During vertebrate embryogenesis, tissue morphology and cell differentiation are constantly influenced by, and responding to, microenvironment cues such as physical forces and associated biochemical signaling processes. Few tissues exemplify the complex interplay of these coexisting processes better than tendons, the extracellular matrix (ECM) rich connective tissues which attach muscle to bone, cartilage, and soft tissues, and coordinate the optimal transfer of force from of muscle contraction to the skeleton. Though in recent years, much has been elucidated about gene regulatory networks coordinating tendon tissue morphogenesis and tendon fibroblast (tenocyte) fate specification/differentiation, very little is known about how heterogenous tenocyte populations sense and respond to muscle contraction forces and uniquely modify their ECM organization and composition in vivo. Current knowledge of tenocyte mechanotransduction has been primarily informed by studies in adult in vivo models, and generally in tendons restricted to mammalian limbs. Zebrafish embryonic tendons provide a particularly useful model to address these research questions, as heterogenous tenocyte populations inhabiting different tendons can easily be studied across developmental timepoints. In this thesis work, I leveraged next generation bulk and single-cell sequencing approaches combined with in vivo functional perturbations and protein binding assays to gain a more defined, holistic understanding of tenocyte-ECM functional interactions during vertebrate development. I investigate tenocyte transcriptional responses to the onset of muscle contraction force during development by conducting RNA sequencing on sorted tenocytes from zebrafish embryos at developmental stages before and after the onset of muscle activity. I show that onset of muscle contractile forces leads to a specific transcriptional change in tenocytes, and using muscle-paralysis perturbation assays confirm that three novel tenocyte subpopulation markers, matn1, klf2a, and mxra5b are differentially expressed. Further, I show that variations in magnitude of muscle contractile force lead to unique transcriptional dynamics in specific tenocyte subpopulations in vivo, suggesting that tenocytes fine-tune ECM gene expression to adapt individual tendons to specific force conditions.
In vivo, tendon-like structures connect muscles not only to bone and cartilage, but to soft tissue such as eye sclera, and transmit varying intensity of forces from muscle contraction. Recent sequencing studies have begun to unravel distinct tenocyte subpopulations but have primarily looked at only gene regulatory networks of tenocytes inhabiting individual tendons or tendon subregions, prompting the question as to whether tenocyte populations are transcriptionally distinct within and between distinct tendons. To understand this tenocyte transcriptional heterogeneity at the level of both inter- and intra-tendon tissues, I conducted single-cell RNA sequencing (scRNAseq) on sorted cranial tenocytes from dissected zebrafish heads. I show for the first time that cranial tenocyte populations transcriptionally cluster into both spatially distinct tendons and functionally distinct intra-tendon regions, and that this clustering is driven not only by generalized patterning genes but also by specific ECM components. I further show that individual intra-tendon attachment zones have distinct ECM transcriptional signatures between tendons, suggesting that a combination of developmental programming and varying levels of force at attachment zones underlies tenocyte heterogeneity. Next, I identify a previously unknown population of Wnt-signaling responsive tenocytes populating multiple tendons in the cranium and show through independent Wnt signaling perturbation assays that a proper balance of Wnt signaling is required for proper patterning of myotendinous junctions of the jaw. Through computational cell-communication inference software, I hypothesize that specific Wnt ligands and receptors are involved in this patterning process. These findings revealed a deeper layer of complexity to the understanding of tendon biology, demonstrating that transcriptional diversity of tenocytes is driven by the dual characteristics of specific attachment zone physiology and the load capacity of the tendon ECM.
Lastly, I investigate how tenocyte fate specification occurs at the level of transcriptional control by identifying putative binding partners to key tenocyte fate determining transcription factors Scleraxis and Mohawk. Through biochemical protein binding assays and immunofluorescence I show that Scleraxis binds a family of Class II helix-loop-helix transcription factor E-box proteins TCF3, TCF4, and TCF 12, and that these proteins are individually sufficient to drive Scleraxis translocation from the cytoplasm to the nucleus. This reveals a novel mechanism for initiation of Scleraxis transcription, which has sweeping implications for further understanding of tenocyte fate determination and maintenance. In this thesis, I have provided unique insights into novel roles for muscle-contraction force on tendon tissue remodeling, tenocyte transcriptional heterogeneity, and lineage specification.
