Loss of BMP2 and BMP4 Signaling in the Dental Epithelium Causes Defective Enamel Maturation and Aberrant Development of Ameloblasts.

BMP signaling is crucial for differentiation of secretory ameloblasts, the cells that secrete enamel matrix. However, whether BMP signaling is required for differentiation of maturation-stage ameloblasts (MA), which are instrumental for enamel maturation into hard tissue, is hitherto unknown. To address this, we used an in vivo genetic approach which revealed that combined deactivation of the Bmp2 and Bmp4 genes in the murine dental epithelium causes development of dysmorphic and dysfunctional MA. These fail to exhibit a ruffled apical plasma membrane and to reabsorb enamel matrix proteins, leading to enamel defects mimicking hypomaturation amelogenesis imperfecta. Furthermore, subsets of mutant MA underwent pathological single or collective cell migration away from the ameloblast layer, forming cysts and/or exuberant tumor-like and gland-like structures. Massive apoptosis in the adjacent stratum intermedium and the abnormal cell-cell contacts and cell-matrix adhesion of MA may contribute to this aberrant behavior. The mutant MA also exhibited severely diminished tissue non-specific alkaline phosphatase activity, revealing that this enzyme's activity in MA crucially depends on BMP2 and BMP4 inputs. Our findings show that combined BMP2 and BMP4 signaling is crucial for survival of the stratum intermedium and for proper development and function of MA to ensure normal enamel maturation.

Distinct and Overlapping Expression Patterns of the Homer Family of Scaffolding Proteins and Their Encoding Genes in Developing Murine Cephalic Tissues.

In mammals Homer1, Homer2 and Homer3 constitute a family of scaffolding proteins with key roles in Ca2+ signaling and Ca2+ transport. In rodents, Homer proteins and mRNAs have been shown to be expressed in various postnatal tissues and to be enriched in brain. However, whether the Homers are expressed in developing tissues is hitherto largely unknown. In this work, we used immunohistochemistry and in situ hybridization to analyze the expression patterns of Homer1, Homer2 and Homer3 in developing cephalic structures. Our study revealed that the three Homer proteins and their encoding genes are expressed in a wide range of developing tissues and organs, including the brain, tooth, eye, cochlea, salivary glands, olfactory and respiratory mucosae, bone and taste buds. We show that although overall the three Homers exhibit overlapping distribution patterns, the proteins localize at distinct subcellular domains in several cell types, that in both undifferentiated and differentiated cells Homer proteins are concentrated in puncta and that the vascular endothelium is enriched with Homer3 mRNA and protein. Our findings suggest that Homer proteins may have differential and overlapping functions and are expected to be of value for future research aiming at deciphering the roles of Homer proteins during embryonic development.

Sonic Hedgehog Signaling Is Required for Cyp26 Expression during Embryonic Development.

Deciphering how signaling pathways interact during development is necessary for understanding the etiopathogenesis of congenital malformations and disease. In several embryonic structures, components of the Hedgehog and retinoic acid pathways, two potent players in development and disease are expressed and operate in the same or adjacent tissues and cells. Yet whether and, if so, how these pathways interact during organogenesis is, to a large extent, unclear. Using genetic and experimental approaches in the mouse, we show that during development of ontogenetically different organs, including the tail, genital tubercle, and secondary palate, Sonic hedgehog (SHH) loss-of-function causes anomalies phenocopying those induced by enhanced retinoic acid signaling and that SHH is required to prevent supraphysiological activation of retinoic signaling through maintenance and reinforcement of expression of the Cyp26 genes. Furthermore, in other tissues and organs, disruptions of the Hedgehog or the retinoic acid pathways during development generate similar phenotypes. These findings reveal that rigidly calibrated Hedgehog and retinoic acid activities are required for normal organogenesis and tissue patterning.

A radical switch in clonality reveals a stem cell niche in the epiphyseal growth plate.

