Ciliary and non-ciliary functions of Rab34 during craniofacial bone development.

The primary cilium is a hair-like projection that controls cell development and tissue homeostasis. Although accumulated studies identify the molecular link between cilia and cilia-related diseases, the underlying etiology of ciliopathies has not been fully understood. In this paper, we determine the function of Rab34, a small GTPase, as a key regulator for controlling ciliogenesis and type I collagen trafficking in craniofacial development. Mechanistically, Rab34 is required to form cilia that control osteogenic proliferation, survival, and differentiation via cilia-mediated Hedgehog signaling. In addition, Rab34 is indispensable for regulating type I collagen trafficking from the ER to the Golgi. These results demonstrate that Rab34 has both ciliary and non-ciliary functions to regulate osteogenesis. Our study highlights the critical function of Rab34, which may contribute to understanding the novel etiology of ciliopathies that are associated with the dysfunction of RAB34 in humans.

IFT20 is critical for collagen biosynthesis in craniofacial bone formation.

Intraflagellar transport (IFT) is essential for assembling primary cilia required for bone formation. Disruption of IFT frequently leads to bone defects in humans. While it has been well studied about the function of IFT in osteogenic cell proliferation and differentiation, little is known about its role in collagen biosynthesis during bone formation. Here we show that IFT20, the smallest IFT protein in the IFT-B complex, is important for collagen biosynthesis in mice. Deletion of Ift20 in craniofacial osteoblasts displayed bone defects in the face. While collagen protein levels are unaffected by loss of Ift20, collagen cross-linking was significantly altered. In both Ift20:Wnt1-Cre and Ift20:Ocn-Cre mice the bones exhibit increased hydroxylysine-aldehyde deived cross-linking, and decreased lysine-aldehyde derived cross-linking. To obtain insight into the molecular mechanisms, we examined the expression levels of telopeptidyl lysyl hydroxylase 2 (LH2), and associated chaperone complexes. The results demonstrated that, while LH2 levels were unaffected by loss of Ift20, its chaperone, FKBP65, was significantly increased in Ift20:Wnt1-Cre and Ift20:Ocn-Cre mouse calvaria as well as femurs. These results suggest that IFT20 plays a pivotal role in collagen biosynthesis by regulating, in part, telopeptidyl lysine hydroxylation and cross-linking in bone. To the best of our knowledge, this is the first to demonstrate that the IFT components control collagen post-translational modifications. This provides a novel insight into the craniofacial bone defects associated with craniofacial skeletal ciliopathies.

Dynamic changes of facial skeletal fractures with time.

To investigate the characteristics of imaging changes with time of facial fractures, patients with facial fractures who had computed tomographic scan were enrolled including 500 patients who were divided into six groups based on the time of scanning: super early (<3 d), early (4-7 d), early-to-medium (8-14 d), medium (15-21d), medium-to-late (22d-2 months) and late stage (>2 months). The data were compared and analyzed. Forty two patients with frontal bone fractures had high-energy impact as the reason of fractures. The fracture line was clear and sharp within one week but blunt and sclerotic due to bone absorption at 2-3 weeks, and might exist for a long time. All patients had soft tissue swelling and paranasal sinus effusion at 1-2 weeks after injury. Air might gather in the adjacent soft tissues and/or intracranially within 3 days of injury if the fracture involved the frontal or other sinuses. Twelve of the 42 patients (28.6%) had intracranial hematoma, and five (11.9%) had epidural effusion. Subarachnoid hemorrhage was mostly absorbed within one week while epidural hematoma was completely absorbed over 3 weeks. Significant changes (P < 0.05) in the fracture lines, effusion of paranasal sinuses, soft tissue swelling and pneumocephalus were observed during the study period. For patients with medial orbital wall fractures, the fracture line was sharp and clear at early stages with concurrent sphenoid sinus effusion, and the fracture line became depressed 3 weeks later with disappearance of sphenoid sinus effusion. Significant changes (P < 0.05) were observed in the sharp fracture line, soft tissue swelling, sphenoid sinus effusion and smooth depression at fracture sites. For nasal fractures, the fracture line was sharp and clear at early stages with concurrent soft tissue swelling which disappeared one week later. The fracture line became smooth three weeks later. A significant (P < 0.05) difference was demonstrated in the changes of fracture line and soft tissue swelling with time. In conclusion, facial fractures have some dynamic alterations with time and identification of these characteristics may help reaching a correct clinical diagnosis with regard to fracture severity and time.

