Genetic interactions between Pax9 and Msx1 regulate lip development and several stages of tooth morphogenesis

Developmental abnormalities of craniofacial structures and teeth often occur sporadically and the underlying genetic defects are not well understood, in part due to unknown gene-gene interactions. Pax9 and Msx1 are co-expressed during craniofacial development, and mice that are single homozygous mutant for either gene exhibit cleft palate and an early arrest of tooth formation. Whereas in vitro assays have demonstrated that protein-protein interactions between Pax9 and Msx1 can occur, it is unclear if Pax9 and Msx1 interact genetically in vivo during development. To address this question, we compounded the Pax9 and Msx1 mutations and observed that double homozygous mutants exhibit an incompletely penetrant cleft lip phenotype. Moreover, in double heterozygous mutants, the lower incisors were consistently missing and we find that transgenic BMP4 expression partly rescues this phenotype. Reduced expression of Shh and Bmp2 indicates that a smaller “incisor field” forms in Pax9^+/−;Msx1^+/− mutants, and dental epithelial growth is substantially reduced after the bud to cap stage transition. This defect is preceded by drastically reduced mesenchymal expression of Fgf3 and Fgf10, two genes that encode known stimulators of epithelial growth during odontogenesis. Consistent with this result, cell proliferation is reduced in both the dental epithelium and mesenchyme of double heterozygous mutants. Furthermore, the developing incisors lack mesenchymal Notch1 expression at the bud stage and exhibit abnormal ameloblast differentiation on both labial and lingual surfaces. Thus, Msx1 and Pax9 interact synergistically throughout lower incisor development and affect multiple signaling pathways that influence incisor size and symmetry. The data also suggest that a combined reduction of PAX9 and MSX1 gene dosage in humans may increase the risk for orofacial clefting and oligodontia.     

The absence of muscle segment homeobox 2 leads to the pyroptosis of ameloblasts by inducing squamous epithelial hyperplasia in the enamel organ

Muscle segment homeobox 2 (MSX2) has been confirmed to be involved in the regulation of early tooth development. However, the role of MSX2 has not been fully elucidated in enamel development. To research the functions of MSX2 in enamel formation, we used a Msx2^−/− (KO) mouse model with no full Msx2 gene. In the present study, the dental appearance and enamel microstructure were detected by scanning electron microscopy and micro-computed tomography. The results showed that the absence of Msx2 resulted in enamel defects, leading to severe tooth wear in KO mice. To further investigate the mechanism behind the phenotype, we performed detailed histological analyses of the enamel organ in KO mice. We discovered that ameloblasts without Msx2 could secrete a small amount of enamel matrix protein in the early stage. However, the enamel epithelium occurred squamous epithelial hyperplasia and partial keratinization in the enamel organ during subsequent developmental stages. Ameloblasts depolarized and underwent pyroptosis. Overall, during the development of enamel, MSX2 affects the formation of enamel by regulating the function of epithelial cells in the enamel organ.     

Severe Extracellular Matrix Abnormalities and Chondrodysplasia in Mice Lacking Collagen Prolyl 4-Hydroxylase Isoenzyme II in Combination with a Reduced Amount of Isoenzyme I

Collagen prolyl 4-hydroxylases (C-P4H-I, C-P4H-II, and C-P4H-III) catalyze formation of 4-hydroxyproline residues required to form triple-helical collagen molecules. Vertebrate C-P4Hs are α₂β₂ tetramers differing in their catalytic α subunits. C-P4H-I is the major isoenzyme in most cells, and inactivation of its catalytic subunit (P4ha1^−/−) leads to embryonic lethality in mouse, whereas P4ha1^+/− mice have no abnormalities. To study the role of C-P4H-II, which predominates in chondrocytes, we generated P4ha2^−/− mice. Surprisingly, they had no apparent phenotypic abnormalities. To assess possible functional complementarity, we established P4ha1^+/−;P4ha2^−/− mice. They were smaller than their littermates, had moderate chondrodysplasia, and developed kyphosis. A transient inner cell death phenotype was detected in their developing growth plates. The columnar arrangement of proliferative chondrocytes was impaired, the amount of 4-hydroxyproline and the T_m of collagen II were reduced, and the extracellular matrix was softer in the growth plates of newborn P4ha1^+/−;P4ha2^−/− mice. No signs of uncompensated ER stress were detected in the mutant growth plate chondrocytes. Some of these defects were also found in P4ha2^−/− mice, although in a much milder form. Our data show that C-P4H-I can to a large extent compensate for the lack of C-P4H-II in proper endochondral bone development, but their combined partial and complete inactivation, respectively, leads to biomechanically impaired extracellular matrix, moderate chondrodysplasia, and kyphosis. Our mouse data suggest that inactivating mutations in human P4HA2 are not likely to lead to skeletal disorders, and a simultaneous decrease in P4HA1 function would most probably be required to generate such a disease phenotype.     

