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. Here we have analysed a mouse model in which expression of Nprl3 has been abolished using homologous recombination. The predominant effect on RNA expression appears to involve genes that regulate protein synthesis and cell cycle, consistent with perturbation of the mTOR pathway. Embryos homozygous for this mutation die towards the end of gestation with a range of cardiovascular defects, including outflow tract abnormalities and ventriculoseptal defects consistent with previous observations, showing that perturbation of the mTOR pathway may affect development of the myocardium. NPRL3 is a candidate gene for harbouring mutations in individuals with developmental abnormalities of the cardiovascular system.

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. The origins of art are therefore much more ancient and lie within Africa, before worldwide human dispersal. The earliest known evidence of 'artistic behaviour' is of human body decoration, including skin colouring with ochre and the use of beads, although both may have had functional origins. Zig-zag and criss-cross patterns, nested curves and parallel lines are the earliest known patterns to have been created separately from the body; their similarity to entopic phenomena (involuntary products of the visual system) suggests a physiological origin. 3D art may have begun with human likeness recognition in natural objects, which were modified to enhance that likeness; some 2D art has also clearly been influenced by suggestive features of an uneven surface. The creation of images from the imagination, or 'the mind's eye', required a seminal evolutionary change in the neural structures underpinning perception; this change would have had a survival advantage in both tool-making and hunting. Analysis of early tool-making techniques suggests that creating 3D objects (sculptures and reliefs) involves their cognitive deconstruction into a series of surfaces, a principle that could have been applied to early sculpture. The cognitive ability to create art separate from the body must have originated in Africa but the practice may have begun at different times in genetically and culturally distinct groups both within Africa and during global dispersal, leading to the regional variety seen in both ancient and recent art. At all stages in the evolution of artistic creativity, stylistic change must have been due to rare, highly gifted individuals.

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. Here we show that by classifying the molecules involved into two groups, stabilizing factors and substrate molecules, complex molecular networks can be modeled by a simple two-species reaction-diffusion model that recapitulates all the known behavior of suture pattern formation. This model reproduces the maintenance of thin sutural tissue at early stages, the later modification of the straight suture to form osseous interdigitations, and the formation of fractal structures. Predictions from the model are in good agreement with experimental observations, indicating that the model captures the essential nature of the interdigitation process.

Expression of the ERK-specific MAP kinase phosphatase PYST1/MKP3 in mouse embryos during morphogenesis and early organogenesis.

Mitogen-activated-protein kinase (MAP kinase) cascades are effector mechanisms for many growth factor signals implicated in developmental processes, including appendage outgrowth and organogenesis. The cascade culminates in extracellular-signal-regulated MAP kinase (ERK), which enters the nucleus. ERK activity reflects the competing actions of upstream activator kinases and inhibitory MAP kinase phosphatases. We have studied embryonic expression of the dual-specificity MAP kinase phosphatase PYST1/MKP3, which is a specific and potent regulator of the ERK class of MAP kinases. We found dynamic patterns of mPyst1 messenger RNA in important signalling centres associated with cell proliferation and patterning in developing mouse embryos, including presegmental paraxial mesoderm, limb bud and branchial arch mesenchyme, midbrain/hindbrain isthmus, and nasal, dental, hair, and mammary placodes. Most of these have been characterised as sites of FGF/FGFR signalling.

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. Development of this incompletely penetrant skull phenotype was dependent on genetic background and sex, with males more often affected. However, these cranial abnormalities were rarely attributable to craniosynostosis, which was only present in 2/364 mutants; more commonly, we found fusion of the premaxillary and/or zygomatic sutures. We also found decreased cortical thickness and bone mineral densities in long bones. We conclude that although both cranial and long bone development is variably affected by the murine Fgfr3(P244R) mutation, coronal craniosynostosis is not reliably reproduced.

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 E11.5, following the onset of ectopic Indian hedgehog (Ihh) expression in the entire distal mesenchyme, except for the zone of polarising activity (ZPA), at E10.5. We show here that limb prepattern, as indicated by expression of Gli3 and Hand2 at E9.5 is unaffected by the mutation. As both Sonic hedgehog (Shh) and Ihh expression are present in Dbf limb buds at E10.5, we generated Dbf/(+);Shh(-/-) mutants to analyse the effects of different patterns of Hedgehog activity on the limb phenotype and molecular differentiation. Dbf/(+) embryos lacking Shh showed postaxial as well as preaxial polydactyly, and the Ihh expression domain extended posteriorly into the domain in which Shh is normally expressed, indicating loss of ZPA identity. Differences in gene expression patterns in wild type, single and compound mutants were associated with differences in Gli3 processing: an increased ratio of Gli3 activator to Gli3 repressor was observed in the anterior half of Dbf/(+) limb buds and in both anterior and posterior halves of compound mutant limb buds at E10.5. To identify the cause of Ihh misregulation in Dbf/(+) mutants, we sequenced approximately 20 kb of genomic DNA around Ihh but found no pathogenic changes. However, Southern blot analysis revealed a approximately 600 kb deletion disrupting or deleting 25 transcripts, starting 50 kb 5' of Ihh and extending away from the gene. The large deletion interval may explain the wide range of abnormalities in Dbf/(+) mutants. However, we did not detect anologous deletions in cases of Laurin-Sandrow syndrome, a human disorder that shows phenotypic similarities to Dbf.