Quercetin Decreased Alveolar Bone Loss and Apoptosis in Experimentally Induced Periodontitis Model in Wistar Rats.

BACKGROUND: Quercetin is a flavonoid which has potent anti-inflammatory, antibacterial, and antioxidant effect. Purpose of this study was to evaluate effects of quercetin on alveolar bone loss and histopathological changes in ligature-induced periodontitis in rats. METHODS: Wistar rats were divided into four experimental groups: non-ligated control (C, n=8) group; periodontitis (P, n=8) group; ligature and low dose quercetin group (75 mg/kg/day quercetin, Q75 group, n=8); ligature and high dose quercetin group (150 mg/kg/day quercetin, Q150 group, n=8). Silk ligatures were placed at gingival margin of lower first molars of mandibular right quadrant. Study duration was 15 days, and animals were sacrificed end of this period. Changes in alveolar bone levels were clinically measured and tissues were immunohistochemically examined, matrix metalloproteinase 8 (MMP 8), inducible nitric oxide synthase (iNOS), tissue inhibitor of metalloproteinase 1 (TIMP 1), Cysteine-aspartic proteases 3 (Caspase 3), and tartrate-resistant acid phosphatase (TRAP) positive osteoclast cells, osteoblast, and neutrophil counts were also determined. RESULTS AND DISCUSSION: Alveolar bone loss was highest in P group, and differences among P, Q75, and Q150 groups were significant. Both doses of quercetin decreased TRAP+ osteoclast cells and increased osteoblast cells. Inflammation in P group was also higher than those of C, Q75, and Q150 groups indicating anti-inflammatory effect of quercetin. iNOS, MMP-8, and caspase-3 levels were highest, and TIMP-1 expression was lowest in P group; differences were statistically significant. CONCLUSION: Within limits of this study, it can be suggested that quercetin administration may reduce alveolar bone loss by increasing osteoblastic activity, decreasing osteoclastic activity, apoptosis, and inflammation in an experimental model of periodontitis.

Osteoimmunology of Oral and Maxillofacial Diseases: Translational Applications Based on Biological Mechanisms.

The maxillofacial skeleton is highly dynamic and requires a constant equilibrium between the bone resorption and bone formation. The field of osteoimmunology explores the interactions between bone metabolism and the immune response, providing a context to study the complex cellular and molecular networks involved in oro-maxillofacial osteolytic diseases. In this review, we present a framework for understanding the potential mechanisms underlying the immuno-pathobiology in etiologically-diverse diseases that affect the oral and maxillofacial region and share bone destruction as their common clinical outcome. These otherwise different pathologies share similar inflammatory pathways mediated by central cellular players, such as macrophages, T and B cells, that promote the differentiation and activation of osteoclasts, ineffective or insufficient bone apposition by osteoblasts, and the continuous production of osteoclastogenic signals by immune and local stromal cells. We also present the potential translational applications of this knowledge based on the biological mechanisms involved in the inflammation-induced bone destruction. Such applications can be the development of immune-based therapies that promote bone healing/regeneration, the identification of host-derived inflammatory/collagenolytic biomarkers as diagnostics tools, the assessment of links between oral and systemic diseases; and the characterization of genetic polymorphisms in immune or bone-related genes that will help diagnosis of susceptible individuals.

Cis-control of Six1 expression in neural crest cells during craniofacial development.

