- Email: [email protected]
Head Development: Craniofacial genetics makes headway
Joy M.
HEAD DEVELOPMENT
RICHMAN RICHMAN
HEAD DEVELOPMENT
Craniofacial genetics makes headway Studies of neural crest migration in animal models, and of human syndromes in which craniofacial development isabnormal, are helping us to understand both prenatal and postnatal development of the head. At the core of craniofacial research is the desire to understand both normal and abnormal development of the head. The head has, however, proven to be a particular challenge to developmental biologists, as many of the patterning mechanisms are unique to this part of the body. The clinical genetic approach, studying inherited syndromes that affect human craniofacial development, is proving to be very useful for understanding human dysmorphology and is also providing new directions for more basic research using animal models. Patterning the cranial neural crest - moving past the hindbrain Neural crest cells play a pivotal role in craniofacial development, as they initially give rise to most of the mesenchyme of the head and face, and ultimately the mesenchyme forms most of the skeletal and connective tissue in the head. It is thought that many craniofacial defects arise from perturbations of the neural crest early in embryonic development. Noden [1], for example, reported that avian cranial neural crest cells transplanted from the midbrain - which normally contribute to the beak - to a more caudal position, migrated into the neck and gave rise to an extra set of beaks, thus following their intrinsic developmental programme. But these results need to be supported by evidence, at the molecular level, that crar;ial neural crest cells are informed of their axial level, and of their ultimate developmental fate, by signals within the neural tube.
Such molecular evidence is starting to be obtained. The focus has been on hindbrain neural crest cells, which make a contribution to the mandible, the cartilage of the neck and the base of the skull [2,3]. The abundance of molecular markers and anatomical landmarks in particular, the motor nuclei of the cranial nerves - make the hindbrain an excellent region for developmental studies. Although there is controversy as to whether all of the rhombomeres give rise to neural crest cells [4,5], there is general agreement that, adjacent to rhombomeres r3 and r5, there are two crest-free regions, and that neural crest cells maintain this segmental pattern during their migration into the branchial arches. Neural crest cells from rhombomeres rl and r2 contribute to the first branchial arch, whereas more caudally positioned neural crest cells from r4 and r6 contribute to the second and third branchial arches, respectively. The first branchial arch later develops two distinct facial swellings, the maxillary prominence which contributes to the upper jaw and the mandibular prominence which forms the entire lower jaw. The more caudal branchial arches give rise to cartilages of the neck. That the hindbrain neural crest is 'prepatterned' has now been confirmed at the molecular level by Prince and Lumsden [6]. The homeobox gene Hoxa-2 - one of the Hox genes that are related to the Drosophila homeotic selector genes and thought to confer segmental identity - is normally expressed in a continuous band up to rhombomere r2, but neural crest cells derived from r2
Fig. 1. The expression of Hoxa-2 (red shading) in a stage 15 chick embryo. (a) A non-manipulated embryo: Hoxa-2 is expressed in the neural tube up to rhombomere r2 and in the second and third branchial arches. (b) An embryo in which rhombomeres r2 and r3 have been grafted in the position of r4 and r5: Hoxa-2 is expressed in the neural crest cells emanating from r6 and more caudal regions; only the third branchial arch contains cells that express Hoxa-2. (c) An embryo in which rhombomeres r4 and r5 have been grafted into the position of r2 and r3: Hoxa-2 is expressed ectopically in neural crest cells derived from the supernumerary r4 and in the adjacent first branchial arch mesenchyme.
© Current Biology 1995, Vol 5 No 4
345
346
Current Biology 1995, Vol 5 No 4 factor receptors FGFR-2 [8] and FGFR-3 (my own group's unpublished results), and the Wnt genes [7,9], homologues of the Drosophila wingless gene that are thought to encode cell-cell signalling molecules. In some cases - such as Wnt-5a [9] - continued expression in the migrating neural crest and facial mesenchyme can be seen. The converse is also observed: precise repression of gene expression in migrating neural crest seems to be required for distinguishing facial neural crest from those cells that contribute to more posterior or dorsal structures [6,10]. Other types of molecule may also be involved in restricting the fate of neural crest cells, such as retinoids [11], metalloendopeptidases [12] or new classes of transcription factors [13]. We may envisage a role for any or all of these molecules in prepatterning of the anterior neural crest cells.
