Gillian M. Morriss-Kay

Growth of the normal skull vault and its alteration in craniosynostosis: insights from human genetics and experimental studies

The mammalian skull vault is constructed principally from five bones: the paired frontals and parietals, and the unpaired interparietal. These bones abut at sutures, where most growth of the skull vault takes place. Sutural growth involves maintenance of a population of proliferating osteoprogenitor cells which differentiate into bone matrix‐secreting osteoblasts. Sustained function of the sutures as growth centres is essential for continuous expansion of the skull vault to accommodate the growing brain. Craniosynostosis, the premature fusion of the cranial sutures, occurs in 1 in 2500 children and often presents challenging clinical problems. Until a dozen years ago, little was known about the causes of craniosynostosis but the discovery of mutations in the MSX2, FGFR1, FGFR2, FGFR3, TWIST1 and EFNB1 genes in both syndromic and non‐syndromic cases has led to considerable insights into the aetiology, classification and developmental pathology of these disorders. Investigations of the biological roles of these genes in cranial development and growth have been carried out in normal and mutant mice, elucidating their individual and interdependent roles in normal sutures and in sutures undergoing synostosis. Mouse studies have also revealed a significant correspondence between the neural crest–mesoderm boundary in the early embryonic head and the position of cranial sutures, suggesting roles for tissue interaction in suture formation, including initiation of the signalling system that characterizes the functionally active suture.

Genetics of craniofacial development and malformation

The head is anatomically the most sophisticated part of the body and its evolution was fundamental to the origin of vertebrates; understanding its development is a formidable problem in biology. A synthesis of embryology, evolution and mouse genetics is shaping our understanding of head development and in this review we discuss its application to studies of human craniofacial malformations. Many of these disorders have their origins in specific embryological processes, including abnormalities of brain patterning, of the migration and fusion of tissues in the face, and of bone differentiation in the skull vault.

Functions of fibroblast growth factors and their receptors.

Fibroblast growth factors were first characterized twenty years ago as mitogens of cultured fibroblasts. Despite a wealth of data from experiments in vitro, insights have begun to emerge only recently on the normal function of these growth factors in mice and humans, as a result of studies of natural and experimental mutations in the factors and their receptors.

De Novo Alu-Element Insertions in FGFR2 Identify a Distinct Pathological Basis for Apert Syndrome

Apert syndrome, one of five craniosynostosis syndromes caused by allelic mutations of fibroblast growth-factor receptor 2 (FGFR2), is characterized by symmetrical bony syndactyly of the hands and feet. We have analyzed 260 unrelated patients, all but 2 of whom have missense mutations in exon 7, which affect a dipeptide in the linker region between the second and third immunoglobulin-like domains. Hence, the molecular mechanism of Apert syndrome is exquisitely specific. FGFR2 mutations in the remaining two patients are distinct in position and nature. Surprisingly, each patient harbors an Alu-element insertion of approximately 360 bp, in one case just upstream of exon 9 and in the other case within exon 9 itself. The insertions are likely to be pathological, because they have arisen de novo; in both cases this occurred on the paternal chromosome. FGFR2 is present in alternatively spliced isoforms characterized by either the IIIb (exon 8) or IIIc (exon 9) domains (keratinocyte growth-factor receptor [KGFR] and bacterially expressed kinase, respectively), which are differentially expressed in mouse limbs on embryonic day 13. Splicing of exon 9 was examined in RNA extracted from fibroblasts and keratinocytes from one patient with an Alu insertion and two patients with Pfeiffer syndrome who had nucleotide substitutions of the exon 9 acceptor splice site. Ectopic expression of KGFR in the fibroblast lines correlated with the severity of limb abnormalities. This provides the first genetic evidence that signaling through KGFR causes syndactyly in Apert syndrome.

Cell lineage in mammalian craniofacial mesenchyme

We have analysed the contributions of neural crest and mesoderm to mammalian craniofacial mesenchyme and its derivatives by cell lineage tracing experiments in mouse embryos, using the permanent genetic markers Wnt1-cre for neural crest and Mesp1-cre for mesoderm, combined with the Rosa26 reporter. At the end of neural crest cell migration (E9.5) the two patterns are reciprocal, with a mutual boundary just posterior to the eye. Mesodermal cells expressing endothelial markers (angioblasts) are found not to respect this boundary; they are associated with the migrating neural crest from the 5-somite stage, and by E9.5 they form a pre-endothelial meshwork throughout the cranial mesenchyme. Mesodermal cells of the myogenic lineage also migrate with neural crest cells, as the branchial arches form. By E17.5 the neural crest-mesoderm boundary in the subectodermal mesenchyme becomes out of register with that of the underlying skeletogenic layer, which is between the frontal and parietal bones. At E13.5 the primordia of these bones lie basolateral to the brain, extending towards the vertex of the skull during the following 4-5 days. We used DiI labelling of the bone primordia in ex-utero E13.5 embryos to distinguish between two possibilities for the origin of the frontal and parietal bones: (1) recruitment from adjacent connective tissue or (2) proliferation of the original primordia. The results clearly demonstrated that the bone primordia extend vertically by intrinsic growth, without detectable recruitment of adjacent mesenchymal cells.

