Melanoblast development coincides with the late emerging cells from the dorsal neural tube in turtle Trachemys scripta.

Ectothermal reptiles have internal pigmentation, which is not seen in endothermal birds and mammals. Here we show that the development of the dorsal neural tube-derived melanoblasts in turtle Trachemys scripta is regulated by similar mechanisms as in other amniotes, but significantly later in development, during the second phase of turtle trunk neural crest emigration. The development of melanoblasts coincided with a morphological change in the dorsal neural tube between stages mature G15 and G16. The melanoblasts delaminated and gathered in the carapacial staging area above the neural tube at G16, and differentiated into pigment-forming melanocytes during in vitro culture. The Mitf-positive melanoblasts were not restricted to the dorsolateral pathway as in birds and mammals but were also present medially through the somites similarly to ectothermal anamniotes. This matched a lack of environmental barrier dorsal and lateral to neural tube and the somites that is normally formed by PNA-binding proteins that block entry to medial pathways. PNA-binding proteins may also participate in the patterning of the carapacial pigmentation as both the migratory neural crest cells and pigment localized only to PNA-free areas.

Loss-of-Function of Gli3 in Mice Causes Abnormal Frontal Bone Morphology and Premature Synostosis of the Interfrontal Suture.

Greig cephalopolysyndactyly syndrome (GCPS) is an autosomal dominant disorder with polydactyly and syndactyly of the limbs and a broad spectrum of craniofacial abnormalities. Craniosynostosis of the metopic suture (interfrontal suture in mice) is an important but rare feature associated with GCPS. GCPS is caused by mutations in the transcription factor GLI3, which regulates Hedgehog signaling. The Gli3 loss-of-function (Gli3(Xt-J/Xt-J)) mouse largely phenocopies the human syndrome with the mice exhibiting polydactyly and several craniofacial abnormalities. Here we show that Gli3(Xt-J/Xt-J) mice exhibit ectopic ossification in the interfrontal suture and in the most severe cases the suture fuses already prior to birth. We show that abnormalities in frontal bones occur early in calvarial development, before the establishment of the interfrontal suture. It provides a model for the metopic suture pathology that can occur in GCPS.

Prevention of premature fusion of calvarial suture in GLI-Kruppel family member 3 (Gli3)-deficient mice by removing one allele of Runt-related transcription factor 2 (Runx2).

Mutations in the gene encoding the zinc finger transcription factor GLI3 (GLI-Kruppel family member 3) have been identified in patients with Grieg cephalopolysyndactyly syndrome in which premature fusion of calvarial suture (craniosynostosis) is an infrequent but important feature. Here, we show that Gli3 acts as a repressor in the developing murine calvaria and that Dlx5, Runx2 type II isoform (Runx2-II), and Bmp2 are expressed ectopically in the calvarial mesenchyme, which results in aberrant osteoblastic differentiation in Gli3-deficient mouse (Gli3(Xt-J/Xt-J)) and resulted in craniosynostosis. At the same time, enhanced activation of phospho-Smad1/5/8 (pSmad1/5/8), which is a downstream mediator of canonical Bmp signaling, was observed in Gli3(Xt-J/Xt-J) embryonic calvaria. Therefore, we generated Gli3;Runx2 compound mutant mice to study the effects of decreasing Runx2 dosage in a Gli3(Xt-J/Xt-J) background. Gli3(Xt-J/Xt-J) Runx2(+/-) mice have neither craniosynostosis nor additional ossification centers in interfrontal suture and displayed a normalization of Dlx5, Runx2-II, and pSmad1/5/8 expression as well as sutural mesenchymal cell proliferation. These findings suggest a novel role for Gli3 in regulating calvarial suture development by controlling canonical Bmp-Smad signaling, which integrates a Dlx5/Runx2-II cascade. We propose that targeting Runx2 might provide an attractive way of preventing craniosynostosis in patients.

Core binding factor beta functions in the maintenance of stem cells and orchestrates continuous proliferation and differentiation in mouse incisors.

