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Expression patterns of Twist and Fgfr1, -2 and -3 in the developing mouse coronal suture suggest a key role for Twist in suture initiation and biogenesis
Mechanisms of Development 91 (2000) 341±345
Gene expression pattern
www.elsevier.com/locate/modo
Expression patterns of Twist and Fgfr1, -2 and -3 in the developing mouse coronal suture suggest a key role for Twist in suture initiation and biogenesis D. Johnson a,b, S. Iseki c,d, A.O.M. Wilkie a,b, G.M. Morriss-Kay c,* a
b
Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK Department of Plastic and Reconstructive Surgery and Oxford Craniofacial Unit, The Radcliffe In®rmary NHS Trust, Woodstock Road, Oxford OX2 6HE, UK c Department of Human Anatomy and Genetics, South Parks Road, Oxford OX1 3QX, UK d Department of Molecular Craniofacial Embryology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan Received 12 August 1999; accepted 18 October 1999
Abstract Sutural growth depends on maintenance of a balance between proliferation of osteogenic stem cells and their differentiation to form new bone, so that the stem cell population is maintained until growth of the skull is complete. The identi®cation of heterozygous mutations in FGFR1, -2 and -3 and TWIST as well as microdeletions of TWIST in human craniosynostosis syndromes has highlighted these genes as playing important roles in maintaining the suture as a growth centre. In contrast to Drosophila, a molecular relationship between human (or other vertebrate) TWIST and FGFR genes has not yet been established. TWIST mutations exert their effect via haploinsuf®ciency whereas FGFR mutations have a gain-of-function mechanism of action. To investigate the biological basis of FGFR signalling pathways in the developing calvarium we compared the expression patterns of Twist with those of Fgfr1, -2 and -3 in the fetal mouse coronal suture over the course of embryonic days 14-18, as the suture is initiated and matures. Our results show that: (1) Twist expression precedes that of Fgfr genes at the time of initiation of the coronal suture; (2) in contrast to Fgfr transcripts, which are localised within and around the developing bone domains, Twist is expressed by the midsutural mesenchyme cells. Twist expression domains show some overlap with those of Fgfr2, which is expressed in the most immature (proliferating) osteogenic tissue. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Twist; Fgfr1; Fgfr2; Fgfr3; Craniosynostosis; Skull; Suture; Mouse fetus; In situ hybridization
1. Introduction The twist gene was originally identi®ed in Drosophila as one of the zygotic genes required for dorso-ventral patterning during embryogenesis; homozygous twist-null embryos fail to generate mesoderm (NuÈsslein-Volhard et al., 1984). The identi®cation of heterozygous mutations in FGFR1, -2 and -3 and TWIST as well as microdeletions of TWIST in human craniosynostosis syndromes (reviewed by Wilkie, 1997; Johnson et al., 1998) has highlighted these genes as playing important roles in maintaining the suture as a growth centre. Evidence that twist is upstream of Fgfr signalling during mesoderm formation in Drosophila comes from the observation that RNA expression of DFR1/htl (an orthologue of mammalian FGFRs) requires twist protein (Shishido et al., 1993). * Corresponding author. Tel.: 144-1865-272-165; fax: 144-1865-272420. E-mail address: [email protected] (G.M. Morriss-Kay)
In cultured mouse calvarial osteoprogenitor cells, Twist expression is down-regulated during differentiation (Murray et al., 1992), suggesting either that it promotes proliferation or that it inhibits differentiation, as in myogenesis (Hebrok et al., 1994). Here we describe the expression pattern of Twist in the fetal mouse coronal suture and compare it with those of Fgfr1, -2 and -3 over the time course E14± E18, as the suture is initiated and matures.
2. Results The mouse coronal suture is ®rst detected at E14. Initiation and maturation progresses from the region closest to the skull base towards the apex, so that in serial transverse sections, as studied here, there is always a greater degree of sutural maturity, including the amount of overlap of the frontal (inner) and parietal (outer) bone plates, in the more basal regions of the suture.
