FAM20A is a golgi-localized Type II transmembrane protein.

Family with sequence similarity 20, member A (FAM20A) is a pseudo-kinase in the secretory pathway and is essential for enamel formation in humans. Here we examine if FAM20A is a membrane-associated protein. We show that the full-length FAM20A can be purified from HEK293 cells transfected with a FAM20A-expresing construct. Further, it is only found in the membrane fraction, but not in the soluble fraction, of cell lysate. Consistently, it is not secreted out of the expressing cells. Moreover, it is co-localized with GM130, a cis-Golgi network marker, and membrane topology analysis indicates that it has its C-terminus oriented towards the lumen of the organelle. Our results support that FAM20A is a Type II transmembrane protein within the secretory compartments.

Intracranial calcification in Fam20c-deficient mice recapitulates human Raine syndrome.

FAM20C (family with sequence similarity 20-member C) is a protein kinase that phosphorylates secretory proteins, including the proteins that are essential to the formation and mineralization of calcified tissues. FAM20C loss-of-function mutations cause Raine syndrome in humans, characterized by generalized osteosclerosis, distinctive craniofacial dysmorphism, along with extensive intracranial calcification. Our previous studies revealed that inactivation of Fam20c in mice led to hypophosphatemic rickets. In this study, we examined the expression of Fam20c in the mouse brain and investigated brain calcification in Fam20c-deficient mice. Reverse transcription polymerase chain reaction (RT-PCR), Western-blotting and in situ hybridization analyses demonstrated the broad expression of Fam20c in the mouse brain tissue. X-ray and histological analyses showed that the global deletion of Fam20c (mediated by Sox2-cre) resulted in brain calcification in mice after postnatal 3 months and that the calcifications were bilaterally distributed within the brain. There was mild perifocal microgliosis as well as astrogliosis around calcospherites. The calcifications were first observed in the thalamus, and later in the forebrain and hindbrain. Furthermore, brain-specific deletion (mediated by Nestin-cre) of Fam20c in mice also led to cerebral calcification at an older age (postnatal 6 months), but no obvious skeletal or dental defects. Our results suggest that the local loss of FAM20C function in the brain may directly account for intracranial calcification. We propose that FAM20C plays an essential role in maintaining normal brain homeostasis and preventing ectopic brain calcification.

Constitutive expression of spliced X-box binding protein 1 inhibits dentin formation in mice.

Upon endoplasmic reticulum (ER) stress, inositol-requiring enzyme 1 (IRE1) is activated, which subsequently converts an unspliced X-box binding protein 1 (XBP1U) mRNA to a spliced mRNA that encodes a potent XBP1S transcription factor. XBP1S is essential for relieving ER stress and secretory cell differentiation. We previously established Twist2-Cre;Xbp1 CS/+ mice that constitutively expressed XBP1S in the Twist2-expressing cells as well as in the cells derived from the Twist2-expressing cells. In this study, we analyzed the dental phenotype of Twist2-Cre;Xbp1 CS/+ mice. We first generated a mutant Xbp1s minigene that corresponds to the recombinant Xbp1 Δ26 allele (the Xbp1 CS allele that has undergone Cre-mediated recombination) and confirmed that the Xbp1s minigene expressed XBP1S that does not require IRE1α activation in vitro. Consistently, immunohistochemistry showed that XBP1S was constitutively expressed in the odontoblasts and other dental pulp cells in Twist2-Cre;Xbp1 CS/+ mice. Plain X-ray radiography and µCT analysis revealed that constitutive expression of XBP1S altered the dental pulp chamber roof- and floor-dentin formation, resulting in a significant reduction in dentin/cementum formation in Twist2-Cre;Xbp1 CS/+ mice, compared to age-matched Xbp1 CS/+ control mice. However, there is no significant difference in the density of dentin/cementum between these two groups of mice. Histologically, persistent expression of XBP1S caused a morphological change in odontoblasts in Twist2-Cre;Xbp1 CS/+ mice. Nevertheless, in situ hybridization and immunohistochemistry analyses showed that continuous expression of XBP1S had no apparent effects on the expression of the Dspp and Dmp1 genes. In conclusion, these results support that sustained production of XBP1S adversely affected odontoblast function and dentin formation.

