Browsing by Author "Hilton, Matthew"

The success of muscle development and regeneration requires cooperation from both myogenic and their supportive niche cells. The muscular niche is complex. At the cellular level it is composed of a broad number of cell types including: endothelial vessels, nerve and nerve-supporting cells, resident immune populations, and a heterogenous group of non-myogenic mesenchymal cells. The non-myogenic mesenchymal cells include pericytes, vascular smooth muscle cells, interstitial tenocyte-like cells, and fibro-adipogenic progenitors (FAPs). Like all members of the muscular niche, this fraction is vital to muscle development and regeneration. Despite their importance to muscle development, regeneration, and homeostasis, detailed identities within non-myogenic mesenchymal cells remain elusive. By understanding the distinct makeup of this population, we can provide a foundation to examine their important regulatory roles in the processes of muscle development, homeostasis, injury and disease.

This thesis utilizes single cell RNA sequencing to establish the populations of non-myogenic mesenchymal cells in developing muscle. Our analysis identified pericytes, vascular smooth muscle cells, and tenocyte-like cell populations while uncovering a new level of heterogeneity in FAPs that not previously appreciated. Despite classical understanding of FAPs as one group, this work found that FAPs were sub-divided into five distinct populations, which compose two trajectories spawning from a common progenitor. This thesis defines the functional differences of each FAP population through a series of experiments including: fluorescence activated cell sorting of various FAP groups, studying their spatial localization on immunofluorescence, and testing the response of different FAPs to multiple injury and disease models.

Separate preliminary work examines the impact of NOTCH signaling in FAPs and the broader non-myogenic mesenchymal cell groups. These studies discovered that NOTCH signaling in the non-myogenic mesenchymal group, but not FAPs specifically, regulates muscular growth and intramuscular adipogenesis. Altogether this thesis advances our understanding of the identity, and role of, non-myogenic mesenchymal cells, in muscle development and regeneration.

Hypertrophic chondrocytes give rise to osteoblasts during skeletal development; however, the process by which these non-mitotic cells make this transition is not well understood. To further understand the cell transition process by which hypertrophic chondrocytes contribute to osteoblasts or other marrow associated cells, we utilized inducible and constitutive hypertrophic chondrocyte lineage tracing and reporter mouse models (Col10a1CreERT2; R26-tdTomatof/+ and Col10a1Cre; R26-tdTomatof/+) in combination with a PDGFRa-H2B-GFP transgenic line, single cell RNA-sequencing, bulk RNA-sequencing, immunofluorescence staining, and cell transplantation assays. Our data demonstrate that hypertrophic chondrocytes undergo a process of dedifferentiation to generate marrow associated SSPCs that serve as a primary source of osteoblasts during skeletal development. These hypertrophic chondrocyte derived SSPCs commit to a CXCL12-abundant reticular (CAR) cell phenotype during skeletal development and demonstrate unique abilities to recruit vasculature and promote bone marrow establishment, in contrast to periosteal-derived SSPCs, while also contributing to the adipogenic lineage. Additionally, to further understand the mechanism that may control this developmental process as well as to confirm its restriction to early development, we utilized a number of NOTCH signaling genetic tools after observation of active NOTCH signaling at the chondro-osseous junction. Histological and microCT studies of NOTCH signaling gain-of-function in hypertrophic chondrocytes (Col10a1Cre; R26-tdTomatof/+; R26-NICD1f/+) revealed a loss of bone phenotype, embryonically and early postnatally, but was recovered by non-hypertrophic chondrocyte derived osteoblasts by 2-months of age. NOTCH signaling loss-of-function in hypertrophic chondrocytes (Col10a1Cre; R26-tdTomatof/+; RBPjkf/f) exhibited no bone/osteoblast changes in early skeletal development. Our data demonstrates that hypertrophic chondrocyte-derived osteoblasts readily contribute to early skeletal development, but can compensated by non-hypertrophic chondrocyte-derived osteoblasts over time.

Osteoarthritis (OA) and post-traumatic OA (PTOA) are prevalent joint disorders and leading causes of chronic pain. While pain is a primary symptom of OA and the reason individuals seek medical attention, the pathology of OA also includes physical manifestations such as articular cartilage and meniscus degeneration, synovial hyperplasia, osteophyte formation, and subchondral bone sclerosis. Despite the extensive socioeconomic burden of OA, effective treatments for both OA-associated joint degeneration and pain are not yet available due to our poor understanding of the mechanisms underlying the disease. The physical manifestations of OA are caused by imbalanced catabolic and anabolic responses and pro-inflammatory changes; however, their connection to pain is not well studied. Because Interleukin-6 (IL-6) is involved in cartilage degradation and conditions of inflammatory pain, we examined whether IL-6 signaling is a driver of both PTOA-associated cartilage degradation and pain. Our data demonstrate that genetic removal of Il6 attenuates PTOA-associated cartilage catabolism, decreases innervation of the knee joint, and reduces nociceptive pain signaling, without improving PTOA-associated subchondral bone sclerosis or chondrocyte apoptosis in male and not female mice. Compared to wild-type controls, the activation of IL-6 downstream mediators, STAT3 and ERK, were reduced in both knees and dorsal root ganglia of Il6-/- male mice following knee injury, while female mice showed a different signaling pattern. Using specific pharmacological inhibitors of STAT and ERK signaling in cartilage and DRG explants, we demonstrated that Janus kinases (JAKs) were critical regulators of STAT and ERK signaling in both cartilage catabolism and pain. STAT3 promoted cartilage catabolism downstream of JAK, but inhibition of STAT3 decreased cartilage anabolism and enhanced pain signals. JAK-dependent ERK activation was important for neurite outgrowth and pain signaling; however, ERK inhibition was less effective than JAK inhibition in reducing cartilage catabolism. Based on the essential role of IL-6 in regulating both cartilage degradation and pain in PTOA, and due to the upregulation of IL-6 in NOTCH-induced OA demonstrated by our lab previously, we examined the necessity of IL-6 at downstream of NOTCH signaling in cartilage degeneration and pain. We generated an alternative less severe NOTCH GOF OA model (AcanCreERT2; R26-NICDf/+), and demonstrated that cartilage-specific removal of Il6 (AcanCreERT2; R26-NICDf/+; Il6f/f) is not sufficient to reduce NOTCH induced joint cartilage degeneration and pain. Collectively, these data demonstrated that IL-6 mediates both cartilage degradation and pain associated with PTOA in a sex-specific manner and provides critical details regarding the tissue-specific contributions of downstream effectors of IL-6 signaling, which are potential therapeutic targets for disease-modifying osteoarthritis drugs.