T-box Transcription RegulatorTbr2Is Essential for the Formation and Maintenance of Opn4/Melanopsin-Expressing Intrinsically Photosensitive Retinal Ganglion Cells

Abstract

Opsin 4 (Opn4)/melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) play a major role in non-imageforming visual system. Although advances have been made in understanding their morphological features and functions, the molecular mechanisms that regulate their formation and survival remain unknown. Previously, we found that mouse T-box brain 2 (Tbr2) (also known as Eomes), a T-box-containing transcription factor, was expressed in a subset of newborn RGCs, suggesting that it is involved in the formation of specific RGC subtypes. In this in vivo study, we used complex mouse genetics, single-cell dye tracing, and behavioral analyses to determine whether Tbr2 regulates ipRGC formation and survival. Our results show the following: (1) Opn4 is expressed exclusively in Tbr2-positive RGCs; (2) no ipRGCs are detected when Tbr2 is genetically ablated before RGC specification; and (3) most ipRGCs are eliminated when Tbr2 is deleted in established ipRGCs. The few remaining ipRGCs display abnormal dendritic morphological features and functions. In addition, some Tbr2-expressing RGCs can activate Opn4 expression on the loss of native ipRGCs, suggesting that Tbr2-expressing RGCs may serve as a reservoir of ipRGCs to regulate the number of ipRGCs and the expression levels of Opn4.

Strict Independence of Parallel and Poly-synaptic Axon-Target Matching during Visual Reflex Circuit Assembly

“…When we used very high intensity blue light or sustained chemogenetic activation of RGCs or OPN neurons, PLR was unaffected in cKOs. This is comparable to what was previously seen for Tbr2 flox/flox :Opn4 Cre/+ :Z/EG mice (Mao et al, 2014). These mice lose 90% of ipRGCs and exhibit only a mild deficit in pupil constriction compared to wildtype mice when using high irradiance levels.…”

“…In order to test this, we sought to eliminate luminance-sensing RGCs at a time before other RGC types begin to innervate the brain. The T-box transcription factor Tbr2 (also known as Eomes) is critical for the survival of a subpopulation of RGCs (Mao et al, 2008; 2014; Sweeney et al, 2014). Like Tph2 Cre , Tbr2 is expressed mainly in the GCL and turns on early in ganglion cell genesis, at ~E14 (Mao et al, 2008).…”

“…Previous work demonstrated that Cre-dependent removal of Tbr2 leads to RGC death, with ipRGCs particularly susceptible and to reduced retinal innervation of several targets, including OPN and mdPPN (Mao et al, 2008; 2014; Sweeney et al, 2014). Here we used the Tph2 Cre line to delete Tbr2 and because of its projection pattern in pretectum as well as its overlap with Tbr2 and luminance-sensing RGCs, the Tbr2 mutation eliminated virtually all retinal input to the OPN and mdPPN without affecting retinal innervation of the neighboring NOT.…”

“…Here we used Cre-dependent deletion of Tbr2, a transcription factor critical for RGC survival (Mao et al, 2008; 2014; Sweeney et al, 2014), to specifically eliminate the early-projecting cohort of luminance-sensing RGCs that connect to PLR centers in the visual brainstem (Figure 1C–1E). We find that even when OPN and mdPPN are rendered devoid of their normal retinal input, the axons of slow-DSGCs destined for eye movement centers continue to avoid PLR targets.…”

Two transcription factors, Pou4f2 and Isl1, are sufficient to specify the retinal ganglion cell fate

“…Nevertheless, we used some of these markers to examine whether the corresponding RGC subtypes were present in the P&I EE retina. Tbr2 (also known as "Eomes") is expressed in a subset of RGCs, from which the intrinsically photosensitive RGCs (ipRGCs) arise (47)(48)(49). At E14.5, by coimmunostaining for Tbr2 and Pou4f2, we observed that Tbr2 was expressed in equivalent numbers of RGCs in the P&I EE retina and the control retina, although its expression was almost completely absent in the Atoh7 tTA/tTA retina (Fig.…”