BACKGROUND: Six1 is a transcriptional factor that plays an important role in embryonic development. Mouse and chick embryos deficient for Six1 have multiple craniofacial anomalies in the facial bones and cartilages. Multiple Six1 enhancers have been identified, but none of them has been reported to be active in the maxillary and mandibular process. RESULTS: We studied two Six1 enhancers in the chick neural crest tissues during craniofacial development. We showed that two evolutionarily conserved enhancers, Six1E1 and Six1E2, act synergistically. Neither Six1E1 nor Six1E2 alone can drive enhancer reporter signal in the maxillary or mandibular processes. However, their combination, Six1E, showed robust enhancer activity in these tissues. Similar reporter signal can also be driven by the mouse homolog of Six1E. Mutations of multiple conserved transcriptional factor binding sites altered the enhancer activity of Six1E, especially mutation of the LIM homeobox binding site, dramatically reduced the enhancer activity, implying that the Lhx protein family be an important regulator of Six1 expression. CONCLUSION: This study, for the first time, described the synergistic activation of two Six1 enhancers in the maxillary and mandibular processes and will facilitate more detailed studies of the regulation of Six1 in craniofacial development.

MOZ directs the distal-less homeobox gene expression program during craniofacial development.

Oral clefts are common birth defects. Individuals with oral clefts who have identical genetic mutations regularly present with variable penetrance and severity. Epigenetic or chromatin-mediated mechanisms are commonly invoked to explain variable penetrance. However, specific examples of these are rare. Two functional copies of the MOZ (KAT6A, MYST3) gene, encoding a MYST family lysine acetyltransferase chromatin regulator, are essential for human craniofacial development, but the molecular role of MOZ in this context is unclear. Using genetic interaction and genomic studies, we have investigated the effects of loss of MOZ on the gene expression program during mouse development. Among the more than 500 genes differentially expressed after loss of MOZ, 19 genes had previously been associated with cleft palates. These included four distal-less homeobox (DLX) transcription factor-encoding genes, Dlx1, Dlx2, Dlx3 and Dlx5 and DLX target genes (including Barx1, Gbx2, Osr2 and Sim2). MOZ occupied the Dlx5 locus and was required for normal levels of histone H3 lysine 9 acetylation. MOZ affected Dlx gene expression cell-autonomously within neural crest cells. Our study identifies a specific program by which the chromatin modifier MOZ regulates craniofacial development.

Whole-exome sequencing identified four loci influencing craniofacial morphology in northern Han Chinese.

Facial shape differences are one of the most significant phenotypes in humans. It is affected largely by skull shape. However, research into the genetic basis of the craniofacial morphology has rarely been reported. The present study aimed to identify genetic variants influencing craniofacial morphology in northern Han Chinese through whole-exome sequencing (WES). Phenotypic data of the volunteers' faces and skulls were obtained through three-dimensional CT scan of the skull. A total of 48 phenotypes (35 facial and 13 cranial phenotypes) were used for the bioinformatics analysis. Four genetic loci were identified affecting the craniofacial shapes. The four candidate genes are RGPD3, IGSF3, SLC28A3, and USP40. Four single-nucleotide polymorphism (SNP) site mutations in RGPD3, IGSF3, and USP40 were significantly associated with the skull shape (p < 1×10-6), and three SNP site mutations in RGPD3, IGSF3, and SLC28A3 were significantly associated with the facial shape (p < 1×10-6). The rs62152530 site mutation in the RGPD3 gene may be closely associated with the nasal length, ear length, and alar width. The rs647711 site mutation in the IGSF3 gene may be closely associated with the nasal length, mandibular width, and width between the mental foramina. The rs10868138 site mutation in the SLC28A3 gene may be associated with the nasal length, alar width, width between tragus, and width between the mental foramina. The rs1048603 and rs838543 site mutations in the USP40 gene may be closely associated with the pyriform aperture width. Our findings provide useful genetic information for the determination of face morphology.

Differential growth of craniofacial and tibial bones to sympathetic hyperactivity-related hypertension in rats.

