OFCD syndrome and extraembryonic defects are revealed by conditional mutation of the Polycomb-group repressive complex 1.1 (PRC1.1) gene BCOR.

BCOR is a critical regulator of human development. Heterozygous mutations of BCOR in females cause the X-linked developmental disorder Oculofaciocardiodental syndrome (OFCD), and hemizygous mutations of BCOR in males cause gestational lethality. BCOR associates with Polycomb group proteins to form one subfamily of the diverse Polycomb repressive complex 1 (PRC1) complexes, designated PRC1.1. Currently there is limited understanding of differing developmental roles of the various PRC1 complexes. We therefore generated a conditional exon 9-10 knockout Bcor allele and a transgenic conditional Bcor expression allele and used these to define multiple roles of Bcor, and by implication PRC1.1, in mouse development. Females heterozygous for Bcor exhibiting mosaic expression due to the X-linkage of the gene showed reduced postnatal viability and had OFCD-like defects. By contrast, Bcor hemizygosity in the entire male embryo resulted in embryonic lethality by E9.5. We further dissected the roles of Bcor, focusing on some of the tissues affected in OFCD through use of cell type specific Cre alleles. Mutation of Bcor in neural crest cells caused cleft palate, shortening of the mandible and tympanic bone, ectopic salivary glands and abnormal tongue musculature. We found that defects in the mandibular region, rather than in the palate itself, led to palatal clefting. Mutation of Bcor in hindlimb progenitor cells of the lateral mesoderm resulted in 2/3 syndactyly. Mutation of Bcor in Isl1-expressing lineages that contribute to the heart caused defects including persistent truncus arteriosus, ventricular septal defect and fetal lethality. Mutation of Bcor in extraembryonic lineages resulted in placental defects and midgestation lethality. Ubiquitous over expression of transgenic Bcor isoform A during development resulted in embryonic defects and midgestation lethality. The defects we have found in Bcor mutants provide insights into the etiology of the OFCD syndrome and how BCOR-containing PRC1 complexes function in development.

Natriuretic peptide system expression in murine and human submandibular salivary glands: a study of the spatial localisation of ANB, BNP, CNP and their receptors.

The natriuretic peptide (NP) system comprises of three ligands, the Atrial Natriuretic Peptide (ANP), Brain Natriuretic peptide (BNP) and C-type Natriuretic peptide (CNP), and three natriuretic peptide receptors, NPRA, NPRB and NPRC. Here we present a comprehensive study of the natriuretic peptide system in healthy murine and human submandibular salivary glands (SMGs). We show CNP is the dominant NP in mouse and human SMG and is expressed together with NP receptors in ducts, autonomic nerves and the microvasculature of the gland, suggesting CNP autocrine signalling may take place in some of these glandular structures. These data suggest the NP system may control salivary gland function during homeostasis through the regulation of electrolyte re-absorption, neural stimulation and/or blood vessel wall contraction/relaxation. We also show abnormal expression of NPRA in the stroma of a subset of human SMGs resected from patients diagnosed with oral squamous cell carcinoma (OSCC) of non-salivary gland origin. This finding warrants further research to investigate a possible correlation between early OSCC invasion and NPRA overexpression.

Molecular regulation of ocular gland development.

The tear film is produced by two ocular glands, the lacrimal glands, which produce the aqueous component of this film, and the meibomian glands, which secrete the lipidic component that is key to reduce evaporation of the watery film at the surface of the eye. Embryonic development of these exocrine glands has been mostly studied in mice, which also develop Harderian glands, a third type of ocular gland whose role is still not well understood. This review provides an update on the signalling pathways, transcription factors andextracellular matrix components that have been shown to play a role in ocular gland development.

Impact of hypofunctional occlusion on upper and lower molars after cessation of root development in adult mice.

