyes (Fig. 2K) and a normal forehead (Fig. 2M). Pdn/Pdn transcriptional repressor after cleavage into two parts. mice exhibited preaxial polydactyly of complete type and syn- According to the positions of mutation, various types of pheno- dactyly both in the fore- and hindlimbs (Fig. 2C,D,G,H) (Hayasaka type appear, such as Greig cephalopolysyndactyly syndrome et al. 1980; Naruse and Kameyama 1982), postaxial polydactyly in (GCPS), Pallister–Hall syndrome (PHS), postaxial polydactyly the forelimbs (Fig. 2C), absence of olfactory bulb (Fig. 2J), hydrocephalus (Naruse et al. 1990), ocular hypertelorism (Fig. 2L), Correspondence: Ichiro Naruse, PhD, School of Health Science, Faculty prominent forehead (Fig. 2N) and retinal coloboma. They die soon of Medicine, Tottori University, Yonago 683-8503, Japan. Email: after birth because of suckling dysfunction (Hongo et al. 2000). The [email protected] similarity of the phenotype between GCPS and Pdn/Pdn is shown Received September 30, 2009; revised and accepted December 7, 2009. in Table 1.

Fig. 1 Phenotype of Greig cephalopolysyndactyly syndrome (GCPS). GCPS exhibits broad nasal root, ocular hypertelorism, macrocephaly (A, B), and polysyndactyly in the hands (C) and feet (D).

GLI3 gene in human and Gli3 gene in mice 2002). SUFU (Suppressor of fused) is essential for regulation of The official name of the GLI-Kruppel family member GLI3 gene is Gli/Ci processing, activity, and localization (Dunaeva et al. 2003). ‘GLI family zinc finger 3’ (Ruppert et al. 1988). Three homologs of Degron N is a specific sequence of amino acids in a protein that Cubitus interruptus (Ci) in Drosophila, Gli1, Gli2 and Gli3,have directs the starting place of degradation positioned in N-terminal been implicated in mediating responses to Sonic hedgehog (SHH) region (Huang et al. 1998). Around 14 cM of mouse chromosome in vertebrates (von Mering and Basler 1999). GLI3 gene is at 7p13 13 is the synteny region with 7p13 on the human chromosome 7 on human chromosome 7 (Ruppert et al. 1990). GLI3 protein has (Pettigrew et al. 1991; Lyon and Kirby 1992). Human GLI3 and been shown to be a sequence-specific DNA binding protein (Kinzler mouse Gli3 have 69% homology in DNA sequence, and 82% and Vogelstein 1990; Ruppert et al. 1990). It has been proposed that homology in amino acid sequence. GLI3 protein plays important roles in the embryonic development Proteins of the GLI family function in the same molecular (Ruppert et al. 1988; Schimmang et al. 1992). pathway as SHH protein. This pathway is essential for early devel- GLI family proteins attach to specific regions of DNA and opment (Ming et al. 1998). It plays a role in cell growth, cell control whether particular genes are turned on or off. Full length specialization and the patterning of structures such as the brain and GLI3 protein works as a transcriptional activator, and N terminal limbs (Genetics Home Reference: http://ghr.nlm.nih.gov). Depend- part of GLI3 protein works as a transcriptional repressor after ing on signals from SHH, the GLI3 protein can either activate or cleavage at nucleotide position 1988 in the protease cleavage site repress other genes such as Emx2, Wnt7b, Wnt8b and Msx (Ueta (Fig. 3A). Human GLI3 gene is constructed with 15 exons, and has et al. 2008; Lallemand et al. 2009). Hill et al. (2009) proposed that 5 zinc finger motifs at nucleotide number 1386–1935, protease unprocessed full-length GLI3 is dispensable for anteroposterior cleavage site, transactivation and CBP-binding regions (TA/CBP) at patterning of the limb bud. Instead, digit identities are most likely 2481–3396, transactivation domain 2 (TA2) at 3132–3966 and defined by GLI3 repressor activity alone. Anteroposterior grading transactivation domain 1 (TA1) at 4128–4740, a-helical region at of GLI3 activity by the action of SHH in digital pattering is 4482–4536 (Kalff-Suske et al. 1999), and the full length of struc- reported by Hill et al. (2009). ture gene is 4743 bp (Fig. 3A) (EMBL ID: AJ250408). CBP, CREB-binding protein, is ubiquitously expressed and is involved in GLI3 and Gli3 genes responsible for Greig the transcriptional coactivation of many different transcription cephalopolysyndactyly syndrome and Pdn mouse factors (Chrivia et al. 1993), and a-helix acts as an activation Different genetic changes involving the GLI3 gene can cause domain (Yoon et al. 1998). GCPS. In some cases, the condition results from a chromosomal Mouse Gli3 gene has also 5 zinc finger motifs at nucleotide abnormality, such as a large deletion or rearrangement of genetic position 1437–1914 and is constructed with 15 exons. It has tran- material, in the region of chromosome 7. In any case, deletion scriptional repressor region at 318–708, Ski binding site at 456– and/or mutations in the 5′ half of the GLI3 gene, in the open reading 1191, SUFU binding site at 984–1014, Degron N (Tsanev et al. frame at nucleotides position 1–1997, cause GCPS (Kang et al. 2009), and the full length of structure gene is 4752 bp (EMBL, ID: 1997; Johnston et al. 2005) (Fig. 4A). GCPS exhibits an autosomal BC145445) (Fig. 3B), and mapped at 14 cM on mouse chromo- dominat trait (Gollop and Fontes 1985). some 13 (EMBL-EBI: http://www.ebi.ac.uk). Ski is a corepressor in Integration of a transposon in the Gli3 gene in a Pdn mouse has transcriptional regulation in the full-length forms of Gli3 (Dai et al. been reported (Thien and Rüther 1999; Naruse et al. 2000), and

