Mammalian Genome 3: 461-463, 1992
enome 9 Springer-VerlagNew YorkInc. 1992
Deletion of GLI3 supports the homology of the human Greig cephalopolysyndactyly syndrome (GCPS) and the mouse mutant extra toes (Xt) Andrea Vortkamp, 1 Thomas Franz, 2 Manfred Gessler, ~ and Karl-Heinz Grzeschik ~ lInstitut ffir Humangenetik der Philipps-Universit/it, Bahnhofstr. 7a, D-3550 Marburg, FRG; 2Anatomisches Institut, Abteilung Neuroanatomie, Universit~tskrankenhaus Eppendorf, Martinistr. 52, D-2000 Hamburg 20, FRG Received February 19, 1992; accepted April 23, 1992
The dominant mouse mutant extra toes (Xt) is characterized by preaxial and postaxial polydactyly of the feet and a white belly spot. An interfrontal bone is present in the skull in 90% of heterozygotes compared with 50% of normal mice. Homozygous Xt/Xt embryos exhibit multiple skeletal defects, extreme polydactyly in both fore- and hindlimbs, and malformations of the brain and the eye (Johnson 1967; Franz and Besecke 1991). Depending on the genetic background, Xt homozygotes die prenatally or perinatally (Johnson 1967). An allelic but recessive syndrome is the anterior digit pattern deformity mutation (add), which is the result of a transgene integration (Pohl et al. 1990). In this case the malformations of the mutants are restricted to the forelimbs. Using DNA probes spanning the add transgene integration site, Pohl and coworkers (1990) could show that at least 80 kbp of surrounding DNA are deleted in Xt mice. On the basis of the similarity of the phenotype, Xt is considered the mouse homolog of the human autosomal dominant Greig cephalopolysyndactyly syndrome, GCPS (Winter and Huson 1988). The latter is characterized by polysyndactyly of hands and feet and mild craniofacial abnormalities (Gollop and Fontes 1985). The gene locus has been pinpointed to human Chromosome (Chr) 7p13 by different translocations and deletions associated with the disorder (Tommerup and Nielsen 1983; Krt~ger et al. 1989; Wagner et al. 1990; Pettigrew et al. 1991; Vortkamp et al. 1991b). Using six hybrids from three GCPS translocation patients, we recently have demonstrated that the zinc finger gene GLI3 (Ruppert et al. 1988, 1990) is disOffprint requests to: K.-H. Grzeschik
rupted in the first third of its coding region by two of the GCPS translocations (Vortkamp et al. 1991a). The third translocation breakpoint was shown to be located approximately 10 kbp downstream of the expressed sequence. Thus, mutations of GLI3 resulting in a reduced gene dosage are probably responsible for the development of GCPS. To characterize the GLI3 gene in Xt mutant mice, we isolated different GLI3 cDNA clones from an 8.5day fetal mouse cDNA library (Fahrner et al. 1987), using segments of the human GLI3 cDNA as probes. The position of the mouse clones relative to the known human GLI3 sequence (Ruppert et al. 1990) was determined by partial sequencing (Fig. la). The distal mouse cDNA clone, pMGli20, corresponds to nucleotides 3232-4734 of the human cDNA. Hybridization with genomic DNA of homozygous Xt/ Xt mice and normal control mice did not show any alteration in the 3' part of the gene (Fig. ld). The proximal mouse cDNA clone pMGlil 1 starts approximately 500 bp upstream of the published GLI3 cDNA and reaches down to nucleotide 415. The probe hybridizes with multiple EcoRI and PstI fragments in normal mouse DNA that are completely deleted in homozygous Xt/Xt embryos (data not shown). To further characterize the deletion boundary within the GLI3 gene, we hybridized the human cDNA fragment pcrGliprox (nucleotides 98-570) with genomic DNA of homozygous Xt/Xt and normal control mice. This probe overlaps with a 300-bp BglII/NotI fragment (pMGlillBN), representing the 3' third of pMGlil 1, but reaches 160 bp further downstream. Like probe pMGlillBN (Fig. lb), pcrGliprox hybridizes with an 8-kbp PstI fragment that is deleted in the DNA of Xt/ Xt embryos. Furthermore, two additional PstI frag-