Long-acting PFI-2 small molecule release and multilayer scaffold design achieve extensive new formation of complex periodontal tissues with unprecedented fidelity.

The faithful engineering of complex human tissues such as the bone/soft tissue/mineralized tissue interface in periodontal tissues requires innovative molecular cues in conjunction with tailored scaffolds. To address the loss of periodontal bone and connective tissues following periodontal disease, we have generated a polydopamine and collagen coated electrospun PLGA-PCL (PP) scaffold enriched with the small molecule mediator PFI-2 (PP-PFI-pDA-COL-PFI). In vitro 3D studies using PDL progenitors revealed that the PP-PFI-pDA-COL-PFI scaffold substantially enhanced Alizarin Red staining, increased Ca/P ratios 4-fold, and stimulated cell proliferation more than 12-fold compared to PP-controls, suggestive of its potential for mineralized tissue engineering. When applied in our experimental periodontitis model, the PP-PFI-pDA-COL-PFI scaffold resulted in a substantial 34% reduction in alveolar bone defect height, a 25% root-length gain in periodontal attachment, and the formation of highly ordered regenerated acellular cementum twice as thick as in controls. Explaining the mechanism of PFI-2 mineralized tissue regeneration in periodontal tissues, PFI-2 inhibited SETD7-mediated ß-Catenin protein methylation and increased ß-Catenin nuclear localization. Together, dual-level PFI-2 incorporation into a degradable, dopamine/collagen coated PLGA/PCL scaffold backbone resulted in the regeneration of the tripartite periodontal complex with unprecedented fidelity, including periodontal attachment and new formation of mineralized tissues in inflamed periodontal environments.

Adenomatoid odontogenic tumor: evidence for a mixed odontogenic tumor.

OBJECTIVE: Adenomatoid odontogenic tumor (AOT) was classified by the World Health Organization as a mixed odontogenic tumor in 1992 and reclassified without a clear rationale as an epithelium-only tumor in 2005. The purpose of this study was to investigate if there was any evidence to suggest AOT might be a mixed odontogenic tumor. STUDY DESIGN: Immunohistochemical studies with nestin, dentin sialophosphoprotein (DSPP), cytokeratin, and vimentin were performed using 21 cases of AOT, and the staining results were analyzed according to the various morphologic patterns seen in AOT. Sirius red stain was used to detect the presence of collagen types I and III in AOT products. RESULTS: Our results showed that 20 of 21 (95.23%), 0 of 21 (0%), 21 of 21 (100%), and 20 of 21 (95.23%) cases expressed nestin, DSPP, cytokeratin, and vimentin, respectively. Some cells in rosette/duct-like structures (RDSs) expressed nestin, vimentin, or both, without cytokeratin. Coexpression of vimentin and cytokeratin or of nestin, cytokeratin, and vimentin was noted in some cells. Sirius red staining was positive in eosinophilic products in RDSs, double-layered spheres, and dentinoids. CONCLUSION: Although most AOT cells appear epithelial, there is a small population of cells expressing mesenchymal proteins and secreting collagen types I and III. This evidence suggests that AOT is a mixed odontogenic tumor.

FAM20C plays a critical role in the development of mouse vertebra.

BACKGROUND CONTEXT: Family with sequence similarity 20-member C (FAM20C) is a protein kinase that is responsible for the phosphorylation of many secretory proteins; however, its roles in spine or vertebra development have not be studied. PURPOSE: The aim of this investigation is to analyze the roles of FAM20C in vertebra development. STUDY DESIGN/SETTING: A mouse study of the Fam20c gene using conditional knockout to assess the effects of its inactivation on vertebra development. METHODS: By breeding Sox2-Cre mice with Fam20cflox/flox mice, Sox2-Cre;Fam20cflox/flox mice (abbreviated as cKO mice) are created. X-ray radiography, resin-casted scanning electron microscopy, Hematoxylin and Eosin staining, safranin O staining, Goldner's Masson trichrome staining, Von Kossa staining, tartrate-resistant alkaline phosphatase staining, immunohistochemistry staining, Western Immunoblotting and real-time PCR were employed to characterize the vertebrae of cKO mice compared to the normal control mice. RESULTS: Inactivation of Fam20c in mice results in remarkable spine deformity, severe morphology and mineralization defects, altered levels of osteoblast differentiation markers, reduction of activity of the Wnt/ß-catenin signaling pathway and reduced level of osteoclastogenesis in the vertebrae. CONCLUSIONS: FAM20C plays an essential role in vertebral development; it may regulate vertebral formation through the Wnt/ß-catenin signaling pathway. CLINICAL SIGNIFICANCE: Mutations in the human FAM20C gene are associated with Raine syndrome. The findings of this study provide valuable clues for the clinical management of Raine syndrome regarding spine manifestations in patients.