Background: Hypofunctional occlusion is known to lead to changes in the length of roots over time. The mechanisms that drive such changes, however, are poorly understood, with most studies concentrating on juvenile rats prior to the arrest of root development. In this article, we investigated the response of the upper and lower first molar roots to lack of occlusion concentrating on time-points after the development of the roots has ceased using the mouse as a model. Mouse molar roots finish development at weaning, much earlier than rat molars, and display a similar pattern of roots in the lower and upper jaw to humans. Methods: Hypofunctional occlusion was achieved in adult mice at 5 and 9 weeks of age by flattening the cusps of the upper first molar. Mice were then sacrificed after 6 and 2 weeks, respectively, along with control littermates. microCT was used to measure root length, alveolar bone height, and the amount of tooth eruption, followed by sectioning to understand the mechanisms behind the changes at the histological level. Results: In the lower first molar, the response to hypofunctional occlusion was characterized by elongation of both the mesial root and its surrounding alveolar bone, while the distal root was unaffected. In contrast, the response of the upper first molar was characterized by over-eruption of the mesial side of the tooth without any significant change in the alveolar bone or root length. From histologic sections, it was clear that increased deposition of cellular cementum played an important role in the changes that occurred in the lower mesial root. Conclusions: In a mouse model, upper and lower molars responded differently to hypofunctional occlusion, with adult mice showing a different response to that previously reported for juvenile rats, highlighting the importance of considering age and tooth position in cases of hypofunction.

Ephrin Ligands and Eph Receptors Show Regionally Restricted Expression in the Developing Palate and Tongue.

The Eph family receptor-interacting (ephrin) ligands and erythropoietin-producing hepatocellular carcinoma (Eph) receptors constitute the largest known family of receptor tyrosine kinases. Ephrin ligands and their receptors form an important cell communication system with widespread roles in normal physiology and disease pathogenesis. In order to investigate potential roles of the ephrin-Eph system during palatogenesis and tongue development, we have characterized the cellular mRNA expression of family members EphrinA1-A3, EphA1-A8, and EphrinB2, EphB1, EphB4 during murine embryogenesis between embryonic day 13.5-16.5 using radioactive in situ hybridization. With the exception of EphA6 and ephrinA3, all genes were regionally expressed during the process of palatogenesis, with restricted and often overlapping domains. Transcripts were identified in the palate epithelium, localized at the tip of the palatal shelves, in the mesenchyme and also confined to the medial epithelium seam. Numerous Eph transcripts were also identified during tongue development. In particular, EphA1 and EphA2 demonstrated a highly restricted and specific expression in the tongue epithelium at all stages examined, whereas EphA3 was strongly expressed in the lateral tongue mesenchyme. These results suggest regulatory roles for ephrin-EphA signaling in development of the murine palate and tongue.

Ring1a/b polycomb proteins regulate the mesenchymal stem cell niche in continuously growing incisors.

Rodent incisors are capable of growing continuously and the renewal of dental epithelium giving rise to enamel-forming ameloblasts and dental mesenchyme giving rise to dentin-forming odontoblasts and pulp cells is achieved by stem cells residing at their proximal ends. Although the dental epithelial stem cell niche (cervical loop) is well characterized, little is known about the dental mesenchymal stem cell niche. Ring1a/b are the core Polycomb repressive complex1 (PRC1) components that have recently also been found in a protein complex with BcoR (Bcl-6 interacting corepressor) and Fbxl10. During mouse incisor development, we found that genes encoding members of the PRC1 complex are strongly expressed in the incisor apical mesenchyme in an area that contains the cells with the highest proliferation rate in the tooth pulp, consistent with a location for transit amplifying cells. Analysis of Ring1a(-/-);Ring1b(cko/cko) mice showed that loss of Ring1a/b postnatally results in defective cervical loops and disturbances of enamel and dentin formation in continuously growing incisors. To further characterize the defect found in Ring1a(-/-);Ring1b(cko/cko) mice, we demonstrated that cell proliferation is dramatically reduced in the apical mesenchyme and cervical loop epithelium of Ring1a(-/-);Ring1b(cko/cko) incisors in comparison to Ring1a(-/-);Ring1b(fl/fl)cre- incisors. Fgf signaling and downstream targets that have been previously shown to be important in the maintenance of the dental epithelial stem cell compartment in the cervical loop are downregulated in Ring1a(-/-);Ring1b(cko/cko) incisors. In addition, expression of other genes of the PRC1 complex is also altered. We also identified an essential postnatal requirement for Ring1 proteins in molar root formation. These results show that the PRC1 complex regulates the transit amplifying cell compartment of the dental mesenchymal stem cell niche and cell differentiation in developing mouse incisors and is required for molar root formation.