Longitudinal bone growth in children is sustained by growth plates, narrow discs of cartilage that provide a continuous supply of chondrocytes for endochondral ossification1. However, it remains unknown how this supply is maintained throughout childhood growth. Chondroprogenitors in the resting zone are thought to be gradually consumed as they supply cells for longitudinal growth1,2, but this model has never been proved. Here, using clonal genetic tracing with multicolour reporters and functional perturbations, we demonstrate that longitudinal growth during the fetal and neonatal periods involves depletion of chondroprogenitors, whereas later in life, coinciding with the formation of the secondary ossification centre, chondroprogenitors acquire the capacity for self-renewal, resulting in the formation of large, stable monoclonal columns of chondrocytes. Simultaneously, chondroprogenitors begin to express stem cell markers and undergo symmetric cell division. Regulation of the pool of self-renewing progenitors involves the hedgehog and mammalian target of rapamycin complex 1 (mTORC1) signalling pathways. Our findings indicate that a stem cell niche develops postnatally in the epiphyseal growth plate, which provides a continuous supply of chondrocytes over a prolonged period.

Cell fate specification in the lingual epithelium is controlled by antagonistic activities of Sonic hedgehog and retinoic acid.

The interaction between signaling pathways is a central question in the study of organogenesis. Using the developing murine tongue as a model, we uncovered unknown relationships between Sonic hedgehog (SHH) and retinoic acid (RA) signaling. Genetic loss of SHH signaling leads to enhanced RA activity subsequent to loss of SHH-dependent expression of Cyp26a1 and Cyp26c1. This causes a cell identity switch, prompting the epithelium of the tongue to form heterotopic minor salivary glands and to overproduce oversized taste buds. At developmental stages during which Wnt10b expression normally ceases and Shh becomes confined to taste bud cells, loss of SHH inputs causes the lingual epithelium to undergo an ectopic and anachronic expression of Shh and Wnt10b in the basal layer, specifying de novo taste placode induction. Surprisingly, in the absence of SHH signaling, lingual epithelial cells adopted a Merkel cell fate, but this was not caused by enhanced RA signaling. We show that RA promotes, whereas SHH, acting strictly within the lingual epithelium, inhibits taste placode and lingual gland formation by thwarting RA activity. These findings reveal key functions for SHH and RA in cell fate specification in the lingual epithelium and aid in deciphering the molecular mechanisms that assign cell identity.

Foxf2 Is Required for Brain Pericyte Differentiation and Development and Maintenance of the Blood-Brain Barrier.

Pericytes are critical for cerebrovascular maturation and development of the blood-brain barrier (BBB), but their role in maintenance of the adult BBB, and how CNS pericytes differ from those of other tissues, is less well understood. We show that the forkhead transcription factor Foxf2 is specifically expressed in pericytes of the brain and that Foxf2(-/-) embryos develop intracranial hemorrhage, perivascular edema, thinning of the vascular basal lamina, an increase of luminal endothelial caveolae, and a leaky BBB. Foxf2(-/-) brain pericytes were more numerous, proliferated faster, and expressed significantly less Pdgfrß. Tgfß-Smad2/3 signaling was attenuated, whereas phosphorylation of Smad1/5 and p38 were enhanced. Tgfß pathway components, including Tgfß2, Tgfßr2, Alk5, and integrins αVß8, were reduced. Foxf2 inactivation in adults resulted in BBB breakdown, endothelial thickening, and increased trans-endothelial vesicular transport. On the basis of these results, FOXF2 emerges as an interesting candidate locus for stroke susceptibility in humans.

Expression patterns and subcellular localization of carbonic anhydrases are developmentally regulated during tooth formation.