Haploinsufficiency of the human homeobox gene ALX4 causes skull ossification defects.

Inherited defects of skull ossification often manifest as symmetric parietal foramina (PFM; MIM 168500). We previously identified mutations of MSX2 in non-syndromic PFM and demonstrated genetic heterogeneity. Deletions of 11p11–p12 (proximal 11p deletion syndrome, P11pDS; MIM 601224; ref. 2) are characterized by multiple exostoses, attributable to haploinsufficiency of EXT2 (refs. 3,4) and PFM. Here we identify ALX4, which encodes a paired-related homeodomain transcription factor, as the PFM disease gene in P11pDS.

Derivation of the mammalian skull vault

This review describes the evolutionary history of the mammalian skull vault as a basis for understanding its complex structure. Current information on the developmental tissue origins of the skull vault bones (mesoderm and neural crest) is assessed for mammals and other tetrapods. This information is discussed in the context of evolutionary changes in the proportions of the skull vault bones at the sarcopterygiantetrapod transition. The dual tissue origin of the skull vault is considered in relation to the molecular mechanisms underlying osteogenic cell proliferation and differentiation in the sutural growth centres and in the proportionate contributions of different sutures to skull growth.

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

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 identification 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 haploinsufficiency 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.

Genetic patterning of the developing mouse tail at the time of posterior neuropore closure

Posterior neuropore (PNP) closure coincides with the end of gastrulation, marking the end of primary neurulation and primary body axis formation. Secondary neurulation and axis formation involve differentiation of the tail bud mesenchyme. Genetic control of the primary‐secondary transition is not understood. We report a detailed analysis of gene expression in the caudal region of day 10 mouse embryos during primary neuropore closure. Embryos were collected at the 27–32 somite stage, fixed, processed for whole mount in situ hybridisation, and subsequently sectioned for a more detailed analysis. Genes selected for study include those involved in the key events of gastrulation and neurulation at earlier stages and more cranial levels. Patterns of expression within the tail bud, neural plate, recently closed neural tube, notochord, hindgut, mesoderm, and surface ectoderm are illustrated and described. Specifically, we report continuity of expression of the genes Wnt5a, Wnt5b, Evx1, Fgf8, RARγ, Brachyury, and Hoxb1from primitive streak and node into subpopulations of the tail bud and caudal axial structures. Within the caudal notochord, developing floorplate, and hindgut, HNF3α, HNF3β, Shh, and Brachyury expression domains correlate directly with known genetic roles and predicted tissue interdependence during induction and differentiation of these structures. The patterns of expression of Wnt5a, Hoxb1, Brachyury, RARγ, and Evx1, together with observations on proliferation, reveal that the caudal mesoderm is organised at a molecular level into distinct domains delineated by longitudinal and transverse borders before histological differentiation. Expression of Wnt5a in the ventral ectodermal ridge supports previous evidence that this structure is involved in epithelial‐mesenchymal interaction. These results provide a foundation for understanding the mechanisms facilitating transition from primary to secondary body axis formation, as well as the factors involved in defective spinal neurulation. Dev. Dyn. 1997;210:431–445.

The Craniofacial Phenotype of the Crouzon Mouse: Analysis of a Model for Syndromic Craniosynostosis Using Three-Dimensional MicroCT

Objective: To characterize the craniofacial phenotype of a mouse model for Crouzon syndrome by a quantitative analysis of skull morphology in mutant and wild-type mice and to compare the findings with skull features observed in humans with Crouzon syndrome. Methods: MicroCT scans and skeletal preparations were obtained on previously described Fgfr2C342Y/+ Crouzon mutant mice and wild-type mice at 6 weeks of age. Three-dimensional coordinate data from biologically relevant landmarks on the skulls were collected. Euclidean Distance Matrix Analysis was used to quantify and compare skull shapes using these landmark data. Results: Obliteration of bilateral coronal sutures was observed in 80% of skulls, and complete synostosis of the sagittal suture was observed in 70%. In contrast, fewer than 40% of lambdoid sutures were found to be fully fused. In each of the 10 Fgfr2C342Y/+ mutant mice analyzed, the presphenoid-basisphenoid synchondrosis was fused. Skull height and width were increased in mutant mice, whereas skull length was decreased. Interorbital distance was also increased in Fgfr2C342Y/+ mice as compared with wild-type littermates. Upper-jaw length was shorter in the Fgfr2C342Y/+ mutant skulls, as was mandibular length. Conclusion: Skulls of Fgfr2C342Y/+ mice differ from normal littermates in a comparable manner with differences between the skulls of humans with Crouzon syndrome and those of unaffected individuals. These findings were consistent across several regions of anatomic interest. Further investigation into the molecular mechanisms underlying the anomalies seen in the Crouzon mouse model is currently under way.