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GLI3 is the gene responsible for Greig cephalopolysyndactyly syndrome (GCPS), Pallister–Hall syndrome (PHS) and Postaxial polydactyly type-A (PAP-A). Genetic polydactyly mice such as Pdn/Pdn (Polydactyly Nagoya), XtH/XtH (Extra toes) and XtJ/XtJ (Extra toes Jackson) are the mouse homolog of GCPS, and Gli3tmlUrtt/Gli3tmlUrt is produced as the mouse homolog of PHS. In the present review, relationships between mutation points of GLI3 and Gli3, and resulting phenotypes in humans and mice are described. It has been confirmed that mutation in the upstream or within the zinc finger domain of the GLI3 gene induces GCPS; that in the post-zinc finger region including the protease cleavage site induces PHS; and that in the downstream of the GLI3 gene induces PAP-A. A mimicking phenomenon was observed in the mouse homolog. Therefore, human GLI3 and mouse Gli3 genes have a common structure, and it is suggested here that mutations in the same functional regions produce similar phenotypes in human and mice. The most important issue might be that GCPS and PHS exhibit an autosomal dominant trait, but mouse homologs, such as Pdn/Pdn, XtH/XtH, XtJ/XtJ and Gli3tmlUrt/Gli3tmlUrt, are autosomal recessive traits in the manifestation of similar phenotypes to human diseases. It is discussed here how the reduced amounts of the GLI3 protein, or truncated mutant GLI3 protein, disrupt development of the limbs, head and face.
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A responsible gene can be identified using blood and/or fibroblast cells of the human birth defects, but it is impossible to know how the phenotypes appear. We can investigate the mechanisms of how the phenotype manifests in the animal homologous disease by observing the phenomena during embryogenesis. In this review, we would like to describe the birth defects caused by the mutation of the GLI3 gene in humans and the Gli3 gene in mice. GLI3 and Gli3 genes have zinc finger domains (transcriptional regulation domains) and are peculiar genes; the full length of the GLI3 protein functions as a transcriptional activator, and the N terminal part functions as a transcriptional repressor after cleavage into two parts.
According to the positions of mutation, various types of phenotype appear, such as Greig cephalopolysyndactyly syndrome (GCPS), Pallister–Hall syndrome (PHS), postaxial polydactyly type-A (PAP-A) and preaxial polydactyly type-IV (PPD-IV). A mimic phenomenon is observed in mice. Investigation of human GLI3 and Gli3 genes has progressed using the knowledge of Cubitus interruptus (Ci) in Drosophila, a homologous gene of GLI3 and Gli3 genes. These genes have been highly conserved in the animal kingdom throughout evolution. Now, a lot of mutant mice of Gli3 gene have been known and knockout mice have been produced. It is expected that the knowledge obtained from mutant and knockout mice will be extrapolated to the manifestation mechanisms in human diseases to understand the diseases caused by the mutations in GLI3 gene.
Phenotype of GCPS
Greig cephalopolysyndactyly syndrome is a disorder that affects the development of the limbs, head and face. The features of this syndrome are highly variable, ranging from very mild to severe. GCPS might characterized by a set of craniofacial defects (e.g. macrocephaly, broad nasal root, ocular hypertelorism and prominent forehead) (Fig. 1A,B), and one or more extra fingers or toes (polydactyly) (Fig. 1C,D) or having an abnormally wide thumb or hallux. The skin between the fingers and toes might exhibit cutaneous syndactyly (Fig. 1C,D) (Greig 1926; Gollop and Fontes 1985; Biesecker, 2009). Rarely, affected individuals have more serious medical problems, including seizures, developmental delay, hydrocephalus and intellectual disability (Biesecker 2009). It is impossible to determine the incidence of GCPS, because reliable clinical criteria and molecular diagnostics are not yet readily available (Biesecker 2008).
Figure 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).