Fig. 2. Scanning electron micrograph of 'A' strain mouse (A/WySn) at developmental stage El 11,by which time the facial prominences are fully developed. On the right side of the photograph, a failure of growth of the medial and lateral nasal prominences has prevented contact between the primordia, and this will lead to a unilateral cleft lip (arrow). MNP, medialnasal prominence; LNP, lateral-nasal prominence; MX, maxillary prominence; MD, mandibular prominence; 2, second branchial arch. stop expressing Hoxa-2 as soon as migration begins, so that cells in the first branchial arch do not express this gene (Fig. la). Neural crest cells originating from r4 continue to express Hoxa-2 during migration and once they have populated the second branchial arch (Fig. la). When r2 was transplanted to the r4 position, emigrating r2 neural crest cells switched off Hoxa-2 expression as usual, implying that the unusual environment did not modify their intrinsic developmental programme (Fig. lb). In the reciprocal experiment, r4-derived neural crest cells that were moved anterior, to the r2 position, continued to express Hoxa-2, again ignoring new environmental cues (Fig. 1c). It should be noted that the ectopic beaks produced in Noden's chick experiments [1] were a result of transplanting midbrain neural crest to the hindbrain, whereas Prince and Lumsden [6] transplanted small regions entirely within the hindbrain. Moreover, Prince and Lumsden [6] sacrificed their embryos a short time after grafting, so that the effects of ectopic gene expression on cartilage morphogenesis could not be determined in their experiments. In contrast to the migratory neural crest cells produced by the hindbrain, those produced anterior to rhombomere r3 do not show overt segmentation. Moreover, none of the 'segment identity' Hox genes is expressed anterior to rhombomere r2, so other molecules must be involved in patterning the anterior neural crest. Other molecules have indeed been reported to show regional patterns of expression in the neural epithelium of the midbrain and forebrain. This is true, for example, of the transcription factors Otx-2, Pax-6 and En-2 [7], the fibroblast growth
Mapping the non-syndromic cleft lip gene inmice After neural crest cells have populated the face, the mesenchyme proliferates and fills the facial prominences. The correct growth and fusion of the fronto-nasal, lateral-nasal, medial-nasal and maxillary prominences is required for normal facial form, and disruption of these processes leads to facial clefting (Fig. 2). One of the year's most important advances in the field of craniofacial biology will surely be the mapping by Juriloff [14,15] of the chromosomal location of the mouse gene in which mutations occur that cause non-syndromic cleft lip. Despite the availability of suitable mouse models - such as 'A' strain mice - for decades, it has been extremely difficult to isolate the genetic causes of cleft lip. The main reason for the intractability of the problem is that the probability of expression of the mutation is modified by many other factors, such as maternal genotype. Juriloff [14,15] overcame these problems by repeated backcrossing of the cleft-lip-causing gene, from a strain of mouse that has naturally occurring cleft lip, into a non-cleft-lip strain. The congenic strain so produced carries a 10 centiMorgan region of DNA that segregates with the cleft-lip phenotype and is located on chromosome 11. Some genes that also map to this region include those encoding retinoic acid receptor o (RARot), Wnt3, the nerve growth factor receptor and the thyroid hormone receptor. The Hoxb cluster, also located on chromosome 11, has recombined with, and is therefore outside of, the candidate region. One of the candidate genes, that encoding RARa, is expressed in migrating neural crest cells, facial mesenchyme and epithelium [16], and continues to be expressed during cytodifferentiation of cartilage and bone. Current understanding of RARat, however, does not support a major role for it in the genesis of nonsyndromic cleft lip. First, RARa is not selectively expressed in regions of the facial prominences where outgrowth or fusion occurs. Second, genetically engineered mice lacking RARa expression do not show craniofacial defects [17]. Although the complete absence of individual RARs may have little effect on embryo development, it is possible that more subtle mutations
DISPATCH may have greater effects on gene function. For example, the RARs are known to dimerize with the related retinoic acid X receptors (RXRs), and a mutation that alters receptor dimerization may affect a wider range of developmental events than a simple null mutation. Indeed, the simultaneous elimination of several different types of RAR in one mouse leads to severe defects in the craniofacial complex, including midline clefts [11]. Finally, Juriloff's congenic mouse data [14,15] suggest the existence of a second locus that suppresses the expression of the cleft phenotype. This would explain the heterogeneous incidence of cleft lip when individual proven carriers are bred with each other. In the next few years, more detailed genetic maps of chromosome 11 will become available, and the identification of the mutations responsible for the cleft phenotype will be feasible. The results from the Juriloff laboratory will certainly advance research on human cleft lip by focusing efforts on homologous regions of the human genome. Genes defective in individuals with craniosynostosis The final phase of cranial morphogenesis involves the differentiation of bone, and coordination of growth between bones. A relatively common problem with bone growth is the premature fusion of the sutures between the flat bones of the skull. The incidence of such 'craniosynostosis' is 1 in 2 500, and over 100 inherited syndromes manifest this trait. Until recently, the cellular and genetic basis of craniosynostosis had remained elusive. Now, however, two groups [18-22] have found that mutations in FGFR genes are associated with four syndromes that cause craniosynostosis. Crouzon syndrome features midface hypoplasia, relative mandibular prognathism and shallow orbits, in addition to craniosynostosis. Jackson-Weiss, Pfeiffer and Apert syndromes share many of the same features, but also include syndactyly and other abnormalities in size and shape of the digits. Each syndrome has been recognized as a distinct entity, and patients with Crouzon syndrome are not born into families with Pfeiffer syndrome, and vice versa [21]. Surprisingly, patients with Crouzon, Jackson-Weiss, Pfeiffer and Apert syndromes have point mutations affecting a similar region of FGFR-2 (isoform IIIc) [18,19,21,22]. Moreover, some Crouzon and Pfeiffer syndrome patients have identical FGFR-2 mutations [21]. This finding was unsettling for clinical geneticists who have always believed each syndrome to be distinct. Further study may reveal other FGFR-2 mutations that are unique to patients with either syndrome. Other Pfeiffer syndrome patients have point mutations affecting the extracellular domain of FGFR-1 [20]. This observation, that mutations of two different FGF receptors can cause Pfeiffer syndrome, can be reconciled in two possible ways. The first possible explanation is that, as the amino-acid residues affected by the mutations causing both types of syndrome have similar locations in the third immunoglobulin-like domains of FGFR-1 and