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(99)00278-6
342
D. Johnson et al. / Mechanisms of Development 91 (2000) 341±345
Fig. 1. (A±H) In situ hybridisation of E14 mouse calvarial transverse (horizontal) frozen sections taken close to the skull base showing expression of Twist, Fgfr1, -2 and -3. (A,E) Twist expression can be seen throughout the subcutaneous tissue and skeletogenic membrane. The arrow identi®es the localised expression of Twist within the skeletogenic membrane at the site of the developing coronal suture. Small arrows identify the strongest expression of Twist in the developing future parietal bone away from the site of the developing coronal suture. Expression in the deepest layer, presumably future dura mater, is seen between the arrowheads. (B,F) Expression of Fgfr1 can be seen throughout the skeletogenic membrane, with no clear indication of the site of the coronal suture at this stage (indicated by the asterisk). (C,G) Expression of Fgfr2 can be seen in the epidermis and the skeletogenic membrane. The arrow indicates reduced expression in an oblique domain between the parietal and frontal domains. (D,H) Expression of Fgfr3 can be seen in the epidermis and the skeletogenic membrane. The arrow indicates reduced pre-sutural expression similar to that of Fgfr2. b, brain; f, frontal bone; p, parietal bone; s, skin. Scale bars: A±D 150 mm; E±H 50 mm.
D. Johnson et al. / Mechanisms of Development 91 (2000) 341±345
343
Fig. 2. (A±H) In situ hybridisation of E16 mouse calvarial transverse frozen sections showing expression of Twist, Fgfr1, -2 and -3, as indicated. (A,E) Twist expression can be seen in the outermost cells of the developing frontal and parietal bone plates. The obliquely placed arrow in A identi®es a focus of strong expression in the midsutural mesenchyme. (B,F) The cells expressing Fgfr1 are closest to the osteoid and do not extend far beyond the ends of the osteoid plates at the suture; there is no Fgfr1 expression in the rounded cells of the midsutural mesenchyme. (C,G) Fgfr2 expression is detected in the population of cells peripheral to those expressing Fgfr1; at the sutural ends of the osteoid, there is a focus of strong expression in a fan-like domain extending into the sutural mesenchyme, except for its central part. (D,H) Fgfr3 is expressed in cells positioned between those expressing Fgfr1 and Fgfr2, probably overlapping both domains. Arrowheads in A±D indicate the endocranial periosteal layer subjacent to the frontal bone. The endocranium is thickened immediately subjacent to the coronal suture (arrow). b, brain; f, frontal bone; p, parietal bone; s, skin; sm, sutural mesenchyme; ec, endocranial layer. Scale bars: A±D 150 mm; E±H 50 mm.
344
D. Johnson et al. / Mechanisms of Development 91 (2000) 341±345
Fig. 3. (A±H) In situ hybridisation of E18 mouse calvarial transverse frozen sections showing expression of Twist, Fgfr1, -2 and -3, as indicated. The endocranial layer is less prominent than on E16 although still slightly thickened immediately subjacent to the suture. The arrows in A indicate the two foci of Twist expression at the sutural edges of the frontal and parietal bones. b, brain; f, frontal bone; p, parietal bone; s, skin. Scale bars: A±D 150 mm; E±H 50 mm.
2.1. E14 expression pattern (Fig. 1) Twist is expressed throughout the developing subcutaneous tissue, and in the developing skeletogenic membrane,
particularly the outer aspect of the future parietal bone domain. Close to the skull base the future coronal suture can be identi®ed as a fusiform swelling of the skeletogenic membrane composed of cells expressing Twist. A few cells
D. Johnson et al. / Mechanisms of Development 91 (2000) 341±345
deep to the endocranial layer (presumably dura mater cells) also show strong Twist expression speci®cally subjacent to the suture (Fig. 1A). Fgfr2 and -3 show strong expression in the epidermis as well as the skeletogenic membrane. The midsutural mesenchyme can be seen to have a lower level of expression of Fgfr2 and -3 than the future bone domains, clearly demarcating the position of the future coronal suture; again this pattern was only apparent in the basal sections. Strong expression of Fgfr1 is detected throughout the skeletogenic membrane, but even in basal sections there is no clear distinction between future sutural and bone domains. In sections taken from higher levels than those illustrated, i.e. closer to the vertex of the skull where differentiation is always less advanced than basally, Fgfr1 and Fgfr3 transcripts are undetectable but low levels of Fgfr2 transcripts are detected uniformly throughout the mesenchymal tissue that will later divide into skeletogenic and meningeal layers. Only Twist gives a strong signal speci®cally within the outer (skeletogenic) layer. 2.2. E16 expression pattern (Fig. 2) Osteoid is now present in the differentiating regions of the frontal and parietal bones and the level of Twist expression in the midsutural mesenchyme is greater than that observed on the two preceding days. The Fgfr expression patterns at E15 (not shown) and E16 correspond to differentiating cells attached to the osteoid (Fgfr1), proliferating cells at the periphery of the bone plates (Fgfr2) and cells in an intermediate position (Fgfr3), as observed by Iseki et al. (1999). Fgfr2-positive cells extend to form a fan-like shape around the sutural ends of the two bones, showing some overlap with the midsutural Twist domain. Comparison of Fgfr1 and Twist expression patterns suggests that their domains may be mutually exclusive. At this stage, the endocranial layer of mesenchymal tissue (endocranium and meninges) that lies immediately subjacent to the skeletogenic membrane is composed of several layers of ¯attened cells, in contrast to the more rounded cells of the midsutural mesenchyme. The endocranium is thicker beneath the coronal suture than beneath the areas of differentiating bone. 2.3. E18 expression pattern (Fig. 3) The overall expression level of Twist in the skeletogenic membrane has decreased compared with previous days. At E17 (not shown) and E18 the Twist expression domain splits into two clearly de®ned zones as the increasing overlap of the frontal and parietal plates separates their sutural borders. The established positional relationship between the expression domains of Twist and the three Fgfr genes is retained.
