Cleft lip and cleft palate in Esrp1 knockout mice is associated with alterations in epithelial-mesenchymal crosstalk.

Cleft lip is one of the most common human birth defects. However, there remain a limited number of mouse models of cleft lip that can be leveraged to characterize the genes and mechanisms that cause this disorder. Crosstalk between epithelial and mesenchymal cells underlies formation of the face and palate, but the basic molecular events mediating this crosstalk remain poorly understood. We previously demonstrated that mice lacking the epithelial-specific splicing factor Esrp1 have fully penetrant bilateral cleft lip and palate. In this study, we further investigated the mechanisms leading to cleft lip as well as cleft palate in both existing and new Esrp1 mutant mouse models. These studies included a detailed transcriptomic analysis of changes in ectoderm and mesenchyme in Esrp1-/- embryos during face formation. We identified altered expression of genes previously implicated in cleft lip and/or palate, including components of multiple signaling pathways. These findings provide the foundation for detailed investigations using Esrp1 mutant disease models to examine gene regulatory networks and pathways that are essential for normal face and palate development - the disruption of which leads to orofacial clefting in human patients.

Novel ANKH mutation in a patient with sporadic craniometaphyseal dysplasia.

Craniometaphyseal dysplasia is caused by mutations in ANKH (ankylosis, progressive homolog [mouse]) in the majority of cases, and all of the reported mutations are single amino acid changes. Genomic DNA from an affected patient, his biological parents, and a sibling was amplified and ANKH was sequenced. The affected patient had a complex heterozygous mutation in exon 7 (c.936T > C, c.938C > G, c.942_953delTGGTTGACGGAA), predicting p.Try290Gln and p.Trp292_Glu295del. We studied the effect of the predicted mutation on the subcellular distribution of ANKH protein. Immunofluorescent labeling of COS-7 cells transduced with normal or mutant Ank (murine progressive ankylosis), showed that normal Ank localized to both the plasma membrane and cytoplasm, whereas mutant Ank was detected only in the cytoplasmic compartment. We propose that this craniometaphyseal dysplasia mutation causes a loss of ANKH protein expression and activity in the plasma membrane as a result of aberrant intracellular protein trafficking.

Roles of Ihh signaling in chondroprogenitor function in postnatal condylar cartilage.

Condylar articular cartilage in mouse temporomandibular joint develops from progenitor cells near the articulating surface that proliferate, undergo chondrogenesis and mature into hypertrophic chondrocytes. However, it remains unclear how these processes are regulated, particularly postnatally. Here we focused on the apical polymorphic layer rich in progenitors and asked whether the phenotype and fate of the cells require signaling by Indian hedgehog (Ihh) previously studied in developing long bones. In condyles in newborn mice, the apical polymorphic/progenitor cell layer was ~10 cell layer-thick and expressed the articular matrix marker Tenascin-C (Tn-C), and the underlying thick cell layer expressed Tn-C as well as the chondrogenic master regulator Sox9. By 1 month, condylar cartilage had gained its full width, but became thinner along its main longitudinal axis and displayed hypertrophic chondrocytes. By 3 months, articular cartilage consisted of a 2-3 cell layer-thick zone of superficial cells and chondroprogenitors expressing both Tn-C and Sox9 and a bottom zone of chondrocytes displaying vertical matrix septa. EdU cell tracing in juvenile mice revealed that conversion of chondroprogenitors into chondrocytes and hypertrophic chondrocytes required about 48 and 72 h, respectively. Notably, EdU injection in 3 month-old mice labeled both progenitors and maturing chondrocytes by 96 h. Conditional ablation of Ihh in juvenile/early adult mice compromised chondroprogenitor organization and function and led to reduced chondroprogenitor and chondrocyte proliferation. The phenotype of mutant condyles worsened over time as indicated by apoptotic chondrocyte incidence, ectopic chondrocyte hypertrophy, chondrocyte column derangement and subchondral bone deterioration. In micromass cultures of condylar apical cells, hedgehog (Hh) treatment stimulated chondrogenesis and alkaline phosphatase (APase) activity, while treatment with HhAntag inhibited both. Our findings indicate that the chondroprogenitor layer is continuously engaged in condylar growth postnatally and its organization and functioning depend on hedgehog signaling.

A New Calcium Silicate-based Bioceramic Material Promotes Human Osteo- and Odontogenic Stem Cell Proliferation and Survival via the Extracellular Signal-regulated Kinase Signaling Pathway.

