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Anterior foregut endoderm (AFE) gives rise to therapeutically relevant cell types in tissues such as the esophagus, salivary glands, lung, thymus, parathyroid and thyroid. Despite its importance, reports describing the generation of AFE from pluripotent stem cells (PSCs) by directed differentiation have mainly focused on the Nkx2.1+ lung and thyroid lineages. Here, we describe a novel protocol to derive a subdomain of AFE, identified by expression of Pax9, from PSCs using small molecules and defined media conditions. We generated a reporter PSC line for isolation and characterization of Pax9+ AFE cells, which when transplanted in vivo, can form several distinct complex AFE-derived epithelia, including mucosal glands and stratified squamous epithelium. Finally, we show that the directed differentiation protocol can be used to generate AFE from human PSCs. Thus, this work both broadens the range of PSC-derived AFE tissues and creates a platform enabling the study of AFE disorders.

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.

Mammalian tooth development depends on activation of odontogenic potential in the presumptive dental mesenchyme by the Msx1 and Pax9 transcription factors. We recently reported that the zinc finger transcription factor Osr2 was expressed in a lingual-to-buccal gradient pattern surrounding the developing mouse molar tooth germs and mice lacking Osr2 developed supernumerary teeth lingual to their molars. We report here generation of a gene-targeted mouse strain that allows conditional inactivation of Pax9 and subsequent activation of expression of Osr2 in the developing tooth mesenchyme from the Pax9 locus. Expression of Osr2 from one copy of the Pax9 gene did not disrupt normal tooth development but was sufficient to suppress supernumerary tooth formation in the Osr2−/− mutant mice. We found that endogenous Osr2 mRNA expression was significantly downregulated in the developing tooth mesenchyme in Pax9del/del mice. Mice lacking both Osr2 and Pax9 exhibited early tooth developmental arrest with significantly reduced Bmp4 and Msx1 mRNA expression in the developing tooth mesenchyme, similar to that in Pax9del/del mutants but in contrast to the rescue of tooth morphogenesis in Msx1−/−Osr2−/− double mutant mice. Furthermore, we found that Osr2 formed stable protein complexes with the Msx1 protein and interacted weakly with the Pax9 protein in co-transfected cells. These data indicate that Osr2 acts downstream of Pax9 and patterns the mesenchymal odontogenic field through protein–protein interactions with Msx1 and Pax9 during early tooth development.

The variation in mandibular morphology of mammals reflects specialisations for different diets. Omnivorous and carnivorous mammals posses large mandibular coronoid processes, while herbivorous mammals have proportionally smaller or absent coronoids. This is correlated with the relative size of the temporalis muscle that forms an attachment to the coronoid process. The role of this muscle attachment in the development of the variation of the coronoid is unclear. By comparative developmental biology and mouse knockout studies, we demonstrate here that the initiation and growth of the coronoid are two independent processes, with initiation being intrinsic to the ossifying bone and growth dependent upon the extrinsic effect of muscle attachment. A necessary component of the intrinsic patterning is identified as the paired domain transcription factor Pax9. We also demonstrate that Sox9 plays a role independent of chondrogenesis in the growth of the coronoid process in response to muscle interaction. The mandibular coronoid process is initiated by intrinsic factors, but later growth is dependent on extrinsic signals from the muscle. These extrinsic influences are hypothesised to be the basis of the variation in coronoid length seen across the mammalian lineage.

Background: Tooth agenesis is one of the most common developmental anomalies in human and the main reasons for its occurrence are still unknown. Mutations of several genes such as PAX9, MSX1, AXIN2, KDF1 and WNT10A have been reported which are associated with non-syndromic tooth agenesis. However, PAX9, MSX1 and WNT10A are commonly reported in the literature. Hence, the aim of this study was to investigate the mutations of these genes in 4 Iranian families with non-syndromic tooth agenesis. Methods: DNA extractions from peripheral blood cells of patients with non-syndromic tooth agenesis from 4 unrelated Iranian families were performed by salting out method, and the candidate genes were amplified then followed by Sanger sequencing method. Results: One missense variant (rs4904210) and 4 Single Nucleotide Polymorphisms (SNPs) (rs2236007, rs12883298, rs12882923 and rs12883049) were found in PAX9 gene. Five variants (rs149370601, rs8670, rs186861426 and rs774949973) including a missense variant (rs36059701) were detected in MSX1 gene and no variants were found in WNT10A gene. Conclusion: All variants were analyzed based on bioinformatics websites and Iranian gene databases, and as a result, it was revealed that variants of PAX9, MSX1 and WNT10A may not play a role in non-syndromic tooth agenesis among Iranian cases.