462
A. Vortkamp et al.: Deletion of GL13 in Xt mice
a)
Fig. 1. The GLI3 gene is partially deleted in Xt
mutants. (a) The different GCPS translocations and the Xt deletion (arrows) are drawn in relation to the human cDNA published by Ruppert and co-workers (1991). The open reading frame ( I Xt-Deietion of GLl3 is indicated by an open box; the 5' end, 3' not yet determined, by a broken line. The posi_[ ].-GLI3-cDNA tions of the cDNA fragments used in this study are marked by shaded boxes. 0r--d) DNA from two homozygous Xt/Xt embryos, a normal ( + / I I pcrGfipro• +) sib and the mouse tumor cell line Rag, was digested with PstI (b,c) and EcoRI (d) and hyI ~pMGii11 [ I pMGli20 bridized with different probes from the GL13 gene. (b) Probe pMGIillBN, a 300-bp BgllI/ r---1 pMGli11BN Not[ fragment ofpMGIil 1 detects an 8-kbp PstI fragment that is deleted in Xt/Xt embryos. This fragment overlaps with the human probe pcrGliprox. (c) Probe pcrGliprox reaches 160 bp further downstream and hybridizes with two additional PstI fragments that are both not altered in the DNAs tested. (d) Hybridization of pMGli20 does not show any indicationfor a mutation in Xt/Xt mice. Xt/Xt breeding pairs in a (C3Hxl01) F2 background were purchased from the MRC Radiobiology Unit, Harwetl, UK. High-molecular-weight DNA was prepared from embryos on day 16 of gestation. Xt/Xt embryos were identified macroscopically by their polydactyly and the malformation of the eye. The DNA was isolated according to the method of Kunkel and co-workers (1982), and Southern blot analysis was carried out under standard conditions (Sambrook et al. 1989). The mouse cDNA probes, pMGlil 1 and pMGli20, which are available upon request, were isolated from an 8.5-day fetal mouse cDNA library (Fahrner et al. 1987), with the human cDNA fragments pcrGliprox and pcrGlidist (Vortkamp et al. 1991a) as probes. + +
,~
m e n t s of 14 k b p a n d 3.8 k b p are d e t e c t e d , b o t h of w h i c h are n o t d e l e t e d in the X t / X t e m b r y o s (Fig. lc). T h u s , the b r e a k p o i n t of the X t d e l e t i o n m u s t be localized b e t w e e n n u c l e o t i d e s 415 a n d 570 of the corr e s p o n d i n g h u m a n c D N A . T h e b r e a k p o i n t is p r o b a b l y l o c a t e d in a n i n t r o n of the G L I 3 g e n e , as h y b r i d i z a t i o n o f E c o R I (not s h o w n ) a n d P s t I (Fig. l b , c ) - d i g e s t e d X t D N A s h o w e d n o r e a r r a n g e d f r a g m e n t s . A t p r e s e n t we do n o t k n o w the size o f the d e l e t i o n , b u t it s e e m s likely that the d e l e t i o n r e g i o n i n c l u d e s 80 k b p of g e n o m i c D N A f r o m the add l o c u s (Pohl et al. 1990) that was s h o w n to b e d e l e t e d in X t . T h e d e l e t i o n o f the 5' r e g i o n o f the G L I 3 c D N A in X t / + mice p r e v e n t s the f o r m a t i o n of a f u n c t i o n a l protein p r o d u c t f r o m o n e allele. T h e r e f o r e , a r e d u c t i o n in g e n e d o s a g e for G L I 3 is the likely c a u s e for the malf o r m a t i o n s s e e n in b o t h , the m o u s e X t m u t a n t a n d the h u m a n G C P S s y n d r o m e , c o n f i r m i n g the h o m o l o g y of these s y n d r o m e s . A s the i n t e g r a t i o n site of the t r a n s g e n e in the a d d m u t a n t has b e e n l o c a l i z e d w i t h i n the X t d e l e t i o n , it will b e of great i n t e r e s t to d e t e r m i n e the p r e c i s e p h y s i c a l r e l a t i o n s h i p b e t w e e n the g e n o m i c G L I 3 l o c u s a n d the a d d i n t e g r a t i o n site. X t m u t a n t mice e x h i b i t a m u c h m o r e s e v e r e p h e n o t y p e c o m p a r e d with the a d d m u t a n t s , l e a v i n g the p o s s i b i l i t y that there are a d d i t i o n a l g e n e s in the X t d e l e t i o n r e g i o n that m a y c o n t r i b u t e to the o b s e r v e d p h e n o t y p e .