Synergistic Interaction of Rnf8 and p53 in the Protection against Genomic Instability and Tumorigenesis

Rnf8 is an E3 ubiquitin ligase that plays a key role in the DNA damage response as well as in the maintenance of telomeres and chromatin remodeling. Rnf8^−/− mice exhibit developmental defects and increased susceptibility to tumorigenesis. We observed that levels of p53, a central regulator of the cellular response to DNA damage, increased in Rnf8^−/− mice in a tissue- and cell type–specific manner. To investigate the role of the p53-pathway inactivation on the phenotype observed in Rnf8^−/− mice, we have generated Rnf8^−/−p53^−/− mice. Double-knockout mice showed similar growth retardation defects and impaired class switch recombination compared to Rnf8^−/− mice. In contrast, loss of p53 fully rescued the increased apoptosis and reduced number of thymocytes and splenocytes in Rnf8^−/− mice. Similarly, the senescence phenotype of Rnf8^−/− mouse embryonic fibroblasts was rescued in p53 null background. Rnf8^−/−p53^−/− cells displayed defective cell cycle checkpoints and DNA double-strand break repair. In addition, Rnf8^−/−p53^−/− mice had increased levels of genomic instability and a remarkably elevated tumor incidence compared to either Rnf8^−/− or p53^−/− mice. Altogether, the data in this study highlight the importance of p53-pathway activation upon loss of Rnf8, suggesting that Rnf8 and p53 functionally interact to protect against genomic instability and tumorigenesis.     

Retinal Changes Precede Visual Dysfunction in the Complement Factor H Knockout Mouse

We previously reported that aged mice lacking complement factor H (CFH) exhibit visual defects and structural changes in the retina. However, it is not known whether this phenotype is age-related or is the consequence of disturbed development. To address this question we investigated the effect of Cfh gene deletion on the retinal phenotype of young and mid-age mice. Cfh ^−/− mouse eyes exhibited thickening of the retina and reduced nuclear density, but relatively normal scotopic and photopic electroretinograms. At 12 months there was evidence of subtle astroglial activation in the Cfh ^−/− eyes, and significant elevation of the complement regulator, decay-accelerating factor (DAF) in Müller cells. In the retinal pigment epithelium (RPE) of young control and Cfh ^−/− animals mitochondria and melanosomes were oriented basally and apically respectively, whereas the apical positioning of melanosomes was significantly perturbed in the mid-age Cfh ^−/− RPE. We conclude that deletion of Cfh in the mouse leads to defects in the retina that precede any marked loss of visual function, but which become progressively more marked as the animals age. These observations are consistent with a lifelong role for CFH in retinal homeostasis.     

p47phox-Nox2-dependent ROS Signaling Inhibits Early Bone Development in Mice but Protects against Skeletal Aging