FGFR-2, they may cause similar functional defects of the two receptors. These domains are involved in determining the receptors' ligand specificity for various members of the ever-growing fibroblast growth factor family [23]. The second possible explanation for the similar phenotypes of the Pfeiffer and Jackson-Weiss syndromes lies in the observation that, in animal models, FGFR-1 and FGFR-2 have been shown to have overlapping expression domains in the limb and head ([8] and my own group's unpublished data); perhaps these similar expression patterns are matched by similar developmental roles for the two receptor types. At present, we have indirect evidence that functional FGF receptors are important for the development of the two body regions affected in Pfeiffer syndrome; some further support for their importance comes from finding that exogenous FGF-2 and FGF-4 can stimulate growth of limb and fronto-nasal mesenchyme ([24-26] and my group's unpublished data). Despite the compelling genetic data, it is far from established that mutations affecting FGFRs cause craniosynostosis. For example, mutations affecting another member of the family, FGFR-3, are associated with achondroplastic dwarfism, a syndrome that does not manifest craniosynostosis [27,28]. Furthermore, other forms of craniosynostosis (such as the Boston type [29]) are associated with mutations affecting a transcription factor (Msx-2), rather than an FGFR. These findings, in combination with the large variations in phenotype between individuals with identical point mutations [21], make it very likely that other genes modify the effects of the FGFR gene mutations. At present, we have little evidence that FGFRs are involved in bone morphogenesis or differentiation, but as a result of the human findings, research efforts will now be directed along these lines. Acknowledgements: I would like to thank D. Juriloff for sharing data prior to publication, and V. Diewert for the photograph shown in Figure 2.
References 1. Noden DM: The role of the neural crest in patterning of avian cranial skeletal, connective and muscle tissues. Dev Biol 1983, 96:144-165. 2. Couly GF, Coltey PM, Le Douarin NM: The triple origin of skull in higher vertebrates: a study in quail-chick chimeras. Development, 1993, 117:409-429. 3. Lumsden A, Sprawson N, Graham A: Segmental origin and migration of neural crest cells inthe hindbrain region of the chick embryo. Development 1991, 113:1281-1292. 4. Bronner-Fraser M: Neural crest cell formation and migration in the
developing embryo. FASEB J 1994, 8:699-706. 5. Graham A, Francis-West P, Brickell P, Lumsden A: The signalling molecule BMP-4 mediates apoptosis inthe rhombencephalic neural crest. Nature 1994, 372:684-686.
6. Prince V, Lumsden A: Hoxa-2 expression in normal and transposed rhombomeres: independent regulation in the neural tube and neural crest. Development 1994, 120:911-923.
7. Bally-Cuif L, Wassef M: Ectopic induction and reorganization of Wnt-1 expression in quail-chick chimeras. Development 1994,
120:3379-3394. 8. Orr-Urtreger
A,
Givo
D,
Yayon
A,
Yarden
Y, Lonai
P:
Developmental expression of two murine fibroblast growth factor receptors, flgand bek. Development 1991, 113:1419-1434. 9. Parr BA, Shea MJ, Vassileva G, McMahon AP: Mouse Wnt genes
exhibit discrete domains of expression inthe early embryonic CNS and limb buds. Development 1993, 119:247-261.
347
348
Current Biology 1995, Vol 5 No 4 10. Echelard Y, Vassileva G, McMahon AP: Cis-acting regulatory sequences governing Wnt-1 expression in the developing mouse CNS. Development 1994, 120:2213-2224. 11. Lohnes D, Mark M, Mendelsohn C, Dolli P, Dierich A, Gorry P, Gansmuller A, Chambon P: Function of the retinoic acid receptors (RARs) during development. (I) Craniofacial and skeletal abnormalities in RAR double mutants. Development 1994, 120:2723-2748. 12. Spencer-Dene B, Thorogood P, Nair S, Kenny AJ, Harris M, Henderson B: Distribution of, and a putative role for, the cellsurface neutral metalloendopeptidases during mammalian craniofacial development. Development 1994, 120:3213-3226. 13. Mohler J, Mahaffey JW, Deutch E, Vani K: Control of Drosophila head segment identity by the BZIP homeotic gene cnc. Development 1995, 121:237-247. 14. Juriloff DM, Mah DG: The major locus for multifactorial nonsyndromic cleft lip maps to mouse chromosome 11. Mamm Genome 1995, in press. 15. Juriloff DM: Genetic analysis of the construction of the AEJ.A congenic strain indicates that nonsyndromic CL(P) in the mouse is caused by two loci with epistatic interaction. Craniofac Genet Dev Biol 1995, in press. 16. Ruberte E, Dolle P, Chambon P, Morriss-Kay G: Retinoic acid receptors and cellular retinoid binding proteins. II. Their differential pattern of transcription during early morphogenesis in mouse embryos. Development 1991, 111:45-60. 17. Lufkin T, Lohnes D, Mark M, Dierich A, Gorr P, Gaub MP, Lemeur M, Chambon P: High postnatal lethality and testis degeneration in retinoic acid receptor-a (RAR-t) mutant mice. Proc Natl Acad Sci USA 1993, 90:7225-7229. 18. Reardon W, Winter RM, Rutland P, Pulleyn LJ,Jones BM, Malcom S: Mutations in the fibroblast growth factor receptor 2 gene cause Crouzon syndrome. Nature Genet 1994, 8:98-103. 19. Jabs EW, Li X, Scott AF, Meyers GC,Chen W, Eccles M, Mao J-l, Charnas LR, Jackson CE, Jaye M: Jackson-Weiss and Crouzon syndromes are allelic with mutations in fibroblast growth factor receptor 2. Nature Genet 1994, 8:275-279. 20. Meunke M, Schell U, Hehr A, Robin NH, Losken W, Schinzel A, Pulleyn LJ, Rutland P, Reardon W, Malcom S, Winter RM: A common mutation in the fibroblast receptor 1 gene in Pfeiffer syndrome. Nature Genet 1994, 8:269-274.