Acknowledgments. We thank B. Hogan for an aliquot of the fetal mouse cDNA library. This work was supported by a grant from the Deutsche Forschungsgemeinschaft.
GCPS-Translocations
References Fahrner, K., Hogan, B.L.M., and Flavell, R.A.: Transcription of H-2 and Qa genes in embryonic and adult mice. EMBO J 6: 12651271, 1987. Franz, T. and Beseke, A.: The development of the eye in homozygous mutants of the extra-toes locus of the mouse. Anat Embryol 184: 335-361, 1991. Gollop, T.R. and Fontes, L.R.: The Greig cephalopolysyndactyly syndrome: report of a family and review of the literature. Am J IVied Genet 22: 59-68, 1985. Johnson, D.R.: Extra-toes: a new mutant gene causing multiple abnormalities in the mouse. J Embryol Exp Morphol 17: 543-581, 1967. Kr/iger, G., G6tz, J., Kvist, U., Dunker, H., Erfurth, F., Pelz, L. and Zech, L.: Greig syndrome in a large kindred due to reciprocal chromosome translocation t(6;7)(q27;p13). Am J Med Genet 32: 411-416, 1989. Kunkel, L.M., Tantravahi, U., Eisenhard, M., and Latt, S.A.: Regional localization on the human X of DNA segments cloned from flow sorted chromosomes. Nucleic Acids Res 10: 1557-1578, 1982. Pettigrew, A.L., Greenberg, F., Caskey, C.T., and Ledbetter, D.H.: Greig syndrome associated with an interstitial deletion of 7p: confirmation of the localization of Greig syndrome to 7p13. Hum Genet 87: 452-456, 1991. Pohl, T.M., Mattei, M.G., and Rfither, U.: Evidence for alMism of the recessive insertional mutation add and the dominant mouse mutation extra-toes (Xt). Development 110: 1153-1157, 1990. Ruppert, J.M., Kinzler, K.W., Wong, A.J., Bigner, S.H., Kao, F.-T., Law, M.I., Seuanez, H.N., O'Brien, S.J., and Vogelstein, B.: The GLI-Krfippel family of human genes. Mol Cell Biol 8: 3104-3113, 1988. Ruppert, J.M., Vogelstein, B., Arheden, K., and Kinzler, K.W.: GLI3 encodes a 190-kilodalton protein with multiple regions of GL1 similarity. Mol Cell Biol I0: 5408-5415, 1990. Sambrook, J., Fritsch, E.G., and Maniatis, T.: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Tommerup, N. and Nielsen, F.: A familial reciprocal translocation
A. Vortkamp et al.: Deletion of GLI3 in Xt mice (3;7)(p21.1;p13) associated with the Greig polysyndactylycraniofacial anomalies syndrome. Am J Med Genet 22: 313-321, 1983. Vortkamp, A., Gessler, M., and Grzeschik, K.-H.: GLI3 zinc finger gene interrupted by translocations in Greig syndrome families. Nature 352: 539-540, 1991a. Vortkamp, A., Thias, U., Gessler, M., Rosenkranz, W., Kroisel, P.M., Tommerup, N., Krfiger, G., G6tz, J., Pelz, L., and Grzeschik, K.-H.: A somatic cell hybrid panel and DNA probes for
463 physical mapping of human chromosome 7p. Genomics 11: 737743, 1991b. Wagner, K., Kroisel, P.M., and Rosenkranz, W.: Molecular and cytogenetic analysis in two patients with microdeletions of 7p and Greig syndrome: hemizygosity for PGAM2 and TCRG genes. Genomics 8: 487-491, 1990. Winter, R.M. and Huson, S.M.: Greig cephalopolysyndactyly syndrome: a possible mouse homologue (Xt-extra toes). A m J Med Genet 31: 793-798, t988.