345
3. Experimental procedures C57BL/6 mouse fetuses (plug day E0) were used. In situ hybridisation was carried out on 12-mm serial transverse frozen sections from fetal mouse heads taken at E14±E18 using 400 ng/ml of digoxygenin-labelled probes, as described previously (Iseki et al., 1997). The following mouse probes were used for in situ hybridisation: (1) Twist ± a 420 bp fragment consisting of exon 2 (non-translated exon); (2) Fgfr1 ± a 3.8 kb fragment (IIIc splice variant); (3) Fgfr2 ± a 1.96 kb fragment including the whole extracellular and transmembrane domains (IIIc splice variant); (4) Fgfr3 ± full length (IIIc splice variant). All were cloned into pBluescript SK(1) (Stratagene). The Fgfr transcripts were reduced to an average size of 200 bases by limited alkaline hydrolysis. Acknowledgements We are grateful to F. Perrin-Schmitt, J. Heath and D. Ornitz for donation of the probes. This study was funded by a Wellcome Clinical Training Fellowship award to D.J., a Wellcome Senior Clinical Fellowship award to A.O.M.W., and an Action Research grant to G.M.M.-K. References Hebrok, M., Wertz, K., Fuchtbauer, E.M., 1994. M-twist is an inhibitor of muscle differentiation. Dev. Biol. 165 (2), 537±544. Iseki, S., Wilkie, A.O.M., Heath, J.K., Ishimaru, T., Eto, K., Morriss-Kay, G.M., 1997. Fgfr2 and osteopontin domains in the developing skull vault are mutually exclusive and can be altered by locally applied FGF2. Development 124, 3375±3384. Iseki, S., Wilkie, A.O.M., Morriss-Kay, G.M., 1999. Fgfr1 and Fgfr2 have distinct differentiation- and proliferation-related roles in the developing mouse skull vault. Development 126(24), 5611±5620. Johnson, D., Horsley, S.W., Moloney, D.M., Oldridge, M., Twigg, S.R.F., Walsh, S., Barrow, M., Njùlstad, P.R., Kunz, J., Ashworth, G.J., Wall, S.A., Kearney, L., Wilkie, A.O.M., 1998. A comprehensive screen for TWIST mutations in patients with craniosynostosis identi®es a new microdeletion syndrome of chromosome band 7p21.1. Am. J. Hum. Genet. 63, 1282±1293. Murray, S.S., Glackin, C.A., Winters, K.A., Gazit, D., Kahn, A.J., Murray, E.J., 1992. Expression of helix-loop-helix regulatory genes during differentiation of mouse osteoblastic cells. J. Bone Miner. Res. 7 (10), 1131±1138. NuÈsslein-Volhard, C., Wieschaus, E., Kluding, H., 1984. Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster I. Zygotic loci on the second chromosome. Roux's Arch. Dev. Biol. 193, 267±282. Shishido, E., Higashijima, S., Saigo, K., 1993. Two FGF-receptor homologues of Drosophila: one is expressed in mesodermal primordium in early embryos. Development 117, 751±761. Wilkie, A.O.M., 1997. Craniosynostosis: genes and mechanisms. Hum. Mol. Genet. 6, 1647±1656.