Bone remodeling is age-dependently regulated and changes dramatically during the course of development. Progressive accumulation of reactive oxygen species (ROS) has been suspected to be the leading cause of many inflammatory and degenerative diseases, as well as an important factor underlying many effects of aging. In contrast, how reduced ROS signaling regulates inflammation and remodeling in bone remains unknown. Here, we utilized a p47^phox knock-out mouse model, in which an essential cytosolic co-activator of Nox2 is lost, to characterize bone metabolism at 6 weeks and 2 years of age. Compared with their age-matched wild type controls, loss of Nox2 function in p47^phox−/− mice resulted in age-related switch of bone mass and strength. Differences in bone mass were associated with increased bone formation in 6-week-old p47^phox−/− mice but decreased in 2-year-old p47^phox−/− mice. Despite decreases in ROS generation in bone marrow cells and p47^phox-Nox2 signaling in osteoblastic cells, 2-year-old p47^phox−/− mice showed increased senescence-associated secretory phenotype in bone compared with their wild type controls. These in vivo findings were mechanistically recapitulated in ex vivo cell culture of primary fetal calvarial cells from p47^phox−/− mice. These cells showed accelerated cell senescence pathway accompanied by increased inflammation. These data indicate that the observed age-related switch of bone mass in p47^phox-deficient mice occurs through an increased inflammatory milieu in bone and that p47^phox-Nox2-dependent physiological ROS signaling suppresses inflammation in aging.     

Accelerated Calvarial Healing in Mice Lacking Toll-Like Receptor 4

The bone and immune systems are closely interconnected. The immediate inflammatory response after fracture is known to trigger a healing cascade which plays an important role in bone repair. Toll-like receptor 4 (TLR4) is a member of a highly conserved receptor family and is a critical activator of the innate immune response after tissue injury. TLR4 signaling has been shown to regulate the systemic inflammatory response induced by exposed bone components during long-bone fracture. Here we tested the hypothesis that TLR4 activation affects the healing of calvarial defects. A 1.8 mm diameter calvarial defect was created in wild-type (WT) and TLR4 knockout (TLR4^−/−) mice. Bone healing was tested using radiographic, histologic and gene expression analyses. Radiographic and histomorphometric analyses revealed that calvarial healing was accelerated in TLR4^−/− mice. More bone was observed in TLR4^−/− mice compared to WT mice at postoperative days 7 and 14, although comparable healing was achieved in both groups by day 21. Bone remodeling was detected in both groups on postoperative day 28. In TLR4^−/− mice compared to WT mice, gene expression analysis revealed that higher expression levels of IL-1β, IL-6, TNF-α,TGF-β1, TGF-β3, PDGF and RANKL and lower expression level of RANK were detected at earlier time points (≤ postoperative 4 days); while higher expression levels of IL-1β and lower expression levels of VEGF, RANK, RANKL and OPG were detected at late time points (> postoperative 4 days). This study provides evidence of accelerated bone healing in TLR4^−/− mice with earlier and higher expression of inflammatory cytokines and with increased osteoclastic activity. Further work is required to determine if this is due to inflammation driven by TLR4 activation.     

Endothelial Msx1 transduces hemodynamic changes into an arteriogenic remodeling response

Collateral remodeling is critical for blood flow restoration in peripheral arterial disease and is triggered by increasing fluid shear stress in preexisting collateral arteries. So far, no arterial-specific mediators of this mechanotransduction response have been identified. We show that muscle segment homeobox 1 (MSX1) acts exclusively in collateral arterial endothelium to transduce the extrinsic shear stimulus into an arteriogenic remodeling response. MSX1 was specifically up-regulated in remodeling collateral arteries. MSX1 induction in collateral endothelial cells (ECs) was shear stress driven and downstream of canonical bone morphogenetic protein–SMAD signaling. Flow recovery and collateral remodeling were significantly blunted in EC-specific Msx1/2 knockout mice. Mechanistically, MSX1 linked the arterial shear stimulus to arteriogenic remodeling by activating the endothelial but not medial layer to a proinflammatory state because EC but not smooth muscle cellMsx1/2 knockout mice had reduced leukocyte recruitment to remodeling collateral arteries. This reduced leukocyte infiltration in EC Msx1/2 knockout mice originated from decreased levels of intercellular adhesion molecule 1 (ICAM1)/vascular cell adhesion molecule 1 (VCAM1), whose expression was also in vitro driven by promoter binding of MSX1.     