enome 9 Springer-VerlagNew YorkInc. 1992
Deletion of GLI3 supports the homology of the human Greig cephalopolysyndactyly syndrome (GCPS) and the mouse mutant extra toes (Xt) Andrea Vortkamp, 1 Thomas Franz, 2 Manfred Gessler, ~ and Karl-Heinz Grzeschik ~ lInstitut ffir Humangenetik der Philipps-Universit/it, Bahnhofstr. 7a, D-3550 Marburg, FRG; 2Anatomisches Institut, Abteilung Neuroanatomie, Universit~tskrankenhaus Eppendorf, Martinistr. 52, D-2000 Hamburg 20, FRG Received February 19, 1992; accepted April 23, 1992
The dominant mouse mutant extra toes (Xt) is characterized by preaxial and postaxial polydactyly of the feet and a white belly spot. An interfrontal bone is present in the skull in 90% of heterozygotes compared with 50% of normal mice. Homozygous Xt/Xt embryos exhibit multiple skeletal defects, extreme polydactyly in both fore- and hindlimbs, and malformations of the brain and the eye (Johnson 1967; Franz and Besecke 1991). Depending on the genetic background, Xt homozygotes die prenatally or perinatally (Johnson 1967). An allelic but recessive syndrome is the anterior digit pattern deformity mutation (add), which is the result of a transgene integration (Pohl et al. 1990). In this case the malformations of the mutants are restricted to the forelimbs. Using DNA probes spanning the add transgene integration site, Pohl and coworkers (1990) could show that at least 80 kbp of surrounding DNA are deleted in Xt mice. On the basis of the similarity of the phenotype, Xt is considered the mouse homolog of the human autosomal dominant Greig cephalopolysyndactyly syndrome, GCPS (Winter and Huson 1988). The latter is characterized by polysyndactyly of hands and feet and mild craniofacial abnormalities (Gollop and Fontes 1985). The gene locus has been pinpointed to human Chromosome (Chr) 7p13 by different translocations and deletions associated with the disorder (Tommerup and Nielsen 1983; Krt~ger et al. 1989; Wagner et al. 1990; Pettigrew et al. 1991; Vortkamp et al. 1991b). Using six hybrids from three GCPS translocation patients, we recently have demonstrated that the zinc finger gene GLI3 (Ruppert et al. 1988, 1990) is disOffprint requests to: K.-H. Grzeschik
rupted in the first third of its coding region by two of the GCPS translocations (Vortkamp et al. 1991a). The third translocation breakpoint was shown to be located approximately 10 kbp downstream of the expressed sequence. Thus, mutations of GLI3 resulting in a reduced gene dosage are probably responsible for the development of GCPS. To characterize the GLI3 gene in Xt mutant mice, we isolated different GLI3 cDNA clones from an 8.5day fetal mouse cDNA library (Fahrner et al. 1987), using segments of the human GLI3 cDNA as probes. The position of the mouse clones relative to the known human GLI3 sequence (Ruppert et al. 1990) was determined by partial sequencing (Fig. la). The distal mouse cDNA clone, pMGli20, corresponds to nucleotides 3232-4734 of the human cDNA. Hybridization with genomic DNA of homozygous Xt/ Xt mice and normal control mice did not show any alteration in the 3' part of the gene (Fig. ld). The proximal mouse cDNA clone pMGlil 1 starts approximately 500 bp upstream of the published GLI3 cDNA and reaches down to nucleotide 415. The probe hybridizes with multiple EcoRI and PstI fragments in normal mouse DNA that are completely deleted in homozygous Xt/Xt embryos (data not shown). To further characterize the deletion boundary within the GLI3 gene, we hybridized the human cDNA fragment pcrGliprox (nucleotides 98-570) with genomic DNA of homozygous Xt/Xt and normal control mice. This probe overlaps with a 300-bp BglII/NotI fragment (pMGlillBN), representing the 3' third of pMGlil 1, but reaches 160 bp further downstream. Like probe pMGlillBN (Fig. lb), pcrGliprox hybridizes with an 8-kbp PstI fragment that is deleted in the DNA of Xt/ Xt embryos. Furthermore, two additional PstI frag-
462
