A journey in the world of craniofacial development: From 1968 to the future.

This article is based on my talk at the meeting "3rd Advances in Craniosynostosis: Basic Science to Clinical Practice", held at University College, London, on 25 August 2023. It describes my contribution, together with that of my research team and external collaborators, to the field of craniofacial development. This began with my PhD research on the effects of excess vitamin A in rat embryos, which led to a study of normal as well as abnormal formation of the cranial neural tube.

The Journal of Anatomy: origin and evolution.

The Journal of Anatomy was launched 150 years ago as the Journal of Anatomy and Physiology, in an age when anatomy and physiology were not regarded as separate disciplines. European science in general was advancing rapidly at the time (it was 7 years after publication of Darwin's Origin of Species), and the recent demise of the Natural History Review meant that there was no English language publication covering these subjects. The founding editors were George Murray Humphry of Cambridge and William Turner of Edinburgh, together with Alfred Newton of Cambridge and Edward Perceval Wright of Dublin (the last two served only for a year).

Nprl3 is required for normal development of the cardiovascular system.

C16orf35 is a conserved and widely expressed gene lying adjacent to the human α-globin cluster in all vertebrate species. In-depth sequence analysis shows that C16orf35 (now called NPRL3) is an orthologue of the yeast gene Npr3 (nitrogen permease regulator 3) and, furthermore, is a paralogue of its protein partner Npr2. The yeast Npr2/3 dimeric protein complex senses amino acid starvation and appropriately adjusts cell metabolism via the TOR pathway.

The evolution of human artistic creativity.

Creating visual art is one of the defining characteristics of the human species, but the paucity of archaeological evidence means that we have limited information on the origin and evolution of this aspect of human culture. The components of art include colour, pattern and the reproduction of visual likeness. The 2D and 3D art forms that were created by Upper Palaeolithic Europeans at least 30,000 years ago are conceptually equivalent to those created in recent centuries, indicating that human cognition and symbolling activity, as well as anatomy, were fully modern by that time.

Mechanism of skull suture maintenance and interdigitation.

Skull sutures serve as growth centers whose function involves multiple molecular pathways. During periods of brain growth the sutures remain thin and straight, later developing complex fractal interdigitations that provide interlocking strength. The nature of the relationship between the molecular interactions and suture pattern formation is not understood.

Skeletal analysis of the Fgfr3(P244R) mouse, a genetic model for the Muenke craniosynostosis syndrome.

Muenke syndrome, defined by heterozygosity for a Pro250Arg substitution in fibroblast growth factor receptor 3 (FGFR3), is the most common genetic cause of craniosynostosis in humans. We have used gene targeting to introduce the Muenke syndrome mutation (equivalent to P244R) into the murine Fgfr3 gene. A rounded skull and shortened snout (often skewed) with dental malocclusion was observed in a minority of heterozygotes and many homozygotes.

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.

Polydactyly in the mouse mutant Doublefoot involves altered Gli3 processing and is caused by a large deletion in cis to Indian hedgehog.

The mouse mutant Doublefoot (Dbf) shows preaxial polydactyly with 6-9 triphalangeal digits in all four limbs and additional abnormalities including a broadened skull, hydrocephalus, and a thickened, kinked tail. The autopod undergoes a characteristic expansion between late embryonic day (E) 10.5 and E11.

A point-wise quantification of asymmetry using deformation fields: application to the study of the Crouzon mouse model.

This paper introduces a novel approach to quantify asymmetry in each point of a surface. The measure is based on analysing displacement vectors resulting from nonrigid image registration. A symmetric atlas, generated from control subjects is registered to a given subject image.

Computational mouse atlases and their application to automatic assessment of craniofacial dysmorphology caused by the Crouzon mutation Fgfr2(C342Y).

Crouzon syndrome is characterized by premature fusion of sutures and synchondroses. Recently, the first mouse model of the syndrome was generated, having the mutation Cys342Tyr in Fgfr2c, equivalent to the most common human Crouzon/Pfeiffer syndrome mutation. In this study, a set of micro-computed tomography (CT) scannings of the skulls of wild-type mice and Crouzon mice were analysed with respect to the dysmorphology caused by Crouzon syndrome.