Carbonic anhydrases (CAs) play fundamental roles in several physiological events, and emerging evidence points at their involvement in an array of disorders, including cancer. The expression of CAs in the different cells of teeth is unknown, let alone their expression patterns during odontogenesis. As a first step towards understanding the role of CAs during odontogenesis, we used immunohistochemistry, histochemistry and in situ hybridization to reveal hitherto unknown dynamic distribution patterns of eight CAs in mice. The most salient findings include expression of CAII/Car2 not only in maturation-stage ameloblasts (MA) but also in the papillary layer, dental papilla mesenchyme, odontoblasts and the epithelial rests of Malassez. We uncovered that the latter form lace-like networks around incisors; hitherto these have been known to occur only in molars. All CAs studied were produced by MA, however CAIV, CAIX and CARPXI proteins were distinctly enriched in the ruffled membrane of the ruffled MA but exhibited a homogeneous distribution in smooth-ended MA. While CAIV, CAVI/Car6, CAIX, CARPXI and CAXIV were produced by all odontoblasts, CAIII distribution displayed a striking asymmetry, in that it was virtually confined to odontoblasts in the root of molars and root analog of incisors. Remarkably, from initiation until near completion of odontogenesis and in several other tissues, CAXIII localized mainly in intracellular punctae/vesicles that we show to overlap with LAMP-1- and LAMP-2-positive vesicles, suggesting that CAXIII localizes within lysosomes. We showed that expression of CAs in developing teeth is not confined to cells involved in biomineralization, pointing at their participation in other biological events. Finally, we uncovered novel sites of CA expression, including the developing brain and eye, the olfactory epithelium, melanoblasts, tongue, notochord, nucleus pulposus and sebaceous glands. Our study provides important information for future single or multiple gene targeting strategies aiming at deciphering the function of CAs during odontogenesis.

The mouse as a developmental model for cleft lip and palate research.

Vertebrate and invertebrate model organisms are essential for deciphering biological processes. One of these, the mouse, proved to be a valuable model for understanding the etiopathogenesis of a vast array of human diseases, including congenital malformations such as orofacial clefting conditions. This small mammal's usefulness in cleft lip and palate research stems not only from the striking anatomical and molecular similarities of lip and palate development between human and mouse embryos, but also from its amenability to experimental and genetic manipulation. Using some recent studies as illustrative examples, this review describes different ways of generating and exploiting mouse models to study normal and abnormal development of the lip and palate. Despite a few surmountable disadvantages of using the mouse, numerous mutants have revealed a growing number of molecular key players and have pointed at a tight and complex molecular control during each step of lip and palate development.

Periodic stripe formation by a Turing mechanism operating at growth zones in the mammalian palate.

We present direct evidence of an activator-inhibitor system in the generation of the regularly spaced transverse ridges of the palate. We show that new ridges, called rugae, that are marked by stripes of expression of Shh (encoding Sonic hedgehog), appear at two growth zones where the space between previously laid rugae increases. However, inter-rugal growth is not absolutely required: new stripes of Shh expression still appeared when growth was inhibited. Furthermore, when a ruga was excised, new Shh expression appeared not at the cut edge but as bifurcating stripes branching from the neighboring stripe of Shh expression, diagnostic of a Turing-type reaction-diffusion mechanism. Genetic and inhibitor experiments identified fibroblast growth factor (FGF) and Shh as components of an activator-inhibitor pair in this system. These findings demonstrate a reaction-diffusion mechanism that is likely to be widely relevant in vertebrate development.

Developmental changes in cellular and extracellular structural macromolecules in the secondary palate and in the nasal cavity of the mouse.

The aim of this study was to analyse the hitherto largely unknown expression patterns of some specific cellular and extracellular molecules during palate and nasal cavity development. We showed that epithelia of the developing palate and the vomerine epithelium express similar sets of structural proteins. With the exception of keratin 15, which becomes barely detectable in the elevated palatal shelves, nearly all of these proteins become upregulated at the presumptive areas of fusion and in the adhering epithelia of the palate and nasal septum. In vivo and in vitro analyses indicated that reduction in the amount of keratin 15 protein is independent of Tgfbeta-Alk5 signalling. Foxa1 expression also highlighted the regionalization of the palatal and nasal epithelia. Owing to the lack of reliable markers of the palatal periderm, the fate of peridermal cells has been controversial. We identified LewisX/stage-specific embryonic antigen-1 as a specific peridermal marker, and showed that numerous peridermal cells remain trapped in the medial epithelial seam (MES). The fate of these cells is probably apoptosis together with the rest of the MES cells, as we provided strong evidence for this event. Heparan sulphate, chondroitin-6-sulphate, and versican displayed dynamically changing distribution patterns. The hitherto-unknown innervation pattern of the developing palate was revealed. These findings may be of value for unravelling the pathogenesis of palatal clefting.

