Graduation Date

Spring 5-5-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Genetics, Cell Biology & Anatomy

First Advisor

Andrew Dudley

Abstract

ABSTRACT

Functional architectures of complex adaptive systems emerge by dynamic control over properties of individual components. During skeletal development, growth plate cartilage matches bone geometries to body plan requisites by spatiotemporally regulating chondrocyte actions. Bone growth potential is managed by the proximodistal patterning of chondrocyte populations into differentiation zones, while growth vectors are specified by the unique columnar arrangement of clonal groups. Chondrocyte organization at both tissue and cell levels is influenced by a cartilage-wide communication network that relies on zone-specific release and interpretation of paracrine signals. Despite genetic characterization of signaling interactions necessary for cartilage maturation, the regulatory mechanisms that couple positional information with polarized chondrocyte activities to coordinate skeletal morphogenesis remain poorly understood. Building on previous kinematic descriptions of column formation, the work contained in this dissertation suggests cytoskeletal regulation mediates crosstalk between long-range signaling and local cell behavior. Rearranging daughter chondrocytes specifically recruit actomyosin contractility to cortical surfaces, indicating a primary role for the actin cytoskeleton as the engine powering column formation kinetics. Disrupted chondrocyte contractility patterns are observed after genetic perturbation of planar cell polarity signaling, and after inhibiting integrin extracellular matrix binding, implicating actomyosin as a sensor able to integrate global with local signaling cues. To gain greater analytical control towards dissecting the mechanochemical patterning systems underlying cartilage architecture, an alginate hydrogel-based model of growth plate was developed. Daughter chondrocytes encapsulated in alginate beads deposit extracellular matrix in anisotropic and hierarchical configurations that resemble myosin localization in vivo, hinting cytoskeletal forces may sculpt the solid-state environment. Single-cell transcriptomic analysis of chondrocytes stimulated with recombinant ligands demonstrates the functionality of the IHH/PTHrP circuit in alginate beads, and points towards a novel role for PTHrP signaling gradients in transcriptional regulation of cytoskeletal and ECM proteins. Basal bead cultures tend towards resting/proliferative phenotypes driven by endogenous PTHrP expression, but activating IHH signaling induces position-dependent gene expression, consistent with a model of zone formation where concentration gradients generate spatial cues. Together, the work suggests that in addition to regulating chondrocyte differentiation, the tissue-wide signaling network in cartilage can influence cell-matrix interactions that may be important for cell behavior, and presents a novel culture model that can be used for future studies investigating how chondrocytes discern positional information to shape the growing tissue.

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