Gene expression pattern
www.elsevier.com/locate/modo
Expression patterns of Twist and Fgfr1, -2 and -3 in the developing mouse coronal suture suggest a key role for Twist in suture initiation and biogenesis D. Johnson a,b, S. Iseki c,d, A.O.M. Wilkie a,b, G.M. Morriss-Kay c,* a
b
Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK Department of Plastic and Reconstructive Surgery and Oxford Craniofacial Unit, The Radcliffe In®rmary NHS Trust, Woodstock Road, Oxford OX2 6HE, UK c Department of Human Anatomy and Genetics, South Parks Road, Oxford OX1 3QX, UK d Department of Molecular Craniofacial Embryology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan Received 12 August 1999; accepted 18 October 1999
Abstract Sutural growth depends on maintenance of a balance between proliferation of osteogenic stem cells and their differentiation to form new bone, so that the stem cell population is maintained until growth of the skull is complete. The identi®cation of heterozygous mutations in FGFR1, -2 and -3 and TWIST as well as microdeletions of TWIST in human craniosynostosis syndromes has highlighted these genes as playing important roles in maintaining the suture as a growth centre. In contrast to Drosophila, a molecular relationship between human (or other vertebrate) TWIST and FGFR genes has not yet been established. TWIST mutations exert their effect via haploinsuf®ciency whereas FGFR mutations have a gain-of-function mechanism of action. To investigate the biological basis of FGFR signalling pathways in the developing calvarium we compared the expression patterns of Twist with those of Fgfr1, -2 and -3 in the fetal mouse coronal suture over the course of embryonic days 14-18, as the suture is initiated and matures. Our results show that: (1) Twist expression precedes that of Fgfr genes at the time of initiation of the coronal suture; (2) in contrast to Fgfr transcripts, which are localised within and around the developing bone domains, Twist is expressed by the midsutural mesenchyme cells. Twist expression domains show some overlap with those of Fgfr2, which is expressed in the most immature (proliferating) osteogenic tissue. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Twist; Fgfr1; Fgfr2; Fgfr3; Craniosynostosis; Skull; Suture; Mouse fetus; In situ hybridization
1. Introduction The twist gene was originally identi®ed in Drosophila as one of the zygotic genes required for dorso-ventral patterning during embryogenesis; homozygous twist-null embryos fail to generate mesoderm (NuÈsslein-Volhard et al., 1984). The identi®cation of heterozygous mutations in FGFR1, -2 and -3 and TWIST as well as microdeletions of TWIST in human craniosynostosis syndromes (reviewed by Wilkie, 1997; Johnson et al., 1998) has highlighted these genes as playing important roles in maintaining the suture as a growth centre. Evidence that twist is upstream of Fgfr signalling during mesoderm formation in Drosophila comes from the observation that RNA expression of DFR1/htl (an orthologue of mammalian FGFRs) requires twist protein (Shishido et al., 1993). * Corresponding author. Tel.: 144-1865-272-165; fax: 144-1865-272420. E-mail address: [email protected] (G.M. Morriss-Kay)
In cultured mouse calvarial osteoprogenitor cells, Twist expression is down-regulated during differentiation (Murray et al., 1992), suggesting either that it promotes proliferation or that it inhibits differentiation, as in myogenesis (Hebrok et al., 1994). Here we describe the expression pattern of Twist in the fetal mouse coronal suture and compare it with those of Fgfr1, -2 and -3 over the time course E14± E18, as the suture is initiated and matures.
2. Results The mouse coronal suture is ®rst detected at E14. Initiation and maturation progresses from the region closest to the skull base towards the apex, so that in serial transverse sections, as studied here, there is always a greater degree of sutural maturity, including the amount of overlap of the frontal (inner) and parietal (outer) bone plates, in the more basal regions of the suture.