A B C D

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I J M N

Fig. 2 Phenotype of Pdn mouse. Pdn/+ mouse exhibited broad thumb of the ﬁrst digit in the forelimb (arrow in A), preaxial polydactyly of distal phalangeal type in the hindlimb (arrows in B and F), normal brain with the olfactory bulb (arrow in I), normal spaced eyes (K), and normal forehead (M). Pdn/Pdn mouse exhibited preaxial polydactyly of complete type and syndactyly both in the fore- and hindlimbs (C, D, G, H), postaxial polydactyly in the forelimb (arrow in C), absence of olfactory bulb (arrow in J), ocular hypertelorism (L), and prominent forehead (N).

we demonstrated in a previous study that 5542 bp of early ret- to approximately 60% of +/+ in the Pdn/+ mice and approxi- rotransposon was inserted into intron 3 in the transcriptional mately 20% of +/+ in the Pdn/Pdn mouse throughout early repressor region of Gli3 gene (Ueta et al. 2002) (Fig. 4B). The embryogenesis (Ueta et al. 2004), and insertion of small pieces of transposon was very similar to Y17106 (Hofmann et al. 1998) taransposon sequence into the mRNA was detected, resulting in (EMBL-EBI: http://www.ebi.ac.uk). Gli3 expression is depressed truncated GLI3 proteins (Thien and Rüther 1999).

Table 1 Comparison of phenotype between Greig cepha- (A): Human GLI3 structure gene lopolysyndactyly syndrome (GCPS) and Pdn/Pdn Transcriptional activator GCPS Pdn mouse 1 bp 1998 bp 3481 bp 4743 bp α-helical Broad macrocephaly Hydrocephalus in the Pdn/Pdn Zinc finger region domain TA/CBP nasal root 4482-4536 1386–1935 2481-3396 Ocular hypertelorism Ocular hypertelorism in the protease TA TA Pdn/Pdn 2 1 cleavage 3132-3966 4128-4740 Prominent forehead Prominent forehead in the site TA/CBP: transactivation and CBP-binding regions Pdn/Pdn Transcriptional repressor TA1: transactivation domain 1 Broad thumb or hallux Broad distal phange of the ﬁrst TA2: transactivation domain 2

digit in the forelimb in the (B): Mouse Gli3 structure gene Pdn/+ Transcriptional activator Preaxial polydactyly in Preaxial polydactyly of the hand complete type in the forelimb Ski binding site 456-1191 in the Pdn/Pdn Zinc finger 1 bp domain 4752 bp 318 708 1437-1914 Postaxila polydactyly in Postaxial polydactyly in the 984-1014 the hand forelimb in the Pdn/Pdn Supposed Supposed Preaxial polydactyly in Preaxial polydactyly of protease transactivation cleavage domain