Mouse models of telomere dysfunction phenocopy skeletal changes found in human age-related osteoporosis

A major medical challenge in the elderly is osteoporosis and the high risk of fracture. Telomere dysfunction is a cause of cellular senescence and telomere shortening, which occurs with age in cells from most human tissues, including bone. Telomere defects contribute to the pathogenesis of two progeroid disorders characterized by premature osteoporosis, Werner syndrome and dyskeratosis congenital. It is hypothesized that telomere shortening contributes to bone aging. We evaluated the skeletal phenotypes of mice with disrupted telomere maintenance mechanisms as models for human bone aging, including mutants in Werner helicase (Wrn^−/−), telomerase (Terc^−/−) and Wrn^−/−Terc^−/− double mutants. Compared with young wild-type (WT) mice, micro-computerized tomography analysis revealed that young Terc^−/− and Wrn^−/−Terc^−/− mice have decreased trabecular bone volume, trabecular number and trabecular thickness, as well as increased trabecular spacing. In cortical bone, young Terc^−/− and Wrn^−/−Terc^−/− mice have increased cortical thinning, and increased porosity relative to age-matched WT mice. These trabecular and cortical changes were accelerated with age in Terc^−/− and Wrn^−/−Terc^−/− mice compared with older WT mice. Histological quantification of osteoblasts in aged mice showed a similar number of osteoblasts in all genotypes; however, significant decreases in osteoid, mineralization surface, mineral apposition rate and bone formation rate in older Terc^−/− and Wrn^−/−Terc^−/− bone suggest that osteoblast dysfunction is a prominent feature of precocious aging in these mice. Except in the Wrn^−/− single mutant, osteoclast number did not increase in any genotype. Significant alterations in mechanical parameters (structure model index, degree of anistrophy and moment of inertia) of the Terc^−/− and Wrn^−/−Terc^−/− femurs compared with WT mice were also observed. Young Wrn^−/−Terc^−/− mice had a statistically significant increase in bone-marrow fat content compared with young WT mice, which remained elevated in aged double mutants. Taken together, our results suggest that Terc^−/− and Wrn^−/−Terc^−/− mutants recapitulate the human bone aging phenotype and are useful models for studying age-related osteoporosis.KEY WORDS: Aging, Bone histomorphometry, Osteoporosis     

Regulation of proliferation in developing human tooth germs by MSX homeodomain proteins and cyclin-dependent kinase inhibitor p19INK4d

Before the secretion of hard dental tissues, tooth germs undergo several distinctive stages of development (dental lamina, bud, cap and bell). Every stage is characterized by specific proliferation patterns, which is regulated by various morphogens, growth factors and homeodomain proteins. The role of MSX homeodomain proteins in odontogenesis is rather complex. Expression domains of genes encoding for murine Msx1/2 during development are observed in tissues containing highly proliferative progenitor cells. Arrest of tooth development in Msx knockout mice can be attributed to impaired proliferation of progenitor cells. In Msx1 knockout mice, these progenitor cells start to differentiate prematurely as they strongly express cyclin-dependent kinase inhibitor p19^INK4d. p19^INK4d induces terminal differentiation of cells by blocking the cell cycle in mitogen-responsive G1 phase. Direct suppression of p19^INK4d by Msx1 protein is, therefore, important for maintaining proliferation of progenitor cells at levels required for the normal progression of tooth development. In this study, we examined the expression patterns of MSX1, MSX2 and p19^INK4d in human incisor tooth germs during the bud, cap and early bell stages of development. The distribution of expression domains of p19^INK4d throughout the investigated period indicates that p19^INK4d plays active role during human tooth development. Furthermore, comparison of expression domains of p19^INK4d with those of MSX1, MSX2 and proliferation markers Ki67, Cyclin A2 and pRb, indicates that MSX-mediated regulation of proliferation in human tooth germs might not be executed by the mechanism similar to one described in developing tooth germs of wild-type mouse.     

Variable Nav1.5 Protein Expression from the Wild-Type Allele Correlates with the Penetrance of Cardiac Conduction Disease in the Scn5a +/? Mouse Model

Background

Loss-of-function mutations in SCN5A, the gene encoding Na_v1.5 Na⁺ channel, are associated with inherited cardiac conduction defects and Brugada syndrome, which both exhibit variable phenotypic penetrance of conduction defects. We investigated the mechanisms of this heterogeneity in a mouse model with heterozygous targeted disruption of Scn5a (Scn5a ^+/− mice) and compared our results to those obtained in patients with loss-of-function mutations in SCN5A.

