Identification of New Regulators of Tendon Development Using the Zebrafish Model

Abstract

Tendons transmit force from muscle to bone, enabling movement; ligaments connect bone to bone, maintaining stability. Despite their importance, tendon and ligament development is relatively uncharacterized. In this dissertation, the zebrafish is introduced as a model to study tendon development; tendon populations in zebrafish are homologous to their force-transmitting counterparts in higher vertebrates. The zebrafish craniofacial tendons and ligaments were identified based on expression analysis of genes enriched in mammalian tendons and ligaments (scleraxis, collagen 1a2 and tenomodulin) or in zebrafish tendon-like myosepta (xirp2a). The craniofacial tendons and ligaments were fate-mapped to arise from cranial neural crest cells (CNCCs). Loss-of-function genetic approaches demonstrated that craniofacial and pectoral fin tendons require muscle for their maintenance and cartilage for their organization, with neither tissues required for their induction. Lastly, adult zebrafish and mammalian tendons and ligaments share similar ultrastructural properties.

The second part of this dissertation sought to understand the processes governing tendon cell induction. A zebrafish chemical screen identified Lovastatin and Simvastatin as compounds that affect craniofacial tendon development. Statins caused a dose-dependent expansion of the craniofacial and pectoral fin tendon programs. The expansion is specific to the tendon and ligament lineages, and is not observed in other musculoskeletal tissues. Chemical rescue and genetic loss-of-function experiments demonstrated that statin-mediated expansion of scleraxisa is specific to the mevalonate pathway and Hmgcr inhibition. Furthermore, the expansion of tendon progenitors is mediated through inhibition of geranylgeranylation, and specifically geranylgeranyltransferase type I (GGTase I). We discovered that this phenotype is due to a change in cell fate specification, and not an increase in cell proliferation. Fate mapping experiments demonstrated that the expanded craniofacial scleraxisa-positive tendon progenitors arise entirely from CNCCs. As statin caused a reduction of CNCC-derived cartilage, statin is speculated to promote expansion of the craniofacial tendon program by directing CNCCs towards a tendon fate through regulation of GGTase I activity. Taken together, this dissertation advances our understanding of vertebrate tendon development. The first part establishes the zebrafish as a model to study tendon development; and the second part explores the regulation of the mevalonate pathway in tendon development.