A. Vortkamp et al.: Deletion of GL13 in Xt mice
a)
Fig. 1. The GLI3 gene is partially deleted in Xt
mutants. (a) The different GCPS translocations and the Xt deletion (arrows) are drawn in relation to the human cDNA published by Ruppert and co-workers (1991). The open reading frame ( I Xt-Deietion of GLl3 is indicated by an open box; the 5' end, 3' not yet determined, by a broken line. The posi_[ ].-GLI3-cDNA tions of the cDNA fragments used in this study are marked by shaded boxes. 0r--d) DNA from two homozygous Xt/Xt embryos, a normal ( + / I I pcrGfipro• +) sib and the mouse tumor cell line Rag, was digested with PstI (b,c) and EcoRI (d) and hyI ~pMGii11 [ I pMGli20 bridized with different probes from the GL13 gene. (b) Probe pMGIillBN, a 300-bp BgllI/ r---1 pMGli11BN Not[ fragment ofpMGIil 1 detects an 8-kbp PstI fragment that is deleted in Xt/Xt embryos. This fragment overlaps with the human probe pcrGliprox. (c) Probe pcrGliprox reaches 160 bp further downstream and hybridizes with two additional PstI fragments that are both not altered in the DNAs tested. (d) Hybridization of pMGli20 does not show any indicationfor a mutation in Xt/Xt mice. Xt/Xt breeding pairs in a (C3Hxl01) F2 background were purchased from the MRC Radiobiology Unit, Harwetl, UK. High-molecular-weight DNA was prepared from embryos on day 16 of gestation. Xt/Xt embryos were identified macroscopically by their polydactyly and the malformation of the eye. The DNA was isolated according to the method of Kunkel and co-workers (1982), and Southern blot analysis was carried out under standard conditions (Sambrook et al. 1989). The mouse cDNA probes, pMGlil 1 and pMGli20, which are available upon request, were isolated from an 8.5-day fetal mouse cDNA library (Fahrner et al. 1987), with the human cDNA fragments pcrGliprox and pcrGlidist (Vortkamp et al. 1991a) as probes. + +
,~
m e n t s of 14 k b p a n d 3.8 k b p are d e t e c t e d , b o t h of w h i c h are n o t d e l e t e d in the X t / X t e m b r y o s (Fig. lc). T h u s , the b r e a k p o i n t of the X t d e l e t i o n m u s t be localized b e t w e e n n u c l e o t i d e s 415 a n d 570 of the corr e s p o n d i n g h u m a n c D N A . T h e b r e a k p o i n t is p r o b a b l y l o c a t e d in a n i n t r o n of the G L I 3 g e n e , as h y b r i d i z a t i o n o f E c o R I (not s h o w n ) a n d P s t I (Fig. l b , c ) - d i g e s t e d X t D N A s h o w e d n o r e a r r a n g e d f r a g m e n t s . A t p r e s e n t we do n o t k n o w the size o f the d e l e t i o n , b u t it s e e m s likely that the d e l e t i o n r e g i o n i n c l u d e s 80 k b p of g e n o m i c D N A f r o m the add l o c u s (Pohl et al. 1990) that was s h o w n to b e d e l e t e d in X t . T h e d e l e t i o n o f the 5' r e g i o n o f the G L I 3 c D N A in X t / + mice p r e v e n t s the f o r m a t i o n of a f u n c t i o n a l protein p r o d u c t f r o m o n e allele. T h e r e f o r e , a r e d u c t i o n in g e n e d o s a g e for G L I 3 is the likely c a u s e for the malf o r m a t i o n s s e e n in b o t h , the m o u s e X t m u t a n t a n d the h u m a n G C P S s y n d r o m e , c o n f i r m i n g the h o m o l o g y of these s y n d r o m e s . A s the i n t e g r a t i o n site of the t r a n s g e n e in the a d d m u t a n t has b e e n l o c a l i z e d w i t h i n the X t d e l e t i o n , it will b e of great i n t e r e s t to d e t e r m i n e the p r e c i s e p h y s i c a l r e l a t i o n s h i p b e t w e e n the g e n o m i c G L I 3 l o c u s a n d the a d d i n t e g r a t i o n site. X t m u t a n t mice e x h i b i t a m u c h m o r e s e v e r e p h e n o t y p e c o m p a r e d with the a d d m u t a n t s , l e a v i n g the p o s s i b i l i t y that there are a d d i t i o n a l g e n e s in the X t d e l e t i o n r e g i o n that m a y c o n t r i b u t e to the o b s e r v e d p h e n o t y p e .