Methodology/Principal Findings

Based on ECG, 10-week-old Scn5a ^+/− mice were divided into 2 subgroups, one displaying severe ventricular conduction defects (QRS interval>18 ms) and one a mild phenotype (QRS≤18 ms; QRS in wild-type littermates: 10–18 ms). Phenotypic difference persisted with aging. At 10 weeks, the Na⁺ channel blocker ajmaline prolonged QRS interval similarly in both groups of Scn5a ^+/− mice. In contrast, in old mice (>53 weeks), ajmaline effect was larger in the severely affected subgroup. These data matched the clinical observations on patients with SCN5A loss-of-function mutations with either severe or mild conduction defects. Ventricular tachycardia developed in 5/10 old severely affected Scn5a ^+/− mice but not in mildly affected ones. Correspondingly, symptomatic SCN5A–mutated Brugada patients had more severe conduction defects than asymptomatic patients. Old severely affected Scn5a ^+/− mice but not mildly affected ones showed extensive cardiac fibrosis. Mildly affected Scn5a ^+/− mice had similar Na_v1.5 mRNA but higher Na_v1.5 protein expression, and moderately larger I_Na current than severely affected Scn5a ^+/− mice. As a consequence, action potential upstroke velocity was more decreased in severely affected Scn5a ^+/− mice than in mildly affected ones.

Conclusions

Scn5a ^+/− mice show similar phenotypic heterogeneity as SCN5A-mutated patients. In Scn5a ^+/− mice, phenotype severity correlates with wild-type Na_v1.5 protein expression.     

Deletion of vascular endothelial growth factor C (VEGF-C) and VEGF-D is not equivalent to VEGF receptor 3 deletion in mouse embryos

Lymphatic vessels play an important role in the regulation of tissue fluid balance, immune responses, and fat adsorption and are involved in diseases including lymphedema and tumor metastasis. Vascular endothelial growth factor (VEGF) receptor 3 (VEGFR-3) is necessary for development of the blood vasculature during early embryogenesis, but later, VEGFR-3 expression becomes restricted to the lymphatic vasculature. We analyzed mice deficient in both of the known VEGFR-3 ligands, VEGF-C and VEGF-D. Unlike the Vegfr3^−/− embryos, the Vegfc^−/−; Vegfd^−/− embryos displayed normal blood vasculature after embryonic day 9.5. Deletion of Vegfr3 in the epiblast, using keratin 19 (K19) Cre, resulted in a phenotype identical to that of the Vegfr3^−/− embryos, suggesting that this phenotype is due to defects in the embryo proper and not in placental development. Interestingly, the Vegfr3^neo hypomorphic mutant mice carrying the neomycin cassette between exons 1 and 2 showed defective lymphatic development. Overexpression of human or mouse VEGF-D in the skin, under the K14 promoter, rescued the lymphatic hypoplasia of the Vegfc^+/− mice in the K14-VEGF-D; Vegfc^+/− compound mice, suggesting that VEGF-D is functionally redundant with VEGF-C in the stimulation of developmental lymphangiogenesis. Our results suggest VEGF-C- and VEGF-D-independent functions for VEGFR-3 in the early embryo.     

Protein Kinase Cα (PKCα) Regulates Bone Architecture and Osteoblast Activity

Bones'' strength is achieved and maintained through adaptation to load bearing. The role of the protein kinase PKCα in this process has not been previously reported. However, we observed a phenotype in the long bones of Prkca^−/− female but not male mice, in which bone tissue progressively invades the medullary cavity in the mid-diaphysis. This bone deposition progresses with age and is prevented by disuse but unaffected by ovariectomy. Castration of male Prkca^−/− but not WT mice results in the formation of small amounts of intramedullary bone. Osteoblast differentiation markers and Wnt target gene expression were up-regulated in osteoblast-like cells derived from cortical bone of female Prkca^−/− mice compared with WT. Additionally, although osteoblastic cells derived from WT proliferate following exposure to estradiol or mechanical strain, those from Prkca^−/− mice do not. Female Prkca^−/− mice develop splenomegaly and reduced marrow GBA1 expression reminiscent of Gaucher disease, in which PKC involvement has been suggested previously. From these data, we infer that in female mice, PKCα normally serves to prevent endosteal bone formation stimulated by load bearing. This phenotype appears to be suppressed by testicular hormones in male Prkca^−/− mice. Within osteoblastic cells, PKCα enhances proliferation and suppresses differentiation, and this regulation involves the Wnt pathway. These findings implicate PKCα as a target gene for therapeutic approaches in low bone mass conditions.     