OBJECTIVE: To evaluate the effect of sympathetic nervous system hyperactivity on craniofacial skeletal growth in growing spontaneously hypertensive rats (SHRs). DESIGN: Craniofacial skeletal growth was compared between male SHR and Wistar-Kyoto rats (WKR) using linear measurements on lateral and transverse cephalometric radiographs at the age of 12 weeks. Tibia length was measured as an index of whole body growth. Body weight　and blood pressure were measured from 3 to 12 weeks of age. Bone microstructure in the mandibular condyle and tibia between the two groups was compared at the age of 12 weeks using microcomputed tomography. RESULTS: The SHRs had a significantly lower body weight than WKRs from 7 weeks of age, and tibial length was significantly smaller in the SHRs than in the WKR at 12 weeks of age. In all SHRs, blood pressure was significantly higher than in WKRs from 3 to 12 weeks of age. Cephalometric analyses revealed decreased measurements of the neurocranium, viscerocranium, and mandible in SHRs, and mandibular growth was most negatively affected in this group. Lastly, in SHRs, microcomputed tomography analyses revealed decreased bone mineral density and bone volume/tissue volume in the mandibular condyle but not in the tibia. CONCLUSION: In growing SHRs, hypertension related to the hyperactivity of the sympathetic nervous system reduced craniofacial skeletal growth more than the growth of the tibia.

Evolvability of the vertebrate craniofacial skeleton.

The skull is a vertebrate novelty. Morphological adaptations of the skull are associated with major evolutionary transitions, including the shift to a predatory lifestyle and the ability to masticate while breathing. These adaptations include the chondrocranium, dermatocranium, articulated jaws, primary and secondary palates, internal choanae, the middle ear, and temporomandibular joint. The incredible adaptive diversity of the vertebrate skull indicates an underlying bauplan that promotes evolvability. Comparative studies in craniofacial development suggest that the craniofacial bauplan includes three secondary organizers, two that are bilaterally placed at the Hinge of the developing jaw, and one situated in the midline of the developing face (the FEZ). These organizers regulate tissue interactions between the cranial neural crest, the neuroepithelium, and facial and pharyngeal epithelia that regulate the development and evolvability of the craniofacial skeleton.

Signals from the brain and olfactory epithelium control shaping of the mammalian nasal capsule cartilage.

Facial shape is the basis for facial recognition and categorization. Facial features reflect the underlying geometry of the skeletal structures. Here, we reveal that cartilaginous nasal capsule (corresponding to upper jaw and face) is shaped by signals generated by neural structures: brain and olfactory epithelium. Brain-derived Sonic Hedgehog (SHH) enables the induction of nasal septum and posterior nasal capsule, whereas the formation of a capsule roof is controlled by signals from the olfactory epithelium. Unexpectedly, the cartilage of the nasal capsule turned out to be important for shaping membranous facial bones during development. This suggests that conserved neurosensory structures could benefit from protection and have evolved signals inducing cranial cartilages encasing them. Experiments with mutant mice revealed that the genomic regulatory regions controlling production of SHH in the nervous system contribute to facial cartilage morphogenesis, which might be a mechanism responsible for the adaptive evolution of animal faces and snouts.

GATA4 as a novel regulator involved in the development of the neural crest and craniofacial skeleton via Barx1.

The role of GATA-binding protein 4 (GATA4) in neural crest cells (NCCs) is poorly defined. Here we showed that mouse NCCs lacking GATA4 exhibited developmental defects in craniofacial bone, teeth, and heart. The defects likely occurred due to decreased cell proliferation at the developmental stage. The in vitro results were consistent with the mouse model. The isobaric tags for relative and absolute quantitation assay revealed that BARX1 is one of the differentially expressed proteins after GATA4 knockdown in NCCs. On the basis of the results of dual-luciferase, electro-mobility shift, and chromatin immunoprecipitation assays, Barx1 expression is directly regulated by GATA4 in NCCs. In zebrafish, gata4 knockdown affects the development of NCCs derivatives. However, the phenotype in zebrafish could be partly rescued by co-injection of gata4 morpholino oligomers and barx1 mRNA. This study identified new downstream targets of GATA4 in NCCs and uncovered additional evidence of the complex regulatory functions of GATA4 in NCC development.