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Phenotype of Pdn mouse
Pdn/+ mice exhibited broad thumbs of the first digit in the forelimb (Fig. 2A), preaxial polydactyly of distal phalangeal type in the hindlimb (Fig. 2B,F) (Hayasaka et al. 1980; Naruse and Kameyama 1982) a normal brain with the olfactory bulb (Fig. 2I), normal spaced eyes (Fig. 2K) and a normal forehead (Fig. 2M). Pdn/Pdn mice exhibited preaxial polydactyly of complete type and syndactyly both in the fore- and hindlimbs (Fig. 2C,D,G,H) (Hayasaka et al. 1980; Naruse and Kameyama 1982), postaxial polydactyly in the forelimbs (Fig. 2C), absence of olfactory bulb (Fig. 2J), hydrocephalus (Naruse et al. 1990), ocular hypertelorism (Fig. 2L), prominent forehead (Fig. 2N) and retinal coloboma. They die soon after birth because of suckling dysfunction (Hongo et al. 2000). The similarity of the phenotype between GCPS and Pdn/Pdn is shown in Table 1.
Figure 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).
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Table 1. Comparison of phenotype between Greig cephalopolysyndactyly syndrome (GCPS) and Pdn/Pdn
|Broad macrocephaly nasal root||Hydrocephalus in the Pdn/Pdn|
|Ocular hypertelorism||Ocular hypertelorism in the Pdn/Pdn|
|Prominent forehead||Prominent forehead in the Pdn/Pdn|
|Broad thumb or hallux||Broad distal phange of the first digit in the forelimb in the Pdn/+|
|Preaxial polydactyly in the hand||Preaxial polydactyly of complete type in the forelimb in the Pdn/Pdn|
|Postaxila polydactyly in the hand||Postaxial polydactyly in the forelimb in the Pdn/Pdn|
|Preaxial polydactyly in the foot||Preaxial polydactyly of complete type in the hindlimb in the Pdn/Pdn|
|Preaxial polydactyly of distal phalangeal type in the hindlimb in the Pdn/+|
|Syndactyly of fingers and toes||Syndactyly of digits in the fore- and hindlimbs in the Pdn/Pdn|
GLI3 gene in human and Gli3 gene in mice
GLI family proteins attach to specific regions of DNA and control whether particular genes are turned on or off. Full length GLI3 protein works as a transcriptional activator, and N terminal part of GLI3 protein works as a transcriptional repressor after cleavage at nucleotide position 1988 in the protease cleavage site (Fig. 3A). Human GLI3 gene is constructed with 15 exons, and has 5 zinc finger motifs at nucleotide number 1386–1935, protease cleavage site, transactivation and CBP-binding regions (TA/CBP) at 2481–3396, transactivation domain 2 (TA2) at 3132–3966 and transactivation domain 1 (TA1) at 4128–4740, α-helical region at 4482–4536 (Kalff-Suske et al. 1999), and the full length of structure gene is 4743 bp (Fig. 3A) (EMBL ID: AJ250408). CBP, CREB-binding protein, is ubiquitously expressed and is involved in the transcriptional coactivation of many different transcription factors (Chrivia et al. 1993), and α-helix acts as an activation domain (Yoon et al. 1998).
Figure 3. Human GLI3 and mouse Gli3 structure genes. (A) Human GLI3 gene has five zinc finger motifs at nucleotide position 1386–1935 (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 α-helical region at 4482–4536 (hatched). Full length of GLI3 gene is 4743 bp. Full length of GLI3 protein works as a transcriptional activator. After cleavage at nucleotide position 1998 in the protease cleavage site, the N-terminal part of the GLI3 protein works as a transcriptional repressor. (B) Mouse Gli3 gene has five zinc finger motifs at nucleotide position 1437–1914 (lined), transcription repressor region at 318–708 (striped), Ski binding site at 456–1191 (dotted), SUFU binding site at 984–1014 (hatched), and Degron N (black). Full length of the structure gene has 4752 bp. Supposed protease cleavage site (mesh) and supposed transactivation domain (grey) were added in the map, provisionally.
