A requirement for Fgfr2 in middle ear development.

The skeletal structure of the mammalian middle ear, which is composed of three endochondral ossicles suspended within a membranous air-filled capsule, plays a critical role in conducting sound. Gene mutations that alter skeletal development in the middle ear result in auditory impairment. Mutations in fibroblast growth factor receptor 2 (FGFR2), an important regulator of endochondral and intramembranous bone formation, cause a spectrum of congenital skeletal disorders featuring conductive hearing loss. Although the middle ear malformations in multiple FGFR2 gain-of-function disorders are clinically characterized, those in the FGFR2 loss-of-function disorder lacrimo-auriculo-dento-digital (LADD) syndrome are relatively undescribed. To better understand conductive hearing loss in LADD, we examined the middle ear skeleton of mice with conditional loss of Fgfr2. We find that decreased auditory function in Fgfr2 mutant mice correlates with hypoplasia of the auditory bulla and ectopic bone growth at sites of tendon/ligament attachment. We show that ectopic bone associated with the intra-articular ligaments of the incudomalleal joint is derived from Scx-expressing cells and preceded by decreased expression of the joint progenitor marker Gdf5. Together, these results identify a role for Fgfr2 in development of the middle ear skeletal tissues and suggest potential causes for conductive hearing loss in LADD syndrome.

Nuclear Fibroblast Growth Factor Receptor Signaling in Skeletal Development and Disease.

PURPOSE OF REVIEW: Fibroblast growth factor receptor (FGFR) signaling regulates proliferation and differentiation during development and homeostasis. While membrane-bound FGFRs play a central role in these processes, the function of nuclear FGFRs is also critical. Here, we highlight mechanisms for nuclear FGFR translocation and the effects of nuclear FGFRs on skeletal development and disease. RECENT FINDINGS: Full-length FGFRs, internalized by endocytosis, enter the nucleus through ß-importin-dependent mechanisms that recognize the nuclear localization signal within FGFs. Alternatively, soluble FGFR intracellular fragments undergo nuclear translocation following their proteolytic release from the membrane. FGFRs enter the nucleus during the cellular transition between proliferation and differentiation. Once nuclear, FGFRs interact with chromatin remodelers to alter the epigenetic state and transcription of their target genes. Dysregulation of nuclear FGFR is linked to the etiology of congenital skeletal disorders and neoplastic transformation. Revealing the activities of nuclear FGFR will advance our understanding of 20 congenital skeletal disorders caused by FGFR mutations, as well as FGFR-related cancers.

TGFß and BMP Dependent Cell Fate Changes Due to Loss of Filamin B Produces Disc Degeneration and Progressive Vertebral Fusions.

Spondylocarpotarsal synostosis (SCT) is an autosomal recessive disorder characterized by progressive vertebral fusions and caused by loss of function mutations in Filamin B (FLNB). FLNB acts as a signaling scaffold by linking the actin cytoskleteon to signal transduction systems, yet the disease mechanisms for SCT remain unclear. Employing a Flnb knockout mouse, we found morphologic and molecular evidence that the intervertebral discs (IVDs) of Flnb-/-mice undergo rapid and progressive degeneration during postnatal development as a result of abnormal cell fate changes in the IVD, particularly the annulus fibrosus (AF). In Flnb-/-mice, the AF cells lose their typical fibroblast-like characteristics and acquire the molecular and phenotypic signature of hypertrophic chondrocytes. This change is characterized by hallmarks of endochondral-like ossification including alterations in collagen matrix, expression of Collagen X, increased apoptosis, and inappropriate ossification of the disc tissue. We show that conversion of the AF cells into chondrocytes is coincident with upregulated TGFß signaling via Smad2/3 and BMP induced p38 signaling as well as sustained activation of canonical and noncanonical target genes p21 and Ctgf. These findings indicate that FLNB is involved in attenuation of TGFß/BMP signaling and influences AF cell fate. Furthermore, we demonstrate that the IVD disruptions in Flnb-/-mice resemble aging degenerative discs and reveal new insights into the molecular causes of vertebral fusions and disc degeneration.