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 Fgfr2(C342Y/+) 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.

A model for the pharmacological treatment of crouzon syndrome.

Objective: Crouzon syndrome is caused by mutations in fibroblast growth factor receptor 2 (FGFR2) leading to constitutive activation of receptors in the absence of ligand binding. The syndrome is characterized by premature fusion of the cranial sutures that leads to abnormal cranium shape, restricted brain growth, and increased intracranial pressure. Surgical remodeling of the cranial vault is currently used to treat affected infants.

Mixed-mode pattern in Doublefoot mutant mouse limb--Turing reaction-diffusion model on a growing domain during limb development.

It has been suggested that the Turing reaction-diffusion model on a growing domain is applicable during limb development, but experimental evidence for this hypothesis has been lacking. In the present study, we found that in Doublefoot mutant mice, which have supernumerary digits due to overexpansion of the limb bud, thin digits exist in the proximal part of the hand or foot, which sometimes become normal abruptly at the distal part. We found that exactly the same behaviour can be reproduced by numerical simulation of the simplest possible Turing reaction-diffusion model on a growing domain.

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.

A mesenchyme-free culture system to elucidate the mechanism of otic vesicle morphogenesis.

The vertebrate inner ear has been extensively studied as a model system of morphogenesis and differentiation. The interactions between epithelium and surrounding mesenchyme have not previously been studied directly, because an appropriate experimental system had not been established. Here we describe a mesenchyme-free culture system of E11.

A gain-of-function mutation of Fgfr2c demonstrates the roles of this receptor variant in osteogenesis.

The b and c variants of fibroblast growth factor receptor 2 (FGFR2) differ in sequence, binding specificity, and localization. Fgfr2b, expressed in epithelia, is required for limb outgrowth and branching morphogenesis, whereas the mesenchymal Fgfr2c variant is required by the osteocyte lineage for normal skeletogenesis. Gain-of-function mutations in human FGFR2c are associated with craniosynostosis syndromes.

Alx4 and Msx2 play phenotypically similar and additive roles in skull vault differentiation.

Alx4 and Msx2 encode homeodomain-containing transcription factors that show a clear functional overlap. In both mice and humans, loss of function of either gene is associated with ossification defects of the skull vault, although the major effect is on the frontal bones in mice and the parietal bones in humans. This study was undertaken to discover whether Alx4 and Msx2 show a genetic interaction in skull vault ossification, and to test the hypothesis that they interact with the pathway that includes the Fgfr genes, Twist1 and Runx2.

Mutations of ephrin-B1 (EFNB1), a marker of tissue boundary formation, cause craniofrontonasal syndrome.

Craniofrontonasal syndrome (CFNS) is an X-linked developmental disorder that shows paradoxically greater severity in heterozygous females than in hemizygous males. Females have frontonasal dysplasia and coronal craniosynostosis (fusion of the coronal sutures); in males, hypertelorism is the only typical manifestation. Here, we show that the classical female CFNS phenotype is caused by heterozygous loss-of-function mutations in EFNB1, which encodes a member of the ephrin family of transmembrane ligands for Eph receptor tyrosine kinases.

Developmental mechanisms underlying polydactyly in the mouse mutant Doublefoot.

The pre-axial polydactylous mouse mutant Doublefoot has 6-9 digits per limb but lacks anteroposterior polarity (there is no biphalangeal digit 1). It differs from other polydactylous mutants in showing normal Shh expression, but polarizing activity (shown by mouse-chick grafting experiments) and hedgehog signalling activity (shown by expression of Ptc1) are present throughout the distal mesenchyme. The Dbf mutation has not yet been identified.

The IIIc alternative of Fgfr2 is a positive regulator of bone formation.

Fibroblast growth factor receptor type 2 (FGFR2) plays major roles in development. Like FGFR1 and FGFR3, it exists as two splice variants, IIIb and IIIc. We have investigated in the mouse the function of FGFR2IIIc, the mesenchymal splice variant of FGFR2.