Enamel Defects Associated With Dentin Sialophosphoprotein Mutation in Mice.

Dentin sialophosphoprotein (DSPP) is an extracellular matrix protein that is highly expressed in odontoblasts, but only transiently expressed in presecretory ameloblasts during tooth development. We previously generated a knockin mouse model expressing a mouse equivalent (DSPP, p.P19L) of human mutant DSPP (p.P17L; referred to as "DsppP19L/+ "), and reported that DsppP19L/+ and DsppP19L/P19L mice manifested a dentin phenotype resembling human dentinogenesis imperfecta (DGI). In this study, we analyzed pathogenic effects of mutant P19L-DSPP on enamel development in DsppP19L/+ and DsppP19L/P19L mice. Micro-Computed Tomography (µCT) analyses of 7-week-old mouse mandibular incisors showed that DsppP19L/P19L mice had significantly decreased enamel volume and/or enamel density at different stages of amelogenesis examined. Acid-etched scanning electron microscopy (SEM) analyses of mouse incisors demonstrated that, at the mid-late maturation stage of amelogenesis, the enamel of wild-type mice already had apparent decussating pattern of enamel rods, whereas only minute particulates were found in DsppP19L/+ mice, and no discernible structures in DsppP19L/P19L mouse enamel. However, by the time that incisor enamel was about to erupt into oral cavity, distinct decussating enamel rods were evident in DsppP19L/+ mice, but only poorly-defined enamel rods were revealed in DsppP19L/P19L mice. Moreover, µCT analyses of the mandibular first molars showed that DsppP19L/+ and DsppP19L/P19L mice had a significant reduction in enamel volume and enamel density at the ages of 2, 3, and 24weeks after birth. Backscattered and acid-etched SEM analyses revealed that while 3-week-old DsppP19L/+ mice had similar pattern of enamel rods in the mandibular first molars as age-matched wild-type mice, no distinct enamel rods were observed in DsppP19L/P19L mice. Yet neither DsppP19L/+ nor DsppP19L/P19L mice showed well-defined enamel rods in the mandibular first molars by the age of 24weeks, as judged by backscattered and acid-etched SEM. In situ hybridization showed that DSPP mRNA level was markedly reduced in the presecretory ameloblasts, but immunohistochemistry revealed that DSP/DSPP immunostaining signals were much stronger within the presecretory ameloblasts in Dspp mutant mice than in wild-type mice. These results suggest that mutant P19L-DSPP protein caused developmental enamel defects in mice, which may be associated with intracellular retention of mutant DSPP in the presecretory ameloblasts.

Effect of high phosphate diet on the formation of dentin in Fam20c-deficient mice.

FAM20C (family with sequence similarity 20-member C), a kinase that phosphorylates secretory proteins, plays essential roles in various biological processes. In humans, mutations in FAM20C gene cause Raine syndrome, an autosomal recessive hereditary disease manifesting a broad spectrum of developmental defects including skeletal and craniofacial deformities. Our previous studies revealed that inactivation of Fam20c in mice led to hypophosphatemic rickets and that high phosphate (hPi) diet significantly improved the development of the skeleton in Fam20c-deficient mice. In this study, we evaluated the effects of hPi diet on the formation of dentin in Fam20c-deficient mice, using plain x-ray radiography, micro-computed tomography (µCT), histology, and immunohistochemistry. Plain x-ray radiography and µCT analyses showed that the hPi diet improved the dentin volume fraction and dentin mineral density of the Fam20c-deficient mice. Histology analyses further demonstrated that the hPi diet dramatically improved the integrity of the mandibular first molars and prevented pulp infection and dental abscesses in Fam20c-deficient mice. Our results support that the hPi diet significantly increased the formation and mineralization of dentin in Fam20c-deficient mice, implying that hypophosphatemia is a significant contributor to the dentin defects in Fam20c-deficient subjects.