TGFß signaling in cartilage development and maintenance.

Members of the transforming growth factor beta (TGFß) superfamily of secreted factors play essential roles in nearly every aspect of cartilage formation and maintenance. However, the mechanisms by which TGFßs transduce their effects in cartilage in vivo remain poorly understood. Mutations in several TGFß family members, their receptors, extracellular modulators, and intracellular transducers have been described, and these usually impact the development of the cartilaginous skeleton. Furthermore, genome-wide association studies have linked components of the (TGFß) superfamily to susceptibility to osteoarthritis. This review focuses on recent discoveries from genetic studies in the mouse regarding the regulation of TGFß signaling in developing growth plate and articular cartilage, as well as the different modes of crosstalk between canonical and noncanonical TGFß signaling. These new insights into TGFß signaling in cartilage may open new prospects for therapies that maintain healthy articular cartilage.

TGFß as a gatekeeper of BMP action in the developing growth plate.

The ligands that comprise the Transforming Growth Factor ß superfamily highly govern the development of the embryonic growth plate. Members of this superfamily activate canonical TGFß and/or BMP (Bone Morphogenetic Protein) signaling pathways. How these pathways interact with one another is an area of active investigation. These two signaling pathways have been described to negatively regulate one another through crosstalk involving Smad proteins, the primary intracellular effectors of canonical signaling. More recently, a mechanism for regulation of the BMP pathway through TGFß and BMP receptor interactions has been described. Here in this review, we demonstrate examples of how TGFß is a gatekeeper of BMP action in the developing growth plate at both the receptor and transcriptional levels.

Homozygous missense variant in BMPR1A resulting in BMPR signaling disruption and syndromic features.

BACKGROUND: The bone morphogenetic protein (BMP) pathway is known to play an imperative role in bone, cartilage, and cardiac tissue formation. Truncating, heterozygous variants, and deletions of one of the essential receptors in this pathway, Bone Morphogenetic Protein Receptor Type1A (BMPR1A), have been associated with autosomal dominant juvenile polyposis. Heterozygous deletions have also been associated with cardiac and minor skeletal anomalies. Populations with atrioventricular septal defects are enriched for rare missense BMPR1A variants. METHODS: We report on a patient with a homozygous missense variant in BMPR1A causing skeletal abnormalities, growth failure a large atrial septal defect, severe subglottic stenosis, laryngomalacia, facial dysmorphisms, and developmental delays. RESULTS: Functional analysis of this variant shows increased chondrocyte death for cells with the mutated receptor, increased phosphorylated R-Smads1/5/8, and loss of Sox9 expression mediated by decreased phosphorylation of p38. CONCLUSION: This homozygous missense variant in BMPR1A appears to cause a distinct clinical phenotype.

CYR61/CCN1 Regulates Sclerostin Levels and Bone Maintenance.

CYR61/CCN1 is a matricellular protein that resides in the extracellular matrix, but serves regulatory rather than structural roles. CYR61/CCN1 is found in mineralized tissues and has been shown to influence bone healing in vivo and osteogenic differentiation in vitro. In this study we generated Cyr61 bone-specific knockout mice to examine the physiological role of CYR61/CCN1 in bone development and maintenance in vivo. Extensive analysis of Cyr61 conditional knockout mice showed a significant decrease in both trabecular and cortical bone mass as compared to WT littermates. Our data suggest that CYR61/CCN1 exerts its effects on mature osteoblast/osteocyte function to modulate bone mass. Specifically, changes were observed in osteocyte/osteoblast expression of RankL, VegfA, and Sost. The increase in RankL expression was correlated with a significant increase in osteoclast number; decreased VegfA expression was correlated with a significant decrease in bone vasculature; increased Sost expression was associated with decreased Wnt signaling, as revealed by decreased Axin2 expression and increased adiposity in the bone marrow. Although the decreased number of vascular elements in bone likely contributes to the low bone mass phenotype in Cyr61 conditional knockout mice, this cannot explain the observed increase in osteoclasts and the decrease in Wnt signaling. We conducted in vitro assays using UMR-106 osteosarcoma cells to explore the role CYR61/CCN1 plays in modulating Sost mRNA and protein expression in osteocytes and osteoblasts. Overexpression of CYR61/CCN1 can suppress Sost expression in both control and Cyr61 knockout cells, and blocking Sost with siRNA can rescue Wnt responsiveness in Cyr61 knockout cells in vitro. Overall, our data suggest that CYR61/CCN1 modulates mature osteoblast and osteocyte function to regulate bone mass through angiogenic effects as well as by modulating Wnt signaling, at least in part through the Wnt antagonist Sost. © 2018 American Society for Bone and Mineral Research.