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. A computational craniofacial atlas was built automatically from the set of wild-type mouse micro-CT volumes using (1) affine and (2) non-rigid image registration. Subsequently, the atlas was deformed to match each subject from the two groups of mice. The accuracy of these registrations was measured by a comparison of manually placed landmarks from two different observers and automatically assessed landmarks. Both of the automatic approaches were within the interobserver accuracy for normal specimens, and the non-rigid approach was within the interobserver accuracy for the Crouzon specimens. Four linear measurements, skull length, height and width and interorbital distance, were carried out automatically using the two different approaches. Both automatic approaches assessed the skull length, width and height accurately for both groups of mice. The non-rigid approach measured the interorbital distance accurately for both groups while the affine approach failed to assess this parameter for both groups. Using the full capability of the non-rigid approach, local displacements obtained when registering the non-rigid wild-type atlas to a non-rigid Crouzon mouse atlas were determined on the surface of the wild-type atlas. This revealed a 0.6-mm bending in the nasal region and a 0.8-mm shortening of the zygoma, which are similar to characteristics previously reported in humans. The most striking finding of this analysis was an angulation of approximately 0.6 mm of the cranial base, which has not been reported in humans. Comparing the two different methodologies, it is concluded that the non-rigid approach is the best way to assess linear skull parameters automatically. Furthermore, the non-rigid approach is essential when it comes to analysing local, non-linear shape differences.

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. A comparison of the resulting displacement vectors on the left and right side of the symmetry plane, gives a point-wise measure of asymmetry. The asymmetry measure was applied to the study of Crouzon syndrome using Micro CT scans of genetically modified mice. Crouzon syndrome is characterised by the premature fusion of cranial sutures, which gives rise to a highly asymmetric growth. Quantification and localisation of this asymmetry is of high value with respect to surgery planning and treatment evaluation. Using the proposed method, asymmetry was calculated in each point of the surface of Crouzon mice and wild-type mice (controls). Asymmetry appeared in similar regions for the two groups but the Crouzon mice were found significantly more asymmetric. The localisation ability of the method was in good agreement with ratings from a clinical expert. Validating the quantification ability is a less trivial task due to the lack of a gold standard. Nevertheless, a comparison with a different, but less accurate measure of asymmetry revealed good correlation.

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.

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. Here we review current understanding of this mutant, and briefly report new results indicating (1) that limb bud expansion is concomitant with ectopic lhh expression and with extension of the posterior high cell proliferation rate into the anterior region, and (2) that the Dbf mutation is epistatic to Shh in the limb.

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. To confirm and extend this evidence, we introduced a Cys342Tyr replacement into Fgfr2c to create a gain-of-function mutation equivalent to a mutation in human Crouzon and Pfeiffer syndromes. Fgfr2c(C342Y/)(+) heterozygote mice are viable and fertile with shortened face, protruding eyes, premature fusion of cranial sutures, and enhanced Spp1 expression in the calvaria. Homozygous mutants display multiple joint fusions, cleft palate, and trachea and lung defects, and die shortly after birth. They show enhanced Cbfa1/Runx2 expression without significant change in chondrocyte-specific Ihh, PTHrP, Sox9, Col2a, or Col10a gene expression. Histomorphometric analysis and bone marrow stromal cell culture showed a significant increase of osteoblast progenitors with no change in osteoclastogenic cells. Chondrocyte proliferation was decreased in the skull base at embryonic day 14.5 but not later. These results suggest that long-term aspects of the mutant phenotype, including craniosynostosis, are related to the Fgfr2c regulation of the osteoblast lineage. The effect on early chondrocyte proliferation but not gene expression suggests cooperation of Fgfr2c with Fgfr3 in the formation of the cartilage model for endochondral bone.