Importance of deubiquitinases in zebrafish craniofacial development.

Deconjugation of ubiquitin and/or ubiqutin-like modified substrates is essential to maintain a sufficient free ubiquitin within the cell. Deubiquitinases (DUBs) play a key role in the process. Besides, DUBs also play several important regulatory roles in cellular processes. However, our knowledge of their developmental roles are limited. The report here aims to study their potential roles in craniofacial development. Based on the previous genome-wide study in 2009, we selected 36 DUBs to perform the morpholino (MO) knockdown in this study, followed by the Alcian blue cartilage staining at 5 days post-fertilization (dpf) larvae to investigate the facial development. Results classified the tested DUBs into three groups, in which 28% showed unchanged phenotype (Class 1); 22% showed mild changes on the branchial arches (Class 2A); 31% had malformation on branchial arches and ethmoid plate (Class 2B); and 19% had severe changes in most of the facial structures (Class 3). Lastly, we used uchl3 morphant as an example to show that our screening data could be useful for further functional studies. To summarize, we identified new craniofacial developmental role of 26 DUBs in the zebrafish.

Expression of Dlx-5 and Msx-1 in Craniofacial Skeletons and Ilia of Rats Treated With Zoledronate.

PURPOSE: Because of the different embryologic origins of the craniofacial skeleton and ilium, differences in gene expression patterns have been observed between the jaw bones and ilium. Distal-less homeobox (Dlx) genes and Msh homeobox genes, particularly Dlx-5 and Msx-1, play major roles in cell differentiation and osteogenesis. The purpose of this study was to investigate the effects of zoledronate (ZOL) on the craniofacial skeleton and ilium by detecting changes in Dlx-5 and Msx-1 expression at both the protein and messenger RNA levels. MATERIALS AND METHODS: A total of 24 female Sprague-Dawley rats were randomly divided into 2 groups: ZOL group (n = 12), in which the rats were injected intraperitoneally with zoledronic acid for 12 weeks, and control group (n = 12), in which the rats were injected with saline solution for 12 weeks. By use of immunohistochemistry, Western blotting, and real-time reverse transcription polymerase chain reaction, the expression levels of Dlx-5 and Msx-1 in the craniofacial skeleton (including the maxilla, mandible, and parietal bone) and ilium were examined. RESULTS: Dlx-5 expression in the maxilla and mandible was increased at the protein and messenger RNA levels in the ZOL group compared with the control group (P < .01). In addition, Msx-1 expression in the maxilla and mandible was decreased in the ZOL group (P < .01). Furthermore, Dlx-5 and Msx-1 expression in the ilium was decreased in the ZOL group (P < .05). However, no significant difference in Dlx-5 or Msx-1 expression in the parietal bone was observed between the 2 groups (P > .05). CONCLUSIONS: Site-specific differences in the effects of ZOL on the craniofacial skeleton and ilium could be explained by differently altered tendencies in Dlx-5 and Msx-1 expression. The jaw bones were more susceptible to the effects of ZOL than the parietal bone and ilium.

Skeletal Changes of an Osteomyocutaneous Facial Allograft Five Years Following Transplantation.

BACKGROUND: More than 30 face transplantations have been performed worldwide, most including part of the facial skeletal framework. In this study, the modifications of the skeletal component of a facial allograft were evaluated. METHODS: Standard head computed tomography (CT) scans, CT angiogram, and bone mineral densitometry were evaluated. Cephalometric analysis was performed. The pre and postoperative CT images were overlapped and the skeletal changes were expressed in a numeric and color-coded scale. The values of the serum calcium, phosphate, vitamin D, alkaline phosphatase, thyroid and parathyroid hormones, TSH, FHS, LH, estradiol, total protein and albumin, serum creatinine, and creatinine clearance were reviewed. RESULTS: At 5 years follow-up the patient was 51 years old, asymptomatic and presented good stability of the Le Fort III component of the allograft. Computed tomography images revealed fibrous union of all fixation sites. There was minimal bone resorption at the osteotomy sites, left infraorbital rim and left maxillary buttress, and anterior maxilla (-0.28 mm). Computed tomography angiogram showed segmental absence at the origin of the left external carotid artery, good opacification of the rest of the external carotid arteries and its branches. Bone mineral densitometry evidenced osteopenia of the spine. The patient presented mild hypoalbuminemia (3.4 g/dL) and perimenopausal hormonal levels. CONCLUSIONS: The skeletal component of the facial allograft was stable over time. Minimal bone resorption was discovered at the level of the left infraorbital rim and anterior maxilla. Transplantation of bone within the facial allograft is a viable reconstructive option.

