Project Grant R01DK141169

DISSECTING DIVERGENT HEMATOPOIETIC STEM AND PROGENITOR CELL ONTOGENY - DISSECTING DIVERGENT HEMATOPOIETIC STEM AND PROGENITOR CELL ONTOGENY DURING DEVELOPMENT PROJECT ABSTRACT 1 THE HEMATOPOIETIC AND IMMUNE LANDSCAPE IS COMPLEX. THE DIVERSITY IS GENERATED AND SUSTAINED BY THE FUNCTION 2 OF HEMATOPOIETIC STEM CELLS (HSCS) AND PROGENITOR CELLS THAT ARISE INDEPENDENTLY DURING EMBRYONIC DEVELOPMENT. 3 HSCS ARE CHARACTERIZED BY THEIR ROBUST, LIFELONG SELF-RENEWAL ABILITY AND DIFFERENTIATION MULTIPOTENCY, WHILE HSC- 4 INDEPENDENT PROGENITOR CAPACITIES AND LIFESPANS ARE MORE LIMITED. HSC AND HSC-INDEPENDENT DERIVED PROGENY 5 CAN HAVE DISTINCTIVE FUNCTIONS WITH DEFECTS IN THE CELLS RESULTING IN DIFFERENT DISEASE STATES. THEIR IMPORTANT AND 6 UNIQUE PROPERTIES ARE INHERENT AND PROGRAMMED DURING ONTOGENY. CURRENTLY, THE FUNCTIONAL DIFFERENCES BETWEEN 7 HSCS AND HSC-INDEPENDENT PROGENITORS ARE LARGELY DISSECTED WITH ASSAYS THAT REQUIRE THE REMOVAL OF CELLS FROM 8 THEIR NATIVE MICROENVIRONMENT, WITH THE MEASURED DIFFERENTIATION OUTPUTS REFLECTING POTENTIAL BUT NOT TRUE 9 ENDOGENOUS FUNCTIONALITY. SEVERAL STUDIES HAVE REVEALED DISCREPANCIES BETWEEN NATIVE AND TRANSPLANTATION- 10 BASED ASSESSMENTS OF HSPC FUNCTION, UNDERSCORING THE CRITICAL IMPORTANCE OF EXAMINING A CELL'S FUNCTION WITHIN 11 ITS NATIVE ENVIRONMENT. WE HAVE DEVELOPED SEVERAL TOOLS IN ZEBRAFISH THAT DISCRIMINATE HSC AND HSC- 12 INDEPENDENT PROGENITOR FUNCTIONS WITHIN THEIR ENDOGENOUS EMBRYONIC/LARVAL ENVIRONMENTS TO FILL THIS KNOWLEDGE 13 GAP. WE ARE NOW POSITIONED TO DEPLOY THE ADVANTAGES OF THE ZEBRAFISH MODEL TO IDENTIFY REGULATORS INFLUENCING 14 THE FORMATION AND FUNCTION OF DIFFERENT HSC/HSC-INDEPENDENT PROGENITOR SUBSETS. IN AIM 1, WE WILL TEST THE 15 HYPOTHESIS THAT THERE COULD BE UNEXPECTED CONNECTIONS AMONG HEMATOPOIETIC AND IMMUNE CELLS AND UNREALIZED 16 DIFFERENTIATION POTENTIALS OF HSC/HSC-INDEPENDENT PROGENITORS. WE WILL USE APPROACHES THAT ALLOW VISUALIZATION 17 AND TRANSCRIPTIONAL CHARACTERIZATION OF HSCS, HSC-INDEPENDENT PROGENITORS, AND THEIR PROGENY THROUGHOUT THE 18 ENTIRE ORGANISM. IN AIM 2, WE WILL TEST THE HYPOTHESIS THAT FUNCTIONAL DIFFERENCES BETWEEN HSCS, HSC- 19 INDEPENDENT PROGENITORS ARISE FROM THEIR DISTINCT SPATIOTEMPORAL ORIGINS AND VIA DISTINCT GENETIC REGULATION. WE 20 WILL UTILIZE TEMPORAL AND SPATIAL LINEAGE TRACING, WHOLE MOUNT IN SITU HYBRIDIZATION, IN VIVO GENETIC SCREENING, AND 21 OUR NEW NATIVE HEMATOPOIESIS FUNCTIONAL ASSAYS TO UNCOVER THE WHEN, WHERE, AND HOW UNIQUELY CONTROLLING THE 22 EMBRYONIC GENERATION OF HSCS, BUT NOT PROGENITORS. IN THIS PROJECT, WE WILL ADDRESS MANY UNRESOLVED QUESTIONS 23 RELATED TO HSC AND HSC-INDEPENDENT PROGENITORS ARISING DURING DEVELOPMENT INCLUDING THE DIFFERENTIATION 24 REPERTOIRE OF THEIR PROGENY, THE CONNECTIONS AMONG LYMPHOID AND MYELOID IMMUNE EFFECTORS FOUND THROUGHOUT 25 THE ORGANISM, AND THE PROGRAMS UNDERLYING THE FORMATION AND FUNCTIONS OF THE DIVERSE STEM AND PROGENITORS 26 ARISING EMBRYONICALLY. THIS WORK WILL PROVIDE FUNDAMENTAL KNOWLEDGE ON THE EARLY ESTABLISHMENT OF HEMATOPOIETIC 27 AND IMMUNE HIERARCHIES RELEVANT TO UNDERSTANDING IMMUNE DISORDERS AND FOR THE DIRECTED PRODUCTION OF HSC AND 28 HSC-INDEPENDENT PROGENITORS AND THEIR FUNCTIONALLY DISTINCT PROGENY FROM HUMAN PLURIPOTENT STEM CELL SOURCES 29 FOR TREATMENT OF A VARIETY OF DISORDERS.