Characterization of Tbr2-expressing Retinal Ganglion Cells

Abstract

Purpose : T-box transcription factor T-brain 2 (Tbr2) is expressed in approximately 18% of all RGCs in mouse retinas. Previously we and others have uncovered that Tbr2 is essential for the formation and maintenance of melanopsin/Opn4-expressing intrinsic photosensitive retinal ganglion cells (ipRGCs), and Tbr2-expressing RGCs can activate Opn4 expression upon native ipRGCs loss, suggesting that Tbr2+ RGCs can serve as a reservoir for ipRGCs during retinal development. However, the developmental and molecular mechanisms controlling the formation of native and reservoir ipRGCs remain unclear. To better understand how Tbr2 regulates this process, we have set out to characterize more Tbr2+ RGCs and to build a Tbr2-mediated genetic regulatory network (GRN).

Methods : To determine the extent to which Tbr2+ RGCs can function as reservoir ipRGCs, we used anti-melanopsin-SAP to ablate ipRGCs/melanopsin-expressing Tbr2+ RGCs in adult mouse retinas. To catalog Tbr2-expressing RGC subtypes, we generated Tbr2^CreERT2:Rosa^iAP and Tbr2^{TauGFP-IRESCreERT2}:Ai9 mouse lines to examine their dendritic morphologies, axonal projections, and intrinsic membrane properties and photosensitivity. To assemble a Tbr2-mediated GRN, we created a Tbr2^Isl1/+ mouse line to uncover Tbr2-regulated genes.

Results : We found that ~33% Tbr2+ RGCs (~6% of all RGCs) were abolished by anti-melanopsin-SAP. By comparing the birth-dating profiles between melanopsin+ ipRGCs and Tbr2+ RGCs, we further found that the early-born Tbr2+ RGCs are more likely to activate Opn4 expression than the late-born ones. The morphological and electrophysiological analyses revealed that only the melanopsin+Tbr2+ RGCs exhibit intrinsic photosensitivity. Tbr2+ RGCs project to the same regions in the brain as ipRGCs. Replacing one allele of Tbr2 with Isl1 (Tbr2^Isl1/+) unexpectedly suppresses Opn4 expression but does not affect survival of Tbr2 expressing cells.

Conclusions : We confirm that a fraction of Tbr2+ RGCs can function as reservoir ipRGCs and Opn4 can be activated in them when the native pool was depleted in adult retinas. Birth-dating data suggest that an early developmental mechanism provides a permissive environment for locking in the Tbr2-Opn4 regulatory cascade. Melanopsin-Tbr2+ RGCs consist of several RGC subtypes, and some of them are morphologically similar to known ipRGC types. Tbr2^Isl1/+ mouse line is being used to reveal Tbr2-regulated network by RNA-seq and ChIP-seq.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.