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Download File Genetic regulation of pulmonary progenitor cell differentiation Maria R. Stupnikov Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2019 © 2019 Maria R. Stupnikov All rights reserved ABSTRACT Genetic regulation of pulmonary progenitor cell differentiation Maria R. Stupnikov The respiratory system represents a major interface between the body and the external environment. Its design includes a tree-like network of conducting tubules (airways) that carries air to millions of alveoli, where gas exchange occurs. The conducting airways are characterized by their great diversity in epithelial cell types with multiple populations of secretory, multiciliated, and neuroendocrine cells. How these different cell types arise and how these populations are balanced are questions still not well understood. Aberrant patterns of airway epithelial differentiation have been described in various human pulmonary diseases, chronic bronchitis, asthma, neuroendocrine hyperplasia of infancy, and others. The goal of this thesis is to investigate mechanisms of regulation of airway epithelial cell fate in the developing lung epithelium. More specifically, these studies focus on Notch signaling and address a long unresolved issue whether the different Notch ligands (Jagged and Delta) have distinct roles in the epithelial differentiation program of the extrapulmonary and intrapulmonary airways. Moreover, these studies investigate the ontogeny of the bHLH transcription factor Ascl1 and identify its targets in the developing airways as potential regulators of neuroepithelial body (NEB) size and maturation. My studies provide evidence that the Notch ligand families Jag and Dll are required for the specification and formation of different cell lineages in the developing airway epithelia. Jag ligands regulate multiciliated versus secretory (club) cell fates but also controls abundance of basal cell progenitors in extrapulmonary airways. Dll ligands regulate pulmonary neuroendocrine versus club cell fates in intrapulmonary airways. Analysis of mouse mutants showed that loss of Jag ligands has minimal impact on the size or abundance of NEBs and their associated secretory cells while loss of Dll ligands results in an expansion of NEB size and associated cells. To gain additional insights into the potential mechanisms of how neuroendocrine cells develop and undergo aberrant hyperplasia, I characterized the global transcriptional profile of embryonic lungs from mice deficient in Ascl1, which lack NEBs and neuroendocrine cells and identified a number of genes associated with neuroendocrine cell development, maturation, and the NEB microenvironment. Among these genes, components of the catecholamine biosynthesis pathway, such as tyrosine hydroxylase (Th), a key enzyme for catecholamine production, were downregulated in Ascl1 null lungs. Subsequent functional analysis using a pharmacological inhibitor of this pathway in lung organ cultures showed expansion of pulmonary neuroendocrine cells and NEB size, an observation of potential relevance in human diseases in which neuroendocrine cells are aberrantly expanded. Together these studies highlight the distinct role of Notch ligands and further implicate Ascl1 targets, as illustrated by catecholamine pathway components, in regulating epithelial cell fate. Further examination of these pathways may provide insights into the pathogenesis and ultimately therapeutic approaches for airway diseases. TABLE OF CONTENTS List of Figures .....................................................................................................................v List of Tables ................................................................................................................... vii List of Abbreviations ..................................................................................................... viii Acknowledgements ........................................................................................................ xiii Dedication .........................................................................................................................xv Chapter One: Embryonic Lung Development and Notch Signaling.............................1 I. Introduction ........................................................................................................1 II. Stages of Murine Lung Development ................................................................2 III. Progenitor Cells of the Developing Epithelium and Differentiation into Cell Lineages .............................................................................................................4 IV. Overview of Conducting Airway Lineages .......................................................4 1. Secretory Cells .............................................................................................8 A. Club Cells......................................................................................8 B. NEB-associated Club Cells ...........................................................9 C. Goblet Cells ..................................................................................9 D. Ionocytes .....................................................................................11 2. Multiciliated Cells ......................................................................................11 3. Pulmonary Neuroendocrine Cells ..............................................................12 4. Basal Cells .................................................................................................13 5. Tuft/Brush Cells .........................................................................................14 V. Mammalian Notch Signaling ...........................................................................15 1. Notch Signaling in Multiciliated and Club Cells .......................................17 i 2. Notch Signaling in PNECs and NEB-CCs ................................................19 3. Notch Signaling in Basal Cells ..................................................................20 Chapter Two: Jagged Control of Tracheal Progenitor Cell Differentiation ..............24 I. Abstract ............................................................................................................24 II. Introduction ......................................................................................................25 III. Materials and Methods .....................................................................................27 1. Mouse Models ............................................................................................27 2. In situ Hybridization ..................................................................................27 3. Immunofluorescence and Immunohistochemistry .....................................27 4. Morphometric Analysis .............................................................................28 IV. Results ..............................................................................................................29 1. Jag1 and Jag2 Have Partially Overlapping Functions in Restricting Basal Cell Expansion ...........................................................................................29 V. Discussion ........................................................................................................33 Chapter Three: Jagged and Delta Ligands Control Distinct Events during Airway Progenitor Cell Differentiation .......................................................................................34 I. Abstract ............................................................................................................34 II. Introduction ......................................................................................................36 III. Materials and Methods .....................................................................................38 1. Mouse Models ............................................................................................38 2. Ascl1 Lineage Trace ...................................................................................39 3. Immunofluorescence and Immunohistochemistry .....................................39 4. Morphometric Analysis .............................................................................40 ii 5. In situ Hybridization ..................................................................................41 6. Quantitative Real-Time PCR .....................................................................42 IV. Results ..............................................................................................................43 1. Jagged Ligands Precede the Appearance of Delta in Differentiating Airway Progenitors ....................................................................................43 2. Jag1 and Jag2 Regulate the Balance of Different Cell Types in Extra- and Intrapulmonary Airways ............................................................................47 3. Jag Ligands have Minimal Effects in the Establishment and Regulation of the NE Microenvironment .........................................................................52 4. Dll Ligands Control the Size of the NEB Microenvironment ...................57 5. Notch Signaling and NEB-associated Club Cells are Preserved in the Absence of Dll Ligands .............................................................................62 6. Preliminary
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