Investigating the Molecular Signaling Pathways Governing Proliferation, Differentiation, and Patterning During Zebrafish Regenerative Osteogenesis
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Upon amputation, zebrafish innately regenerate lost or damaged bone by precisely positioning injury-induced, lineage-restricted osteoblast progenitors (pObs). While substantial progress has been made in identifying the cellular and molecular mechanisms underlying this fascinating process, the cell-specific function of these pathways is poorly understood. Understanding how molecular signals initiate osteoblast dedifferentiation, balance progenitor renewal and re-differentiation, and control bone shape during regeneration are of paramount importance for developing human therapies. We show that fin amputation induces a Wnt/β-catenin-dependent epithelial to mesenchymal transformation (EMT) of osteoblasts to generate proliferative Runx2+ pObs. Localized Wnt/β-catenin signaling maintains this progenitor population towards the distal tip of the regenerative blastema. As they become proximally displaced, pObs upregulate sp7 and subsequently mature into re-epithelialized Runx2-/sp7+ osteoblasts that extend pre-existing bone. Autocrine Bone Morphogenetic Protein (BMP) signaling promotes osteoblast differentiation by activating sp7 expression and counters Wnt by inducing Dickkopf-related Wnt antagonists. As such, opposing activities of Wnt and BMP coordinate the simultaneous demand for growth and differentiation during bone regeneration. Previous studies have implicated Hedgehog/Smoothened (Hh/Smo) signaling in controlling the re-establishment of stereotypically branched bony rays during fin regeneration. Using a photoconvertible patched2 reporter, we resolve active Hh/Smo output to a narrow distal regenerate zone comprising pObs and neighboring migratory basal epidermal cells. Hh/Smo activity is driven by epidermal Sonic hedgehog a (Shha) rather than Ob-derived Indian hedgehog a (Ihha), which instead uses non-canonical signaling to support bone maturation. Using high-resolution imaging and BMS-833923, a uniquely effective Smo inhibitor, we show that Shha/Smo promotes branching by escorting pObs into split groups that mirror transiently divided clusters of Shha-expressing epidermis. Epidermal cellular protrusions directly contact pObs only where an otherwise occluding basement membrane remains incompletely assembled. These intimate interactions progressively generate physically separated pOb pools that then regenerate independently to collectively re-form a now branched bone. Our studies elucidate a signaling network model that provides a conceptual framework to understand innate bone repair and regeneration mechanisms and rationally design regenerative therapeutics. This dissertation includes previously published co-authored material.