Ellis Van Creveld2 is Required for Postnatal Craniofacial Bone Development.

Ellis-van Creveld (EvC) syndrome is a genetic disorder with mutations in either EVC or EVC2 gene. Previous case studies reported that EvC patients underwent orthodontic treatment, suggesting the presence of craniofacial bone phenotypes. To investigate whether a mutation in EVC2 gene causes a craniofacial bone phenotype, Evc2 knockout (KO) mice were generated and cephalometric analysis was performed. The heads of wild type (WT), heterozygous (Het) and homozygous Evc2 KO mice (1-, 3-, and 6-week-old) were prepared and cephalometric analysis based on the selected reference points on lateral X-ray radiographs was performed. The linear and angular bone measurements were then calculated, compared between WT, Het and KO and statistically analyzed at each time point. Our data showed that length of craniofacial bones in KO was significantly lowered by ∼20% to that of WT and Het, the growth of certain bones, including nasal bone, palatal length, and premaxilla was more affected in KO, and the reduction in these bone length was more significantly enhanced at later postnatal time points (3 and 6 weeks) than early time point (1 week). Furthermore, bone-to-bone relationship to cranial base and cranial vault in KO was remarkably changed, i.e. cranial vault and nasal bone were depressed and premaxilla and mandible were developed in a more ventral direction. Our study was the first to show the cause-effect relationship between Evc2 deficiency and craniofacial defects in EvC syndrome, demonstrating that Evc2 is required for craniofacial bone development and its deficiency leads to specific facial bone growth defect. Anat Rec, 299:1110-1120, 2016. © 2016 Wiley Periodicals, Inc.

Testicular receptor 2, Nr2c1, is associated with stem cells in the developing olfactory epithelium and other cranial sensory and skeletal structures.

Comparative genomic analysis of the nuclear receptor family suggests that the testicular receptor 2, Nr2c1, undergoes positive selection in the human-chimpanzee clade based upon a significant increase in nonsynonymous compared to synonymous substitutions. Previous in situ analyses of Nr2c1 lacked the temporal range and spatial resolution necessary to characterize cellular expression of this gene from early to mid gestation, when many nuclear receptors are key regulators of tissue specific stem or progenitor cells. Thus, we asked whether Nr2c1 protein is associated with stem cell populations in the mid-gestation mouse embryo. Nr2c1 is robustly expressed in the developing olfactory epithelium. Its expression in the olfactory epithelium shifts from multiple progenitor classes at early stages to primarily transit amplifying cells later in olfactory epithelium development. In the early developing central nervous system, Nr2c1 is limited to the anterior telencephalon/olfactory bulb anlagen, coincident with Nestin-positive neuroepithelial stem cells. Nr2c1 is also seen in additional cranial sensory specializations including cells surrounding the mystacial vibrissae, the retinal pigment epithelium and Scarpa's ganglion. Nr2c1 was also detected in a subset of mesenchymal cells in developing teeth and cranial bones. The timing and distribution of embryonic expression suggests that Nr2c1 is primarily associated with the early genesis of mammalian cranial sensory neurons and craniofacial skeletal structures. Thus, Nr2c1 may be a candidate for mediating parallel adaptive changes in cranial neural sensory specializations such as the olfactory epithelium, retina and mystacial vibrissae and in non-neural craniofacial features including teeth.