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. We generated Alx4(+/-)/Msx2(+/-) double heterozygous mutant mice, interbred them to produce compound genotypes and analysed the genotype-phenotype relationships. Loss of an increasing number of alleles correlated with an incremental exacerbation of the skull vault defect; loss of Alx4 function had a marginally greater effect than loss of Msx2 and also affected skull thickness. In situ hybridization showed that Alx4 and Msx2 are expressed in the cranial skeletogenic mesenchyme and in the growing calvarial bones. Studies of the coronal suture region at embryonic day (E)16.5 revealed that Alx4 expression was decreased, but not abolished, in Msx2(-/-) mutants, and vice versa; expression of Fgfr2 and Fgfr1, but not Twist1, was reduced in both mutants at the same stage. Runx2 expression was unaffected in the coronal suture; in contrast, expression of the downstream ossification marker Spp1 was delayed. Double homozygous pups showed substantial reduction of alkaline phosphatase expression throughout the mineralized skull vault; they died at birth due to defects of the heart, lungs and diaphragm not previously associated with Alx4 or Msx2. Our observations suggest that Alx4 and Msx2 are partially functionally redundant, acting within a network of transcription factors and signalling events that regulate the rate of osteogenic proliferation and differentiation at a stage after the commitment of mesenchymal stem cells to osteogenesis.

Tissue origins and interactions in the mammalian skull vault.

During mammalian evolution, expansion of the cerebral hemispheres was accompanied by expansion of the frontal and parietal bones of the skull vault and deployment of the coronal (fronto-parietal) and sagittal (parietal-parietal) sutures as major growth centres. Using a transgenic mouse with a permanent neural crest cell lineage marker, Wnt1-Cre/R26R, we show that both sutures are formed at a neural crest-mesoderm interface: the frontal bones are neural crest-derived and the parietal bones mesodermal, with a tongue of neural crest between the two parietal bones. By detailed analysis of neural crest migration pathways using X-gal staining, and mesodermal tracing by DiI labelling, we show that the neural crest-mesodermal tissue juxtaposition that later forms the coronal suture is established at E9.5 as the caudal boundary of the frontonasal mesenchyme. As the cerebral hemispheres expand, they extend caudally, passing beneath the neural crest-mesodermal interface within the dermis, carrying with them a layer of neural crest cells that forms their meningeal covering. Exposure of embryos to retinoic acid at E10.0 reduces this meningeal neural crest and inhibits parietal ossification, suggesting that intramembranous ossification of this mesodermal bone requires interaction with neural crest-derived meninges, whereas ossification of the neural crest-derived frontal bone is autonomous. These observations provide new perspectives on skull evolution and on human genetic abnormalities of skull growth and ossification.

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. Fgfr2IIIc is expressed in early mesenchymal condensates and in the periosteal collar around the cartilage models; later it is expressed in sites of both endochondral and intramembranous ossification. A translational stop codon inserted into exon 9 disrupted the synthesis of Fgfr2IIIc without influencing the localized transcription of Fgfr2IIIb, the epithelial Fgfr2 variant. The recessive phenotype of Fgfr2IIIc(-/-) mice was characterized initially by delayed onset of ossification, with continuing deficiency of ossification in the sphenoid region of the skull base. During subsequent stages of skeletogenesis, the balance between proliferation and differentiation was shifted towards differentiation, leading to premature loss of growth, synostosis in certain sutures of the skull base and in the coronal suture of the skull vault, with dwarfism in the long bones and axial skeleton. The retarded ossification was correlated with decrease in the localized transcription of the osteoblast markers secreted phosphoprotein 1 (Spp1) and Runx2/Cbfa1. A decrease in the domain of transcription of the chondrocyte markers Ihh and PTHrP (Pthlh) corresponded with a decrease in their transcripts in the proliferative and hypertrophic chondrocyte zones. These results suggest that Fgfr2IIIc is a positive regulator of ossification affecting mainly the osteoblast, but also the chondrocyte, lineages. This role contrasts with the negative role of Fgfr3, although recent reports implicate FGF18, a ligand for FGFR3IIIc and FGFR2IIIc, as a co-ordinator of osteogenesis via these two receptors.

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. In mice, the orthologous Efnb1 gene is expressed in the frontonasal neural crest and demarcates the position of the future coronal suture. Although EFNB1 is X-inactivated, we did not observe markedly skewed X-inactivation in either blood or cranial periosteum from females with CFNS, indicating that lack of ephrin-B1 does not compromise cell viability in these tissues. We propose that in heterozygous females, patchwork loss of ephrin-B1 disturbs tissue boundary formation at the developing coronal suture, whereas in males deficient in ephrin-B1, an alternative mechanism maintains the normal boundary. This is the only known mutation in the ephrin/Eph receptor signaling system in humans and provides clues to the biogenesis of craniosynostosis.