Congratulations to Connor Leek, PhD, and Elahe Ganji, PhD, who have successfully defended their dissertation work!

Connor defended his dissertation work on June 29th 2021.

Elahe defended her dissertation work on July 13th 2021.

Abstract for Connor's dissertation:

The musculoskeletal system depends on mechanical load from skeletal muscle to develop and function. Bone ridges are skeletal superstructures found on the periosteal surface of bone and are typically where tendons attach. 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 deltoid muscle loading. In mouse embryos with global deletion of fibroblast growth factor 9 (Fgf9null), the size of the DT is notably enlarged and attached to a shorter humerus. Our laboratory 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 Fgf9null 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 Fgf9null 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 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 at P0 compared to WT littermates (visualized using fluorescent in situ hybridization) as well as decreased expression of mitochondria-associated, 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 enlargement of the mature DT. Additionally, skeletal muscle-specific deletion of Fgf9 positively correlated with an increased number of muscle acetylcholine receptor clusters as well as muscle contractility and innervation-related gene expression. These findings established a relationship between Fgf9 expression in skeletal muscle and a bony 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 ScxCre. I found that ScxCre; Fgfr1/2 conditional knockout mice developed enlarged DTs and other superstructure phenotypes compared to age-matched WT mice. Together, these findings support the functional role of FGF signaling as a negative 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 as well as the role of FGF9 in skeletal muscle metabolism, including mitochondrial function, lipid biosynthesis, and proton transport. Fgf9 and these downstream effectors have a potential contribution towards ligand-based connections between muscle and bone.

Abstract for Elahe's dissertation:

Entheses are connective tissues that connect tendon to bone, two vastly different hierarchical materials with different structural and mechanical properties. As a result of this material mismatch, entheses are prone to local peaks in mechanical stress (stress concentrations) that increase their susceptibility to overuse injuries, especially during rapid growth (postnatal maturation) and in young athletes. The enthesis matures postnatally in a mechanoadaptive process, similar to the growing bone, and forms a graded transition to dampen the stress concentrations at the attachment site. Despite decades of research, the key biological and mechanoadaptive processes that govern the adaptation of the enthesis under repeated loading and onset of injury during its postnatal maturation remain unknown.

The objective of this research was to investigate the role of mechanical and biological cues on the mechanoadaptation of growing and adult entheses. I did that through four main aims: (1) developing and confirming the feasibility and repeatability of a novel non-invasive in vivo model for repeated loading of the tendon and enthesis, using optogenetics; (2) investigating the age-dependent mechanicallyinduced structural and functional adaptation of the enthesis during growth and adulthood; (3) exploring the structural and functional relationships and possible mechanisms of damage (i.e., disrupted interdigitation vs. collagen denaturation) in disruption of the toughening mechanism of the maturing enthesis; and (4) identifying FGFs signaling, a known mediator of bone growth, as a critical regulator of the structural gradation, and therefore, mechanical properties of maturing entheses.

This study is innovative in taking an interdisciplinary approach to put forth a novel model for skeletal adaptation to loading during growth, elucidating the structural adaptation of maturing and adult enthesis under repeated loading, and proposing new biological pathways involved in the mechanoadaptation of the maturing enthesis. The results and tools developed in this work can be used to investigate the adaptation of the enthesis by investigating the mechanobiology of enthesis formation, with the longterm goal of improving the diagnosis and treatment of overuse injuries in maturing attachment.