the foot complete type in the hindlimb Degron N site in the Pdn/Pdn Transcriptional repressor region Preaxial polydactyly of distal binding site SUFU phalangeal type in the Transcriptional repressor hindlimb in the Pdn/+ Fig. 3 Human GLI3 and mouse Gli3 structure genes. (A) Human GLI3 Syndactyly of fingers Syndactyly of digits in the fore- gene has five zinc finger motifs at nucleotide position 1386–1935 and toes and hindlimbs in the Pdn/Pdn (lined), followed by protease cleavage site (mesh), followed by transactivation and CBP-binding regions (TA/CBP) at 2481–3396 (dotted), transactivation domain TA2 at 3132–3966 (grey), transactivation domain TA1 at 4128–4740 (grey), and a-helical region at 4482–4536 (hatched). Full length of GLI3 gene is 4743 bp. Full Pallister–Hall syndrome and postaxial polydactyly type-A length of GLI3 protein works as a transcriptional activator. After caused by the mutations in GLI3 gene cleavage at nucleotide position 1998 in the protease cleavage site, Pallister–Hall syndrome is a disorder that affects the development the N-terminal part of the GLI3 protein works as a transcriptional of many parts of the body. Most people with this condition exhibit repressor. (B) Mouse Gli3 gene has five zinc finger motifs at nucle- polydactyly and cutaneous syndactyly. An abnormal growth in the otide position 1437–1914 (lined), transcription repressor region at brain called a hypothalamic hamartoma is characteristic of this 318–708 (striped), Ski binding site at 456–1191 (dotted), SUFU binding site at 984–1014 (hatched), and Degron N (black). Full disorder. In many cases, this hamartoma does not cause any medical length of the structure gene has 4752 bp. Supposed protease cleav- problems; however, some hypothalamic hamartomas lead to sei- age site (mesh) and supposed transactivation domain (grey) were zures or hormone abnormalities that can be life-threatening in added in the map, provisionally. infancy. Other features of PHS include bifid epiglottis, imperforate anus and kidney abnormalities. The signs and symptoms of this of the upper and/or lower extremities. The extra digit is well formed disorder vary from mild to severe (Biesecker 2009). This condition and articulates with the fifth digit having metacarpal/metatarsal, is very rare; its prevalence is unknown (Genetics Home Reference: and, therefore, it is usually functional. PAP-A has been known to be http://ghr.nlm.nih.gov). induced by the mutation at nucleotide position 2292 in the post-zinc Most of the mutations responsible for PHS occur near the middle finger region of the GLI3 gene (Radhakrishna et al. 1997; Kalff- of the GLI3 gene, the protease cleavage site (Kang et al. 1997; Suske et al. 1999) (Fig. 4A). Johnston et al. 2005) (Fig. 4A). Johnston et al. (2005) suggest that Another form of polydactyly, PPD-IV, can also result from muta- mutations in the open reading frame nucleotide position 1998–3481 tions in the GLI3 gene. People with this condition have extra primarily cause PHS. These genetic changes typically create a pre- digits next to the thumb or hallux and cutaneous syndactyly mature stop signal in the instructions for making the GLI3 protein, (Radhakrishna et al. 1999). PPD-IV can also be characterized by resulting in the production of the truncated protein. Unlike the extra digits in other positions in the hands or feet. The pattern of full-length GLI3 protein, which can activate target genes, the trun- polydactyly seen with PPD-IV is similar to that of GCPS, and some cated protein can only repress the expression of target genes. researchers suggest that PPD-IV might be a very mild form of Although this defect clearly disrupts aspects of embryonic devel- GCPS (Genetics Home Reference: http://ghr.nlm.nih.gov) opment, it is not known how the altered function of GLI3 leads to (Fig. 4A). the varied signs and symptoms of PHS (Biesecker 2009) (Genetics Home Reference: http://ghr.nlm.nih.gov). Mutant mouse of the Gli3 gene Postaxial polydactyly type-A is an autosomal dominant trait, In addition to the Pdn mouse, a lot of mutant mice of the Gli3 gene characterized by an extra digit in the postaxial and/or preaxial side are known. XtH (Extra toes) (Gli3Xt–H) mice are a result of a spon-

