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Bone and muscle interact 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.AUTHOR SUMMARYIn humans, genetic variants (DNA sequences that vary amongst individuals) have been identified that appear to influence bone and skeletal muscle mass. However, how single genes and genetic variants exert dual influence on both tissues is not well understood. In this study, we found a gene called wnt16 is necessary for specifying the size and shape of muscle and bone during development in zebrafish. Moreover, we disentangled how wnt16 affects both tissues: distinct cellular populations adjacent to muscle and bone secrete Wnt16, where it acts as a signal this guides the size and shape of each tissue. This is important because in humans, genetic variants near the WNT16 gene have effects on both bone- and muscle-related traits. This study expands our understanding of the role of WNT16 in bone and muscle development, and helps to explain how genetic variants near WNT16 affect traits for both tissues. Moreover, WNT16 is actively being explored as a target for osteoporosis therapies; our study could have implications with regard to the potential to target WNT16 to treat bone and muscle simultaneously.

SummaryVertebrate skeletal muscles are composed of both slow-twitch and fast-twitch fiber types. How the differentiation of distinct fiber types is activated during embryogenesis is not well characterized. Skeletal muscle differentiation is initiated by the activity of the myogenic basic helix-loop-helix (bHLH) transcription factors Myf5, Myod1, Myf6, and Myog. Myod1 functions as a muscle master regulatory factor and directly activates muscle differentiation genes, including those specific to both slow and fast muscle fibers. Our previous studies showed that Pbx TALE-class homeodomain proteins bind with Myod1 on the promoter of the zebrafish fast muscle genemylpfaand are required for proper activation ofmylpfaexpression and the fast-twitch muscle-specific differentiation program in zebrafish embryos. Pbx proteins have also been shown to bind regulatory regions of muscle differentiation genes in mammalian muscle cells in culture. Here, we use new zebrafish mutant strains to confirm the essential roles of zebrafish Pbx factors in embryonic fast muscle differentiation. Furthermore, we examine the requirements forPbxgenes in mouse embryonic skeletal muscle differentiation, an area that has not been investigated in the mammalian embryo. RemovingPbx1function from skeletal muscle inMyf5Cre/+;Pbx1fl/flmouse embryos has minor effects on embryonic muscle development. However, concomitantly deletingPbx2function inMyf5Cre/+;Pbx1fl/fl;Pbx2-/-mouse embryos causes delayed activation and reduced expression of fast muscle differentiation genes. In the mouse,Pbx1/Pbx2-dependent fast muscle genes closely match those that have been previously shown to be dependent on murineSix1andSix4. This work establishes evolutionarily conserved requirements for Pbx factors in embryonic fast muscle differentiation. Our studies are revealing how Pbx homeodomain proteins help direct specific cellular differentiation pathways.

SummaryDuchenne muscular dystrophy (DMD) is a severe neuromuscular disorder and is one of the most common muscular dystrophies. There are currently few effective therapies to treat the disease, although many small-molecule approaches are being pursued. Specific histone deacetylase inhibitors (HDACi) can ameliorate DMD phenotypes in mouse and zebrafish animal models and have also shown promise for DMD in clinical trials. However, beyond these HDACi, other classes of epigenetic small molecules have not been broadly and systematically studied for their benefits for DMD. Here, we performed a novel chemical screen of a library of epigenetic compounds using the zebrafishdmdmodel. We identified candidate pools of epigenetic compounds that improve skeletal muscle structure indmdzebrafish. We then identified a specific combination of two drugs, oxamflatin and salermide, that significantly rescueddmdzebrafish skeletal muscle degeneration. Furthermore, we validated the effects of oxamflatin and salermide in an independent laboratory. Our results provide novel, effective methods for performing a combination small-molecule screen in zebrafish. Our results also add to the growing evidence that epigenetic small molecules may be promising candidates for treating DMD.

Whole-genome and exome sequencing efforts are increasingly identifying candidate genetic variants associated with human disease. However, predicting and testing the pathogenicity of a genetic variant remains challenging. Genome editing allows for the rigorous functional testing of human genetic variants in animal models. Congenital heart defects (CHDs) are a prominent example of a human disorder with complex genetics. An inherited sequence variant in the human PBX3 gene (PBX3 p.A136V) has previously been shown to be enriched in a CHD patient cohort, indicating that the PBX3 p.A136V variant could be a modifier allele for CHDs. Pbx genes encode three-amino-acid loop extension (TALE)-class homeodomain-containing DNA-binding proteins with diverse roles in development and disease, and are required for heart development in mouse and zebrafish. Here, we used CRISPR-Cas9 genome editing to directly test whether this Pbx gene variant acts as a genetic modifier in zebrafish heart development. We used a single-stranded oligodeoxynucleotide to precisely introduce the human PBX3 p.A136V variant in the homologous zebrafish pbx4 gene (pbx4 p.A131V). We observed that zebrafish that are homozygous for pbx4 p.A131V are viable as adults. However, the pbx4 p.A131V variant enhances the embryonic cardiac morphogenesis phenotype caused by loss of the known cardiac specification factor, Hand2. Our study is the first example of using precision genome editing in zebrafish to demonstrate a function for a human disease-associated single nucleotide variant of unknown significance. Our work underscores the importance of testing the roles of inherited variants, not just de novo variants, as genetic modifiers of CHDs. Our study provides a novel approach toward advancing our understanding of the complex genetics of CHDs., Summary: This study uses genome editing in zebrafish to demonstrate that a human DNA sequence variant of unknown significance might contribute to the complex genetics of congenital heart defects.