Determinants of multi-scale patterning in growth plate cartilage

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

Illuminating underappreciated mechanisms of receptor regulation in human lymphocytes.

A thorough understanding of receptor regulation is imperative to predict expression in varying contexts of disease or treatment. Lymphocyte surface receptors are often used as biomarkers and drug targets, making them particularly important for study. For receptors of debated functionality, such as the Fc receptor for IgM (FcMR), understanding regulation can also help to predict expression in vivo to supplement hypotheses of biological roles. Various mechanisms exist for altering receptor surface expression, including direct feedback mechanisms such as ligand-induced endocytosis and broader mechanisms such as transcriptional and translational control. In this dissertation, we explore selected underappreciated mechanisms of lymphocyte receptor regulation. Specifically, we investigate the effects of cell culture conditions on FcMR availability and the potential for global regulation of lymphocyte receptors via isoform variation. FcMR is a constitutively expressed Fc receptor on human T cells, though its function there remains debated. It was previously thought that FcMR was kept low in circulation by FcMR-IgM complex internalization. However, we found that FcMR expression was independent of IgM levels in culture and was higher on direct ex vivo stained lymphocytes than in processed PBMC. Instead, increasing cell culture density inhibited FcMR expression in an apparent cell-contact mediated mechanism, suggesting higher circulating expression of FcMR than previously appreciated and a primary role for FcMR in cell-scarce environments. When next attempting to investigate the potential for isoform-based regulation of FcMR in lymphocytes, we found no applicable isoform-level references. We thus decided to fill this gap using Pacific Biosciences Isoform Sequencing (Iso-Seq) and developed the first Iso-Seq reference transcriptomes of human lymphocytes and activated CD4 T cells. In these references, we discovered many potentially novel transcripts, including end-variant transcripts that only differed from annotated counterparts on their 5’ or 3’ end. Using plasmids designed to express novel CXCR5 end-variant isoforms in a HEK293T cell system, we further validated the potential for novel 5’ end-variants to affect both mRNA stability and protein expression. The studies presented here provide valuable contributions to the understanding of lymphocyte receptor regulation by positing novel regulatory mechanisms that lay the groundwork for many future studies

Conserved stromal-immune cell circuits secure B cell homeostasis and function

B cell zone reticular cells (BRCs) form stable microenvironments that direct efficient humoral immunity with B cell priming and memory maintenance being orchestrated across lymphoid organs. However, a comprehensive understanding of systemic humoral immunity is hampered by the lack of knowledge of global BRC sustenance, function and major pathways controlling BRC-immune cell interactions. Here we dissected the BRC landscape and immune cell interactome in human and murine lymphoid organs. In addition to the major BRC subsets underpinning the follicle, including follicular dendritic cells, PI16$^{+}$ RCs were present across organs and species. As well as BRC-produced niche factors, immune cell-driven BRC differentiation and activation programs governed the convergence of shared BRC subsets, overwriting tissue-specific gene signatures. Our data reveal that a canonical set of immune cell-provided cues enforce bidirectional signaling programs that sustain functional BRC niches across lymphoid organs and species, thereby securing efficient humoral immunity

A Tale of Two Direction Codes in Rat Retrosplenial Cortex: Uncovering the Neural Basis of Spatial Orientation in Complex Space

Head direction (HD) cells only become active whenever a rat faces one direction and stay inactive when it faces others, producing a unimodal activity distribution. Working together in a network, HD cells are considered the neural basis supporting a sense of direction. The retrosplenial cortex (RSC) is part of the HD circuit and contains neurons that express multiple spatial signals, including a pattern of bipolar directional tuning – as recently reported in rats exploring a rotationally symmetric two-compartment space. This suggests an unexplored mechanism of the neural compass. In this thesis, I investigated whether the association between the two-way firing symmetry and twofold environment symmetry reveals a general environment symmetry-encoding property of these RSC neurons. I recorded RSC neurons in environments having onefold, twofold and fourfold symmetry. The current study showed that RSC HD cells maintained a consistent global signal, whereas other RSC directional cells showed multi-fold symmetric firing patterns that reflected environment symmetry, not just globally (across all sub-compartments) but also locally (within each sub-compartment). The analyses also showed that the pattern was independent of egocentric boundary vector coding but represented an allocentric spatial code. It means that these RSC cells use environmental cues to organise multiple singular tuning curves which sometimes are combined to form a multidirectional pattern, likely via an interaction with the global HD signal. Thus, both local and global environment symmetry are encoded by local firing patterns in subspaces. This interestingly suggests cognitive mapping and abstraction of space beyond immediate perceptual bounds in RSC. The data generated from this study provides important insights for modelling of direction computation. Taken together, I discuss how having two types of direction codes in RSC may help us to orient more accurately and flexibly in complex and ambiguous space