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Mouse Gli3 gene has also 5 zinc finger motifs at nucleotide position 1437–1914 and is constructed with 15 exons. It has transcriptional repressor region at 318–708, Ski binding site at 456–1191, SUFU binding site at 984–1014, Degron N (Tsanev et al. 2009), and the full length of structure gene is 4752 bp (EMBL, ID: BC145445) (Fig. 3B), and mapped at 14 cM on mouse chromosome 13 (EMBL-EBI: http://www.ebi.ac.uk). Ski is a corepressor in transcriptional regulation in the full-length forms of Gli3 (Dai et al. 2002). SUFU (Suppressor of fused) is essential for regulation of Gli/Ci processing, activity, and localization (Dunaeva et al. 2003). Degron N is a specific sequence of amino acids in a protein that directs the starting place of degradation positioned in N-terminal region (Huang et al. 1998). Around 14 cM of mouse chromosome 13 is the synteny region with 7p13 on the human chromosome 7 (Pettigrew et al. 1991; Lyon and Kirby 1992). Human GLI3 and mouse Gli3 have 69% homology in DNA sequence, and 82% homology in amino acid sequence.
Proteins of the GLI family function in the same molecular pathway as SHH protein. This pathway is essential for early development (Ming et al. 1998). It plays a role in cell growth, cell specialization and the patterning of structures such as the brain and limbs (Genetics Home Reference: http://ghr.nlm.nih.gov). Depending on signals from SHH, the GLI3 protein can either activate or repress other genes such as Emx2, Wnt7b, Wnt8b and Msx (Ueta et al. 2008; Lallemand et al. 2009). Hill et al. (2009) proposed that unprocessed full-length GLI3 is dispensable for anteroposterior patterning of the limb bud. Instead, digit identities are most likely defined by GLI3 repressor activity alone. Anteroposterior grading of GLI3 activity by the action of SHH in digital pattering is reported by Hill et al. (2009).
GLI3 and Gli3 genes responsible for Greig cephalopolysyndactyly syndrome and Pdn mouse
Different genetic changes involving the GLI3 gene can cause GCPS. In some cases, the condition results from a chromosomal abnormality, such as a large deletion or rearrangement of genetic material, in the region of chromosome 7. In any case, deletion and/or mutations in the 5′ half of the GLI3 gene, in the open reading frame at nucleotides position 1–1997, cause GCPS (Kang et al. 1997; Johnston et al. 2005) (Fig. 4A). GCPS exhibits an autosomal dominat trait (Gollop and Fontes 1985).
Figure 4. Syndrome caused by the mutations in GLI3 gene and mutation points in the Gli3 gene in the mutant mice. (A) Mutations in the 5′ half of GLI3 gene in the open reading frame nucleotide position 1–1997, which includes zinc finger domain (lined), cause GCPS. Most of the mutations responsible for PHS occur near the middle of the GLI3 gene, the protease cleavage site (mesh). It was reported that mutations in the open reading frame nucleotide position 1998–3481 caused primarily PHS. PAP-A has been known to be induced by the mutation in the post-zinc finger region of GLI3 gene. It was reported that PPD-IV is induced by a mutation in the downstream of the post-zinc finger region in the GLI3 gene. GCPS, Greig cephalopolysyndactyly syndrome; PAP−A, postaxial polydactyly type-A; PHS, Pallister–Hall syndrome; PPD-IV, preaxial polydactyly type-IV. (B) Pdn has a transposon in the intron 3 in the transcriptional repressor region (striped), upstream of the zinc finger domain of the Gli3 gene. XtH has a deletion of A2 region on chromosome 13, including the Gli3 gene. Deletion of 51.5 Kbp from the first zinc finger motif was found in XtJ mice. They exhibit a similar phenotype to GCPS. The molecular change in Gli3Mos1 is a point mutation in exon 8 at nucleotide position 1148. Gli3tm1.1Alj is a targeted knockout mouse removing exon 8 in the upstream of the zinc finger domain. Gli3tm1Urt mice produce truncated protein of 699 amino acid (nucleotide position 2097), and exhibit similar phenotype to PHS. Gli3tm2Blnw has a mutation in the genomic sequence, including nucleotide position 2538–2742, downstream of the post-zinc finger region, and exhibits a similar phenotype to PPD-IV. Gli3tm1Blnw has a deletion in nucleotide position 2025–2229.