p63 and IRF6: brothers in arms against cleft palate.

Cleft lip and cleft palate, which can also occur together as cleft lip and palate, are frequent and debilitating congenital malformations, with complex geneses that have both genetic and environmental factors implicated. Mutations in the genes encoding the p53 homolog p63 and interferon regulatory factor 6 (IRF6) are major causes of cleft lip and cleft palate, but the molecular and cellular mechanisms underlying this have not been clear. However, in this issue of the JCI, Thomason et al. and Moretti et al. independently show that p63 and IRF6 operate within a regulatory loop to coordinate epithelial proliferation and differentiation during normal palate development. Disruption of this loop as a result of mutations in p63 or IRF6 causes congenital clefting.

Shh pathway activation is present and required within the vertebrate limb bud apical ectodermal ridge for normal autopod patterning.

Expression of Sonic Hedgehog (Shh) in the posterior mesenchyme of the developing limb bud regulates patterning and growth of the developing limb by activation of the Hedgehog (Hh) signaling pathway. Through the analysis of Shh and Hh signaling target genes, it has been shown that activation in the limb bud mesoderm is required for normal limb development to occur. In contrast, it has been stated that Hh signaling in the limb bud ectoderm cannot occur because components of the Hh signaling pathway and Hh target genes have not been found in this tissue. However, recent array-based data identified both the components necessary to activate the Hh signaling pathway and targets of this pathway in the limb bud ectoderm. Using immunohistochemistry and various methods of detection for targets of Hh signaling, we found that SHH protein and targets of Hh signaling are present in the limb bud ectoderm including the apex of the bud. To directly test whether ectodermal Hh signaling was required for normal limb patterning, we removed Smo, an essential component of the Hh signaling pathway, from the apical ectodermal ridge (AER). Loss of functional Hh signaling in the AER resulted in disruption of the normal digit pattern and formation of additional postaxial cartilaginous condensations. These data indicate that contrary to previous accounts, the Hh signaling pathway is present and required in the developing limb AER for normal autopod development.

Expression patterns of the Tmem16 gene family during cephalic development in the mouse.

Tmem16a, Tmem16c, Tmem16f, Tmem16h and Tmem16k belong to the newly identified Tmem16 gene family encoding eight-pass transmembrane proteins. We have analyzed the expression patterns of these genes during mouse cephalic development. In the central nervous system, Tmem16a transcripts were abundant in the ventricular neuroepithelium, whereas the other Tmem16 family members were readily detectable in the subventricular zone and differentiating fields. In the rostral spinal cord, Tmem16f expression was highest in the motor neuron area. In the developing eye, the highest amounts of Tmem16a transcripts were detected in the lens epithelium, hyaloid plexus and outer layer of the retina, while the other family members were abundant in the retinal ganglionic cell layer. Interestingly, throughout development, Tmem16a expression in the inner ear was robust and restricted to a subset of cells within the epithelium, which at later stages formed the organ of Corti. The stria vascularis was particularly rich in Tmem16a and Tmem16f mRNA. Other sites of Tmem16 expression included cranial nerve and dorsal root ganglia, meningeal precursors and the pituitary. Tmem16c and Tmem16f transcripts were also patent in the submandibular autonomic ganglia. A conspicuous feature of Tmem16a was its expression along the walls of blood vessels as well as in cells surrounding the trigeminal and olfactory nerve axons. In organs developing through epithelial-mesenchymal interactions, such as the palate, tooth and tongue, the above five Tmem16 family members showed interesting dynamic expression patterns as development proceeded. Finally and remarkably, osteoblasts and chondrocytes were particularly loaded with Tmem16a, Tmem16c and Tmem16f transcripts.

