Loss of cped1 does not affect bone and lean mass in zebrafish.

Human genetic studies have nominated Cadherin-like and PC-esterase Domain-containing 1 ( CPED1 ) as a candidate target gene mediating bone mineral density (BMD) and fracture risk heritability. Recent efforts to define the role of CPED1 in bone in mouse and human models have revealed complex alternative splicing and inconsistent results arising from gene targeting, making its function difficult to interpret. To better understand the role of CPED1 in adult bone mass and morphology, we turned to zebrafish, an emerging model for orthopaedic research. We analyzed two different cped1 mutant lines and performed deep phenotyping to characterize more than 200 measures of adult vertebral, craniofacial, and lean tissue morphology. We also examined alternative splicing of zebrafish cped1 and gene expression in various cell/tissue types. Our studies fail to support an essential role of cped1 in adult zebrafish bone. Specifically, homozygous mutants for both cped1 mutant alleles, which are expected to result in loss-of-function and impact all cped1 isoforms, exhibited no significant differences in the measures examined when compared to their respective wildtype controls, suggesting that cped1 does not significantly contribute to these traits. We identified sequence differences in critical residues of the catalytic triad between the zebrafish and mouse orthologs of CPED1, and discuss how these differences, as well as distinct alternative splicing, could underlie different functions of CPED1 orthologs in the two species. Our studies demonstrate that cped1 is not required for normal adult zebrafish bone mass, lean mass, or bone and lean mass morphology, adding to evidence that variants at 7q31.31 can act independently of CPED1 to influence BMD and fracture risk.

Multiple Mechanisms Explain Genetic Effects at the CPED1-WNT16 Bone Mineral Density Locus.

PURPOSE OF REVIEW: Chromosome region 7q31.31, also known as the CPED1-WNT16 locus, is robustly associated with BMD and fracture risk. The aim of the review is to highlight experimental studies examining the function of genes at the CPED1-WNT16 locus. RECENT FINDINGS: Genes that reside at the CPED1-WNT16 locus include WNT16, FAM3C, ING3, CPED1, and TSPAN12. Experimental studies in mice strongly support the notion that Wnt16 is necessary for bone mass and strength. In addition, roles for Fam3c and Ing3 in regulating bone morphology in vivo and/or osteoblast differentiation in vitro have been identified. Finally, a role for wnt16 in dually influencing bone and muscle morphogenesis in zebrafish has recently been discovered, which has brought forth new questions related to whether the influence of WNT16 in muscle may conspire with its influence in bone to alter BMD and fracture risk.

Systematic analysis of cilia characteristics and Hedgehog signaling in five immortal cell lines.

Dysfunction of the primary cilium, a microtubule-based signaling organelle, leads to genetic conditions called ciliopathies. Hedgehog (Hh) signaling is mediated by the primary cilium in vertebrates and is therefore implicated in ciliopathies; however, it is not clear which immortal cell lines are the most appropriate for modeling pathway response in human disease; therefore, we systematically evaluated Hh in five commercially available, immortal mammalian cell lines: ARPE-19, HEK293T, hTERT RPE-1, NIH/3T3, and SH-SY5Y. Under proper conditions, all of the cell lines ciliated adequately for our subsequent experiments, except for SH-SY5Y which were excluded from further analysis. hTERT RPE-1 and NIH/3T3 cells relocalized Hh pathway components Smoothened (SMO) and GPR161 and upregulated Hh target genes in response to pathway stimulation. In contrast, pathway stimulation did not induce target gene expression in ARPE-19 and HEK293T cells, despite SMO and GPR161 relocalization. These data indicate that human hTERT RPE-1 cells and murine NIH/3T3 cells, but not ARPE-19 and HEK293T cells, are suitable for modeling the role of Hh signaling in ciliopathies.

wnt16 regulates spine and muscle morphogenesis through parallel signals from notochord and dermomyotome.

Bone and muscle are coupled through developmental, mechanical, paracrine, and autocrine signals. Genetic variants at the CPED1-WNT16 locus are dually associated with bone- and muscle-related traits. While Wnt16 is necessary for bone mass and strength, this fails to explain pleiotropy at this locus. Here, we show wnt16 is required for spine and muscle morphogenesis in zebrafish. In embryos, wnt16 is expressed in dermomyotome and developing notochord, and contributes to larval myotome morphology and notochord elongation. Later, wnt16 is expressed at the ventral midline of the notochord sheath, and contributes to spine mineralization and osteoblast recruitment. Morphological changes in wnt16 mutant larvae are mirrored in adults, indicating that wnt16 impacts bone and muscle morphology throughout the lifespan. Finally, we show that wnt16 is a gene of major effect on lean mass at the CPED1-WNT16 locus. Our findings indicate that Wnt16 is secreted in structures adjacent to developing bone (notochord) and muscle (dermomyotome) where it affects the morphogenesis of each tissue, thereby rendering wnt16 expression into dual effects on bone and muscle morphology. This work expands our understanding of wnt16 in musculoskeletal development and supports the potential for variants to act through WNT16 to influence bone and muscle via parallel morphogenetic processes.