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Integration of a transposon in the Gli3 gene in a Pdn mouse has been reported (Thien and Rüther 1999; Naruse et al. 2000), and we demonstrated in a previous study that 5542 bp of early retrotransposon was inserted into intron 3 in the transcriptional repressor region of Gli3 gene (Ueta et al. 2002) (Fig. 4B). The transposon was very similar to Y17106 (Hofmann et al. 1998) (EMBL-EBI: http://www.ebi.ac.uk). Gli3 expression is depressed to approximately 60% of +/+ in the Pdn/+ mice and approximately 20% of +/+ in the Pdn/Pdn mouse throughout early embryogenesis (Ueta et al. 2004), and insertion of small pieces of taransposon sequence into the mRNA was detected, resulting in truncated GLI3 proteins (Thien and Rüther 1999).
Pallister–Hall syndrome and postaxial polydactyly type-A caused by the mutations in GLI3 gene
Pallister–Hall syndrome is a disorder that affects the development of many parts of the body. Most people with this condition exhibit polydactyly and cutaneous syndactyly. An abnormal growth in the brain called a hypothalamic hamartoma is characteristic of this disorder. In many cases, this hamartoma does not cause any medical problems; however, some hypothalamic hamartomas lead to seizures or hormone abnormalities that can be life-threatening in infancy. Other features of PHS include bifid epiglottis, imperforate anus and kidney abnormalities. The signs and symptoms of this disorder vary from mild to severe (Biesecker 2009). This condition is very rare; its prevalence is unknown (Genetics Home Reference: http://ghr.nlm.nih.gov).
Most of the mutations responsible for PHS occur near the middle of the GLI3 gene, the protease cleavage site (Kang et al. 1997; Johnston et al. 2005) (Fig. 4A). Johnston et al. (2005) suggest that mutations in the open reading frame nucleotide position 1998–3481 primarily cause PHS. These genetic changes typically create a premature stop signal in the instructions for making the GLI3 protein, resulting in the production of the truncated protein. Unlike the full-length GLI3 protein, which can activate target genes, the truncated protein can only repress the expression of target genes. Although this defect clearly disrupts aspects of embryonic development, it is not known how the altered function of GLI3 leads to the varied signs and symptoms of PHS (Biesecker 2009) (Genetics Home Reference: http://ghr.nlm.nih.gov).
Postaxial polydactyly type-A is an autosomal dominant trait, characterized by an extra digit in the postaxial and/or preaxial side of the upper and/or lower extremities. The extra digit is well formed and articulates with the fifth digit having metacarpal/metatarsal, and, therefore, it is usually functional. PAP-A has been known to be induced by the mutation at nucleotide position 2292 in the post-zinc finger region of the GLI3 gene (Radhakrishna et al. 1997; Kalff-Suske et al. 1999) (Fig. 4A).
Another form of polydactyly, PPD-IV, can also result from mutations in the GLI3 gene. People with this condition have extra digits next to the thumb or hallux and cutaneous syndactyly (Radhakrishna et al. 1999). PPD-IV can also be characterized by extra digits in other positions in the hands or feet. The pattern of polydactyly seen with PPD-IV is similar to that of GCPS, and some researchers suggest that PPD-IV might be a very mild form of GCPS (Genetics Home Reference: http://ghr.nlm.nih.gov) (Fig. 4A).