Rodent incisors grow continuously throughout life, and epithelial progenitor cells are supplied from stem cells in the cervical loop. We report that epithelial Runx genes are involved in the maintenance of epithelial stem cells and their subsequent continuous differentiation and therefore growth of the incisors. Core binding factor ß (Cbfb) acts as a binding partner for all Runx proteins, and targeted inactivation of this molecule abrogates the activity of all Runx complexes. Mice deficient in epithelial Cbfb produce short incisors and display marked underdevelopment of the cervical loop and suppressed epithelial Fgf9 expression and mesenchymal Fgf3 and Fgf10 expression in the cervical loop. In culture, FGF9 protein rescues these phenotypes. These findings indicate that epithelial Runx functions to maintain epithelial stem cells and that Fgf9 may be a target gene of Runx signaling. Cbfb mutants also lack enamel formation and display downregulated Shh mRNA expression in cells differentiating into ameloblasts. Furthermore, Fgf9 deficiency results in a proximal shift of the Shh expressing cell population and ectopic FGF9 protein suppresses Shh expression. These findings indicate that Shh as well as Fgf9 expression is maintained by Runx/Cbfb but that Fgf9 antagonizes Shh expression. The present results provide the first genetic evidence that Runx/Cbfb genes function in the maintenance of stem cells in developing incisors by activating Fgf signaling loops between the epithelium and mesenchyme. In addition, Runx genes also orchestrate continuous proliferation and differentiation by maintaining the expression of Fgf9 and Shh mRNA.

Gli3Xt-J/Xt-J mice exhibit lambdoid suture craniosynostosis which results from altered osteoprogenitor proliferation and differentiation.

Gli3 is a zinc-finger transcription factor whose activity is dependent on the level of hedgehog (Hh) ligand. Hh signaling has key roles during endochondral ossification; however, its role in intramembranous ossification is still unclear. In this study, we show that Gli3 performs a dual role in regulating both osteoprogenitor proliferation and osteoblast differentiation during intramembranous ossification. We discovered that Gli3Xt-J/Xt-J mice, which represent a Gli3-null allele, exhibit craniosynostosis of the lambdoid sutures and that this is accompanied by increased osteoprogenitor proliferation and differentiation. These cellular changes are preceded by ectopic expression of the Hh receptor Patched1 and reduced expression of the transcription factor Twist1 in the sutural mesenchyme. Twist1 is known to delay osteogenesis by binding to and inhibiting the transcription factor Runx2. We found that Runx2 expression in the lambdoid suture was altered in a pattern complimentary to that of Twist1. We therefore propose that loss of Gli3 results in a Twist1-, Runx2-dependent expansion of the sutural osteoprogenitor population as well as enhanced osteoblastic differentiation which results in a bony bridge forming between the parietal and interparietal bones. We show that FGF2 will induce Twist1, normalize osteoprogenitor proliferation and differentiation and rescue the lambdoid suture synostosis in Gli3Xt-J/Xt-J mice. Taken together, we define a novel role for Gli3 in osteoblast development; we describe the first mouse model of lambdoid suture craniosynostosis and show how craniosynostosis can be rescued in this model.

Convergent signalling through Fgfr2 regulates divergent craniofacial morphogenesis.

Fibroblast growth factor receptor 2 (Fgfr2) has two splice variants IIIb and IIIc, which are unique in function and localization. Signalling through Fgfr2IIIb controls epithelial-mesenchymal interactions, which regulate morphogenesis during the development of several organs including the palate and tooth. In this study, we confirm that molar tooth development in Fgfr2IIIb(-/-) mice is arrested early in development and that the molar teeth of Fgf10(-/-) mice develop through all the normal stages of morphogenesis. We show that the molar phenotype of Fgfr2IIIb(-/-) mice is, in part, owing to reduced cell proliferation in both epithelial and mesenchymal compartments. We also show that the developing molar teeth of Fgf10(-/-) mice exhibit reduced cell proliferation. However, this reduction is not sufficient to arrest molar development. Recent evidence has indicated that Fgfr2IIIb/Fgf10 signalling is active in the calvaria in some pathological situations as heterozygous deletion of Fgfr2 exon IIIc in mice leads to ectopic expression of Fgfr2IIIb in the calvarial bones and causes craniosynostosis. Here, we investigate the mRNA expression of Fgfr2IIIb and Fgfr2IIIc as well as their ligands Fgf3, -7 and -10 in the developing murine tooth, palate and calvaria. We show that Fgf7 is expressed in the calvarial mesenchyme adjacent to the developing frontal bone and Fgf10 is expressed by osteoprogenitors in the developing frontal bone condensation. Taken together, we highlight the overlapping roles of Fgfr2IIIb/Fgf10 signalling in controlling epithelial-mesenchymal interactions during normal palate and tooth morphogenesis and how elevated signalling through Fgfr2IIIb/Fgf10 solely within the mesenchyme can result in abnormal calvarial morphogenesis.