Developmental stalling and organ-autonomous regulation of morphogenesis.

Timing of organ development during embryogenesis is coordinated such that at birth, organ and fetal size and maturity are appropriately proportioned. The extent to which local developmental timers are integrated with each other and with the signaling interactions that regulate morphogenesis to achieve this end is not understood. Using the absolute requirement for a signaling pathway activity (bone morphogenetic protein, BMP) during a critical stage of tooth development, we show that suboptimal levels of BMP signaling do not lead to abnormal morphogenesis, as suggested by mutants affecting BMP signaling, but to a 24-h stalling of the intrinsic developmental clock of the tooth. During this time, BMP levels accumulate to reach critical levels whereupon tooth development restarts, accelerates to catch up with development of the rest of the embryo and completes normal morphogenesis. This suggests that individual organs can autonomously control their developmental timing to adjust their stage of development to that of other organs. We also find that although BMP signaling is critical for the bud-to-cap transition in all teeth, levels of BMP signaling are regulated differently in multicusped teeth. We identify an interaction between two homeodomain transcription factors, Barx1 and Msx1, which is responsible for setting critical levels of BMP activity in multicusped teeth and provides evidence that correlates the levels of Barx1 transcriptional activity with cuspal complexity. This study highlights the importance of absolute levels of signaling activity for development and illustrates remarkable self-regulation in organogenesis that ensures coordination of developmental processes such that timing is subordinate to developmental structure.

Coordination of epithelial branching and salivary gland lumen formation by Wnt and FGF signals.

Branching morphogenesis is a molecularly conserved mechanism that is adopted by several organs, such as the lung, kidney, mammary gland and salivary gland, to maximize the surface area of a tissue within a small volume. Branching occurs through repetitive clefting and elongation of spherical epithelial structures, called endbuds, which invade the surrounding mesenchyme. In the salivary gland, lumen formation takes place alongside branching morphogenesis, but in a controlled manner, so that branching is active at the distal ends of epithelial branches while lumen formation initiates at the proximal ends, and spreads distally. We present here data showing that interaction between FGF signaling and the canonical (ß-catenin dependent) and non-canonical branches of Wnt signaling coordinates these two processes. Using the Axin2(lacZ) reporter mice, we find Wnt/ß-catenin signaling activity first in the mesenchyme and later, at the time of lumen formation, in the ductal epithelium. Gain and loss of function experiments reveal that this pathway exerts an inhibitory effect on salivary gland branching morphogenesis. We have found that endbuds remain devoid of Wnt/ß-catenin signaling activity, a hallmark of ductal structures, through FGF-mediated inhibition of this pathway. Our data also show that FGF signaling has a major role in the control of lumen formation by preventing premature hollowing of epithelial endbuds and slowing down the canalization of presumptive ducts. Concomitantly, FGF signaling strongly represses the ductal marker Cp2l1, most likely via repression of Wnt5b and non-canonical Wnt signaling. Inhibition of canonical and non-canonical Wnt signaling in endbuds by FGF signaling occurs at least in part through sFRP1, a secreted inhibitor of Wnt signaling and downstream target of FGF signaling. Altogether, these findings point to a key function of FGF signaling in the maintenance of an undifferentiated state in endbud cells by inhibition of a ductal fate.

Barx1-mediated inhibition of Wnt signaling in the mouse thoracic foregut controls tracheo-esophageal septation and epithelial differentiation.

