Germline Saturation Mutagenesis Induces Skeletal Phenotypes in Mice

Proper embryonic and postnatal skeletal development require coordination of myriad complex molecular mechanisms. Disruption of these processes, through genetic mutation, contributes to variation in skeletal development. We developed a high-throughput N-ethyl-N-nitrosourea (ENU)-induced saturation mutagenesis skeletal screening approach in mice to identify genes required for proper skeletal development. Here, we report initial results from live-animal X-ray and dual-energy X-ray absorptiometry (DXA) imaging of 27,607 G3 mice from 806 pedigrees, testing the effects of 32,198 coding/splicing mutations in 13,020 genes. A total of 39.7% of all autosomal genes were severely damaged or destroyed by mutations tested twice or more in the homozygous state. Results from our study demonstrate the feasibility of in vivo mutagenesis to identify mouse models of skeletal disease. Furthermore, our study demonstrates how ENU mutagenesis provides opportunities to create and characterize putative hypomorphic mutations in developmentally essential genes. Finally, we present a viable mouse model and case report of recessive skeletal disease caused by mutations in FAM20B. Results from this study, including engineered mouse models, are made publicly available via the online Mutagenetix database. © 2021 American Society for Bone and Mineral Research (ASBMR).     

Effects of Deletion of ERα in Osteoblast‐Lineage Cells on Bone Mass and Adaptation to Mechanical Loading Differ in Female and Male Mice

Estrogen receptor alpha (ERα) has been implicated in bone's response to mechanical loading in both males and females. ERα in osteoblast lineage cells is important for determining bone mass, but results depend on animal sex and the cellular stage at which ERα is deleted. We demonstrated previously that when ERα is deleted from mature osteoblasts and osteocytes in mixed‐background female mice, bone mass and strength are decreased. However, few studies exist examining the skeletal response to loading in bone cell–specific ERαKO mice. Therefore, we crossed ERα floxed (ERα^fl/fl) and osteocalcin‐Cre (OC‐Cre) mice to generate animals lacking ERα in mature osteoblasts and osteocytes (pOC‐ERαKO) and littermate controls (LC). At 10 weeks of age, the left tibia was loaded in vivo for 2 weeks. We analyzed bone mass through micro‐CT, bone formation rate by dynamic histomorphometry, bone strength from mechanical testing, and osteoblast and osteoclast activity by serum chemistry and immunohistochemistry. ERα in mature osteoblasts differentially regulated bone mass in males and females. Compared with LC, female pOC‐ERαKO mice had decreased cortical and cancellous bone mass, whereas male pOC‐ERαKO mice had equal or greater bone mass than LC. Bone mass results correlated with decreased compressive strength in pOC‐ERαKO female L₅ vertebrae and with increased maximum moment in pOC‐ERαKO male femora. Female pOC‐ERαKO mice responded more to mechanical loading, whereas the response of pOC‐ERαKO male animals was similar to their littermate controls. © 2015 American Society for Bone and Mineral Research. © 2015 American Society for Bone and Mineral Research.     

Control of Bone Anabolism in Response to Mechanical Loading and PTH by Distinct Mechanisms Downstream of the PTH Receptor

Osteocytes integrate the responses of bone to mechanical and hormonal stimuli by poorly understood mechanisms. We report here that mice with conditional deletion of the parathyroid hormone (PTH) receptor 1 (Pth1r) in dentin matrix protein 1 (DMP1)‐8kb–expressing cells (cKO) exhibit a modest decrease in bone resorption leading to a mild increase in cancellous bone without changes in cortical bone. However, bone resorption in response to endogenous chronic elevation of PTH in growing or adult cKO mice induced by a low calcium diet remained intact, because the increased bone remodeling and bone loss was indistinguishable from that exhibited by control littermates. In contrast, the bone gain and increased bone formation in cancellous and cortical bone induced by daily injections of PTH and the periosteal bone apposition induced by axial ulna loading were markedly reduced in cKO mice compared to controls. Remarkably, however, wild‐type (WT) control littermates and transgenic mice overexpressing SOST injected daily with PTH exhibit similar activation of Wnt/β‐catenin signaling, increased bone formation, and cancellous and cortical bone gain. Taken together, these findings demonstrate that Pth1r in DMP1‐8kb–expressing cells is required to maintain basal levels of bone resorption but is dispensable for the catabolic action of chronic PTH elevation; and it is essential for the anabolic actions of daily PTH injections and mechanical loading. However, downregulation of Sost/sclerostin, previously shown to be required for bone anabolism induced by mechanical loading, is not required for PTH‐induced bone gain, showing that other mechanisms downstream of the Pth1r in DMP1‐8kb–expressing cells are responsible for the hormonal effect. © 2016 American Society for Bone and Mineral Research.     

Loss of Sprouty Produces a Ciliopathic Skeletal Phenotype in Mice Through Upregulation of Hedgehog Signaling

The Sprouty family is a highly conserved group of intracellular modulators of receptor tyrosine kinase (RTK)-signaling pathways, which have been recently linked to primary cilia. Disruptions in the structure and function of primary cilia cause inherited disorders called ciliopathies. We aimed to evaluate Sprouty2 and Sprouty4 gene-dependent alterations of ciliary structure and to focus on the determination of its association with Hedgehog signaling defects in chondrocytes. Analysis of the transgenic mice phenotype with Sprouty2 and Sprouty4 deficiency revealed several defects, including improper endochondral bone formation and digit patterning, or craniofacial and dental abnormalities. Moreover, reduced bone thickness and trabecular bone mass, skull deformities, or chondroma-like lesions were revealed. All these pathologies might be attributed to ciliopathies. Elongation of the ciliary axonemes in embryonic and postnatal growth plate chondrocytes was observed in Sprouty2^−/− and Sprouty2^+/−/Sprouty4^−/− mutants compared with corresponding littermate controls. Also, cilia-dependent Hedgehog signaling was upregulated in Sprouty2/4 mutant animals. Ptch1 and Ihh expression were upregulated in the autopodium and the proximal tibia of Sprouty2^−/−/Sprouty4^−/− mutants. Increased levels of the GLI3 repressor (GLI3R) form were detected in Sprouty2/4 mutant primary fibroblast embryonic cell cultures and tissues. These findings demonstrate that mouse lines deficient in Sprouty proteins manifest phenotypic features resembling ciliopathic phenotypes in multiple aspects and may serve as valuable models to study the association between overactivation of RTK and dysfunction of primary cilia during skeletogenesis. © 2021 American Society for Bone and Mineral Research (ASBMR).