Constitutive expression of spliced XBP1 causes perinatal lethality in mice.

Upon endoplasmic reticulum (ER) stress, inositol-requiring enzyme 1 (IRE1) is activated and catalyzes nonconventional splicing of an unspliced X-box binding protein 1 (XBP1U) mRNA to yield a spliced XBP1 (XBP1S) mRNA that encodes a potent XBP1S transcription factor. XBP1S is a key mediator of the IRE1 branch that is essential for alleviating ER stress. We generated a novel mouse strain (referred to as "Xbp1CS/+ " mice) that constitutively expressed XBP1S after Cre recombinase-mediated recombination. Further breeding of these mice with Twist2 Cre recombinase (Twist2-Cre) knock-in mice generated Twist2-Cre;Xbp1CS/+ mice. Most Twist2-Cre;Xbp1CS/+ mice died shortly after birth. Reverse-transcription polymerase chain reaction (RT-PCR) showed that constitutive expression of XBP1S occurred in various mouse tissues examined, but not in the brain. Immunohistochemistry confirmed that although the immunostaining signals for total XBP1 (XBP1U and XBP1S) were found in the calvarial bones in both Twist2-Cre;Xbp1CS/+ and control mice, the signals for XBP1S were only detected in the Twist2-Cre;Xbp1CS/+ mice, but not in the control mice. These results suggest that a precise control of XBP1S production is essential for normal mouse development.

Construction of a Cu-Sn Heterojunction Interface Derived from a Schottky Junction in Cu@Sn/rGO Composites as a Highly Efficient Dielectric Microwave Absorber.

Developing high-performance dielectric absorbers, low filler loading, and a broad absorption band remains a great challenge for wireless data communication systems, household appliances, local area network, and so on. Herein, we report a facile green method to design and fabricate a copper-coated tin/reduced graphene oxide (Cu@Sn/rGO) composites with a heterojunction obtained by modifying a Schottky junction. The unique heterojunction can enable an appropriate balance between impedance and strong loss capacity. Meanwhile, it can not only promote the carrier migration but also obtain the rich interfaces. Consequently, a Cu@Sn/rGO composite with a heterojunction exhibits superior absorption intensity, far surpassing that of other absorbing materials reported. With a weight content of only 5 wt %, the maximum absorptivity reaches -49.19 dB at 6.08 GHz, and an effective absorption bandwidth (RL < -10 dB) of 13.94 GHz is achieved when the absorber's thickness ranges from 1.7 to 5.5 mm. This study provides new insights into the design and synthesis of a novel microwave absorption material with lightweight, smaller filler loading, and strong reflection loss.

Noggin inhibition of mouse dentinogenesis.

OBJECTIVES: The Bone Morphogenetic Proteins (BMPs) direct tooth development and still express in the adult tooth. We hypothesized that inhibition of BMP function would therefore disrupt dentinogenesis by differentiated odontoblasts. METHODS: We generated mice overexpressing the BMP-inhibitory protein Noggin in differentiated odontoblasts and osteocytes under control of a Dmp1 promoter-driven cre transgene. We compared the dentin phenotype in these mice with that in WT littermates and in mice with a Smad4 odontoblast/osteocyte knockout mediated by the same cre and therefore lacking all BMP and Tgfß signaling in the same tissues. RESULTS: Three-month-old first molars from both Noggin-expressing and Smad4-deleted mice showed decreased dentin volume with enlarged pulp cavities, and both displayed less organized and mineralized dentinal tubules compared to WT. The Smad4-ablated phenotype was more severe. While dentin sialophosphoprotein (DSPP) and bone sialoprotein (BSP) were decreased in the dentin of both lines, dentin matrix protein 1 (DMP1) was sharply increased in Noggin-expressing teeth. CONCLUSIONS: The phenotypes we observed in Noggin-overexpressing and Smad4-conditional knockout teeth resemble the phenotype of Dentinogenesis Imperfecta (DGI) type III. Our results show that BMPs regulate post-natal dentinogenesis and that BMP-inhibitory proteins like Noggin play a role in that regulation. The increased severity of the Smad4 phenotype indicates that Tgfß ligands, in addition to BMPs, play a crucial role in post-developmental dentinogenesis.