21. Rutland P, Pulleyn LJ, Reardon W, Baraitser M, Haward R, Jones B, Malcolm S, Winter RM, Oldridge M, Slaney SF et al.: Identical mutations in the FGFR-2 gene cause both Pfeiffer and Crouzon syndrome phenotypes. Nature Genet 1995, 9:173-176. 22. Wilkie AOM, Slaney SF, Oldridge M, Poole MD, Ashworth GJ, Hockley AD, Hayward RD, David DJ, Pulleyn L, Rutland P: Apert syndrome results from localized mutations of FGFR2 and is allelic with Crouzon syndrome. Nature Genet 1995, 9:165-172. 23. Miller K, Rizzino A: Developmental regulation and signal transduction pathways of fibroblast growth factors and their receptors. In Modern Cell Biology, Vol 14. Edited by Nilsen-Hamilton M. Toronto: Wiley-Liss Inc.; 1994:19-49. 24. Fallon JF, Lopez A, Ros MA, Savage MP, Olwin BB, Simandl B: FGF-2: apical ectodermal ridge growth signal for chick limb development. Science 1994, 264:104-107. 25. Niswander L, Tickle C, Vogel A, Booth I, Martin GR: FGF-4 replaces the apical ectodermal ridge and directs outgrowth and patterning of the limb. Cell 1993, 75:579-587. 26. Richman JM, Crosby Z: Differential growth of facial primordia in chick embryos: responses of facial mesenchyme to basic fibroblast growth factor (bFGF) and serum in micromass culture. Development 1990, 109:341-348. 27. Rousseau F, Bonaventure J, Legeal-Mallet L, Pelet A, Rozet J-M, Maroteaux P, Le Merrer M, Munnich A: Mutations in the gene encoding fibroblast growth factor receptor-3 in achondroplasia. Nature 1994, 371:252-254. 28. Shiang R, Thompson LM, Zhu Y-Z, Church DM, Fieder TJ,Bocian M, Winokur ST, Wasmuth JJ: Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia. Cell 1994, 78:35-342. 29. Jabs EW, Muller U, Li X, Ma L, Luo W, Haworth IS,Klisak I, Sparkes R, Warman ML, Muliken JB, et al.: A mutation in the homeodomain of the human MSX2 gene in a family affected with autosomal dominant craniosynostosis. Cell 1993, 75:443-450.
Joy M. Richman, Department of Clinical Dental Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, Canada V6T 1Z3.
THE FEBRUARY 1995 ISSUE (VOL. 5, NO. 1) OF CURRENT OPINION IN NEUROBIOLOGY included the following reviews, edited by Friedrich Bonhoeffer and Joshua R. Sanes, on Development: Control of neuronal development in Caenorhabditis elegans by Anne Duggan and Martin Chalfie Neuromuscular development in Drosophila: insights from embryos and pupae by Joyce Fernandes and Haig Keshishian Intrinsic and extrinsic factors regulating vertebrate neurogenesis by Anne L. Calof Remodeling of the insect nervous system by Richard B. Levine, David B. Morton and Linda L. Restifo Molecular genetics of neuronal adhesion by Ulrich Muller and Robert Kypta Molecular genetics of neuronal survival by Immaculada Silos-Santiago, Laura J.S. Greenlund, Eugene M. Johnson Jr and William D. Snider Development of the zebrafish nervous system: genetic analysis and manipulation by John Y. Kuwada Targeting of mRNAs to subsynaptic microdomains in dendrites by Oswald Steward Assembly of the postsynaptic apparatus by Elizabeth D. Apel and John P. Merlie The sensory-motor role of growth cone filopodia by S.B. Kater and Vincent Rehder Repulsive and inhibitory signals by RogerJ. Keynes and Geoffrey M.W. Cook Guidance and induction of branch formation in developing axons by target-derived diffusible factors by Timothy E. Kennedy and Marc Tessier-Lavigne Development of layers, maps and modules by Alfred Gierer and Christian M. Muller Neuronal coupling and uncoupling in the developing nervous system by Karl Kandler and Lawrence C. Katz Activity-dependent remodeling of connections in the mammalian visual system by Karina S. Cramer and Mriganka Sur
HEAD DEVELOPMENT
RICHMAN RICHMAN
HEAD DEVELOPMENT
Craniofacial genetics makes headway Studies of neural crest migration in animal models, and of human syndromes in which craniofacial development isabnormal, are helping us to understand both prenatal and postnatal development of the head. At the core of craniofacial research is the desire to understand both normal and abnormal development of the head. The head has, however, proven to be a particular challenge to developmental biologists, as many of the patterning mechanisms are unique to this part of the body. The clinical genetic approach, studying inherited syndromes that affect human craniofacial development, is proving to be very useful for understanding human dysmorphology and is also providing new directions for more basic research using animal models. Patterning the cranial neural crest - moving past the hindbrain Neural crest cells play a pivotal role in craniofacial development, as they initially give rise to most of the mesenchyme of the head and face, and ultimately the mesenchyme forms most of the skeletal and connective tissue in the head. It is thought that many craniofacial defects arise from perturbations of the neural crest early in embryonic development. Noden [1], for example, reported that avian cranial neural crest cells transplanted from the midbrain - which normally contribute to the beak - to a more caudal position, migrated into the neck and gave rise to an extra set of beaks, thus following their intrinsic developmental programme. But these results need to be supported by evidence, at the molecular level, that crar;ial neural crest cells are informed of their axial level, and of their ultimate developmental fate, by signals within the neural tube.