Acknowledgments. We thank B. Hogan for an aliquot of the fetal mouse cDNA library. This work was supported by a grant from the Deutsche Forschungsgemeinschaft.
GCPS-Translocations
References Fahrner, K., Hogan, B.L.M., and Flavell, R.A.: Transcription of H-2 and Qa genes in embryonic and adult mice. EMBO J 6: 12651271, 1987. Franz, T. and Beseke, A.: The development of the eye in homozygous mutants of the extra-toes locus of the mouse. Anat Embryol 184: 335-361, 1991. Gollop, T.R. and Fontes, L.R.: The Greig cephalopolysyndactyly syndrome: report of a family and review of the literature. Am J IVied Genet 22: 59-68, 1985. Johnson, D.R.: Extra-toes: a new mutant gene causing multiple abnormalities in the mouse. J Embryol Exp Morphol 17: 543-581, 1967. Kr/iger, G., G6tz, J., Kvist, U., Dunker, H., Erfurth, F., Pelz, L. and Zech, L.: Greig syndrome in a large kindred due to reciprocal chromosome translocation t(6;7)(q27;p13). Am J Med Genet 32: 411-416, 1989. Kunkel, L.M., Tantravahi, U., Eisenhard, M., and Latt, S.A.: Regional localization on the human X of DNA segments cloned from flow sorted chromosomes. Nucleic Acids Res 10: 1557-1578, 1982. Pettigrew, A.L., Greenberg, F., Caskey, C.T., and Ledbetter, D.H.: Greig syndrome associated with an interstitial deletion of 7p: confirmation of the localization of Greig syndrome to 7p13. Hum Genet 87: 452-456, 1991. Pohl, T.M., Mattei, M.G., and Rfither, U.: Evidence for alMism of the recessive insertional mutation add and the dominant mouse mutation extra-toes (Xt). Development 110: 1153-1157, 1990. Ruppert, J.M., Kinzler, K.W., Wong, A.J., Bigner, S.H., Kao, F.-T., Law, M.I., Seuanez, H.N., O'Brien, S.J., and Vogelstein, B.: The GLI-Krfippel family of human genes. Mol Cell Biol 8: 3104-3113, 1988. Ruppert, J.M., Vogelstein, B., Arheden, K., and Kinzler, K.W.: GLI3 encodes a 190-kilodalton protein with multiple regions of GL1 similarity. Mol Cell Biol I0: 5408-5415, 1990. Sambrook, J., Fritsch, E.G., and Maniatis, T.: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Tommerup, N. and Nielsen, F.: A familial reciprocal translocation
A. Vortkamp et al.: Deletion of GLI3 in Xt mice (3;7)(p21.1;p13) associated with the Greig polysyndactylycraniofacial anomalies syndrome. Am J Med Genet 22: 313-321, 1983. Vortkamp, A., Gessler, M., and Grzeschik, K.-H.: GLI3 zinc finger gene interrupted by translocations in Greig syndrome families. Nature 352: 539-540, 1991a. Vortkamp, A., Thias, U., Gessler, M., Rosenkranz, W., Kroisel, P.M., Tommerup, N., Krfiger, G., G6tz, J., Pelz, L., and Grzeschik, K.-H.: A somatic cell hybrid panel and DNA probes for
463 physical mapping of human chromosome 7p. Genomics 11: 737743, 1991b. Wagner, K., Kroisel, P.M., and Rosenkranz, W.: Molecular and cytogenetic analysis in two patients with microdeletions of 7p and Greig syndrome: hemizygosity for PGAM2 and TCRG genes. Genomics 8: 487-491, 1990. Winter, R.M. and Huson, S.M.: Greig cephalopolysyndactyly syndrome: a possible mouse homologue (Xt-extra toes). A m J Med Genet 31: 793-798, t988.