FAM20A is essential for amelogenesis, but is dispensable for dentinogenesis.

Mutations in the gene encoding family with sequence similarity 20, member A (FAM20A) caused amelogenesis imperfecta (AI), in humans. However, the roles of FAM20A in amelogenesis and dentinogenesis are poorly understood. In this study, we generated a Fam20a knockout (Sox2-Cre;Fam20afl/fl) mouse model by crossing Fam20afl/fl mice with Sox2-Cre transgenic mice, in which Fam20a was ablated in both dental epithelium and dental mesenchyme. We found that these mice developed an enamel phenotype that resembles human AI associated with FAM20A mutations, but did not have apparent dentin defects. The secretory stage ameloblasts in the mandibular incisors from the Sox2-Cre;Fam20afl/fl mice were shorter and detached from the enamel matrix, and subsequently lost their polarity, became disorganized and formed numerous spherical extracellular matrices in place of normal enamel. At the molecular level, the Sox2-Cre;Fam20afl/fl mice displayed dramatically reduced expression levels of the genes encoding the enamel matrix proteins, but unaltered levels of the genes encoding the dentin matrix proteins. Moreover, Fam20a ablation resulted in a great decrease in FAM20C protein level, but it did not alter the intracellular localization of FAM20C protein in ameloblasts and odontoblasts. These results indicate that FAM20A is essential for amelogenesis, but is dispensable for dentinogenesis.

Inactivation of FAM20B causes cell fate changes in annulus fibrosus of mouse intervertebral disc and disc defects via the alterations of TGF-ß and MAPK signaling pathways.

Intervertebral disc (IVD) disorder is often caused by the defect of annulus fibrosus (AF), especially that of the outer AF. Studies about the mechanisms governing the development of the outer AF are needed for a better understanding of pathogenesis of IVD defects. Glycosaminoglycans (GAGs) are essential components of extracellular matrix (ECM) in AF. FAM20B is a newly identified xylose kinase that catalyzes the biosynthesis of GAGs. In this study, we created Fam20B conditional knockout (cKO) mice in which FAM20B was inactivated in type I collagen-expressing cells, the main type of cells in the outer AF of IVD. The cKO mice showed severe spine deformity and remarkable IVD defects associated with AF malformation. The AF of cKO mice had a lower level of chondroitin sulfate and heparan sulfate, and the outer AF cells lost their normal fibroblast-like morphology and acquired chondrocyte phenotypes, expressing a higher level of Sox 9 and type II collagen along with a reduced level of type I collagen. The level of phospho-Smad 2 and phospho-Smad 3, and that of scleraxis, a downstream target molecule of canonical TGF-ß signaling pathway were significantly lower in the AF of cKO mice. The AF in cKO mice also manifested altered levels in the molecules associated with the activations of MAPK pathway; the changes included the increase of phospho-P38 and phospho-ERK and a decrease of phospho-JNK. These results indicate that FAM20B plays an essential role in the development of AF by regulating the TGF-ß signaling and MAPK signaling pathways.

Loss of Fam20c causes defects in the acinar and duct structure of salivary glands in mice.

Family with sequence similarity 20­member C (FAM20C), a recently characterized Golgi kinase, performs numerous biological functions by phosphorylating more than 100 secreted proteins. However, the role of FAM20C in the salivary glands remains undefined. The present study demonstrated that FAM20C is mainly located in the cytoplasm of duct epithelial cells in the salivary glands. Fam20cf/f; Mmtv­Cre mice were created in which Fam20c was inactivated in the salivary gland cells and observed that the number of ducts and the ductal cross­sectional area increased significantly, while the number of acinar cells was reduced. The granular convoluted tubules (GCTs) exhibited an accumulation of aberrant secretory granules, along with a reduced expression and altered distribution patterns of ß nerve growth factor, α­amylase and bone morphogenetic protein (BMP) 4. This abnormality suggested that the GCT cells were immature and exhibited defects in developmental and secretory functions. In accordance with the morphological alterations and the reduced number of acinar cells, FAM20C deficiency in the salivary glands significantly decreased the salivary flow rate. The Na+, Cl- and K+ concentrations in the saliva were all significantly increased due to dysfunction of the ducts. Furthermore, Fam20c deficiency significantly increased BMP2 and BMP7 expression, decreased BMP4 expression, and attenuated the canonical and noncanonical BMP signaling pathways in the salivary glands. Collectively, the results of the present study demonstrate that FAM20C is a key regulator of acinar and duct structure and duct maturation and provide a novel avenue for investigating novel therapeutic targets for oral diseases including xerostomia.

