Molecular Diversity of Intrinsically Photosensitive Ganglion Cells

Abstract

14Intrinsically photosensitive retinal ganglion cells (ipRGCs) are rare mammalian photoreceptors 15 essential for non-image-forming vision functions, such as circadian photoentrainment and the 16 pupillary light reflex. They comprise multiple subtypes distinguishable by morphology, physiology, 17 projections, and levels of expression of melanopsin (Opn4), their photopigment. The molecular 18 programs that differentiate ipRGCs from other ganglion cells and ipRGC subtypes from one 19 another remain elusive. Here, we present comprehensive gene expression profiles of early 20 postnatal and adult mouse ipRGCs purified from two lines of reporter mice marking different sets 21 of ipRGC subtypes. We find dozens of novel genes highly enriched in ipRGCs. We reveal that 22 Rasgrp1 and Tbx20 are selectively expressed in subsets of ipRGCs, though these molecularly 23 defined groups imperfectly match established ipRGC subtypes. We demonstrate that the ipRGCs 24 regulating circadian photoentrainment are unexpectedly diverse at the molecular level. Our 25 findings reveal unexpected complexity in gene expression patterns across mammalian ipRGC 26 subtypes.

Distribution of prototypical primary cilia markers in subtypes of retinal ganglion cells

“…The OPN4‐GFP transgenic mouse line has been shown to label ipRGCs. This line, however, is reported to only visualize M1, M2, and M3 as the amount of OPN4 expression varies between the ipRGC subgroups (Berg et al., 2018). OPN4‐GFP‐positive cells colocalized with RBPMS staining (Figure 4c), and 17% ± 0% of OPN4‐GFP cells were positively labeled for Arl13b and 54% ± 7% for AC3 (Figure 4d).…”

“…C57BL/6J mice were purchased from The Jackson Laboratories (stock # 000664). Genetically defined RGC transgenic mice Trhr‐GFP (Gong et al., 2003; Rivlin‐Etzion et al., 2011), Hoxd10‐GFP (Dhande et al., 2013), OPN4‐GFP (Berg et al., 2018), and Cdh3‐GFP (Osterhout et al., 2011; Quattrochi et al., 2019) were a gift from Dr. Andrew Huberman's Lab (Stanford). Mice were kept under a 12‐h light/dark cycle and had free access to food and water.…”

Single-Cell Profiles of Retinal Ganglion Cells Differing in Resilience to Injury Reveal Neuroprotective Genes

“…Our previous studies identified 4 T-RGC and 4 F-RGC types, but this new approach revealed a fifth type within each subclass (C9 and C32, respectively; Figures 2C, 2D, and S2B). For ipRGCs, we discovered markers for M1 (C33 and 40), M2 (C31), and M4 (C43) types, including some that divide M1-RGCs (identified by high levels of Opn4 and expression of Adcyap1; Hannibal et al, 2002) into two types (M1a, M1b), as well as an additional cluster (C22) that could correspond to the morphologically and physiologically characterized M3, M5, or M6 types (Figure 2E) (Quattrochi et al, 2019;Schmidt et al, 2011;Stabio et al, 2018;Berg et al, 2019). For ooDSGCs, most of which are Cartpt+, we identified the nasal-preferring type (N-ooDSGC) by expression of Mmp17, but only a single cluster (C16) expressed Col25a1, a marker of both D-and V-ooDSGCs (Kay et al, 2011).…”

Subtype-specific survival and regeneration of retinal ganglion cells in response to injury

“…For instance, M1 ipRGCs can be distinguished by expression of adenylate cyclase activating polypeptide 1 (Adcyap1) and the absence of the neuromedin B (Nmb) gene while an additional M1 type can be identified by the co-expression of Adcyap1 and Nmb (Tran et al, 2019). M2 subtype can be identified by the presence of Spp1 and T-box transcription factor (Tbx20), known to be selectively expressed in ipRGCs and greatly enriched in M2 over other ipRGC types (Berg et al, 2019). M4/alpha ON-S RGCs were found to be distinguished by the expression of Spp1 and interleukin one receptor accessory protein like 2 (Il1rapl2) while all other ipRGC types by serpin family E member 2 (Serpine2) and cadherin related family member 1 (Cdhr1) (Tran et al, 2019).…”