Lrp4 modulates extracellular integration of cell signaling pathways in development.

The extent to which cell signaling is integrated outside the cell is not currently appreciated. We show that a member of the low-density receptor-related protein family, Lrp4 modulates and integrates Bmp and canonical Wnt signalling during tooth morphogenesis by binding the secreted Bmp antagonist protein Wise. Mouse mutants of Lrp4 and Wise exhibit identical tooth phenotypes that include supernumerary incisors and molars, and fused molars. We propose that the Lrp4/Wise interaction acts as an extracellular integrator of epithelial-mesenchymal cell signaling. Wise, secreted from mesenchyme cells binds to BMP's and also to Lrp4 that is expressed on epithelial cells. This binding then results in the modulation of Wnt activity in the epithelial cells. Thus in this context Wise acts as an extracellular signaling molecule linking two signaling pathways. We further show that a downstream mediator of this integration is the Shh signaling pathway.

The etiopathogenesis of cleft lip and cleft palate: usefulness and caveats of mouse models.

Cleft lip and cleft palate are frequent human congenital malformations with a complex multifactorial etiology. These orofacial clefts can occur as part of a syndrome involving multiple organs or as isolated clefts without other detectable defects. Both forms of clefting constitute a heavy burden to the affected individuals and their next of kin. Human and mouse facial traits are utterly dissimilar. However, embryonic development of the lip and palate are strikingly similar in both species, making the mouse a model of choice to study their normal and abnormal development. Human epidemiological and genetic studies are clearly important for understanding the etiology of lip and palate clefting. However, our current knowledge about the etiopathogenesis of these malformations has mainly been gathered throughout the years from mouse models, including those with mutagen-, teratogen- and targeted mutation-induced clefts as well as from mice with spontaneous clefts. This review provides a comprehensive description of the numerous mouse models for cleft lip and/or cleft palate. Despite a few weak points, these models have revealed a high order of molecular complexity as well as the stringent spatiotemporal regulations and interactions between key factors which govern the development of these orofacial structures.

Abnormal hair development and apparent follicular transformation to mammary gland in the absence of hedgehog signaling.

We show that removing the Shh signal tranducer Smoothened from skin epithelium secondarily results in excess Shh levels in the mesenchyme. Moreover, the phenotypes we observe reflect decreased epithelial Shh signaling, yet increased mesenchymal Shh signaling. For example, the latter contributes to exuberant hair follicle (HF) induction, while the former depletes the resulting follicular stem cell niches. This disruption of the niche apparently also allows the remaining stem cells to initiate hair formation at inappropriate times. Thus, the temporal structure of the hair cycle may depend on the physical structure of the niche. Finally, we find that the ablation of epithelial Shh signaling results in unexpected transformations: the follicular outer root sheath takes on an epidermal character, and certain HFs disappear altogether, having adopted a strikingly mammary gland-like fate. Overall, our study uncovers a multifaceted function for Shh in sculpting and maintaining the integrity and identity of the developing HF.

Molecular control of secondary palate development.

Compared with the embryonic development of other organs, development of the secondary palate is seemingly simple. However, each step of palatogenesis, from initiation until completion, is subject to a tight molecular control that is governed by epithelial-mesenchymal interactions. The importance of a rigorous molecular regulation of palatogenesis is reflected when loss of function of a single protein generates cleft palate, a frequent malformation with a complex etiology. Genetic studies in humans and targeted mutations in mice have identified numerous factors that play key roles during palatogenesis. This review highlights the current understanding of the molecular and cellular mechanisms involved in normal and abnormal palate development with special respect to recent advances derived from studies of mouse models.

Expression of Pit2 sodium-phosphate cotransporter during murine odontogenesis is developmentally regulated.

