Project Grant R01EY036173

IDENTIFICATION OF GENE REGULATORY NETWORKS CONTROLLING TEMPORAL PATTERNING IN RETINAL PROGENITOR CELLS AND NEUROGENIC MULLER GLIA - PROJECT SUMMARY: OVER THE COURSE OF DEVELOPMENTAL NEUROGENESIS, RETINAL PROGENITOR CELLS SHOW PROGRESSIVE CHANGES IN THEIR ABILITY TO GENERATE SPECIFIC CELL TYPES, AND BECOME PROGRESSIVELY MORE LIKELY TO UNDERGO BOTH NEUROGENIC AND TERMINAL DIVISIONS. BOTH INTRINSIC AND EXTRINSIC MECHANISM CONTROL THESE PROCESSES, WHICH ARE COLLECTIVELY REFERRED TO AS TEMPORAL PATTERNING. ALTHOUGH THE IDENTITY AND MECHANISM OF ACTION OF THE GENE REGULATORY NETWORKS THAT CONTROL THIS PROCESS HAS BEEN LARGELY UNKNOWN, WE HAVE RECENTLY USED SINGLE-CELL RNA-SEQ AND ATAC-SEQ TO IDENTIFY TWO SEPARATE TRANSCRIPTIONAL REGULATORY NETWORKS THAT RESPECTIVELY PROMOTE EXPRESSION OF GENES SPECIFICALLY EXPRESSED IN EITHER EARLY OR LATE-STAGE RETINAL PROGENITOR CELLS, WHILE REPRESSING GENES SPECIFIC TO THE OTHER STAGE. IN ADDITION, MANY OF THESE TRANSCRIPTION FACTORS ARE PREDICTED TO DIRECTLY ACTIVATE OR INHIBIT GENES THAT REGULATE CELL CYCLE PROGRESSION, NOTCH SIGNALING AND/OR NEUROGENESIS. WE PROPOSE TO DETERMINE WHETHER THESE TRANSCRIPTION FACTORS ARE NECESSARY AND SUFFICIENT TO CONTROL TEMPORAL PROFILING IN RETINAL PROGENITOR CELLS. WE WILL CONDUCT BOTH GAIN AND LOSS OF FUNCTION ANALYSIS OF TRANSCRIPTION FACTORS S THAT ARE TOP CANDIDATES FOR SPECIFYING EARLY OR LATE-STAGE TEMPORAL IDENTITY, AND PROFILE RESULTING CHANGES IN CELL COMPOSITION, GENE EXPRESSION, AND CHROMATIN CONFORMATION. WE WILL THEN INVESTIGATE THE MECHANISM OF ACTION OF TRANSCRIPTION FACTORS THAT SHOW CLEAR PHENOTYPES IN FURTHER DETAIL, DETERMINING HOW THEY REGULATE DEVELOPMENTAL COMPETENCE, PROLIFERATION, AND NEUROGENESIS. FINALLY, WE WILL TEST WHETHER GAIN OR LOSS OF FUNCTION OF THESE TRANSCRIPTION FACTORS CAN REPROGRAM NEUROGENIC RETINAL MULLER GLIA INTO EARLY-STAGE RETINAL PROGENITOR CELLS. WE ANTICIPATE THAT THESE STUDIES WILL BOTH IDENTIFY MOLECULAR MECHANISMS CONTROLLING TEMPORAL PATTERNING IN RETINAL PROGENITORS, AS WELL AS NEW TECHNIQUES FOR IMPROVING THE DIRECTED DIFFERENTIATION OF EARLY- BORN CELL TYPES SUCH AS RETINAL GANGLION CELLS AND CONE PHOTORECEPTOR RECEPTORS, FOR USE IN CELL-BASED THERAPIES FOR TREATING BLINDING DISEASES.