(A): Syndrome caused by the mutations in GLI3 gene with eight or nine digits, hemimelia, abnormalities of the brain, spinal cord and sense organs, and edema (Johnson 1967; Franz H Transcriptional activator 1994). Southern blot analysis of genomic DNA from +/+, Xt /+ and XtH/XtH mice showed that at least part of the Gli3 gene (cDNA 1 bp 1998 bp 3481 bp 4743 bp homologous with a 5′ part of the human GLI3 gene) is deleted in Protease XtH mice (Schimmang et al. 1992; Vortkamp et al. 1992). Recently, cleavage α-helical Zinc finger H H site TA/CBP region it was determined that Xt /Xt is a null mutation and has a large domain deletion in the A2 region of chromosome 13 including the Gli3 gene TA TA 2 1 (Genestine et al. 2007). GCPS GCPS GCPS PHS PAP-A PPD-IV The XtJ (Extra toes Jackson) (Gli3Xt-J) mouse is the result of a spontaneous mutation (Dickie 1967). Deletion of more than 30 Kbp Transcriptional repressor from the first zinc finger motif in the Gli3 gene was found by Hui (B): Mutation points in the Gli3 gene in the mutant mice and Joyner (1993). After the report by Hui and Joyner, deletion of J PPD-IV 51.5 Kbp from the first zinc finger motif was determined in Xt (Maynard et al. 2002) (Fig. 4B). The phenotype of XtJ heterozy- tm2Blnw Gli3 gotes is very similar to that of XtH. XtJ homozygotes (XtJ/XtJ) die 367-368 Gli3tm1.1Alj Replaced with Deletion Gli3tm1Blnw human cDNA within 2 days after birth or in utero with a wide range of abnor- of exon 8 Deletion at containing mutation malities, including gross polydactyly and syndactyly in the fore- 1026-1242 2025-2229 at 2538-2742 1 bp 4752 bp and hindlimbs and gross malformations of the brain (Hui and Joyner 1993). XtJ/XtJ exhibits more severe polysyndactyly and brain abnormalities than Pdn/Pdn, including arhinencephaly (Naruse Gli3Mos1 Gli3tm1Urt et al. 2001). Pdn 1148 2097 Gli3Xt-2H, Gli3Xt-3H, Gli3Xt-4H, Gli3Xt-5H and Gli3Xt-6H are reported as Integration Xt J H Xt of a trans- the alleles of Xt (Gli3 ) based on the data from linkage analysis PHS Deletion of 51.5 Kbp poson into from the first zinc and similar phenotypes (Batchelor et al. 1966; Lyon et al. 1967). intron 3 finger motif These mutations were induced by radiation and mutation points are GCPS GCPS not yet defined. Gli3Xt-7H is a spontaneous mutation and reported as H Xt H Xt-H Deletion of A2 region on chromosome 13 including Gli3 an allele of Xt (Gli3 ) based on a similar phenotype (Lyon et al. 1967). The mutation point is not yet defined (Mouse Genome Infor- GCPS matics: http://www.informatics.jax.org). Mos1 Fig. 4 Syndrome caused by the mutations in GLI3 gene and mutation Gli3 was discovered in a N-ethyl N-nitrosourea mutagenesis points in the Gli3 gene in the mutant mice. (A) Mutations in the 5′ screening (Matera et al. 2008). The molecular change is a point half of GLI3 gene in the open reading frame nucleotide position mutation in exon 8 substituting adenine for cytosine at nucleotide 1–1997, which includes zinc finger domain (lined), cause GCPS. position 1148, resulting in replacement of tyrosine by a stop codon Most of the mutations responsible for PHS occur near the middle of (Fig. 4B). Gli3tm1.1Alj is a targeted knockout mouse removing exon 8 the GLI3 gene, the protease cleavage site (mesh). It was reported and leading to a frameshift mutation upstream of the zinc finger that mutations in the open reading frame nucleotide position 1998– TgBR 3481 caused primarily PHS. PAP-A has been known to be induced domain (Blaess et al. 2008) (Fig. 4B). Gli3 is induced by a by the mutation in the post-zinc finger region of GLI3 gene. It was transgenic insertion (Rachel et al. 2002). Evidence that this reported that PPD-IV is induced by a mutation in the downstream of mutant represented a remutation at the Gli3 locus was provided the post-zinc finger region in the GLI3 gene. GCPS, Greig cepha- through mapping data and complementation testing with XtH lopolysyndactyly syndrome; PAP-A, postaxial polydactyly type-A; (Gli3Xt-H). PHS, Pallister–Hall syndrome; PPD-IV, preaxial polydactyly type- To make a Gli3tm1Urt mouse, the Gli3 gene was disrupted by IV. (B) Pdn has a transposon in the intron 3 in the transcriptional repressor region (striped), upstream of the zinc finger domain of the targeting of a PGK-neo cassette to exon 1 via homologous recom- Gli3 gene. XtH has a deletion of A2 region on chromosome 13, bination, resulting in a frameshift and premature translation termi- including the Gli3 gene. Deletion of 51.5 Kbp from the first zinc nation of Gli3. The mutant protein truncates at amino acid 699 with finger motif was found in XtJ mice. They exhibit a similar pheno- 21 additional mutant amino acids before the stop codon. The change type to GCPS. The molecular change in Gli3Mos1 is a point mutation tm1.1Alj in amino acid 699 (nucleotide position 2097), in the post-zinc finger in exon 8 at nucleotide position 1148. Gli3 is a targeted knock- region, induced fetal death in the homozygote (Fig. 4B). The out mouse removing exon 8 in the upstream of the zinc finger domain. Gli3tm1Urt mice produce truncated protein of 699 amino homozygote exhibited central polydactyly, imperforate anus, gas- acid (nucleotide position 2097), and exhibit similar phenotype to trointestinal, epiglottis and larynx defects, abnormal kidney, and PHS. Gli3tm2Blnw has a mutation in the genomic sequence, including absence of adrenal glands, showing a similar phenotype to nucleotide position 2538–2742, downstream of the post-zinc finger Pallister–Hall syndrome (Böse et al. 2002). To make Gli3tm1Blnw,a tm1Blnw region, and exhibits a similar phenotype to PPD-IV. Gli3 has vector was designed to delete 68 residues, resulting in the fusion of a deletion in nucleotide position 2025–2229. residue 675–743 (nucleotide position 2025–2229), including the protease cleavage site (Fig. 4B). This deletion caused a half reduc- tion in the GLI3 repressor levels and a slight increased activity of full-length mutant protein in the limb. Homozygotes exhibit one to taneous mutation, found by Lyon et al. (1967), and the phenotype of two extra partial digits in the anterior of the limb, while heterozy- XtH/XtH was described by Johnson (Johnson 1967). Heterozygotes gotes die soon after birth and display seven digits. Gli3tm1Blnw/ have varying numbers of extra digits on the preaxial side in the fore- Gli3tm1Blnw does not exhibit PHS-like phenotypes (Wang et al. and hindlimbs. Occasionally there is a postaxial rudimentary digit. 2007a). They are fully viable and fertile. Homozygotes die in utero or at To make Gli3tm2Blnw, genomic sequences including 2538–2742 of birth with multiple abnormalities, including paddle-shaped feet the Gli3 gene (transcriptional activator region) were replaced with