Such molecular evidence is starting to be obtained. The focus has been on hindbrain neural crest cells, which make a contribution to the mandible, the cartilage of the neck and the base of the skull [2,3]. The abundance of molecular markers and anatomical landmarks in particular, the motor nuclei of the cranial nerves - make the hindbrain an excellent region for developmental studies. Although there is controversy as to whether all of the rhombomeres give rise to neural crest cells [4,5], there is general agreement that, adjacent to rhombomeres r3 and r5, there are two crest-free regions, and that neural crest cells maintain this segmental pattern during their migration into the branchial arches. Neural crest cells from rhombomeres rl and r2 contribute to the first branchial arch, whereas more caudally positioned neural crest cells from r4 and r6 contribute to the second and third branchial arches, respectively. The first branchial arch later develops two distinct facial swellings, the maxillary prominence which contributes to the upper jaw and the mandibular prominence which forms the entire lower jaw. The more caudal branchial arches give rise to cartilages of the neck. That the hindbrain neural crest is 'prepatterned' has now been confirmed at the molecular level by Prince and Lumsden [6]. The homeobox gene Hoxa-2 - one of the Hox genes that are related to the Drosophila homeotic selector genes and thought to confer segmental identity - is normally expressed in a continuous band up to rhombomere r2, but neural crest cells derived from r2
Fig. 1. The expression of Hoxa-2 (red shading) in a stage 15 chick embryo. (a) A non-manipulated embryo: Hoxa-2 is expressed in the neural tube up to rhombomere r2 and in the second and third branchial arches. (b) An embryo in which rhombomeres r2 and r3 have been grafted in the position of r4 and r5: Hoxa-2 is expressed in the neural crest cells emanating from r6 and more caudal regions; only the third branchial arch contains cells that express Hoxa-2. (c) An embryo in which rhombomeres r4 and r5 have been grafted into the position of r2 and r3: Hoxa-2 is expressed ectopically in neural crest cells derived from the supernumerary r4 and in the adjacent first branchial arch mesenchyme.
© Current Biology 1995, Vol 5 No 4
345
346
Current Biology 1995, Vol 5 No 4 factor receptors FGFR-2 [8] and FGFR-3 (my own group's unpublished results), and the Wnt genes [7,9], homologues of the Drosophila wingless gene that are thought to encode cell-cell signalling molecules. In some cases - such as Wnt-5a [9] - continued expression in the migrating neural crest and facial mesenchyme can be seen. The converse is also observed: precise repression of gene expression in migrating neural crest seems to be required for distinguishing facial neural crest from those cells that contribute to more posterior or dorsal structures [6,10]. Other types of molecule may also be involved in restricting the fate of neural crest cells, such as retinoids [11], metalloendopeptidases [12] or new classes of transcription factors [13]. We may envisage a role for any or all of these molecules in prepatterning of the anterior neural crest cells.