High-Phosphate Diet Improved the Skeletal Development of Fam20c-Deficient Mice.

FAM20C (family with sequence similarity 20 - member C) is a protein kinase that phosphorylates secretory proteins, including the proteins that are essential to the formation and mineralization of calcified tissues. Previously, we reported that inactivation of Fam20c in mice led to hypophosphatemic rickets/osteomalacia along with increased circulating fibroblast growth factor 23 (FGF23) levels and dental defects. In this study, we examined whether a high-phosphate (hPi) diet could rescue the skeletal defects in Fam20c-deficient mice. Fam20c conditional knockout (cKO) mice were generated by crossing female Fam20c-floxed mice (Fam20cfl/fl) with male Sox2-Cre;Fam20cfl/+ mice. The pregnant female Fam20cfi/fl mice were fed either a normal or hPi diet until the litters were weaned. The cKO and control offspring were continuously given a normal or hPi diet for 4 weeks after weaning. Plain X-ray radiography, micro-CT, histology, immunohistochemistry (FGF23, DMP1, OPN, and SOX9), and in situ hybridization (type II and type X collagen) analyses were performed to evaluate the effects of an hPi diet on the mouse skeleton. Plain X-ray radiography and micro-CT radiography analyses showed that the hPi diet improved the shape and mineral density of the Fam20c-deficient femurs/tibiae, and rescued the growth plate defects in the long bone. Histology analyses further demonstrated that an hPi diet nearly completely rescued the growth plate-widening defects in the long bone and restored the expanded hypertrophic zone to nearly normal width. These results suggested that the hPi diet significantly improved the skeletal development of the Fam20c-deficient mice, implying that hypophosphatemia partially contributed to the skeletal defects in Fam20c-deficient subjects.

Cobalt-Iron Oxide Nanoarrays Supported on Carbon Fiber Paper with High Stability for Electrochemical Oxygen Evolution at Large Current Densities.

Here, we demonstrate that nonprecious CoFe-based oxide nanoarrays exhibit excellent electrocatalytic activity and superior stability for electrochemical oxygen evolution reaction (OER) at large current densities (>500 mA cm-2). Carbon fiber paper (CFP) with three-dimensional macroporous structure, excellent corrosion resistance, and high electrical properties is used as the support material to prevent surface passivation during the long-term process of OER. Through a facile method of hydrothermal synthesis and thermal treatment, vertically aligned arrays of spinel Co xFe3- xO4 nanostructures are homogeneously grown on CFP. The morphology and the Fe-doping content of the CoFe oxide nanoarrays can be controlled by the Fe3+ concentration in the precursor solution. The arrays of CoFe oxide nanosheets (NSs) grown on CFP (Co2.3Fe0.7O4-NSs/CFP) deliver lower Tafel slope (34.3 mV dec-1) than CoFe oxide nanowire (NW) arrays grown on CFP (Co2.7Fe0.3O4-NWs/CFP) in alkaline solution, owing to higher Fe-doping content and larger effective specific surface area. The Co2.3Fe0.7O4-NSs/CFP electrode exhibits excellent stability for OER at large current densities in alkaline solution. Moreover, the morphology and structure of CoFeO nanoarrays are well preserved after long-term testing, indicating the high stability for OER.

Inactivation of Fam20b in the neural crest-derived mesenchyme of mouse causes multiple craniofacial defects.