0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(99)00278-6
342
D. Johnson et al. / Mechanisms of Development 91 (2000) 341±345
Fig. 1. (A±H) In situ hybridisation of E14 mouse calvarial transverse (horizontal) frozen sections taken close to the skull base showing expression of Twist, Fgfr1, -2 and -3. (A,E) Twist expression can be seen throughout the subcutaneous tissue and skeletogenic membrane. The arrow identi®es the localised expression of Twist within the skeletogenic membrane at the site of the developing coronal suture. Small arrows identify the strongest expression of Twist in the developing future parietal bone away from the site of the developing coronal suture. Expression in the deepest layer, presumably future dura mater, is seen between the arrowheads. (B,F) Expression of Fgfr1 can be seen throughout the skeletogenic membrane, with no clear indication of the site of the coronal suture at this stage (indicated by the asterisk). (C,G) Expression of Fgfr2 can be seen in the epidermis and the skeletogenic membrane. The arrow indicates reduced expression in an oblique domain between the parietal and frontal domains. (D,H) Expression of Fgfr3 can be seen in the epidermis and the skeletogenic membrane. The arrow indicates reduced pre-sutural expression similar to that of Fgfr2. b, brain; f, frontal bone; p, parietal bone; s, skin. Scale bars: A±D 150 mm; E±H 50 mm.
D. Johnson et al. / Mechanisms of Development 91 (2000) 341±345
343
Fig. 2. (A±H) In situ hybridisation of E16 mouse calvarial transverse frozen sections showing expression of Twist, Fgfr1, -2 and -3, as indicated. (A,E) Twist expression can be seen in the outermost cells of the developing frontal and parietal bone plates. The obliquely placed arrow in A identi®es a focus of strong expression in the midsutural mesenchyme. (B,F) The cells expressing Fgfr1 are closest to the osteoid and do not extend far beyond the ends of the osteoid plates at the suture; there is no Fgfr1 expression in the rounded cells of the midsutural mesenchyme. (C,G) Fgfr2 expression is detected in the population of cells peripheral to those expressing Fgfr1; at the sutural ends of the osteoid, there is a focus of strong expression in a fan-like domain extending into the sutural mesenchyme, except for its central part. (D,H) Fgfr3 is expressed in cells positioned between those expressing Fgfr1 and Fgfr2, probably overlapping both domains. Arrowheads in A±D indicate the endocranial periosteal layer subjacent to the frontal bone. The endocranium is thickened immediately subjacent to the coronal suture (arrow). b, brain; f, frontal bone; p, parietal bone; s, skin; sm, sutural mesenchyme; ec, endocranial layer. Scale bars: A±D 150 mm; E±H 50 mm.
344
D. Johnson et al. / Mechanisms of Development 91 (2000) 341±345
Fig. 3. (A±H) In situ hybridisation of E18 mouse calvarial transverse frozen sections showing expression of Twist, Fgfr1, -2 and -3, as indicated. The endocranial layer is less prominent than on E16 although still slightly thickened immediately subjacent to the suture. The arrows in A indicate the two foci of Twist expression at the sutural edges of the frontal and parietal bones. b, brain; f, frontal bone; p, parietal bone; s, skin. Scale bars: A±D 150 mm; E±H 50 mm.
2.1. E14 expression pattern (Fig. 1) Twist is expressed throughout the developing subcutaneous tissue, and in the developing skeletogenic membrane,
particularly the outer aspect of the future parietal bone domain. Close to the skull base the future coronal suture can be identi®ed as a fusiform swelling of the skeletogenic membrane composed of cells expressing Twist. A few cells
D. Johnson et al. / Mechanisms of Development 91 (2000) 341±345
deep to the endocranial layer (presumably dura mater cells) also show strong Twist expression speci®cally subjacent to the suture (Fig. 1A). Fgfr2 and -3 show strong expression in the epidermis as well as the skeletogenic membrane. The midsutural mesenchyme can be seen to have a lower level of expression of Fgfr2 and -3 than the future bone domains, clearly demarcating the position of the future coronal suture; again this pattern was only apparent in the basal sections. Strong expression of Fgfr1 is detected throughout the skeletogenic membrane, but even in basal sections there is no clear distinction between future sutural and bone domains. In sections taken from higher levels than those illustrated, i.e. closer to the vertex of the skull where differentiation is always less advanced than basally, Fgfr1 and Fgfr3 transcripts are undetectable but low levels of Fgfr2 transcripts are detected uniformly throughout the mesenchymal tissue that will later divide into skeletogenic and meningeal layers. Only Twist gives a strong signal speci®cally within the outer (skeletogenic) layer. 2.2. E16 expression pattern (Fig. 2) Osteoid is now present in the differentiating regions of the frontal and parietal bones and the level of Twist expression in the midsutural mesenchyme is greater than that observed on the two preceding days. The Fgfr expression patterns at E15 (not shown) and E16 correspond to differentiating cells attached to the osteoid (Fgfr1), proliferating cells at the periphery of the bone plates (Fgfr2) and cells in an intermediate position (Fgfr3), as observed by Iseki et al. (1999). Fgfr2-positive cells extend to form a fan-like shape around the sutural ends of the two bones, showing some overlap with the midsutural Twist domain. Comparison of Fgfr1 and Twist expression patterns suggests that their domains may be mutually exclusive. At this stage, the endocranial layer of mesenchymal tissue (endocranium and meninges) that lies immediately subjacent to the skeletogenic membrane is composed of several layers of ¯attened cells, in contrast to the more rounded cells of the midsutural mesenchyme. The endocranium is thicker beneath the coronal suture than beneath the areas of differentiating bone. 2.3. E18 expression pattern (Fig. 3) The overall expression level of Twist in the skeletogenic membrane has decreased compared with previous days. At E17 (not shown) and E18 the Twist expression domain splits into two clearly de®ned zones as the increasing overlap of the frontal and parietal plates separates their sutural borders. The established positional relationship between the expression domains of Twist and the three Fgfr genes is retained.
