Cleft palate is one of the most common birth defects in humans. Whereas gene knockout studies in mice have shown that both the Osr2 and Pax9 transcription factors are essential regulators of palatogenesis, little is known about the molecular mechanisms involving these transcription factors in palate development. We report here that Pax9 plays a crucial role in patterning the anterior-posterior axis and outgrowth of the developing palatal shelves. We found that tissue-specific deletion of Pax9 in the palatal mesenchyme affected Shh expression in palatal epithelial cells, indicating that Pax9 plays a crucial role in the mesenchyme-epithelium interactions during palate development. We found that expression of the Bmp4, Fgf10, Msx1 and Osr2 genes is significantly downregulated in the developing palatal mesenchyme in Pax9 mutant embryos. Remarkably, restoration of Osr2 expression in the early palatal mesenchyme through a Pax9Osr2KI allele rescued posterior palate morphogenesis in the absence of Pax9 protein function. Our data indicate that Pax9 regulates a molecular network involving the Bmp4, Fgf10, Shh and Osr2 pathways to control palatal shelf patterning and morphogenesis.

Previous studies have suggested that Bmp4 is a key Msx1-dependent mesenchymal odontogenic signal for driving tooth morphogenesis through the bud-to-cap transition. Whereas all tooth germs were arrested at the bud stage in Msx1–/– mice, we show that depleting functional Bmp4 mRNAs in the tooth mesenchyme, through neural crest-specific gene inactivation in Bmp4f/f;Wnt1Cre mice, caused mandibular molar developmental arrest at the bud stage but allowed maxillary molars and incisors to develop to mineralized teeth. We found that expression of Osr2, which encodes a zinc finger protein that antagonizes Msx1-mediated activation of odontogenic mesenchyme, was significantly upregulated in the molar tooth mesenchyme in Bmp4f/f;Wnt1Cre embryos. Msx1 heterozygosity enhanced maxillary molar developmental defects whereas Osr2 heterozygosity partially rescued mandibular first molar morphogenesis in Bmp4f/f;Wnt1Cre mice. Moreover, in contrast to complete lack of supernumerary tooth initiation in Msx1–/–Osr2–/– mice, Osr2–/–Bmp4f/f;Wnt1Cre compound mutant mice exhibited formation and subsequent arrest of supernumerary tooth germs that correlated with downregulation of Msx1 expression in the tooth mesenchyme. In addition, we found that the Wnt inhibitors Dkk2 and Wif1 were much more abundantly expressed in the mandibular than maxillary molar mesenchyme in wild-type embryos and that Dkk2 expression was significantly upregulated in the molar mesenchyme in Bmp4f/f;Wnt1Cre embryos, which correlated with the dramatic differences in maxillary and mandibular molar phenotypes in Bmp4f/f;Wnt1Cre mice. Together, these data indicate that Bmp4 signaling suppresses tooth developmental inhibitors in the tooth mesenchyme, including Dkk2 and Osr2, and synergizes with Msx1 to activate mesenchymal odontogenic potential for tooth morphogenesis and sequential tooth formation.

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Slow vascularization rate is considered one of the main drawbacks of scaffolds used in wound healing. Several efforts, including cellular and acellular technologies, have been made to induce vascular growth in scaffolds. However, thus far, there is no established technology for inducing vascular growth. The aim of this study was to promote the vascularization capacities of scaffolds by seeding adipose-derived stem cells (ADSCs) on them and to compare the vascularization capacities of different scaffolds seeded with ADSCs. Two kinds of extracellular matrix scaffolds (small intestinal submucosa [SIS] and acellular dermal matrix [ADM]) and a kind of composite scaffold (collagen-chondroitin sulfate-hyaluronic acid [Co-CS-HA]) were selected. Subcutaneous implantation analysis showed that the vascularization capacity of SIS and ADM was greater than that of Co-CS-HA. ADSCs seeded in SIS and ADM secreted greater amounts of vascular endothelial growth factor than those seeded in Co-CS-HA. In a murine skin injury model, ADSC-seeded scaffolds enhanced the angiogenesis and wound healing rate compared with the nonseeded scaffolds. Moreover, ADSC-SIS and ADSC-ADM had greater vascularization capacity than that of ADSC-Co-CS-HA. Taken together, these results suggest that ADSCs could be used as a cell source to promote the vascularization capacities of scaffolds. The vascularization capacities of ADSC-seeded scaffolds were influenced by both the vascularization capacities of the scaffolds themselves and their effects on the angiogenic potential of ADSCs; the combination of extracellular matrix scaffolds and ADSCs exhibited synergistic angiogenesis promoting effects.

Clefts of the palate and/or lip are among the most common human craniofacial malformations and involve multiple genetic and environmental factors. Defects can only be corrected surgically and require complex life-long treatments. Our studies utilized the well-characterized mouse model with a consistent cleft palate phenotype to test small-molecule Wnt agonist therapies. We show that the absence of Pax9 alters the expression of Wnt pathway genes including and , proven antagonists of Wnt signaling. The functional interactions between Pax9 and Dkk1 are shown by the genetic rescue of secondary palate clefts in embryos. The controlled intravenous delivery of small-molecule Wnt agonists (Dkk inhibitors) into pregnant mice restored Wnt signaling and led to the growth and fusion of palatal shelves, as marked by an increase in cell proliferation and osteogenesis, while other organ defects were not corrected. This work underscores the importance of Pax9-dependent Wnt signaling in palatogenesis and suggests that this functional upstream molecular relationship can be exploited for the development of therapies for human cleft palates that arise from single-gene disorders.

In Streaming Feature Selection (SFS), new features are sequentially considered for addition to a predictive model. When the space of potential features is large, SFS offers many advantages over traditional feature selection methods, which assume that all features are known in advance. Features can be generated dynamically, focusing the search for new features on promising subspaces, and overfitting can be controlled by dynamically adjusting the threshold for adding features to the model. We describe α-investing, an adaptive complexity penalty method for SFS which dynamically adjusts the threshold on the error reduction required for adding a new feature. α-investing gives false discovery ratestyle guarantees against overfitting. It differs from standard penalty methods such as AIC, BIC or RIC, which always drastically over-or under-fit in the limit of infinite numbers of non-predictive features. Empirical results show that SFS is competitive with much more compute-intensive feature selection methods such as stepwise regression, and allows feature selection on problems with over a million potential features.

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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 locus 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.