Biology subjects

Charles, J. F.

Publications and source records attributed to Charles, J. F..

4 recordsLinked to original sources

bioRxiv2026

Site-, strain- and mutation-specific osteopetrosis phenotypes in mice with Clcn7 variants

Background: Autosomal dominant osteopetrosis type II (ADOII) is a rare bone disorder caused primarily by heterozygous mutations in CLCN7 and characterized by increased bone mass yet fragility and marked phenotypic heterogeneity among affected individuals. The biological factors underlying this variability remain poorly understood. We investigated the effects of age, sex, genetic background, and mutation type on bone phenotypes in murine models of ADOII carrying heterozygous Clcn7 F318L/+ or G213R/+ mutations. Methods: Bone microarchitecture was assessed by micro-computed tomography in vertebrae and femora from female and male mice across multiple ages (12, 24, and 52 weeks) and strain backgrounds (FVB/NJ, C57BL/6, and 129). Vertebral biomechanical properties were estimated by micro-finite element analysis, while femoral mechanical properties were measured using three-point bending. Results: Clcn7 F318L/+ mice exhibited significantly increased trabecular bone compared with wild-type littermates at vertebral and femoral sites across the lifespan in both sexes. The magnitude of the vertebral trabecular phenotype increased with age, whereas age-related effects in the femur were more modest and site dependent. Vertebral micro-finite element analysis predicted greater stiffness and ultimate force in F318L/+ mice irrespective of age. Cortical thickness was modestly increased, particularly in female mice, but femoral mechanical properties were largely unchanged. The relative increase in vertebral trabecular bone was similar on the FVB/NJ and C57BL/6 backgrounds in males but greater on C57BL/6 in females, whereas the femoral trabecular and cortical responses were markedly attenuated on C57BL/6 in both sexes. In G213R/+ mice, the relative trabecular phenotype was similar on the C57BL/6 and 129 backgrounds, except for a greater vertebral phenotype in 129 males. Likewise, comparison of F318L/+ and G213R/+ mice demonstrated comparable vertebral phenotypes, although mutation-specific differences emerged at the femur, particularly in males. Conclusion: Collectively, these findings demonstrate that heterozygous Clcn7 mutations produce a robust osteopetrotic phenotype characterized primarily by increased trabecular bone mass that is present regardless of sex, age, genetic background, and mutation type. Age and genetic background modify its magnitude in a site- and sex-specific manner, suggesting that local factors may contribute more strongly to phenotypic variation than the specific disease-causing Clcn7 mutation. These results provide insight into determinants of phenotypic heterogeneity in ADOII and establish a framework for future mechanistic studies of osteopetrotic bone quality and fragility.

physiology↗

bioRxiv2022

En1 and Lmx1b do not recapitulate embryonic dorsal-ventral limb patterning functions during mouse digit tip regeneration

The mouse digit tip is a complex tissue that is capable of regeneration after amputation. How the regenerated digit tip is patterned is unknown, but a long-standing hypothesis in the field of regeneration proposes that developmental patterning mechanisms are re-used during regeneration. The digit tip bone exhibits strong dorsal-ventral (DV) polarity, so we focus on Engrailed 1 (En1) and LIM homeobox transcription factor 1B (Lmx1b), two well-studied transcription factors necessary for DV patterning during limb development. We investigate if En1 and Lmx1b are re-expressed during regeneration in a developmental-like spatially restricted pattern, and if they direct DV morphology of the regenerated digit tip. We find that both En1 and Lmx1b are expressed in the regenerating mouse digit tip epithelium and mesenchyme, respectively, but without DV polarity. We use conditional genetics and quantitative analysis of digit tip bone morphology to determine that genetic deletion of En1 or Lmx1b in adult digit tip regeneration modestly reduces bone regeneration but does not affect DV patterning of the regenerate. Collectively, our data suggest that while En1 and Lmx1b are re-expressed during mouse digit tip regeneration, they do not define the DV axis during regeneration.

developmental biology↗

bioRxiv2021

LGR6 is necessary for attaining peak bone mass and regulates osteogenesis through differential ligand use

Leucine-rich repeat containing G-protein-coupled receptor 6 (LGR6) is a marker of osteoprogenitor cells and is dynamically expressed during in vitro osteodifferentation of mouse and human mesenchymal stem cells (MSCs). While the Lgr6 genomic locus has been associated with osteoporosis in human cohorts, the precise molecular function of LGR6 in osteogenesis and maintenance of bone mass are not yet known. In this study, we performed in vitro Lgr6 knockdown and overexpression experiments in murine osteoblastic cells and find decreased Lgr6 levels results in reduced osteoblast proliferation, differentiation, and mineralization. Consistent with these data, overexpression of Lgr6 in these cells leads to significantly increased proliferation and osteodifferentiation. To determine whether these findings are recapitulated in vivo, we performed microCT and ex vivo osteodifferentiation analyses using our newly generated CRISPR-Cas9 mediated Lgr6 mouse knockout allele (Lgr6-KO). We find that ex vivo osteodifferentiation of Lgr6-KO primary MSCs is significantly reduced, and 8 week-old Lgr6-KO mice have less trabecular bone mass as compared to Lgr6 wildtype controls, indicating that Lgr6 is necessary for normal osteogenesis and to attain peak bone mass. Toward mechanism, we analyzed in vitro signaling in the context of two LGR6 ligands, RSPO2 and MaR1. We find that RSPO2 stimulates LGR6-mediated WNT/{beta}-catenin signaling whereas MaR1 stimulates LGR6-mediated cAMP activity, suggesting two ligand-dependent functions for LGR6 receptor signaling during osteogenesis. Collectively, this study reveals that Lgr6 is necessary for wildtype levels of proliferation and differentiation of osteoblasts, and achieving peak bone mass.

developmental biology↗

bioRxiv2020

Obesity-linked PPARγ S273 phosphorylation promotes insulin resistance through Growth Differentiation Factor 3

Overnutrition and obesity promote adipose tissue dysfunction, often leading to systemic insulin resistance. The thiazolidinediones (TZDs) are a potent class of insulin-sensitizing drugs and ligands of PPAR{gamma} that improve insulin sensitivity, but their use is limited due to significant side effects. Recently, we demonstrated a mechanism by which TZDs improve insulin sensitivity distinct from receptor agonism and adipogenesis: reversal of obesity-linked phosphorylation of PPAR{gamma} at Serine 273. However, the role of this modification has not been tested genetically. Here we demonstrate that mice encoding an allele of PPAR{gamma} which cannot be phosphorylated at S273 are protected from insulin resistance, without exhibiting differences in body weight or TZD-associated side effects. Indeed, hyperinsulinemic-euglycemic clamp experiments confirm improved insulin sensitivity, as evidenced by increased whole-body glucose uptake. RNA-seq experiments reveal PPAR{gamma} S273 phosphorylation specifically enhances transcription of Gdf3, a BMP family member. Ectopic expression of Gdf3 is sufficient to induce insulin resistance in lean, healthy mice. We find that Gdf3 can impact metabolism by inhibition of BMP signaling. Together, these results highlight the diabetogenic role of PPAR{gamma} S273 phosphorylation and focuses attention on a putative target, Gdf3.

cell biology↗