Decreased Bone Formation Explains Osteoporosis in a Genetic Mouse Model of Hemochromatosiss

Osteoporosis may complicate iron overload diseases such as genetic hemochromatosis. However, molecular mechanisms involved in the iron-related osteoporosis remains poorly understood. Recent in vitro studies support a role of osteoblast impairment in iron-related osteoporosis. Our aim was to analyse the impact of excess iron in Hfe^-/- mice on osteoblast activity and on bone microarchitecture. We studied the bone formation rate, a dynamic parameter reflecting osteoblast activity, and the bone phenotype of Hfe^−/− male mice, a mouse model of human hemochromatosis, by using histomorphometry. Hfe^−/− animals were sacrificed at 6 months and compared to controls. We found that bone contains excess iron associated with increased hepatic iron concentration in Hfe^−/− mice. We have shown that animals with iron overload have decreased bone formation rate, suggesting a direct impact of iron excess on active osteoblasts number. For bone mass parameters, we showed that iron deposition was associated with bone loss by producing microarchitectural impairment with a decreased tendency in bone trabecular volume and trabecular number. A disorganization of trabecular network was found with marrow spaces increased, which was confirmed by enhanced trabecular separation and star volume of marrow spaces. These microarchitectural changes led to a loss of connectivity and complexity in the trabecular network, which was confirmed by decreased interconnectivity index and increased Minkowski’s fractal dimension. Our results suggest for the first time in a genetic hemochromatosis mouse model, that iron overload decreases bone formation and leads to alterations in bone mass and microarchitecture. These observations support a negative effect of iron on osteoblast recruitment and/or function, which may contribute to iron-related osteoporosis.     

MCP/CCR2 Signaling Is Essential for Recruitment of Mesenchymal Progenitor Cells during the Early Phase of Fracture Healing

Objective

The purpose of this study was to investigate chemokine profiles and their functional roles in the early phase of fracture healing in mouse models.

Methods

The expression profiles of chemokines were examined during fracture healing in wild-type (WT) mice using a polymerase chain reaction array and histological staining. The functional effect of monocyte chemotactic protein-1 (MCP-1) on primary mouse bone marrow stromal cells (mBMSCs) was evaluated using an in vitro migration assay. MCP-1^−/− and C-C chemokine receptor 2 (CCR2)^−/− mice were fractured and evaluated by histological staining and micro-computed tomography (micro-CT). RS102895, an antagonist of CCR2, was continuously administered in WT mice before or after rib fracture and evaluated by histological staining and micro-CT. Bone graft exchange models were created in WT and MCP-1^−/− mice and were evaluated by histological staining and micro-CT.

Results

MCP-1 and MCP-3 expression in the early phase of fracture healing were up-regulated, and high levels of MCP-1 and MCP-3 protein expression observed in the periosteum and endosteum in the same period. MCP-1, but not MCP-3, increased migration of mBMSCs in a dose-dependent manner. Fracture healing in MCP-1^−/− and CCR2^−/− mice was delayed compared with WT mice on day 21. Administration of RS102895 in the early, but not in the late phase, caused delayed fracture healing. Transplantation of WT-derived graft into host MCP-1^−/− mice significantly increased new bone formation in the bone graft exchange models. Furthermore, marked induction of MCP-1 expression in the periosteum and endosteum was observed around the WT-derived graft in the host MCP-1^−/− mouse. Conversely, transplantation of MCP-1^−/− mouse-derived grafts into host WT mice markedly decreased new bone formation.

Conclusions

MCP-1/CCR2 signaling in the periosteum and endosteum is essential for the recruitment of mesenchymal progenitor cells in the early phase of fracture healing.