345
3. Experimental procedures C57BL/6 mouse fetuses (plug day E0) were used. In situ hybridisation was carried out on 12-mm serial transverse frozen sections from fetal mouse heads taken at E14±E18 using 400 ng/ml of digoxygenin-labelled probes, as described previously (Iseki et al., 1997). The following mouse probes were used for in situ hybridisation: (1) Twist ± a 420 bp fragment consisting of exon 2 (non-translated exon); (2) Fgfr1 ± a 3.8 kb fragment (IIIc splice variant); (3) Fgfr2 ± a 1.96 kb fragment including the whole extracellular and transmembrane domains (IIIc splice variant); (4) Fgfr3 ± full length (IIIc splice variant). All were cloned into pBluescript SK(1) (Stratagene). The Fgfr transcripts were reduced to an average size of 200 bases by limited alkaline hydrolysis. Acknowledgements We are grateful to F. Perrin-Schmitt, J. Heath and D. Ornitz for donation of the probes. This study was funded by a Wellcome Clinical Training Fellowship award to D.J., a Wellcome Senior Clinical Fellowship award to A.O.M.W., and an Action Research grant to G.M.M.-K. References Hebrok, M., Wertz, K., Fuchtbauer, E.M., 1994. M-twist is an inhibitor of muscle differentiation. Dev. Biol. 165 (2), 537±544. Iseki, S., Wilkie, A.O.M., Heath, J.K., Ishimaru, T., Eto, K., Morriss-Kay, G.M., 1997. Fgfr2 and osteopontin domains in the developing skull vault are mutually exclusive and can be altered by locally applied FGF2. Development 124, 3375±3384. Iseki, S., Wilkie, A.O.M., Morriss-Kay, G.M., 1999. Fgfr1 and Fgfr2 have distinct differentiation- and proliferation-related roles in the developing mouse skull vault. Development 126(24), 5611±5620. Johnson, D., Horsley, S.W., Moloney, D.M., Oldridge, M., Twigg, S.R.F., Walsh, S., Barrow, M., Njùlstad, P.R., Kunz, J., Ashworth, G.J., Wall, S.A., Kearney, L., Wilkie, A.O.M., 1998. A comprehensive screen for TWIST mutations in patients with craniosynostosis identi®es a new microdeletion syndrome of chromosome band 7p21.1. Am. J. Hum. Genet. 63, 1282±1293. Murray, S.S., Glackin, C.A., Winters, K.A., Gazit, D., Kahn, A.J., Murray, E.J., 1992. Expression of helix-loop-helix regulatory genes during differentiation of mouse osteoblastic cells. J. Bone Miner. Res. 7 (10), 1131±1138. NuÈsslein-Volhard, C., Wieschaus, E., Kluding, H., 1984. Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster I. Zygotic loci on the second chromosome. Roux's Arch. Dev. Biol. 193, 267±282. Shishido, E., Higashijima, S., Saigo, K., 1993. Two FGF-receptor homologues of Drosophila: one is expressed in mesodermal primordium in early embryos. Development 117, 751±761. Wilkie, A.O.M., 1997. Craniosynostosis: genes and mechanisms. Hum. Mol. Genet. 6, 1647±1656.