Chris Healy

Embryonic expression of the chicken Sox2, Sox3 and Sox11 genes suggests an interactive role in neuronal development

Three chicken Sox (SRY-like box) genes have been identified that show an interactive pattern of expression in the developing embryonic nervous system. cSox2 and cSox3 code for related proteins and both are predominantly expressed in the immature neural epithelium of the entire CNS of HH stage 10 to 34 embryos. cSox11 is related to cSox2 and cSox3 only by virtue of containing an SRY-like HMG-box sequence but shows extensive homology with Sox-4 at its C-terminus. cSox11 is expressed in the neural epithelium but is transiently upregulated in maturing neurons after they leave the neural epithelium. These patterns of expression suggest that Sox genes play a role in neural development and that the developmental programme from immature to mature neurons may involve switching of Sox gene expression. cSox11 also exhibits a lineage restricted pattern of expression in the peripheral nervous system.

REGULATION AND ROLE OF SOX9 IN CARTILAGE FORMATION

The HMG‐domain transcription factor Sox9 is a known regulator of the type II collagen gene, a major developmentally regulated protein of cartilage. In order to place Sox9 function in skeletogenesis we have investigated the regulation and misexpression of Sox9 in avian embryos. Application of exogenous BMP2 to chick limbs resulted in upregulation of Sox9, concomitant with induction of ectopic cartilage. Ectopic expression of the BMP antagonist Noggin in the limb resulted in loss of Sox9 expression from the developing digits, indicating that Sox9 expression during chondrogenesis is BMP dependent. Misexpression of Sox9 in vivo resulted in ectopic cartilage formation in limbs and in vitro was able to change the aggregation properties of limb mesenchymal cells, suggesting that Sox9 functions at the level of mesenchymal cell condensation. Misexpression of Sox9 in dermomyotomal cells, which normally give rise to the axial musculature and dermis, can result in the diversion of these cells from their normal fates towards the cartilage differentiation programme. These cells not only express type II collagen, but also Pax1, a marker of ventral fate in the developing somite. This suggests that the cell fate decision to follow the cartilage differentiation pathway is regulated at an early stage by Sox9. Dev Dyn 1999;215:69–78.

Reduced dosage of ERF causes complex craniosynostosis in humans and mice and links ERK1/2 signaling to regulation of osteogenesis

The extracellular signal–related kinases 1 and 2 (ERK1/2) are key proteins mediating mitogen-activated protein kinase signaling downstream of RAS: phosphorylation of ERK1/2 leads to nuclear uptake and modulation of multiple targets. Here, we show that reduced dosage of ERF, which encodes an inhibitory ETS transcription factor directly bound by ERK1/2 (refs. 2,3,4,5,6,7), causes complex craniosynostosis (premature fusion of the cranial sutures) in humans and mice. Features of this newly recognized clinical disorder include multiple-suture synostosis, craniofacial dysmorphism, Chiari malformation and language delay. Mice with functional Erf levels reduced to ∼30% of normal exhibit postnatal multiple-suture synostosis; by contrast, embryonic calvarial development appears mildly delayed. Using chromatin immunoprecipitation in mouse embryonic fibroblasts and high-throughput sequencing, we find that ERF binds preferentially to elements away from promoters that contain RUNX or AP-1 motifs. This work identifies ERF as a novel regulator of osteogenic stimulation by RAS-ERK signaling, potentially by competing with activating ETS factors in multifactor transcriptional complexes.

Regionalisation of early head ectoderm is regulated by endoderm and prepatterns the orofacial epithelium

The oral epithelium becomes regionalised proximodistally early in development, and this is reflected by the spatial expression of signalling molecules such as Fgf8 and Bmp4. This regionalisation is responsible for regulating the spatial expression of genes in the underlying mesenchyme. These genes are required for the spatial patterning of bone, cartilage orofacial development and, in mammals, teeth. The mechanism and timing of this important regionalisation during head epithelium development are not known. Using lipophilic dyes to fate map the oral epithelium in chick embryos, we show that the cells that will occupy the epithelium of the distal and the proximal mandible primordium already occupy different spatial locations in the developing head ectoderm prior to the formation of the first pharyngeal arch and neural crest migration. Moreover, the ectoderm cells fated to become proximal oral epithelium express Fgf8 and this expression requires the presence of endoderm. Thus, the first fundamental patterning process in jaw morphogenesis is controlled by the early separation of specific areas of ectoderm that are regulated by ectoderm-endoderm interactions, and does not involve neural crest cells.

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 Fgfr3P244R mutation, coronal craniosynostosis is not reliably reproduced. Developmental Dynamics 238:331–342, 2009.