© 2010 The Authors Journal compilation © 2010 Japanese Teratology Society 6 I. Naruse et al. the corresponding human cDNA sequence containing the mutation while, it was speculated that truncated mutant GLI3 protein, having (Wang et al. 2007b) (Fig. 4B). Gli3tm2Blnw produced only a GLI3 only repressor function, might induce the phenotypes both in repressor form, and heterozygote exhibited mild preaxial polysyn- humans and mice. dactyly and a partial loss of digit identity similar to human disease PPD-IV (Wang et al. 2007b). Gli3add (anterior digit-pattern deformity) is the result of multi- ACKNOWLEDGMENTS copy transgene integration, and unknown mouse sequences are This study was supported by Grants-in-Aid for Scientific Research cointegrated with the transgene. Homozygotes exhibit altered (20591297) from the Ministry of Education, Culture, Sports, thumb and sometimes an extra phalanx in the anterior part (Pohl Science and Technology, Japan. et al. 1990) (Mouse Genome Informatics: http://www.informatics. jax.org). REFERENCES Investigation of mouse homolog to understand human disease Batchelor AL, Phillips RJ, Searle AG (1966) A comparison of the We have considered that Pdn/Pdn is a mouse homolog of GCPS mutagenic effectiveness of chronic neutron- and gamma-irradiation of (Naruse and Keino 1995; Naruse et al. 2005). Then, we speculated mouse spermatogonia. Mutat Res 3: 218–229. that the telencephalic dysmorphogenetic mechanisms by the altered Biesecker LG (2008) The Greig cephalopolysyndactyly syndrome. Orpha- signaling pathway accompanied with depressed expression of Gli3 net J Rare Dis 3: 10. gene and truncated GLI3 protein in the Pdn/Pdn might be extrapo- Biesecker LG (2009) Greig Cephalopolysyndactyly Syndrome. GENE lated to those in GCPS (Ueta et al. 2008). Using the transposon Reviews (http://www.ncbi.nlm.nih.gov). integrated into intron 3 of Gli3 gene in the Pdn mouse, a quick Blaess S, Stephen D, Joyner AL (2008) Gli3 coordinates three-dimensional patterning and growth of the tectum and cerebellum by integrating Shh genotyping method was developed to discriminate +/+, Pdn/+ and and Fgf8 signaling. Development 135: 2093–2103. Pdn/Pdn embryos. It allowed us to investigate the signaling Böse J, Grotewold L, Rüther U (2002) Pallister-Hall syndrome phenotype in pathway in the early brain morphogenesis in the Pdn/Pdn embryos mice mutant for Gli3. Humana Mol Genet 11: 1129–1135. (Ueta et al. 2008). We have suggested that the Pdn/Pdn mouse, Büscher D, Rüther U (1998) Expression profile of Gli family members which exhibits polydactyly and various brain abnormalities, might and Shh in normal and mutant mouse limb development. 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