Mesenchymal cells underlying the definitive endoderm in vertebrate animals play a vital role in digestive and respiratory organogenesis. Although several signaling pathways are implicated in foregut patterning and morphogenesis, and despite the clinical importance of congenital tracheal and esophageal malformations in humans, understanding of molecular mechanisms that allow a single tube to separate correctly into the trachea and esophagus is incomplete. The homoebox gene Barx1 is highly expressed in prospective stomach mesenchyme and required to specify this organ. We observed lower Barx1 expression extending contiguously from the proximal stomach domain, along the dorsal anterior foregut mesenchyme and in mesenchymal cells between the nascent esophagus and trachea. This expression pattern exactly mirrors the decline in Wnt signaling activity in late development of the adjacent dorsal foregut endoderm and medial mainstem bronchi. The hypopharynx in Barx1(-/-) mouse embryos is abnormally elongated and the point of esophago-tracheal separation shows marked caudal displacement, resulting in a common foregut tube that is similar to human congenital tracheo-esophageal fistula and explains neonatal lethality. Moreover, the Barx1(-/-) esophagus displays molecular and cytologic features of respiratory endoderm, phenocopying abnormalities observed in mouse embryos with activated ß-catenin. The zone of canonical Wnt signaling is abnormally prolonged and expanded in the proximal Barx1(-/-) foregut. Thus, as in the developing stomach, but distinct from the spleen, Barx1 control of thoracic foregut specification and tracheo-esophageal septation is tightly associated with down-regulation of adjacent Wnt pathway activity.

Introduction to salivary glands: structure, function and embryonic development.

Salivary glands are a group of organs secreting a watery substance that is of utmost importance for several physiological functions ranging from the protection of teeth and surrounding soft tissues to the lubrication of the oral cavity, which is crucial for speech and perception of food taste. Salivary glands are complex networks of hollow tubes and secretory units that are found in specific locations of the mouth and which, although architecturally similar, exhibit individual specificities according to their location. This chapter focuses on the embryonic development of vertebrate salivary glands, which has been classically studied in the mouse model system since the 1950s. We describe here where, when and how major salivary glands develop in the lower jaw of the mouse embryo. Key mechanisms involved in this process are discussed, including reciprocal tissue interactions between epithelial and mesenchymal cells, epithelial branching morphogenesis and coordinated cell death and cell proliferation.

Independent functions and mechanisms for homeobox gene Barx1 in patterning mouse stomach and spleen.

Homeobox genes convey positional information in embryos and their role in patterning the mammalian gut is a topic of considerable interest. Barx1 is expressed selectively in fetal stomach mesenchyme and directs differentiation of overlying endoderm. Recombinant tissue cultures and study of young mouse embryos previously suggested that Barx1 controls expression of secreted Wnt antagonists, which suppress endodermal Wnt signaling, to enable stomach epithelial differentiation. We overcame mid-gestational lethality of Barx1(-/-) mouse embryos and report here the spectrum of anomalies in a distinctive and unprecedented model of gastrointestinal homeotic transformation. Using various mouse models, we confirm the importance of attenuated Wnt signaling in stomach development and the role of Barx1 in suppressing endodermal Wnt activity. Absence of Barx1 also results in fully penetrant defects in positioning and expansion of the spleen, an organ that originates within the mesothelial lining of the stomach. Barx1 is absent from the spleen primordium but highly expressed in the mesogastrium, indicating an indirect effect on spleen development. However, our results argue against a role for Wnt antagonism in genesis of the spleen. Mouse spleen development relies on several homeodomain transcriptional regulators that are expressed in the spleen primordium. Loss of Barx1 does not affect expression of any of these genes but notably reduces expression of Wt1, a transcription factor implicated in spleen morphogenesis and expressed in the mesothelium. These observations place Barx1 proximally within a Wt1 pathway of spleen development and reveal how a homeotic regulator employs different molecular mechanisms to mold neighboring organs.

Satb2 haploinsufficiency phenocopies 2q32-q33 deletions, whereas loss suggests a fundamental role in the coordination of jaw development.