INTRODUCTION: The purpose of this study was to investigate odontogenic and osteogenic cell adhesion, proliferation, and survival on the surface of a newly developed bioceramic material (EndoSequence Root Repair Material [RRM]; Brasseler USA, Savannah, GA) and compare it with mineral trioxide aggregate (gray MTA) (ProRoot MTA; Dentsply Tulsa Dental, Tulsa, OK). A potential role of extracellular signal-regulated kinase (ERK) signaling in the RRM/MTA-induced cellular activities was also investigated. METHODS: Human bone marrow mesenchymal stem cells, periodontal ligament stem cells, and dental pulp stem cells were cultured on RRM- or MTA-coated slides. Cell proliferation was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays after 1, 3, and 5 days of growth. Cell survival was assessed under serum starvation (0.5% and 0.2% serum) using MTT assays. RRM and MTA surface characteristics and cell morphology were studied using a scanning electron microscope. The role of ERK signaling in RRM/MTA-induced cell proliferation/survival was studied using an ERK-specific inhibitor. RESULTS: All cell types firmly attached to RRM- and MTA-coated plates. The coated surfaces had a granular appearance under the scanning electron microscope. Compared with those grown on uncoated plates, the cells on MTA/RRM-coated plates appeared healthy and smaller. Cell proliferation was significantly higher on RRM/MTA-coated surfaces (2- to 3-fold in cell number). The mitogenic effect on periodontal ligament stem cells and dental pulp stem cells was more pronounced with RRM than MTA (49% and 26% higher, respectively), but human bone marrow mesenchymal stem cells responded to both materials similarly. In serum-deprived conditions, significantly more cells (2- to 3-fold) survived on RRM/MTA surfaces. The cells grown on RRM/MTA surfaces showed sustained up-regulation of ERK phosphorylation, and blocking ERK signaling with U0126 significantly reduced RRM- and MTA-dependent cell survival. CONCLUSIONS: MTA and RRM are biocompatible and promote cell proliferation and survival in an ERK-dependent manner.

Osteophyte formation and matrix mineralization in a TMJ osteoarthritis mouse model are associated with ectopic hedgehog signaling.

The temporomandibular joint (TMJ) is a diarthrodial joint that relies on lubricants for frictionless movement and long-term function. It remains unclear what temporal and causal relationships may exist between compromised lubrication and onset and progression of TMJ disease. Here we report that Proteoglycan 4 (Prg4)-null TMJs exhibit irreversible osteoarthritis-like changes over time and are linked to formation of ectopic mineralized tissues and osteophytes in articular disc, mandibular condyle and glenoid fossa. In the presumptive layer of mutant glenoid fossa's articulating surface, numerous chondrogenic cells and/or chondrocytes emerged ectopically within the type I collagen-expressing cell population, underwent endochondral bone formation accompanied by enhanced Ihh expression, became entrapped into temporal bone mineralized matrix, and thereby elicited excessive chondroid bone formation. As the osteophytes grew, the roof of the glenoid fossa/eminence became significantly thicker and flatter, resulting in loss of its characteristic concave shape for accommodation of condyle and disc. Concurrently, the condyles became flatter and larger and exhibited ectopic bone along their neck, likely supporting the enlarged condylar heads. Articular discs lost their concave configuration, and ectopic cartilage developed and articulated with osteophytes. In glenoid fossa cells in culture, hedgehog signaling stimulated chondrocyte maturation and mineralization including alkaline phosphatase, while treatment with hedgehog inhibitor HhAntag prevented such maturation process. In sum, our data indicate that Prg4 is needed for TMJ integrity and long-term postnatal function. In its absence, progenitor cells near presumptive articular layer and disc undergo ectopic chondrogenesis and generate ectopic cartilage, possibly driven by aberrant activation of Hh signaling. The data suggest also that the Prg4-null mice represent a useful model to study TMJ osteoarthritis-like degeneration and clarify its pathogenesis.

Effects of FGF-2/-9 in calvarial bone cell cultures: differentiation stage-dependent mitogenic effect, inverse regulation of BMP-2 and noggin, and enhancement of osteogenic potential.

Systemically administered fibroblast growth factors (FGFs) show anabolic effects on bone formation in animals, whereas in vitro cell culture studies have demonstrated that FGFs block mineralized bone nodule formation. These apparently contradictory outcomes indicate that the nature of FGF action is complex and that the biological effect of FGFs may depend on the differentiation stage of osteoblasts, interaction with other cytokines, or the length and mode of exposure to factors. Thus, we have utilized primary calvarial bone cell populations at different maturation phases to determine their responses to 2, FGF-9, and BMP-2, the factors expressed in bone. FGF-2 and FGF-9 stimulated proliferation of the cell populations consisting of more mature osteoblasts, but not those with undifferentiated precursor cells. Continuous treatment with FGF-2/-9 inhibited expression of several osteoblast marker genes and mineralization. However, brief pretreatment with FGF-2/-9 or sequential treatment with FGF-2/-9 followed by BMP-2 led to marked stimulation of mineralization, suggesting that FGFs enhance the intrinsic osteogenic potential. Furthermore, FGF-2 and FGF-9 increased expression of other osteogenic factors BMP-2 and TGFbeta-1. Meanwhile, blocking endogenous FGF signaling, using a virally transduced dominant-negative FGF receptor (FgfR), resulted in drastically reduced expression of the BMP-2 gene, demonstrating for the first time that endogenous FGF/FgfR signaling is a positive upstream regulator of the BMP-2 gene in calvarial osteoblasts. In contrast, expression of a BMP antagonist noggin was inhibited by FGF-2 and FGF-9. Thus, collective data from this study suggest that FGF/FgfR signaling enhances the intrinsic osteogenic potential by selectively expanding committed osteogenic cell populations as well as inversely regulating BMP-2 and noggin gene expression.