A regulatory relationship between Tbx1 and FGF signaling during tooth morphogenesis and ameloblast lineage determination.

The Tbx1 gene is a transcriptional regulator involved in the DiGeorge syndrome, which affects normal facial and tooth development. Several clinical reports point to a common enamel defect in the teeth of patients with DiGeorge syndrome. Here, we have analyzed the expression, regulation, and function of Tbx1 during mouse molar development. Tbx1 expression is restricted to epithelial cells that give rise to the enamel producing ameloblasts and correlates with proliferative events. Tbx1 expression in epithelium requires mesenchyme-derived signals: dental mesenchyme induces expression of Tbx1 in recombined dental and non-dental epithelia. Bead implantation experiments show that FGF molecules are able to maintain epithelial Tbx1 expression during odontogenesis. Expression of Tbx1 in dental epithelium of FGF receptor 2b(-/-) mutant mice is downregulated, showing a genetic link between FGF signaling and Tbx1 in teeth. Forced expression of Tbx1 in dental explants activates amelogenin expression. These results indicate that Tbx1 expression in developing teeth is under control of FGF signaling and correlates with determination of the ameloblast lineage.

Cell fate specification during calvarial bone and suture development.

In this study we have addressed the fundamental question of what cellular mechanisms control the growth of the calvarial bones and conversely, what is the fate of the sutural mesenchymal cells when calvarial bones approximate to form a suture. There is evidence that the size of the osteoprogenitor cell population determines the rate of calvarial bone growth. In calvarial cultures we reduced osteoprogenitor cell proliferation; however, we did not observe a reduction in the growth of parietal bone to the same degree. This discrepancy prompted us to study whether suture mesenchymal cells participate in the growth of the parietal bones. We found that mesenchymal cells adjacent to the osteogenic fronts of the parietal bones could differentiate towards the osteoblastic lineage and could become incorporated into the growing bone. Conversely, mid-suture mesenchymal cells did not become incorporated into the bone and remained undifferentiated. Thus mesenchymal cells have different fate depending on their position within the suture. In this study we show that continued proliferation of osteoprogenitors in the osteogenic fronts is the main mechanism for calvarial bone growth, but importantly, we show that suture mesenchyme cells can contribute to calvarial bone growth. These findings help us understand the mechanisms of intramembranous ossification in general, which occurs not only during cranial and facial bone development but also in the surface periosteum of most bones during modeling and remodeling.

Expression patterns of Hedgehog signalling pathway members during mouse palate development.

Hedgehog signalling regulates morphogenesis of many developing organs. Sonic hedgehog (Shh) signalling has been shown to regulate the growth and morphogenesis of the palatal shelves prior to their elevation and fusion. Here, we show that Shh expression is limited to a thickened palatal oral epithelium prior to palatal shelf elevation. After palatal shelf elevation above the tongue, Shh is expressed only in small areas of thickened palatal oral epithelium that corresponded to developing rugae. The receptor Ptc1 and a regulator of Hh signalling Hhip1 are expressed in the mesenchyme adjacent to the palatal oral epithelium so that the highest level of transcripts localize to the palatal mesenchyme surrounding the Shh-expressing thickened epithelium. Smoothened and transcriptional effectors Gli1-3, and Hh regulator Gas1 are expressed widely in the palatal mesenchyme. No differences were found in the expression patterns of Hh pathway members along the anterior-posterior axis of the developing palate.

Craniofacial anomalies: from development to molecular pathogenesis.

Advances in developmental biology combined with progress in human genetics are helping us decipher how the craniofacial region develops and how the consequences of misdirected development result in malformation. This review describes the molecular etiology of a number of craniofacial developmental anomalies. The more common craniofacial anomalies cleft lip and palate and craniosynostosis, as well as cleidocranial dysplasia, hemifacial microsomia, holoprosencephaly, enlarged parietal foramina, Treacher Collins syndrome and cherubism are discussed.