The glycosaminoglycan (GAG) chains attached to the core proteins of proteoglycans exert multiple roles, such as enriching signal molecules and regulating the binding of ligands to the corresponding receptors. A newly identified kinase - family with sequence similarity 20 member B (FAM20B) - is essential for the formation of GAG chains. The FAM20B protein phosphorylates the initial xylose on the side chain of a serine residue in the protein. Although the GAG chains of proteoglycans are believed to be indispensable during craniofacial development, there are few reports on their exact functions in craniofacial organogenesis. In this study, by mating Wnt1-cre mice with Fam20b-floxed mice (Fam20bflox/flox), we created Wnt1-Cre;Fam20bflox/flox mice in which Fam20b is ablated in the neural crest-derived mesenchyme. The Wnt1-Cre;Fam20bflox/flox mice died immediately after birth because of complete cleft palates. In addition to cleft palate, Wnt1-Cre;Fam20bflox/flox mice also manifested tongue elevation, micrognathia, microcephaly, suture widening, and reduced mineralization in the calvaria, facial bones, and temporomandibular joint. These findings indicate that the proteoglycans formed through the catalysis of FAM20B are essential for the morphogenesis and mineralization of the craniofacial complex.

Transgenic expression of dentin phosphoprotein (DPP) partially rescued the dentin defects of DSPP-null mice.

Mutations in the dentin sialophosphoprotein (DSPP) gene cause dentinogenesis imperfecta. After synthesis, DSPP is proteolytically processed into NH2- and COOH-terminal fragments. The NH2-terminal fragment of DSPP is highly glycosylated but not phosphorylated, whereas the COOH-terminal fragment (named "dentin phosphoprotein" or "DPP") is highly phosphorylated but not glycosylated. These two fragments are believed to perform distinct roles in dentin formation. To analyze the functions of DPP in dentinogenesis, we created "Dspp-/-;DPP Tg mice", which expressed transgenic DPP driven by a Type I collagen promoter but lacked the endogenous Dspp gene. We characterized the dentin of the Dspp-/-;DPP Tg mice using X-ray radiography, histology, scanning electron microscopy, double fluorochrome labeling, immunohistochemistry and in situ hybridization. Micro-computed tomography analyses revealed that at postnatal 6 months, the transgenic expression of DPP increased the dentin thickness of the Dspp-null mice by 97.1% and restored the dentin material density by 29.5%. Histological analyses showed that the Dspp-null mice manifested an abnormal widening of the predentin while the predentin in Dspp-/-;DPP Tg mice was narrower than in the Dspp-null mice. Scanning electron microscopy analyses showed that the dentinal tubules in the Dspp-/-;DPP Tg mice were better organized than in the Dspp-null mice. The double fluorochrome labeling analyses demonstrated that the dentin mineral deposition rate in the Dspp-/-;DPP Tg mice was significantly improved compared to that in the Dspp-null mice. These findings indicate that the transgenic expression of DPP partially rescued the dentin defects of the DSPP-null mice, suggesting that DPP may promote dentin formation and that the coordinated actions between DPP and the NH2-terminal fragment of DSPP may be necessary for dentinogenesis.

The role of bone morphogenetic proteins 2 and 4 in mouse dentinogenesis.

OBJECTIVE: The bone morphogenetic proteins (BMPs) play crucial roles in tooth development. However, several BMPs retain expression in the dentin of the fully patterned and differentiated tooth. We hypothesized that BMP signaling therefore plays a role in the function of the differentiated odontoblast, the job of which is to lay down and mineralize the dentin matrix. DESIGN: We generated mice deficient in Bmp2 and 4 using a dentin matrix protein 1 (Dmp1) promoter-driven cre recombinase that was expressed in differentiated odontoblasts. RESULTS: The first and second molars of these Bmp2 and Bmp4 double conditional knockout (DcKO) mice displayed reduced dentin and enlarged pulp chambers compared to cre-negative littermate controls. DcKO mouse dentin in first molars was characterized by small, disorganized dentinal fibers, a wider predentin layer, and reduced expression of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), and bone sialoprotein (BSP). DcKO mouse odontoblasts demonstrated increased type I collagen mRNA production, indicating that the loss of BMP signaling altered the rate of collagen gene expression in these cells. Bmp2 and Bmp4 single Dmp1-cre knockout mice displayed no discernable dentin phenotype. CONCLUSIONS: These data demonstrate that BMP signaling in differentiated odontoblasts is necessary for proper dentin production in mature teeth.