Project Grant R01MH134993

UNDERSTANDING GLIAL FATE RESTRICTION IN HUMANS - ABSTRACT THIS PROPOSAL AIMS TO DETERMINE THE MECHANISMS THAT LEAD TO THE GENERATION OF ASTROCYTES IN THE DEVELOPING HUMAN CORTEX. ASTROCYTES ARE THE MOST ABUNDANT CELL TYPE IN THE ADULT HUMAN BRAIN, MEDIATING VITAL FUNCTIONS INCLUDING MYELINATION, AXONAL GUIDANCE, SYNAPTOGENESIS, AND MAINTENANCE OF THE BLOOD-BRAIN BARRIER. DURING DEVELOPMENT, CONTROL OVER THE SPATIO-TEMPORAL PATTERNS OF THE PRODUCTION AND NUMBERS OF ASTROCYTES IS NECESSARY FOR PROPER BRAIN FUNCTION. THE DEVELOPMENTAL POTENTIAL OF NEOCORTICAL PROGENITORS EXHIBITS EVOLUTIONARY DIVERGENCE ACROSS MAMMALIAN CLADES, LEADING TO PRIMATE-SPECIFIC CELL TYPES, SUCH AS INTERLAMINAR ASTROCYTES. PROGRESS IN STUDYING HUMAN ASTROCYTIC DEVELOPMENT IS LIMITED BY THE ETHICAL AND PRACTICAL CHALLENGES IN WORKING WITH HUMAN FETAL TISSUE AND FUNDAMENTAL DIFFERENCES BETWEEN MAMMALIAN MODELS AND HUMANS. CONSEQUENTLY, HOW THE DEVELOPMENTAL POTENTIAL OF PROGENITOR CELLS OF THE CORTEX IS RESTRICTED TO GENERATE ASTROCYTES REMAINS UNKNOWN. ADDRESSING THIS QUESTION IS ESSENTIAL FOR UNDERSTANDING HUMAN BRAIN DEVELOPMENT AND WILL LAY THE FOUNDATION FOR UNCOVERING THE CAUSES AND DEVISING NEW TREATMENT STRATEGIES FOR ASTROCYTE-RELATED HUMAN DISEASES IN MOUSE, ASTROCYTIC PROGENITORS ARISE ONLY AFTER CORTICAL NEURONS FROM THE SIX LAYERS ARE GENERATED. THE COMMON HYPOTHESIS WAS THAT, SIMILARLY, ASTROCYTES ARISE FROM OUTER RADIAL GLIA IN HUMANS IN THE SECOND TRIMESTER OF DEVELOPMENT AFTER THE LAYERS OF THE CORTEX ARE ALREADY FORMED. PRELIMINARY WORK OBTAINED THROUGH THE ANALYSIS OF SINGLE-CELL GENE EXPRESSION DATA FROM THE DEVELOPING HUMAN CORTEX, IN VITRO EMBRYONIC STEM CELL EXPERIMENTS, AND IN VITRO TO IN VIVO MAPPING SHOWS THAT THE ASTROCYTIC LINEAGE IN HUMANS ARISES VERY EARLY BEFORE THE CORTICAL LAYERS ARE FORMED AND LIKELY FROM THE NEUROEPITHELIUM OF THE FOREBRAIN. COMPUTATIONAL ANALYSIS OF THE FETAL TISSUE SINGLE-CELL EXPRESSION DATA AND PRELIMINARY EXPERIMENTS IMPLICATE CELL-TO-CELL CONTACT-DEPENDENT SIGNALING THROUGH YAP AND NOTCH PATHWAYS IN DETERMINING THE ASTROGLIOGENIC FATE OF THE PROGENITORS. FURTHER, PRELIMINARY COMPUTATIONAL ANALYSES OF HUMAN FETAL DATA IDENTIFY CRITICAL TRANSCRIPTION FACTORS INVOLVED IN THIS FATE DECISION. THE PROPOSAL AIMS TO DETERMINE THE DEVELOPMENTAL POTENTIAL AND THE LINEAGE DECISIONS OF THE NEWLY IDENTIFIED ASTROGLIOGENIC PROGENITORS OVER 9 MONTHS. THIS WILL BE ACCOMPLISHED BY TRACING THE LINEAGE DECISIONS OF PROGENITORS IN THE VALIDATED IN VITRO SYSTEM USING VIRAL BAR-CODED VIRAL LIBRARIES AND CLONAL ANALYSES AND MAPPING THE RESULTS, THROUGH DATA ANALYSIS, ONTO IN VIVO HUMAN FETAL DATA AS WELL AS THROUGH IMMUNOSTAINING OF CRYOSECTIONED FETAL TISSUE OBTAINED AT DIFFERENT TIME POINTS. THE PROPOSAL FURTHER AIMS TO DETERMINE THE ROLE OF YAP AND NOTCH SIGNALING AND THE COMPUTATIONALLY IMPLICATED TRANSCRIPTION FACTORS IN GENERATING THESE NEWLY IDENTIFIED PROGENITORS. THIS IS ACCOMPLISHED THROUGH NOVEL HUMAN EMBRYONIC STEM CELL LINES AND LIVE IMAGING TO MONITOR THE DYNAMICS OF THESE SIGNALING PATHWAYS, CHEMICAL, AND GENETIC PERTURBATIONS THAT ALLOW MODULATION OF PATHWAY ACTIVITY, AND INDUCIBLE CRISPRI TO KNOCK DOWN SPECIFIC GENES WHILE MONITORING THEIR EFFECTS ON THE GENERATION AND PATTERNING OF THE PROGENITORS.