Amacrine cells (ACs) are the principle inhibitory neurons of the inner retina, with at least 45 morphologically distinct subtypes that can be distinguished by the size, shape, and stratification of their dendritic arbors within the inner plexiform layer (IPL). Most ACs do not have axons, and instead integrate signaling across their dendritic arbors. This unique arrangement means that form and function are intimately related in ACs, perhaps more so than in any other neuron type. However, we know virtually nothing about how individual AC subtypes are specified during development, or how their adopt their stereotyped morphologies. We hypothesize that selectively expressed transcription factors (TFs) are well poised to direct subtype-specific genetic programs that specify and direct the morphological maturation of ACs. Within the AC population, the homeodomain TF Isl1 is only expressed in starburst amacrine cells (SACs) and Gbx2 is only expressed in a previously unidentified population of medium-field non-GABAeric, non-Glycinergic ACs (MF-nGnGs). Our preliminary results show that early deletion of Isl1 or Gbx2 from AC precursors results in defects in SAC and MF-nGnG subtype specification, respectively. Later deletion of Isl1 or Gbx2 in post-migratory SACs or MF-nGnGs alters their dendritic morphology and stratification patterns. Therefore, Isl1 and Gbx2 appear to be required for the initial specification and the subsequent morphological maturation of their respective AC subtypes. We will test this in the following Specific Aims: 1) Determine whether Isl1 and Gbx2 are necessary and sufficient for AC subtype specification and maturation. We will use conditional loss- and gain-of function approaches to determine whether Isl1 and Gbx2 are both necessary and sufficient to initiate and maintain terminal differentiation in SACs and MF-nGnGs, respectively. Furthermore, we will use a combination of genomic approaches (RNASeq, ATACseq) to identify the Isl1 and Gbx2 gene regulator networks in the respective AC subtypes. 2) Define how Isl1 and Gbx2 regulate SAC and MF-nGnG morphology and functional connectivity. We will first identify how the loss of Isl1 in SACs and Gbx2 in MF-nGnGs affects the development of their stereotyped dendritic morphology and stratification patterns. Then we will test candidate effector genes and pathways for their involvement in the establishment of dendritic arbors. Finally, we will determine how the loss of Isl1 and Gbx2 affects the functional connectivity of SACs and MF-nGnGs in visual circuits. Together, these experiments are expected to reveal how selectively expressed transcription factors specify AC subtypes and drive terminal differentiation programs that endow ACs with their unique morphological properties. We expect that these findings will reveal principles that are broadly applicable across the developing nervous system.
/PUBLIC HEALTH RELEVANCE The mammalian retina is comprised of hundreds of distinct neuronal subtypes that are precisely assembled into functional circuits. This project examines how selectively expressed transcription factors are involved in the specification and morphological maturation of specific neuronal subtypes in the retina during development. These studies will provide important insight that can be used to guide regenerative therapies in the nervous system following injury or disease.