Mutant mouse of the Gli3 gene
In addition to the Pdn mouse, a lot of mutant mice of the Gli3 gene are known. XtH (Extra toes) (Gli3Xt–H) mice are a result of a spontaneous mutation, found by Lyon et al. (1967), and the phenotype of XtH/XtH was described by Johnson (Johnson 1967). Heterozygotes have varying numbers of extra digits on the preaxial side in the fore- and hindlimbs. Occasionally there is a postaxial rudimentary digit. They are fully viable and fertile. Homozygotes die in utero or at birth with multiple abnormalities, including paddle-shaped feet with eight or nine digits, hemimelia, abnormalities of the brain, spinal cord and sense organs, and edema (Johnson 1967; Franz 1994). Southern blot analysis of genomic DNA from +/+, XtH/+ and XtH/XtH mice showed that at least part of the Gli3 gene (cDNA homologous with a 5′ part of the human GLI3 gene) is deleted in XtH mice (Schimmang et al. 1992; Vortkamp et al. 1992). Recently, it was determined that XtH/XtH is a null mutation and has a large deletion in the A2 region of chromosome 13 including the Gli3 gene (Genestine et al. 2007).
The XtJ (Extra toes Jackson) (Gli3Xt-J) mouse is the result of a spontaneous mutation (Dickie 1967). Deletion of more than 30 Kbp from the first zinc finger motif in the Gli3 gene was found by Hui and Joyner (1993). After the report by Hui and Joyner, deletion of 51.5 Kbp from the first zinc finger motif was determined in XtJ (Maynard et al. 2002) (Fig. 4B). The phenotype of XtJ heterozygotes is very similar to that of XtH. XtJ homozygotes (XtJ/XtJ) die within 2 days after birth or in utero with a wide range of abnormalities, including gross polydactyly and syndactyly in the fore- 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 et al. 2001).
Gli3Xt-2H, Gli3Xt-3H, Gli3Xt-4H, Gli3Xt-5H and Gli3Xt-6H are reported as the alleles of XtH (Gli3Xt) based on the data from linkage analysis and similar phenotypes (Batchelor et al. 1966; Lyon et al. 1967). These mutations were induced by radiation and mutation points are not yet defined. Gli3Xt-7H is a spontaneous mutation and reported as an allele of XtH (Gli3Xt-H) based on a similar phenotype (Lyon et al. 1967). The mutation point is not yet defined (Mouse Genome Informatics: http://www.informatics.jax.org).
Gli3Mos1 was discovered in a N-ethyl N-nitrosourea mutagenesis screening (Matera et al. 2008). The molecular change is a point mutation in exon 8 substituting adenine for cytosine at nucleotide position 1148, resulting in replacement of tyrosine by a stop codon (Fig. 4B). Gli3tm1.1Alj is a targeted knockout mouse removing exon 8 and leading to a frameshift mutation upstream of the zinc finger domain (Blaess et al. 2008) (Fig. 4B). Gli3TgBR is induced by a transgenic insertion (Rachel et al. 2002). Evidence that this mutant represented a remutation at the Gli3 locus was provided through mapping data and complementation testing with XtH (Gli3Xt-H).
To make a Gli3tm1Urt mouse, the Gli3 gene was disrupted by targeting of a PGK-neo cassette to exon 1 via homologous recombination, resulting in a frameshift and premature translation termination of Gli3. The mutant protein truncates at amino acid 699 with 21 additional mutant amino acids before the stop codon. The change in amino acid 699 (nucleotide position 2097), in the post-zinc finger region, induced fetal death in the homozygote (Fig. 4B). The homozygote exhibited central polydactyly, imperforate anus, gastrointestinal, epiglottis and larynx defects, abnormal kidney, and absence of adrenal glands, showing a similar phenotype to Pallister–Hall syndrome (Böse et al. 2002). To make Gli3tm1Blnw, a vector was designed to delete 68 residues, resulting in the fusion of residue 675–743 (nucleotide position 2025–2229), including the protease cleavage site (Fig. 4B). This deletion caused a half reduction in the GLI3 repressor levels and a slight increased activity of full-length mutant protein in the limb. Homozygotes exhibit one to two extra partial digits in the anterior of the limb, while heterozygotes die soon after birth and display seven digits. Gli3tm1Blnw/Gli3tm1Blnw does not exhibit PHS-like phenotypes (Wang et al. 2007a).
