Roles of Tbr1 in retinal ganglion cell subtype formation

Abstract

Purpose : The developmental and molecular mechanisms controlling the formation and maturation of the diverse RGC subtypes remains unclear. Here, we provide evidence showing that T-box transcription factor T-brain 1 (Tbr1) is expressed in a subset of RGCs in mouse retinas and may play key roles in the formation and maintenance of two morphologically and functionally distinct RGC subtypes.

Methods : Molecular tools (ISH, IF, and qRT-PCR) were used to detect the spatiotemporal expression patterns of Tbr1 in developing and mature mouse retinas. To determine Tbr1-expressing RGC subtypes, we generated Tbr1^CreERT2:Pou4f1^CKOAP and Tbr1^CreERT2:Ai9 mouse lines and conducted sparse-labeling and dye-filling in Tbr1-expressing RGCs, respectively. Alkaline phosphatase staining on the tamoxifen-activated Tbr1^CreERT2: Pou4f1^CKOAP retinas was used to reveal the overall Tbr1+ RGC subtypes. NBT dye-filling in individual Tbr1+ RGCs in tamoxifen-activated Tbr1^CreERT2:Ai9 retinas was used to examine the detailed dendritic morphologies of these RGCs. Furthermore, we conducted loose patch-clamp recording on individual Tbr1+ RGCs to determine their light-responses. To determine the central projection of Tbr1+ RGCs, we examined the GFP signal in brain sections from Tbr1^TauGFP mice.

Results : We detected Tbr1 expression in a subset of RGCs, starting from E14.5. By sparse labeling and dye-filling, we revealed two morphologically distinct Tbr1+ RGC subtypes, including the JAM-B RGC and a RGC subtype with symmetric dendritic ramification pattern. The terminal dendrites of both RGC subtypes stratify slightly above the ChAT OFF band in the IPL. The second RGC subtype exhibited sluggish OFF response, and preferred medium size spots with both positive and negative contrast. They are not direction-selective RGCs, and showed ON responses to annuli. The GFP signals in Tbr1^TauGFP brain sections were detected in superior colliculus and dorsal lateral geniculate nucleus.

Conclusions : In summary, we have demonstrated that Tbr1 expression marks two RGC subtypes, suggesting that RGC subtypes may be established by intrinsically programmed transcriptional regulatory mechanism. These mouse lines will be used to study the developmental timing and relationship of these two RGC subtypes during retinogenesis. Furthermore, Tbr1^flox allele will be used to determine whether and how Tbr1 regulates the formation and maintenance of these two RGC subtypes.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.