Regulation of Twist, Snail, and Id1 is conserved between the developing murine palate and tooth.

The development of both the tooth and palate requires coordinated bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) signalling between epithelial and mesenchymal tissues. Here, we demonstrate that transcription factors Twist and Snail are downstream targets of FGF signalling, that Id1 and Msx2 are downstream targets of BMP signalling, and that Msx1 is regulated by both signalling pathways during tooth and palate development. We show that Twist and Snail expression in the mesenchyme is regulated by the overlying epithelium and that exogenous FGF4 in tooth and FGF2 in palate can mimic this regulation in isolated mesenchymal explants. Ids act in a dominant-negative manner to inhibit the function of other transcription factors such as Twist and Snail. FGF and BMP signalling can regulate development antagonistically, and we suggest that FGF-regulated Twist and Snail and BMP-regulated Id1 may mediate these antagonistic effects during both tooth and palate development.

Foxc1 integrates Fgf and Bmp signalling independently of twist or noggin during calvarial bone development.

Calvarial bone and suture development is under complex regulation where bone morphogenetic protein (Bmp) and fibroblast growth factor (Fgf) signalling interact with Msx2/Twist and Noggin and regulate frontal bone primordia proliferation and suture fusion, respectively. We have shown previously that the winged helix transcription factor Foxc1, which is necessary for calvarial bone development, is required for the Bmp regulation of Msx2. We now show that FGF2 regulates the expression of Foxc1, indicating that Foxc1 integrates Bmp and Fgf signalling pathways. We also show that Foxc1 is not needed for the acquisition of osteogenic potential or for the differentiation of osteoblasts. The expression of Fgf receptors and Twist were normal in Foxc1-deficient calvarial mesenchyme, and ectopic FGF2 was able to induce the expression Osteopontin. Furthermore, we demonstrate that Foxc1 does not participate in the regulation of Noggin expression. Our findings indicate that Foxc1 integrates the Bmp and Fgf signalling pathways independently of Twist or Noggin. This signalling network is essential for the correct patterning and growth of calvarial bones.

Delineation of mechanisms and regions of dosage imbalance in complex rearrangements of 1p36 leads to a putative gene for regulation of cranial suture closure.

Structural chromosome abnormalities have aided in gene identification for over three decades. Delineation of the deletion sizes and rearrangements allows for phenotype/genotype correlations and ultimately assists in gene identification. In this report, we have delineated the precise rearrangements in four subjects with deletions, duplications, and/or triplications of 1p36 and compared the regions of imbalance to two cases recently published. Fluorescence in situ hybridization (FISH) analysis revealed the size, order, and orientation of the duplicated/triplicated segments in each subject. We propose a premeiotic model for the formation of these complex rearrangements in the four newly ascertained subjects, whereby a deleted chromosome 1 undergoes a combination of multiple breakage-fusion-bridge (BFB) cycles and inversions to produce a chromosome arm with a complex rearrangement of deleted, duplicated and triplicated segments. In addition, comparing the six subjects' rearrangements revealed a region of overlap that when triplicated is associated with craniosynostosis and when deleted is associated with large, late-closing anterior fontanels. Within this region are the MMP23A and -B genes. We show MMP23 gene expression at the cranial sutures and we propose that haploinsufficiency results in large, late-closing anterior fontanels and overexpression results in craniosynostosis. These data emphasize the important role of cytogenetics in investigating and uncovering the etiologies of human genetic disease, particularly cytogenetic imbalances that reveal potentially dosage-sensitive genes.

Disruption of Fgf10/Fgfr2b-coordinated epithelial-mesenchymal interactions causes cleft palate.

Classical research has suggested that early palate formation develops via epithelial-mesenchymal interactions, and in this study we reveal which signals control this process. Using Fgf10-/-, FGF receptor 2b-/- (Fgfr2b-/-), and Sonic hedgehog (Shh) mutant mice, which all exhibit cleft palate, we show that Shh is a downstream target of Fgf10/Fgfr2b signaling. Our results demonstrate that mesenchymal Fgf10 regulates the epithelial expression of Shh, which in turn signals back to the mesenchyme. This was confirmed by demonstrating that cell proliferation is decreased not only in the palatal epithelium but also in the mesenchyme of Fgfr2b-/- mice. These results reveal a new role for Fgf signaling in mammalian palate development. We show that coordinated epithelial-mesenchymal interactions are essential during the initial stages of palate development and require an Fgf-Shh signaling network.

