The musculoskeletal system depends on mechanical load from skeletal muscle to form bone and respond to motion. Bone ridges are skeletal superstructures found on the periosteal surface of bone and are typically where tendons attach muscle to bone. One of the most prominent bone ridges in the murine skeleton is the deltoid tuberosity (DT), which fails to maintain its size and shape in the absence of muscle loading. In mouse embryos with global deletion of fibroblast growth factor 9 (Fgf9^null), the size of the DT is notably enlarged and attached to a shorter humerus. We and others have shown that Fgf9 is primarily expressed in the surrounding soft tissue including skeletal muscle, implicating the role of FGF9 in muscle-bone crosstalk. The goal of this dissertation work was to identify the role of global, muscle-specific, and tendon-specific FGF signaling in the development and growth of bone ridge superstructures like the DT.

My research was organized in four distinct research aims that measured the growth of bone ridge size using transgenic mouse strains. The first aim was to quantify the enlargement of the DT in Fgf9^null embryos. I developed a method to quantify bone ridge size using whole-mount staining of embryonic mouse forelimbs. This aim also established that the DTs of Fgf9^null embryos started to grow larger and faster than the DTs of WT embryos around embryonic day (E) 16.5. In aim 2, I showed that the global loss of Fgf9 led to increased chondrocyte hypertrophy and reduced cell proliferation at the DT attachment site. Global loss of Fgf9 led to increased expression of Gli1, Sox9, and Fgf18 in and around the DT at E16.5 as well as decreased expression of Sost and Sox9 at P0 compared to WT littermates (visualized using fluorescent in situ hybridization) as well as decreased expression of mitochondria-, proton-transport, and metabolism-associated genes in skeletal muscle but not bone (measured using bulk RNA sequencing). In aim 3, I showed that inducible deletion of Fgf9 in skeletal muscle throughout development led to the enlargement of the mature DT. Additionally, skeletal muscle-specific deletion of Fgf9 positively correlated with an increased number of muscle acetylcholine receptor clusters. These findings established a relationship between Fgf9 expression in skeletal muscle and an osteogenic phenotype. For the fourth aim, I compared DT size and shape in the postnatal skeleton of mice with a tendon-specific knockout of both Fgfr1/2 using Scx-Cre. I found that Scx-Cre;​ Fgfr1/2 conditional knockout mice developed enlarged DTs and other superstructure phenotypes compared to age-matched WT mice. The quantification of enlarged bone ridges in Fgf9^null, Fgf9^cKO, and Fgfr^DKO mice support the functional role of FGF signaling as a down regulator of bone ridge size and shape.

This dissertation work identified the role of FGF signaling in muscle-bone crosstalk by studying bone ridge development with tissue-specific transgenic mouse strains. Future work in this area could explore the broader role of FGF signaling in DT development and the role of FGF9 in skeletal muscle metabolism, including mitochondrial function, lipid biosynthesis, and proton transport. In addition, Fgf9 and these downstream effectors have a potential contribution towards ligand-based connections between muscle and bone.