Browsing by Author "Tata, Purushothama Rao"

Abstract

Adult zebrafish demonstrate a remarkable capacity to regenerate heart tissue following injury, and thus have served as a valuable model for developing our understanding of cardiac repair and regeneration. Recent work has identified and characterized multiple cardiac mitogens all of which can drive cardiomyocyte (CM) division in the absence of injury. Despite these impressive responses, little is known regarding the shared specific molecular mechanisms of CM proliferation that lie downstream of these unique ligand-receptor interactions. Here, we found that the tumor suppressor p53 was significantly suppressed during regeneration which correlated with increases in the transcription of p53’s primary negative regulator Mdm2. Using established and newly generated transgenic lines we demonstrated that experimentally altering cellular p53 levels affects CM proliferation. Inducible overexpression of the cardiac mitogens Nrg1 and Vegfaa demonstrated similar findings with increased mdm2 transcription and p53 suppression during regeneration along with augmented CM proliferation with loss of p53. Furthermore, we observed significant overlap between gata4 and mdm2 gene expression domains during development, following heart injury, and with mitogen stimulation suggesting potential interactions between these two genes.  Our findings indicate a novel injury and mitogen-induced function of Mdm2 to repress p53 during zebrafish heart regeneration. Here we also investigated the presence of additional cardiac mitogens, specifically HB-EGF, an ErbB ligand. We found that both HB-EGF paralogs are both present in the zebrafish heart and are both transcriptionally upregulated near the site of injury. A newly generated set of novel HB-EGF transgenic reporters, knock-outs, and overexpression lines will further investigate the importance of these early findings and HB-EGF signaling which will add to our understanding of heart regeneration.

Lung epithelium, the lining that covers the inner surface of the respiratory tract, is directly exposed to the environment and thus susceptible to airborne toxins, irritants, and pathogen-induced damages. In adult mammalian lungs, epithelial cells are generally quiescent but can respond rapidly to repair damaged tissues. Evidence from experimental injury models in rodents and human clinical samples has led to the identification of these regenerative cells, as well as pathological metaplastic states specifically associated with different forms of damages. The primary alveolar stem cell, alveolar type-2 cells (AT2s) are sparsely distributed and make up only 5% of the surface area. Despite this organization, AT2s are still able to maintain tissue homeostasis and achieve efficient repair after injury. However, the underlying mechanisms of stem cell activation, injury response, and subsequent cell-cell communication signals mediating resolution of injury and restoration of alveolar homeostasis remain elusive. Additionally, modulation of these regenerative cells for therapeutic potential has not been well established, primarily due to a lack of viable gene editing tools and vehicles for gene delivery to the alveolar stem cells, and neighboring niche. First, to better target the lung alveolus, we screened and identified cell-type specific adeno-associated virus (AAV) serotypes, enabling efficient targeting and gene expression of exogenous genes in alveolar stem cells as well neighboring mesenchyme. These tools were also capable for both in vitro and in vivo gene editing, forgoing the need for development of complex genetic mouse models as well as enabling diverse, viral-based screens. Second, using 3-dimensional, thick tissue imaging we reveal that a single AT2 cell can enface multiple alveolar compartments by virtue of a unique, multi-apical domain architecture. Lineage tracing and live imaging coupled with genetic and AAV-mediated selective ablation of AT2s was used to show robust recovery of AT2 numbers and distribution via clonal proliferation and migration, even after three successive rounds of ablation. Clonal tracing revealed that a single AT2 can differentiate to cover multiple alveolar cups. During injury repair, AT2s dynamically reorganize their apical domains to facilitate either migration or differentiation. Single- cell transcriptome profiling, genetic and pharmacologic disruption of actin dynamics, and evaluation of multiple physiologically relevant disease states identified the roles of actin cytoskeleton, cell migration, and multi-apical domains in AT2 recovery and regenerative potency. Lastly, using cell-type specific ablation of alveolar type 1 cells (AT1s), we identified novel mechanisms of epithelium-mediated signaling to mesenchymal fibroblasts, thereby uncovering novel mechanisms of fibrosis initiation and progression. Modulation of AT1 ablation dynamics preferentially drives fibrosis or, in contrast, emphysema, both at histological and physiological levels, as assessed by whole body plethysmography. Single-cell sequencing identified the epithelial and mesenchymal cell identities involved in regenerative processes, as well as identification of a PDGFA signaling axis between AT1s and resident alveolar fibroblasts necessary for fibroblast maintenance. We demonstrate that modulation of these signaling pathways during lung regeneration could enhance fibrosis or convert fibrosis to emphysema. In sum, the work presented herein both develops functional tools for perturbation of alveolar stem cells, as well as an improved understanding of alveolar architecture, stem cell dynamics during injury repair, homeostatic intercellular signaling, and mechanisms of disease progression.