Project Grant R01EY035324

TRANSCRIPTIONAL CONTROL OF NEURONAL POSITION AND CONNECTION IN THE RETINA - PROJECT SUMMARY/ABSTRACTOR IN THE RETINA, VARIOUS NEURONAL TYPES ARE CONFINED TO THREE SOMATIC LAYERS AND CONNECTED IN TWO PLEXIFORM LAYERS. DEFECTS IN NEURONAL POSITIONING OR CONNECTIONS RESULT IN VISION IMPAIRMENT OR BLINDNESS. WHILE MIGRATION OF NEURONS TO THEIR DESTINED LAYERS IS WELL STUDIED, THE INFLUENCE OF SEGREGATED NUCLEAR LAYERS ON THE FORMATION OF NEURONAL CONNECTIONS REMAINS UNCLEAR. THIS PROJECT AIMS TO DELINEATE THE TRANSCRIPTIONAL CONTROL OF NEURONAL POSITION AND CONNECTION. WE USE STARBURST AMACRINE CELLS (SACS)-THE CHOLINERGIC INTERNEURONS IN THE RETINA- AS A MODEL TO ADDRESS THIS QUESTION. SACS ARE CRUCIAL COMPONENTS OF THE DIRECTION-SELECTIVE (DS) CIRCUIT AND CONSIST OF TWO CLOSELY RELATED SUBTYPES. OFF SACS IN THE INNER NUCLEAR LAYER (INL) ESTABLISH DENDRITIC STRATIFICATIONS AND CONNECTIONS IN THE OFF LAYER (S2) OF THE INNER PLEXIFORM LAYER (IPL), WHEREAS ON SACS IN THE GANGLION CELL LAYER (GCL) RAMIFY THEIR DENDRITES IN THE ON LAYER (S4) OF THE IPL. MY PREVIOUS RESEARCH IDENTIFIED THE TRANSCRIPTION FACTOR FEZF1 THAT IS SPECIFICALLY EXPRESSED BY ON SACS AND DETERMINES THEIR SOMATIC POSITIONS IN THE GCL. IN THE FEZF1 CONSTITUTIVE KNOCKOUT (FEZF1-/-), ON SACS ARE MISLOCALIZED TO THE INL ALONGSIDE OFF SACS. TO OVERCOME THE NEONATAL LETHALITY ASSOCIATED WITH FEZF1-/-, WE GENERATED A NEW FEZF1 CONDITIONAL ALLELE TO FACILITATE THE STUDY OF POSTNATAL DEVELOPMENT OF SACS. WE MADE THREE OBSERVATIONS: (1) SIMILAR TO FEZF1-/-, EARLY EMBRYONIC DELETION OF FEZF1 RESULTS IN MISLOCALIZATION OF ON SACS TO THE GCL, BUT LATE DELETION ALLOWS THEM TO REMAIN IN THE GCL (2) IN BOTH EARLY AND LATE FEZF1 DELETIONS, SAC DENDRITES NO LONGER SEPARATE INTO TWO DISTINCT LAYERS BUT REMAIN INTERMINGLED. (3) FOLLOWING ALTERED SAC DENDRITIC STRATIFICATION, ON AND OFF DIRECTION-SELECTIVE GANGLION CELLS (OODSGCS) AND ON DSGCS REDIRECT THEIR DENDRITIC ARBORS FROM MAKING CONTACTS WITH SACS IN S2 AND S4 (OODSGCS) OR S4 (ON DSGCS) TO CONTACTING S2 ALONE. THESE FINDINGS COLLECTIVELY SUPPORT THE CENTRAL HYPOTHESIS THAT FEZF1 SEQUENTIALLY CONTROLS THE SOMATIC POSITION, DENDRITIC STRATIFICATION, AND SYNAPTIC CONNECTIONS OF SACS. WE THEREFORE PROPOSE TWO SPECIFIC AIMS. AIM 1 AIMS TO DETERMINE HOW FEZF1 MEDIATES DENDRITIC STRATIFICATION OF SACS. WE WILL ELUCIDATE THE MORPHOLOGICAL CHANGES OF DEVELOPING SACS VIA IMAGE-TRACING AND ASSESS THE SUFFICIENCY OF FEZF1 IN RESCUING THE DENDRITIC DEFECTS VIA IN UTERO ELECTROPORATION. WE WILL DETERMINE THE REGULATORY NETWORK OF FEZF1 USING COMBINED ATAC-SEQ AND RNA-SEQ ASSAYS. LASTLY, WE WILL TEST THE ROLE OF REPULSIVE MOLECULES SLIT2/NELL2/ROBO2 IN SEPARATING THE DENDRITIC ARBORS BETWEEN ON AND OFF SACS. AIM 2 AIMS TO DETERMINE HOW THE ALTERED DENDRITIC STRATIFICATION OF SACS REDIRECTS DENDRITIC INNERVATION OF OODSGCS AND ON DSGCS. WE WILL USE GENETIC LABELING AND IMAGE-TRACING TO DETERMINE THE SPECIFICITY OF TARGETING DEFECTS TO DSGC TYPES BUT NOT OTHER RGC TYPES. WE WILL THEN USE RNA-SEQ AND AAV-MEDIATED GENETIC MANIPULATION TO DETERMINE THE MOLECULAR MECHANISM THAT MEDIATES THE DENDRITIC MISTARGETING OF DSGCS. THIS PROJECT WILL COMPREHENSIVELY DISSECT THE TRANSCRIPTIONAL CONTROL OF NEURONAL POSITIONING AND CONNECTION FORMATION OF SACS AND GENERATE A NEW MOLECULAR BASIS FOR THE ASSEMBLY OF THE DIRECTION-SELECTIVE CIRCUIT.