Fig. 2. Scanning electron micrograph of 'A' strain mouse (A/WySn) at developmental stage El 11,by which time the facial prominences are fully developed. On the right side of the photograph, a failure of growth of the medial and lateral nasal prominences has prevented contact between the primordia, and this will lead to a unilateral cleft lip (arrow). MNP, medialnasal prominence; LNP, lateral-nasal prominence; MX, maxillary prominence; MD, mandibular prominence; 2, second branchial arch. stop expressing Hoxa-2 as soon as migration begins, so that cells in the first branchial arch do not express this gene (Fig. la). Neural crest cells originating from r4 continue to express Hoxa-2 during migration and once they have populated the second branchial arch (Fig. la). When r2 was transplanted to the r4 position, emigrating r2 neural crest cells switched off Hoxa-2 expression as usual, implying that the unusual environment did not modify their intrinsic developmental programme (Fig. lb). In the reciprocal experiment, r4-derived neural crest cells that were moved anterior, to the r2 position, continued to express Hoxa-2, again ignoring new environmental cues (Fig. 1c). It should be noted that the ectopic beaks produced in Noden's chick experiments [1] were a result of transplanting midbrain neural crest to the hindbrain, whereas Prince and Lumsden [6] transplanted small regions entirely within the hindbrain. Moreover, Prince and Lumsden [6] sacrificed their embryos a short time after grafting, so that the effects of ectopic gene expression on cartilage morphogenesis could not be determined in their experiments. In contrast to the migratory neural crest cells produced by the hindbrain, those produced anterior to rhombomere r3 do not show overt segmentation. Moreover, none of the 'segment identity' Hox genes is expressed anterior to rhombomere r2, so other molecules must be involved in patterning the anterior neural crest. Other molecules have indeed been reported to show regional patterns of expression in the neural epithelium of the midbrain and forebrain. This is true, for example, of the transcription factors Otx-2, Pax-6 and En-2 [7], the fibroblast growth
Mapping the non-syndromic cleft lip gene inmice After neural crest cells have populated the face, the mesenchyme proliferates and fills the facial prominences. The correct growth and fusion of the fronto-nasal, lateral-nasal, medial-nasal and maxillary prominences is required for normal facial form, and disruption of these processes leads to facial clefting (Fig. 2). One of the year's most important advances in the field of craniofacial biology will surely be the mapping by Juriloff [14,15] of the chromosomal location of the mouse gene in which mutations occur that cause non-syndromic cleft lip. Despite the availability of suitable mouse models - such as 'A' strain mice - for decades, it has been extremely difficult to isolate the genetic causes of cleft lip. The main reason for the intractability of the problem is that the probability of expression of the mutation is modified by many other factors, such as maternal genotype. Juriloff [14,15] overcame these problems by repeated backcrossing of the cleft-lip-causing gene, from a strain of mouse that has naturally occurring cleft lip, into a non-cleft-lip strain. The congenic strain so produced carries a 10 centiMorgan region of DNA that segregates with the cleft-lip phenotype and is located on chromosome 11. Some genes that also map to this region include those encoding retinoic acid receptor o (RARot), Wnt3, the nerve growth factor receptor and the thyroid hormone receptor. The Hoxb cluster, also located on chromosome 11, has recombined with, and is therefore outside of, the candidate region. One of the candidate genes, that encoding RARa, is expressed in migrating neural crest cells, facial mesenchyme and epithelium [16], and continues to be expressed during cytodifferentiation of cartilage and bone. Current understanding of RARat, however, does not support a major role for it in the genesis of nonsyndromic cleft lip. First, RARa is not selectively expressed in regions of the facial prominences where outgrowth or fusion occurs. Second, genetically engineered mice lacking RARa expression do not show craniofacial defects [17]. Although the complete absence of individual RARs may have little effect on embryo development, it is possible that more subtle mutations
DISPATCH may have greater effects on gene function. For example, the RARs are known to dimerize with the related retinoic acid X receptors (RXRs), and a mutation that alters receptor dimerization may affect a wider range of developmental events than a simple null mutation. Indeed, the simultaneous elimination of several different types of RAR in one mouse leads to severe defects in the craniofacial complex, including midline clefts [11]. Finally, Juriloff's congenic mouse data [14,15] suggest the existence of a second locus that suppresses the expression of the cleft phenotype. This would explain the heterogeneous incidence of cleft lip when individual proven carriers are bred with each other. In the next few years, more detailed genetic maps of chromosome 11 will become available, and the identification of the mutations responsible for the cleft phenotype will be feasible. The results from the Juriloff laboratory will certainly advance research on human cleft lip by focusing efforts on homologous regions of the human genome. Genes defective in individuals with craniosynostosis The final phase of cranial morphogenesis involves the differentiation of bone, and coordination of growth between bones. A relatively common problem with bone growth is the premature fusion of the sutures between the flat bones of the skull. The incidence of such 'craniosynostosis' is 1 in 2 500, and over 100 inherited syndromes manifest this trait. Until recently, the cellular and genetic basis of craniosynostosis had remained elusive. Now, however, two groups [18-22] have found that mutations in FGFR genes are associated with four syndromes that cause craniosynostosis. Crouzon syndrome features midface hypoplasia, relative mandibular prognathism and shallow orbits, in addition to craniosynostosis. Jackson-Weiss, Pfeiffer and Apert syndromes share many of the same features, but also include syndactyly and other abnormalities in size and shape of the digits. Each syndrome has been recognized as a distinct entity, and patients with Crouzon syndrome are not born into families with Pfeiffer syndrome, and vice versa [21]. Surprisingly, patients with Crouzon, Jackson-Weiss, Pfeiffer and Apert syndromes have point mutations affecting a similar region of FGFR-2 (isoform IIIc) [18,19,21,22]. Moreover, some Crouzon and Pfeiffer syndrome patients have identical FGFR-2 mutations [21]. This finding was unsettling for clinical geneticists who have always believed each syndrome to be distinct. Further study may reveal other FGFR-2 mutations that are unique to patients with either syndrome. Other Pfeiffer syndrome patients have point mutations affecting the extracellular domain of FGFR-1 [20]. This observation, that mutations of two different FGF receptors can cause Pfeiffer syndrome, can be reconciled in two possible ways. The first possible explanation is that, as the amino-acid residues affected by the mutations causing both types of syndrome have similar locations in the third immunoglobulin-like domains of FGFR-1 and