Different sodium-dependent inorganic phosphate (P(i)) uptake mechanisms play a major role in cellular P(i) homeostasis. The function and detailed distribution patterns of the type III Na(+)-phosphate cotransporter, PiT-2, in different organs during development are still largely unknown. We therefore examined the temporospatial expression patterns of Pit2 during murine odontogenesis. Odontoblasts were always devoid of Pit2 expression, whereas a transient, but strong, expression was detected in young secretory ameloblasts. However, the stratum intermedium and, later on, the papillary layer and cells of the subodontoblastic layer, exhibited high levels of Pit2 mRNA, which increased gradually as the tooth matured. Hormonal treatment or P(i) starvation of tooth germs in vitro did not alter Pit2 levels or patterns of expression, indicating mechanisms of regulation different from those of PiT-1 or other cell types. PiT-2 also functions as a retroviral receptor, and functional membrane-localized protein was confirmed throughout the dental papilla/pulp by demonstrating cellular permissiveness to infection by a gammaretrovirus that uses PiT-2 as a receptor. The distinct pattern of Pit2 expression during odontogenesis suggests that its P(i)-transporter function may be important for homeostasis of dental cells and not specifically for mineralization of the dental extracellular matrices. The expression of viral receptors in enamel-forming cells and the dental pulp may be of pathological significance.

Fate-mapping of the epithelial seam during palatal fusion rules out epithelial-mesenchymal transformation.

During palatogenesis, fusion of the palatine shelves is a crucial event, the failure of which results in the birth defect, cleft palate. The fate of the midline epithelial seam (MES), which develops transiently upon contact of the two palatine shelves, is still strongly debated. Three major mechanisms underlying the regression of the MES upon palatal fusion have been proposed: (1) apoptosis has been evidenced by morphological and molecular criteria; (2) epithelial-mesenchymal transformation has been suggested based on ultrastructural and lipophilic dye cell labeling observations; and (3) migration of MES cells toward the oral and nasal areas has been proposed following lipophilic dye cell labeling. To verify whether epithelial-mesenchymal transformation of MES cells takes place during murine palatal fusion, we used the Cre/lox system to genetically mark Sonic hedgehog- and Keratin-14-expressing palatal epithelial cells and to identify their fate in vivo. Our analyses provide conclusive evidence that rules out the occurrence of epithelial-mesenchymal transformation of MES cells.

Nuclear factor 1-C2 is regulated by prolactin and shows a distinct expression pattern in the mouse mammary epithelial cells during development.

We have previously demonstrated that the transcription factor nuclear factor (NF)1-C2 plays an important role in the mammary gland for the activation of the tumor suppressor gene p53. It also activates the milk genes carboxyl ester lipase and whey acidic protein, implying that NF1-C2 participates both in the establishment of a functional gland and in protection of the gland against tumorigenesis during proliferation. In this study, we have developed a new sensitive NF1-C2-specific antiserum for immunohistochemical analyses of the NF1-C2 distribution during mammary gland development. We show that the NF1-C2 protein is present in the epithelial compartment at the virgin stage and throughout mammary gland development. However, in the lactation stage the NF1-C2 protein levels strongly decreased, and many epithelial nuclei stained negative. In situ hybridization shows that NF1-C2 transcripts are expressed in the whole epithelium at pregnancy as well as the lactation stage, indicating that the reduction in protein levels is posttranscriptionally regulated. At involution, the NF1-C2 proteins are back to high levels. Based on studies using NMuMG cells and mammary tissue from heterozygous prolactin receptor knockout mice, we also demonstrate that prolactin has a direct effect in the maintenance of the NF1-C2 protein levels in the mammary epithelial nuclei at the virgin stage and during pregnancy. Hence, we have identified another transcription factor in the mammary gland, besides signal transducer and activator of transcription 5, through which prolactin may control mammary gland development. Furthermore, our data suggest a link between prolactin and p53 in the mammary gland, through NF1-C2.