Msx2 and Twist cooperatively control the development of the neural crest-derived skeletogenic mesenchyme of the murine skull vault.

The flat bones of the vertebrate skull vault develop from two migratory mesenchymal cell populations, the cranial neural crest and paraxial mesoderm. At the onset of skull vault development, these mesenchymal cells emigrate from their sites of origin to positions between the ectoderm and the developing cerebral hemispheres. There they combine, proliferate and differentiate along an osteogenic pathway. Anomalies in skull vault development are relatively common in humans. One such anomaly is familial calvarial foramina, persistent unossified areas within the skull vault. Mutations in MSX2 and TWIST are known to cause calvarial foramina in humans. Little is known of the cellular and developmental processes underlying this defect. Neither is it known whether MSX2 and TWIST function in the same or distinct pathways. We trace the origin of the calvarial foramen defect in Msx2 mutant mice to a group of skeletogenic mesenchyme cells that compose the frontal bone rudiment. We show that this cell population is reduced not because of apoptosis or deficient migration of neural crest-derived precursor cells, but because of defects in its differentiation and proliferation. We demonstrate, in addition, that heterozygous loss of Twist function causes a foramen in the skull vault similar to that caused by loss of Msx2 function. Both the quantity and proliferation of the frontal bone skeletogenic mesenchyme are reduced in Msx2-Twist double mutants compared with individual mutants. Thus Msx2 and Twist cooperate in the control of the differentiation and proliferation of skeletogenic mesenchyme. Molecular epistasis analysis suggests that Msx2 and Twist do not act in tandem to control osteoblast differentiation, but function at the same epistatic level.

Progression of calvarial bone development requires Foxc1 regulation of Msx2 and Alx4.

Calvarial bones form by direct ossification of mesenchyme. This requires condensation of mesenchymal cells which then proliferate and differentiate into osteoblasts. Congenital hydrocephalus (ch) mutant mice lack the forkhead/winged helix transcription factor Foxc1. In ch mutant mice, calvarial bones remain rudimentary at the sites of initial osteogenic condensations. In this study, we have localized the ossification defect in ch mutants to the calvarial mesenchyme, which lacks the expression of transcription factors Msx2 and Alx4. This lack of expression is associated with a reduction in the proliferation of osteoprogenitor cells. We have previously shown that BMP induces Msx2 in calvarial mesenchyme (Development 125, 1241-1251, 1998). Here, we show that BMP also induces Alx4 in this tissue. We also show that BMP-induced expression of Msx2 and Alx4 requires Foxc1. We therefore suggest that Foxc1 regulates BMP-mediated osteoprogenitor proliferation and that this regulation is required for the progression of osteogenesis beyond the initial condensations in calvarial bone development.

Molecular mechanisms in calvarial bone and suture development, and their relation to craniosynostosis.

The development and growth of the skull is a co-ordinated process involving many different tissues that interact with each other to form a complex end result. When normal development is disrupted, debilitating pathological conditions, such as craniosynostosis (premature calvarial suture fusion) and cleidocranial dysplasia (delayed suture closure), can result. It is known that mutations in the fibroblast growth factor receptors 1, 2, and 3(FGFR1, 2, and 3), as well as the transcription factors MSX2 and TWIST cause craniosynostosis, and that mutations in the transcription factor RUNX2 (CBFA1) cause cleidocranial dysplasia. However, relatively little is known about the development of the calvaria: where and when these genes are active during normal calvarial development, how these genes may interact in the developing calvaria, and the disturbances that may occur to cause these disorders. In this work an attempt has been made to address some of these questions from a basic biological perspective. The expression patterns of the above-mentioned genes in the developing mouse skull are detailed. The microdissection and in vitro culture techniques have begun the task of identifying Fgfrs, Msx2, and Twist interacting in intricate signalling pathways that if disrupted could lead to craniosynostosis.