The recent identification of SATB2 as a candidate gene responsible for the craniofacial dysmorphologies associated with deletions and translocations at 2q32-q33, one of only three regions of the genome for which haploinsufficiency has been significantly associated with isolated cleft palate, led us to investigate the in vivo functions of murine Satb2. We find that, similar to the way in which SATB2 is perceived to act in humans, craniofacial defects due to haploinsufficiency of Satb2, including cleft palate (in approximately 25% of cases), phenocopy those seen with 2q32-q33 deletions and translocations in humans. Full functional loss of Satb2 results in amplification of these defects and leads both to increased apoptosis in the craniofacial mesenchyme where Satb2 is usually expressed and to changes in the pattern of expression of three genes implicated in the regulation of craniofacial development in humans and mice: Pax9, Alx4, and Msx1. The Satb2-dosage sensitivity in craniofacial development is conspicuous--along with its control of cell survival, pattern of expression, and reversible functional modification by SUMOylation, it suggests that Satb2/SATB2 function in craniofacial development may prove to be more profound than has been anticipated previously. Because jaw development is Satb2-dosage sensitive, the regulators of Satb2 expression and posttranslational modification become of critical importance both ontogenetically and evolutionarily, especially since such regulators plausibly play undetected roles in jaw and palate development and in the etiology of craniofacial malformations.

Barx1 and evolutionary changes in feeding.

During mouse embryonic development, the Barx1 homeobox gene is expressed in the mesenchymal cells of molar teeth and stomach. During early stages of molar development, Barx1 has an instructive role, directing the as yet undetermined ectomesenchymal cells in the proximal region of the jaws to follow a multicuspid tooth developmental pathway. We review here recent results showing an absence of stomach tissue in Barx1 mutant mice. The data strongly suggest that in the presumptive stomach mesenchyme Barx1 acts to attenuate Wnt signalling allowing digestive tract endoderm to differentiate into a highly specialized stomach epithelium. In the light of these new data, we discuss the possibility that evolutionary changes in the Barx1 gene could have simultaneously altered the dentition and the digestive system, therefore positioning Barx1 as a key gene in the evolution of mammals.

The stomach mesenchymal transcription factor Barx1 specifies gastric epithelial identity through inhibition of transient Wnt signaling.

Inductive interactions between gut endoderm and the underlying mesenchyme pattern the developing digestive tract into regions with specific morphology and functions. The molecular mechanisms behind these interactions are largely unknown. Expression of the conserved homeobox gene Barx1 is restricted to the stomach mesenchyme during gut organogenesis. Using recombinant tissue cultures, we show that Barx1 loss in the mesenchyme prevents stomach epithelial differentiation of overlying endoderm and induces intestine-specific genes instead. Additionally, Barx1 null mouse embryos show visceral homeosis, with intestinal gene expression within a highly disorganized gastric epithelium. Barx1 directs mesenchymal cell expression of two secreted Wnt antagonists, sFRP1 and sFRP2, and these factors are sufficient replacements for Barx1 function. Canonical Wnt signaling is prominent in the prospective gastric endoderm prior to epithelial differentiation, and its inhibition by Barx1-dependent signaling permits development of stomach-specific epithelium. These results define a transcriptional and signaling pathway of inductive cell interactions in vertebrate organogenesis.

Expression of the Hedgehog antagonists Rab23 and Slimb/betaTrCP during mouse tooth development.

The sonic hedgehog signalling peptide has been demonstrated to play an important role in the growth and patterning of several organs including the tooth. Inappropriate activation of Shh signalling in the embryo causes various patterning defects and complex regulation of this pathway is important during normal development. A growing list of diverse antagonists have been identified that restrict Shh signalling in the embryo, however, only Ptc1, Gas1 and Hip1 have been studied during tooth development. We have examined the expression pattern of the putative antagonists Rab23 and Slimb/betaTrCP during early murine odontogenesis and find that these molecules are expressed in the developing tooth. Interestingly, Rab23 demonstrates contrasting expression domains in the incisor and molar dentition during the cap stage, being restricted to the mesenchymal compartment of molar teeth and the epithelium of the enamel knot in incisor teeth. These findings provide the first evidence of distinct regulatory pathways for Shh in teeth of different classes.

Neural crest contribution to mammalian tooth formation.

