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P&F Grant Awards

Year 5

Grant # 10

Genetic Basis of Musculoskeletal Disease: Emphasis on Zebrafish Models of Late-Onset Scoliosis and Tendon Development

PI: Lilianna Solnica-Krezel, Ph.D.,


Specific Aims

In humans, adolescent idiopathic scoliosis (AIS) is defined as a lateral curvature of the spine, free of vertebral malformations, with unknown etiology and occurring near the onset of puberty (Weinstein et al., 2008). Analysis of pedigrees and genome wide association studies (GWAS) of AIS patients are suggestive of multiple chromosomal loci exhibiting both dominant and recessive characteristics (Wise et al., 2008). Genetically tractable animal models of AIS will facilitate the discovery of the genetic, cellular, and tissue level factors that contribute to the pathology of late-onset scoliosis. The zebrafish, Danio rerio, is one of the preeminent vertebrate model systems for modeling disease pathology in a variety of organs. For example, the zebrafish is a robust system for both forward- and reversegenetic approaches, allowing for the discovery of genes essential for a process of interest, or for targeted gene disruptions, respectively. In addition, the large collection of zebrafish mutants and transgenic lines, along with the external development of numerous transparent embryos, afford effective dissection of molecular genetic pathways. The goal of this proposed investigation is to further the discovery of the genetic causes of skeletal deformities – with particular emphasis on late-onset scoliosis – using zebrafish. In addition, we propose to develop novel transgenic animals that will allow us to visualize the development and homeostasis of tenocytes/tendons in vivo. Importantly, we generated a novel dominant zebrafish mutant, Druk, displaying many corollaries to AIS in humans, including progressive curvature of the axial skeleton starting in larval fish (~15 days post fertilization), without any malformations of initial vertebrae development. This model of lateonset scoliosis provides us with a defined time window to focus on new screening efforts for additional scoliosis mutants. We already have established modern fish facilities, and robust screening methods to uncover both recessive and dominant mutations causing scoliosis. We also have generated large numbers of highly mutagenized founder fish that will afford an effective screen for new scoliosis phenotypes. In fact, during our pilot screen (~3 months), we have uncovered one dominant mutant exhibiting late-onset scoliosis (Stl24); two dominant phenotypes exhibiting vertebral fusions and dwarfism (Squat 1; Squat 2); and two putative recessive late-onset scoliosis mutations (ep71; ep108). The elucidation of the genetic lesions underlying these mutant phenotypes will illuminate the cellular and molecular etiology of late-onset skeletal defects. The formation of scoliosis is hypothesized to involve structural defects of musculoskeletal tissues; the bone, muscles or connective tissues. Interestingly, the surgical resection of myotendinous connections of the posterior ends of caudal ribs, including the costo-vertebral joints, can induce scoliosis in young rabbits (Langenskiold and Michelsson, 1961). In addition to a potential role in the formation of scoliosis, the development and homeostasis of tendon-bone insertions is a critical topic in developmental biology. We posit that the zebrafish model provides a promising model with which to study the development of tendons in realtime due to the ease of direct, long-term in vivo imaging available in the externally developing and optically transparent zebrafish embryos. The development of fluorescent tendon marker transgenics will greatly facilitate dissecting the normal development of tendons and the mechanisms of tendon-bone interactions during injury and repair. Finally, the establishment of these tendon-marking transgenics will enable effective genetic screening for the pathways and molecular agents that direct the development and maintenance tendons in a vertebrate organism. Here, we propose to build upon our initial investigations of the late-onset scoliosis zebrafish model, as well as to test a hypothesis involving the role of tendons in the development of late-onset scoliosis. We propose to address the following specific aims in this pilot study: Aim 1: We will carry out phenotypic and molecular characterization of novel mutants presenting with musculoskeletal defects, with emphasis on models of late-onset scoliosis. • Aim 1A: Determine the genetic lesions associated with mutants found in the late-onset scoliosis screen using next generation sequencing at the Genome Technology Access Center (WUSM). • Aim 1B: Assay the quality of the vertebral skeleton in late-onset scoliosis mutants using both histological and μ-CT imaging techniques at the Musculoskeletal Research Center (WUMRC). Aim 2: Generate tendon-specific transgenic zebrafish lines using BAC recombineering targeting genes that are essential for tendon development and repair. Our pilot study will identify conserved genes controlling the formation of late-onset scoliosis. Moreover, it will establish tools for imaging tendon development in wild-type and late-onset scoliosis mutant animals.