The expression of the homeobox gene Uncx4.1 in the somite is restricted to the caudal half of the newly formed somite and sclerotome. Here we show that mice with a targeted mutation of the Uncx4.1 gene exhibit defects in the axial skeleton and ribs. In the absence of Uncx4.1, pedicles of the neural arches and proximal ribs are not formed. In addition, dorsal root ganglia are disorganized. Histological and marker analysis revealed that Uncx4.1 is not necessary for somite segmentation. It is required to maintain the condensation of the caudal half-sclerotome, from which the missing skeletal elements are derived. The loss of proximal ribs in Pax1/Pax9 double mutants and the data presented here argue for a role of Uncx4.1 upstream of Pax9 in the caudolateral sclerotome. Our results further indicate that Uncx4.1 may be involved in the differential cell adhesion properties of the somite.

Editor—Hypodontia, congenitally missing teeth, is common in modern man. The teeth most often missing in populations of European origin are the upper lateral incisors and second premolars. The condition is known to have a strong genetic component. At present two mutated genes in humans, MSX1 1 and PAX9 ,2 are known to cause missing permanent teeth. Mutations in MSX1 can also cause orofacial clefting.3 Several experimental and clinical studies indicate that other genetic components are also involved.4-8 Hypodontia is also often seen in syndromes, particularly in those which present with other ectodermal anomalies,9 and in non-syndromic patients with cleft lip/alveolus with or without cleft palate. The population prevalence of the common incisor-premolar hypodontia (IPH, MIM 106600) is 8-10% in healthy European children. Some or all third molar teeth are missing in one-fifth of the population,10 and missing third molars are seen in varying combinations in IPH patients and/or family members.11Family studies also indicate that peg shaped upper lateral incisors, impacted canines, rotated bicuspids, and short root anomaly (SRA) are caused by the same genetic components that cause missing incisors and premolars.12-15 The condition is inherited as an autosomal dominant trait16 with reduced penetrance and is mostly restricted to the permanent dentition. When a large number of teeth (>6) are congenitally missing, the term used is oligodontia (MIM 6044625). The prevalence of oligodontia in European populations is estimated at 0.08%,17 but this figure also includes syndromic patients. We describe 37 Finnish patients from 34 families with several lower incisors and upper lateral permanent incisors congenitally missing. In half of the patients, the corresponding deciduous teeth had either been missing or peg shaped. An atopic condition had been diagnosed in two thirds of the patients. Occurrence of the trait within …

Objective: Tooth loss is a common problem and since current tooth replacement methods cannot counter balance with biological tooth structures, regenerating natural tooth structures has become an ideal goal. A challenging problem in tooth regeneration is to find a proper clinically feasible cell to seed.This study was designed to investigate the odontogenic potential of human bone marrow mesenchymal stem cells (HBMSCs) for seeding in tooth regeneration. Materials and Methods: In this experimental study, three pregnant Sprague Dawley (SD) rats were used at the eleventh embryonic day and rat fetuses were removed surgically using semilunar flap under general anesthesia. The primary mandible was cut using a stereomicroscope. The epithelial and mesenchymal components were separated and the dissected oral epithelium was cultured for 3 days. We used flow cytometry analysis to confirm presence of mesenchymal stem cells and not hematopoietic cells and to demonstrate the presence of oral epithelium. Bone marrow mesenchymal stem cells (BMSCs) and cultured oral epithelium were then co-cultured for 14 days. BMSCs cultured alone were used as controls. Expression of two odontogenic genes Pax9 and DMP1 was assessed using quantitative reverse transcription- polymerase chain reaction (RT-PCR). Results: Expression of two odontogenic genes, Pax9 and DMP1, were detected in BMSCs co-cultured with oral epithelium but not in the control group. Conclusion: Expression of Pax9 and DMP1 by human BMSCs in the proximity of odontogenic epithelium indicates odontogenic potential of these cells.