Author ORCID Identifier

https://orcid.org/0000-0003-4159-7125

Year of Publication

2020

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture, Food and Environment

Department/School/Program

Veterinary Science

First Advisor

Dr. James N. MacLeod

Abstract

At the presumptive sites of future synovial joints during mammalian skeletogenesis, articular cartilage develops from interzone located between the cartilaginous anlagen of bones. Thus, two types of cartilaginous tissues differentiate in close proximity. While anlagen cartilage is transient, progressing through endochondral ossification to form bones, articular cartilage is stable and functions throughout life to facilitate both low friction movement and load distribution. Despite important life-long functional properties, articular cartilage has a very limited intrinsic ability to repair structural defects. On the other hand, structural lesions in bones generally heal well by forming a cartilaginous callus and recapitulating endochondral ossification to repair fractures and other defects. Therefore, understanding the comparative aspects of interzone and anlagen cell differentiation may provide novel insights into emergent cell-based therapies to support articular cartilage regeneration. The objective of this dissertation research was to compare patterns of gene expression between equine interzone and anlagen cells across multiple post-induction time points to test the hypothesis that chondrogenic differentiation of these two cell lines is directed to articular and hypertrophic developmental pathways, respectively. The first part of the study was conducted using microfluidic RT-qPCR to analyze a selected panel of 93 genes. The data provided evidence that genes involved in transcriptional regulation and signaling transduction are differentially expressed as early as 1.5 hours after the start of chondrogenic induction, followed at later time points by effector genes such as those encoding cartilage matrix proteins. Then, RNA sequencing was used to expand the analyses at selected time points to a whole transcriptome level. A pilot single cell RNA sequencing experiment further described the two chondrogenic pathways characterizing subpopulations of these skeletal cell lines. Taken together, the results demonstrated that interzone and analgen cells respond very quickly but in different ways to the same inductive signals. Important regulatory mechanisms are likely activated almost immediately, within a few hours, after chondrogenic induction. These differential regulatory responses progress to cell type-specific profiles of effector genes that result in the two different cartilaginous tissues.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2020.328

Funding Information

Morris Animal Foundation (2016-2020)

USDA-NRSP8 National Research Support Project 8 National Animal Genome Research Program (2016-2020)

Lourie Foundation (2015-2020)

Gluck Equine Research Foundation endowments (2015-2020)

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