To make Gli3tm2Blnw, genomic sequences including 2538–2742 of the Gli3 gene (transcriptional activator region) were replaced with the corresponding human cDNA sequence containing the mutation (Wang et al. 2007b) (Fig. 4B). Gli3tm2Blnw produced only a GLI3 repressor form, and heterozygote exhibited mild preaxial polysyndactyly 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 multicopy transgene integration, and unknown mouse sequences are cointegrated with the transgene. Homozygotes exhibit altered thumb and sometimes an extra phalanx in the anterior part (Pohl et al. 1990) (Mouse Genome Informatics: http://www.informatics.jax.org).
Investigation of mouse homolog to understand human disease
We have considered that Pdn/Pdn is a mouse homolog of GCPS (Naruse and Keino 1995; Naruse et al. 2005). Then, we speculated that the telencephalic dysmorphogenetic mechanisms by the altered signaling pathway accompanied with depressed expression of Gli3 gene and truncated GLI3 protein in the Pdn/Pdn might be extrapolated to those in GCPS (Ueta et al. 2008). Using the transposon integrated into intron 3 of Gli3 gene in the Pdn mouse, a quick genotyping method was developed to discriminate +/+, Pdn/+ and Pdn/Pdn embryos. It allowed us to investigate the signaling pathway in the early brain morphogenesis in the Pdn/Pdn embryos (Ueta et al. 2008). We have suggested that the Pdn/Pdn mouse, which exhibits polydactyly and various brain abnormalities, might be a suitable material to investigate the signaling pathway during the morphogenesis of limbs and brain.
The mutation point of Gli3tm1Urt/Gli3tmlUrt is at nucleotide position 2097, the post-zinc finger region, and exhibits a similar phenotype to PHS (Böse et al. 2002), suggesting that nucleotide position 2097 in mice might be in the protease cleavage site (Figs 3B,4B). Gli3tm1Urt/Gli3tm1Urt was investigated to elucidate the relationship between the PHS-like polydactyly and the pathogenic mode of the action of the truncated GLI3 protein at nucleotide position 2097 (Hill et al. 2007, 2009). These reports provide evidence that unprocessed full-length GLI3 is dispensable for anteroposterior patterning of the limb bud, but digit identities are most likely defined by GLI3 repressor activity alone. Gli3tm2Blnw has a mutation in 2538–2742, the transcriptional activator region of Gli3 gene, resulting in a similar phenotype to PPD-IV (Wang et al. 2007b). Therefore, human GLI3 and mouse Gli3 genes have almost the same construction and functions. It was suggested that mutation positions in which the same function exists produce similar phenotypes in humans and mice.
The most important issue may be that human diseases such as GCPS and PHS are an autosomal dominant trait, but mouse homologs such as Pdn/Pdn, XtJ/XtJ and Gli3tm1Urt/Gli3tmlUrt are autosomal recessive in the manifestation of similar phenotypes. In the autosomal dominant case, only half the normal amount of this protein is available to control the expression of target genes during embryogenesis. It remains unclear how the reduced amounts of the GLI3 protein, or truncated mutant GLI3 protein, disrupt development of the limbs, head and face. We expect that it will be cleared in future research that the disease appears in spite of having half the amount of normal GLI3 protein in humans; however, complete loss of normal GLI3 protein causes similar phenotypes in mice. Meanwhile, it was speculated that truncated mutant GLI3 protein, having only repressor function, might induce the phenotypes both in humans and mice.