FGFR-2, they may cause similar functional defects of the two receptors. These domains are involved in determining the receptors' ligand specificity for various members of the ever-growing fibroblast growth factor family [23]. The second possible explanation for the similar phenotypes of the Pfeiffer and Jackson-Weiss syndromes lies in the observation that, in animal models, FGFR-1 and FGFR-2 have been shown to have overlapping expression domains in the limb and head ([8] and my own group's unpublished data); perhaps these similar expression patterns are matched by similar developmental roles for the two receptor types. At present, we have indirect evidence that functional FGF receptors are important for the development of the two body regions affected in Pfeiffer syndrome; some further support for their importance comes from finding that exogenous FGF-2 and FGF-4 can stimulate growth of limb and fronto-nasal mesenchyme ([24-26] and my group's unpublished data). Despite the compelling genetic data, it is far from established that mutations affecting FGFRs cause craniosynostosis. For example, mutations affecting another member of the family, FGFR-3, are associated with achondroplastic dwarfism, a syndrome that does not manifest craniosynostosis [27,28]. Furthermore, other forms of craniosynostosis (such as the Boston type [29]) are associated with mutations affecting a transcription factor (Msx-2), rather than an FGFR. These findings, in combination with the large variations in phenotype between individuals with identical point mutations [21], make it very likely that other genes modify the effects of the FGFR gene mutations. At present, we have little evidence that FGFRs are involved in bone morphogenesis or differentiation, but as a result of the human findings, research efforts will now be directed along these lines. Acknowledgements: I would like to thank D. Juriloff for sharing data prior to publication, and V. Diewert for the photograph shown in Figure 2.
References 1. Noden DM: The role of the neural crest in patterning of avian cranial skeletal, connective and muscle tissues. Dev Biol 1983, 96:144-165. 2. Couly GF, Coltey PM, Le Douarin NM: The triple origin of skull in higher vertebrates: a study in quail-chick chimeras. Development, 1993, 117:409-429. 3. Lumsden A, Sprawson N, Graham A: Segmental origin and migration of neural crest cells inthe hindbrain region of the chick embryo. Development 1991, 113:1281-1292. 4. Bronner-Fraser M: Neural crest cell formation and migration in the
developing embryo. FASEB J 1994, 8:699-706. 5. Graham A, Francis-West P, Brickell P, Lumsden A: The signalling molecule BMP-4 mediates apoptosis inthe rhombencephalic neural crest. Nature 1994, 372:684-686.
6. Prince V, Lumsden A: Hoxa-2 expression in normal and transposed rhombomeres: independent regulation in the neural tube and neural crest. Development 1994, 120:911-923.
7. Bally-Cuif L, Wassef M: Ectopic induction and reorganization of Wnt-1 expression in quail-chick chimeras. Development 1994,
120:3379-3394. 8. Orr-Urtreger
A,
Givo
D,
Yayon
A,
Yarden
Y, Lonai
P:
Developmental expression of two murine fibroblast growth factor receptors, flgand bek. Development 1991, 113:1419-1434. 9. Parr BA, Shea MJ, Vassileva G, McMahon AP: Mouse Wnt genes
exhibit discrete domains of expression inthe early embryonic CNS and limb buds. Development 1993, 119:247-261.
347
348
Current Biology 1995, Vol 5 No 4 10. Echelard Y, Vassileva G, McMahon AP: Cis-acting regulatory sequences governing Wnt-1 expression in the developing mouse CNS. Development 1994, 120:2213-2224. 11. Lohnes D, Mark M, Mendelsohn C, Dolli P, Dierich A, Gorry P, Gansmuller A, Chambon P: Function of the retinoic acid receptors (RARs) during development. (I) Craniofacial and skeletal abnormalities in RAR double mutants. Development 1994, 120:2723-2748. 12. Spencer-Dene B, Thorogood P, Nair S, Kenny AJ, Harris M, Henderson B: Distribution of, and a putative role for, the cellsurface neutral metalloendopeptidases during mammalian craniofacial development. Development 1994, 120:3213-3226. 13. Mohler J, Mahaffey JW, Deutch E, Vani K: Control of Drosophila head segment identity by the BZIP homeotic gene cnc. Development 1995, 121:237-247. 14. Juriloff DM, Mah DG: The major locus for multifactorial nonsyndromic cleft lip maps to mouse chromosome 11. Mamm Genome 1995, in press. 15. Juriloff DM: Genetic analysis of the construction of the AEJ.A congenic strain indicates that nonsyndromic CL(P) in the mouse is caused by two loci with epistatic interaction. Craniofac Genet Dev Biol 1995, in press. 16. Ruberte E, Dolle P, Chambon P, Morriss-Kay G: Retinoic acid receptors and cellular retinoid binding proteins. II. Their differential pattern of transcription during early morphogenesis in mouse embryos. Development 1991, 111:45-60. 17. Lufkin T, Lohnes D, Mark M, Dierich A, Gorr P, Gaub MP, Lemeur M, Chambon P: High postnatal lethality and testis degeneration in retinoic acid receptor-a (RAR-t) mutant mice. Proc Natl Acad Sci USA 1993, 90:7225-7229. 18. Reardon W, Winter RM, Rutland P, Pulleyn LJ,Jones BM, Malcom S: Mutations in the fibroblast growth factor receptor 2 gene cause Crouzon syndrome. Nature Genet 1994, 8:98-103. 19. Jabs EW, Li X, Scott AF, Meyers GC,Chen W, Eccles M, Mao J-l, Charnas LR, Jackson CE, Jaye M: Jackson-Weiss and Crouzon syndromes are allelic with mutations in fibroblast growth factor receptor 2. Nature Genet 1994, 8:275-279. 20. Meunke M, Schell U, Hehr A, Robin NH, Losken W, Schinzel A, Pulleyn LJ, Rutland P, Reardon W, Malcom S, Winter RM: A common mutation in the fibroblast receptor 1 gene in Pfeiffer syndrome. Nature Genet 1994, 8:269-274.