Fuz Mutant Mice Reveal Shared Mechanisms between Ciliopathies and FGF-Related Syndromes

Summary Ciliopathies are a broad class of human disorders with craniofacial dysmorphology as a common feature. Among these is high arched palate, a condition that affects speech and quality of life. Using the ciliopathic Fuz mutant mouse, we find that high arched palate does not, as commonly suggested, arise from midface hypoplasia. Rather, increased neural crest expands the maxillary primordia. In Fuz mutants, this phenotype stems from dysregulated Gli processing, which in turn results in excessive craniofacial Fgf8 gene expression. Accordingly, genetic reduction of Fgf8 ameliorates the maxillary phenotypes. Similar phenotypes result from mutation of oral-facial-digital syndrome 1 (Ofd1), suggesting that aberrant transcription of Fgf8 is a common feature of ciliopathies. High arched palate is also a prevalent feature of fibroblast growth factor (FGF) hyperactivation syndromes. Thus, our findings elucidate the etiology for a common craniofacial anomaly and identify links between two classes of human disease: FGF-hyperactivation syndromes and ciliopathies.

Caspase-7 participates in differentiation of cells forming dental hard tissues

Apoptosis during tooth development appears dependent on the apoptotic executioner caspase‐3, but not caspase‐7. Instead, activated caspase‐7 has been found in differentiated odontoblasts and ameloblasts, where it does not correlate with apoptosis. To further investigate these findings, the mouse incisor was used as a model. Analysis of caspase‐7‐deficient mice revealed a significant thinner layer of hard tissue in the adult incisor. Micro computed tomography scan confirmed this decrease in mineralized tissues. These data strongly suggest that caspase‐7 might be directly involved in functional cell differentiation and regulation of the mineralization of dental matrices.

Caspase-7 in molar tooth development

OBJECTIVES The primary enamel knot (PEK) is a population of cells that shows spatio-temporal restricted apoptosis during tooth development. It has been shown that caspase-9 and Apaf-1 are essential for apoptosis in the PEK as well as the central caspase-3. Caspase-7, as another executioner member in the caspase machinery, is considered to have caspase-3 like properties. DESIGN The aim of this study was to detect caspase-7 activation during molar tooth development with a special focus on the cells of the PEK and to correlate the expression with the pattern of apoptosis and caspase-3 activation. Apoptosis in the PEK was investigated in caspase-7 deficient mice to examine the functional consequence of loss of this specific caspase. In addition, odontoblasts and ameloblasts, which are known to undergo cell death during their secretory and maturation stages, were investigated. RESULTS Cleaved caspase-7 was found in the apoptotic region of the PEK, however, caspase-7-deficient mice still possessed apoptotic cells in the PEK in a similar distribution to the wild type. Caspase-7 is therefore not essential for apoptosis in the PEK. Notably, cleaved caspase-7-positive cells were found at later stages in odontoblasts and ameloblasts, but expression did not correlate with apoptosis in these tissues. CONCLUSIONS The results indicate a non-essential apoptotic role of caspase-7 in the PEK apoptosis but suggest also possible non-apoptotic functions for caspase-7 in tooth development.

Activated WNT signaling in postnatal SOX2-positive dental stem cells can drive odontoma formation.

In common with most mammals, humans form only two dentitions during their lifetime. Occasionally, supernumerary teeth develop in addition to the normal complement. Odontoma represent a small group of malformations containing calcified dental tissues of both epithelial and mesenchymal origin, with varying levels of organization, including tooth-like structures. The specific cell type responsible for the induction of odontoma, which retains the capacity to re-initiate de novo tooth development in postnatal tissues, is not known. Here we demonstrate that aberrant activation of WNT signaling by expression of a non-degradable form of β-catenin specifically in SOX2-positive postnatal dental epithelial stem cells is sufficient to generate odontoma containing multiple tooth-like structures complete with all dental tissue layers. Genetic lineage-tracing confirms that odontoma form in a similar manner to normal teeth, derived from both the mutation-sustaining epithelial stem cells and adjacent mesenchymal tissues. Activation of the WNT pathway in embryonic SOX2-positive progenitors results in ectopic expression of secreted signals that promote odontogenesis throughout the oral cavity. Significantly, the inductive potential of epithelial dental stem cells is retained in postnatal tissues, and up-regulation of WNT signaling specifically in these cells is sufficient to promote generation and growth of ectopic malformations faithfully resembling human odontoma.

Characterisation of the genomic structure of chick Fgf8

The Fgf8 gene encodes a series of secreted signalling molecules important in the normal development of the face, brain and limbs. The genomic structure of the chick Fgf8 gene has been analysed and compared to the human and mouse sequences. Divergence between the chick, human and mouse genomic structure was observed. Data indicates that the long alternatively spliced form of exon 1b observed in mouse and exon 1c observed in human and mouse do not exist in the chick Fgf8 gene. RT-PCR analysis indicates that chick Fgf8, like its mouse and human counterpart is alternatively spliced. This data along with the genomic structure data indicates that in the chick there are only two isoforms of Fgf8. This is in contrast to the human and mouse, where evidence suggests that there are 4 and 8 isoforms, respectively. Approximately 400 bp of intron 1d is highly conserved between chick, human and mouse genomic sequences. Using TRANSFAC possible conserved regulatory element binding sites within this domain were identified.