Regulating Craniofacial Development at the 3' End: MicroRNAs and Their Function in Facial Morphogenesis.

Defects in craniofacial development represent a majority of observed human birth defects, occurring at a rate as high as 1:800 live births. These defects often occur due to changes in neural crest cell (NCC) patterning and development and can affect non-NCC-derived structures due to interactions between NCCs and the surrounding cell types. Proper craniofacial development requires an intricate array of gene expression networks that are tightly controlled spatiotemporally by a number of regulatory mechanisms. One of these mechanisms involves the action of microRNAs (miRNAs), a class of noncoding RNAs that repress gene expression by binding to miRNA recognition sequences typically located in the 3' UTR of target mRNAs. Recent evidence illustrates that miRNAs are crucial for vertebrate facial morphogenesis, with changes in miRNA expression leading to facial birth defects, including some in complex human syndromes such as 22q11 (DiGeorge Syndrome). In this review, we highlight the current understanding of miRNA biogenesis, the roles of miRNAs in overall craniofacial development, the impact that loss of miRNAs has on normal development and the requirement for miRNAs in the development of specific craniofacial structures, including teeth. From these studies, it is clear that miRNAs are essential for normal facial development and morphogenesis, and a potential key in establishing new paradigms for repair and regeneration of facial defects.

YPEL1 overexpression in early avian craniofacial mesenchyme causes mandibular dysmorphogenesis by up-regulating apoptosis.

BACKGROUND: The YPEL (Yippee-like) gene family comprises five highly conserved members (YPEL1-5), but their biological function remains largely unknown. Early studies of YPEL1 function suggested that it plays a role in the development of structures derived from the pharyngeal arches. Human YPEL1 localises to distal chromosome 22q11.2 and copy number changes at this locus lead to diverse phenotypes that include facial dysmorphism, facial asymmetry, and palatal anomalies comprising the distal 22q11.2 deletion/duplication syndromes (OMIM 611867). We therefore investigated the role of chick YPEL1 in craniofacial development using ex vivo and in vivo approaches in the avian model. RESULTS: We found that retroviral-mediated in vivo overexpression of YPEL1 causes abnormal mandibular morphogenesis associated with increased apoptosis and involvement of the BMP/MSX pathway. CONCLUSIONS: Our results suggest that YPEL1 expression is regulated by bone morphogenetic protein signaling and suggest a role for YPEL1 in the pathogenesis of the craniofacial abnormalities observed in humans with distal chromosome 22q11.2 deletions or duplications.

Epigenetically Modified Bone Marrow Stromal Cells in Silk Scaffolds Promote Craniofacial Bone Repair and Wound Healing.

Epigenetic regulation of gene expression is a central mechanism that governs cell stemness, determination, commitment, and differentiation. It has been recently found that PHF8, a major H4K20/H3K9 demethylase, plays a critical role in craniofacial and bone development. In this study, we hypothesize that PHF8 promotes osteoblastogenesis by epigenetically regulating the expression of a nuclear matrix protein, special AT-rich sequence-binding protein 2 (SATB2) that plays pivotal roles in skeletal patterning and osteoblast differentiation. Our results showed that expression levels of PHF8 and SATB2 in preosteoblasts and bone marrow stromal cells (BMSCs) increased simultaneously during osteogenic induction. Overexpressing PHF8 in these cells upregulated the expression of SATB2, Runx2, osterix, and bone matrix proteins. Conversely, knockdown of PHF8 reduced the expression of these genes. Furthermore, ChIP assays confirmed that PHF8 specifically bound to the transcription start site (TSS) of the SATB2 promoter, and the expression of H3K9me1 at the TSS region of SATB2 decreased in PHF8 overexpressed group. Implantation of the BMSCs overexpressing PHF8 with silk protein scaffolds promoted bone regeneration in critical-sized defects in mouse calvaria. Taken together, our results demonstrated that PHF8 epigenetically modulates SATB2 activity, triggering BMSCs osteogenic differentiation and facilitating bone formation and regeneration in biodegradable silk scaffolds.