21. Rutland P, Pulleyn LJ, Reardon W, Baraitser M, Haward R, Jones B, Malcolm S, Winter RM, Oldridge M, Slaney SF et al.: Identical mutations in the FGFR-2 gene cause both Pfeiffer and Crouzon syndrome phenotypes. Nature Genet 1995, 9:173-176. 22. Wilkie AOM, Slaney SF, Oldridge M, Poole MD, Ashworth GJ, Hockley AD, Hayward RD, David DJ, Pulleyn L, Rutland P: Apert syndrome results from localized mutations of FGFR2 and is allelic with Crouzon syndrome. Nature Genet 1995, 9:165-172. 23. Miller K, Rizzino A: Developmental regulation and signal transduction pathways of fibroblast growth factors and their receptors. In Modern Cell Biology, Vol 14. Edited by Nilsen-Hamilton M. Toronto: Wiley-Liss Inc.; 1994:19-49. 24. Fallon JF, Lopez A, Ros MA, Savage MP, Olwin BB, Simandl B: FGF-2: apical ectodermal ridge growth signal for chick limb development. Science 1994, 264:104-107. 25. Niswander L, Tickle C, Vogel A, Booth I, Martin GR: FGF-4 replaces the apical ectodermal ridge and directs outgrowth and patterning of the limb. Cell 1993, 75:579-587. 26. Richman JM, Crosby Z: Differential growth of facial primordia in chick embryos: responses of facial mesenchyme to basic fibroblast growth factor (bFGF) and serum in micromass culture. Development 1990, 109:341-348. 27. Rousseau F, Bonaventure J, Legeal-Mallet L, Pelet A, Rozet J-M, Maroteaux P, Le Merrer M, Munnich A: Mutations in the gene encoding fibroblast growth factor receptor-3 in achondroplasia. Nature 1994, 371:252-254. 28. Shiang R, Thompson LM, Zhu Y-Z, Church DM, Fieder TJ,Bocian M, Winokur ST, Wasmuth JJ: Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia. Cell 1994, 78:35-342. 29. Jabs EW, Muller U, Li X, Ma L, Luo W, Haworth IS,Klisak I, Sparkes R, Warman ML, Muliken JB, et al.: A mutation in the homeodomain of the human MSX2 gene in a family affected with autosomal dominant craniosynostosis. Cell 1993, 75:443-450.
Joy M. Richman, Department of Clinical Dental Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, Canada V6T 1Z3.
THE FEBRUARY 1995 ISSUE (VOL. 5, NO. 1) OF CURRENT OPINION IN NEUROBIOLOGY included the following reviews, edited by Friedrich Bonhoeffer and Joshua R. Sanes, on Development: Control of neuronal development in Caenorhabditis elegans by Anne Duggan and Martin Chalfie Neuromuscular development in Drosophila: insights from embryos and pupae by Joyce Fernandes and Haig Keshishian Intrinsic and extrinsic factors regulating vertebrate neurogenesis by Anne L. Calof Remodeling of the insect nervous system by Richard B. Levine, David B. Morton and Linda L. Restifo Molecular genetics of neuronal adhesion by Ulrich Muller and Robert Kypta Molecular genetics of neuronal survival by Immaculada Silos-Santiago, Laura J.S. Greenlund, Eugene M. Johnson Jr and William D. Snider Development of the zebrafish nervous system: genetic analysis and manipulation by John Y. Kuwada Targeting of mRNAs to subsynaptic microdomains in dendrites by Oswald Steward Assembly of the postsynaptic apparatus by Elizabeth D. Apel and John P. Merlie The sensory-motor role of growth cone filopodia by S.B. Kater and Vincent Rehder Repulsive and inhibitory signals by RogerJ. Keynes and Geoffrey M.W. Cook Guidance and induction of branch formation in developing axons by target-derived diffusible factors by Timothy E. Kennedy and Marc Tessier-Lavigne Development of layers, maps and modules by Alfred Gierer and Christian M. Muller Neuronal coupling and uncoupling in the developing nervous system by Karl Kandler and Lawrence C. Katz Activity-dependent remodeling of connections in the mammalian visual system by Karina S. Cramer and Mriganka Sur