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Vertebrate skeletons are an intricate framework of bony and cartilaginous structures that form through carefully orchestrated developmental processes, guided by interacting genetic pathways that regulate cellular differentiation, migration, and tissue morphogenesis. The specific timing and localisation of gene expression shapes the diverse array of skeletal elements, from the flexible cartilages of the embryonic stage to the hardened bones that provide structural support in adulthood, and the joints and connective tissues that articulate the musculoskeletal system. This thesis aims to use the zebrafish (Danio rerio) as a model organism to study the role and regulation of three genes in controlling musculoskeletal development from larvae to adulthood: nkx3.2, gdf5, and mkx. In the first study, we used CRISPR/Cas9 genome editing to knock out nkx3.2 and characterise the resulting mutant phenotypes, including a jaw joint fusion and occipital and vertebral defects. In the second study, we extended the phenotypic characterisation of nkx3.2 mutants into the skeleton-associated soft tissues using a novel synchrotron-based tomographic imaging technique and revealed a series of defects in the jaw musculature, Weberian ligaments, and fluid-filled sacs of the ear. In the third study, we identified and functionally characterised a novel cis-regulatory element responsible for driving nkx3.2 expression in the early developing jaw joint, with its presence and activity being highly conserved in jawed vertebrates but absent in jawless vertebrates. In the fourth study, we examined the role of gdf5 in skeletal development by generating a knockout mutant line, finding striking defects in fin radial development including a clear endoskeletal disc segmentation phenotype resulting in a complete absence of posterior radials in the pectoral fin. Finally, in the fifth study, we studied the regulation of Mkx, an important factor in tendon and ligament development, and identified a novel enhancer with different species-dependent activity patterns. In summary, this thesis contributes to our understanding of the derived and conserved functions of Nkx3.2, Gdf5, and Mkx in the development of the vertebrate skeleton and associated connective tissues, and provides a novel high-resolution 3D imaging method for future studies.