The cranial neural crest cells, which are specialized cells of neural origin, are central to the process of mammalian tooth development. They are the only source of mesenchyme able to sustain tooth development, and give rise not only to most of the dental tissues, but also to the periodontium, the surrounding tissues that hold teeth in position. Tooth organogenesis is regulated by a series of interactions between cranial neural crest cells and the oral epithelium. In the development of a tooth, the epithelium covering the inside of the developing oral cavity provides the first instructive signals. Signaling molecules secreted by the oral epithelium 1) establish large cellular fields competent to form a specific tooth shape (mono- or multicuspid) along a proximodistal axis; 2) define an oral (capable of forming teeth) and non-oral mesenchyme along a rostrocaudal axis; and 3) position the sites of future tooth development. The critical information to model tooth shape resides later in the neural crest-derived mesenchyme. Cranial neural crest cells ultimately differentiate into highly specialized cell types to produce mature dental organs. Some cranial neural crest cells located in the dental pulp, however, maintain plasticity in their developmental potential up to postnatal life, offering new prospects for regeneration of dental tissues.

Restriction of sonic hedgehog signalling during early tooth development.

The signalling peptide encoded by the sonic hedgehog gene is restricted to localised thickenings of oral epithelium, which mark the first morphological evidence of tooth development, and is known to play a crucial role during the initiation of odontogenesis. We show that at these stages in the murine mandibular arch in the absence of epithelium, the Shh targets Ptc1 and Gli1 are upregulated in diastema mesenchyme, an edentulous region between the sites of molar and incisor tooth formation. This ectopic expression is not associated with Shh transcription but with Shh protein, undetectable in the presence of epithelium. These findings suggest that, in diastema mesenchyme, restriction of Shh activity is dependent upon the overlying epithelium. This inhibitory activity was demonstrated by the ability of transplanted diastema epithelium to downregulate Ptc1 in tooth explants, and for isolated diastema mesenchyme to express Ptc1. A candidate inhibitor in diastema mesenchyme is the glycosylphosphatidylinositol-linked membrane glycoprotein Gas1. Gas1 is normally expressed throughout mandibular arch mesenchyme; however, in the absence of epithelium this expression was downregulated specifically in the diastema where ectopic Shh protein was identified. Although Shh signalling has no effect upon Gas1 expression in mandibular arch mesenchyme, overexpression of Gas1 results in downregulation of ectopic Ptc1. Therefore, control of the position of tooth initiation in the mandibular arch involves a combination of Shh signalling at sites where teeth are required and antagonism in regions destined to remain edentulous.

Directed expression of the HIV-1 accessory protein Vpu in Drosophila fat-body cells inhibits Toll-dependent immune responses.

Human immunodeficiency virus 1 (HIV-1) expresses several accessory proteins that manipulate various host-cell processes to achieve optimum replicative efficiency. One of them, viral protein U (Vpu), has been shown to interfere with the cellular degradation machinery through interaction with SCF(beta-TrCP) complexes. To learn more about Vpu function in vivo, we used the genetically tractable fruit fly, Drosophila melanogaster. Our results show that the directed expression of Vpu, but not the non-phosphorylated form, Vpu2/6, in fat-body cells affects Drosophila antimicrobial responses. In flies, the Toll and Imd pathways regulate antimicrobial-peptide gene expression. We show that Vpu specifically affects Toll pathway activation by inhibiting Cactus degradation. Given the conservation of the Toll/nuclear factor-kappa B (NF-kappa B) signalling pathways between flies and mammals, our results suggest a function for Vpu in the inhibition of host NF-kappa B-mediated innate immune defences and provide a powerful genetic approach for studying Vpu inhibition of NF-kappa B signalling in vivo.

Normal and abnormal dental development.

Teeth are vertebrate organs that arise from complex and progressive interactions between an ectoderm, the oral epithelium and an underlying mesenchyme. During their early development, tooth germs exhibit many morphological and molecular similarities with other developing epithelial appendages, such as hair follicles, mammary and salivary glands, lungs, kidneys, etc. The developing mouse tooth germ, which is an experimentally accessible model for organogenesis, provides a powerful tool for elucidating the molecular mechanisms that control the development of these organs. Dentition patterning also provides a unique model for understanding how different shapes of teeth arise in different regions of the jaws. We review here the main signalling networks mediating the epithelial-mesenchymal interactions involved in tooth morphogenesis and patterning.