Development of bipolar interneuron subtypes within the retinal space-time continuum

Abstract

Development of the retina requires precise production of more than 100 neuronal subtypes. How does such diversity emerge during development at the perfect time and place to create functional circuitry? Recent molecular studies have expanded our knowledge of existing neuronal diversity, generating quantitative definitions of cell identities based on combinatorial expression of RNA markers. Recently, 15 types of mouse retinal bipolar interneurons have been characterized based on distinct transcriptional and morphological profiles, making them an excellent model for studying neuronal subtype development. However, studying their development has been technically limited due to lack of methods for simultaneously measuring multiple RNA markers within single cells in situ. In Chapter 1, we develop a method for co-detecting many RNA and DNA markers in situ to enable quantitative profiling of single cells in tissue. In Chapter 2, we apply this method to simultaneously classify all subtypes of bipolar interneurons within mouse retinal sections to create a comprehensive map of subtype genesis across space and time. Combining multiplexed detection of 16 RNA markers with timed delivery of EdU and BrdU, we analyzed more than 30,000 single cells in full retinal sections to classify all bipolar subtypes and their birthdates. We found that bipolar subtype birthdates are ordered and follow a centrifugal developmental axis. Spatial analysis revealed a striking wave pattern of bipolar subtype birthdates with hotspots of local genesis, and lineage analyses suggest clonal restriction on homotypic subtype production. These results inspired a hierarchical model of subtype genesis, with the wave pattern of birthdates arising from tiered retinal lineages unfolding in space and time.