The type I BMP receptor ACVR1/ALK2 is required for chondrogenesis during development.

Bone morphogenetic proteins (BMPs) are crucial regulators of chondrogenesis. BMPs transduce their signals through three type I receptors: BMPR1A, BMPR1B, and ACVR1/ALK2. Fibrodysplasia ossificans progressiva (FOP), a rare disorder characterized by progressive ossification of connective tissue, is caused by an activating mutation in Acvr1 (the gene that encodes ACVR1/ALK2). However, there are few developmental defects associated with FOP. Thus, the role of ACVR1 in chondrogenesis during development is unknown. Here we report the phenotype of mice lacking ACVR1 in cartilage. Acvr1(CKO) mice are viable but exhibit defects in the development of cranial and axial structures. Mutants exhibit a shortened cranial base, and cervical vertebrae are hypoplastic. Acvr1(CKO) adult mice develop progressive kyphosis. These morphological defects were associated with decreased levels of Smad1/5 and p38 activation, and with reduced rates of chondrocyte proliferation in vertebral cartilage. We also tested whether ACVR1 exerts coordinated functions with BMPR1A and BMPR1B through analysis of double mutants. Acvr1/Bmpr1a and Acvr1/Bmpr1b mutant mice exhibited generalized perinatal lethal chondrodysplasia that was much more severe than in any of the corresponding mutant strains. These findings demonstrate that ACVR1 is required for chondrocyte proliferation and differentiation, particularly in craniofacial and axial elements, but exerts coordinated functions with both BMPR1A and BMPR1B throughout the developing endochondral skeleton.

Whole-mount skeletal staining.

The first step in almost every investigation of skeletal phenotypes is analysis of whole-mount skeletal preparations. Whole-mount skeletal staining permits evaluation of the shapes and sizes of skeletal elements in their appropriate locations. The technique is thus the major method for detecting changes in skeletal patterning. Because cartilage and bone can be distinguished by differential staining, this technique is also a powerful means to assess the pace of skeletal maturation. This protocol covers staining of the pre- and postnatal mouse skeleton using Alcian blue and Alizarin red to identify cartilage and bone, respectively.

GATA4 is essential for bone mineralization via ERα and TGFß/BMP pathways.

Osteoporosis is a disease characterized by low bone mass, leading to an increased risk of fragility fractures. GATA4 is a zinc-finger transcription factor that is important in several tissues, such as the heart and intestines, and has recently been shown to be a pioneer factor for estrogen receptor alpha (ERα) in osteoblast-like cells. Herein, we demonstrate that GATA4 is necessary for estrogen-mediated transcription and estrogen-independent mineralization in vitro. In vivo deletion of GATA4, driven by Cre-recombinase in osteoblasts, results in perinatal lethality, decreased trabecular bone properties, and abnormal bone development. Microarray analysis revealed GATA4 suppression of TGFß signaling, necessary for osteoblast progenitor maintenance, and concomitant activation of BMP signaling, necessary for mineralization. Indeed, pSMAD1/5/8 signaling, downstream of BMP signaling, is decreased in the trabecular region of conditional knockout femurs, and pSMAD2/3, downstream of TGFß signaling, is increased in the same region. Together, these experiments demonstrate the necessity of GATA4 in osteoblasts. Understanding the role of GATA4 to regulate the tissue specificity of estrogen-mediated osteoblast gene regulation and estrogen-independent bone differentiation may help to develop therapies for postmenopausal osteoporosis.