Abstract
Purpose :
The molecular factors that specify and maintain retinal ganglion cell (RGC) circuitry in the adult mouse remain a mystery. By using a mouse knockout of Tbr2 (Tbr2CreER; Tbr2fl/+) and ectopic Tbr2 expression, this study investigates the role of the transcription factor Tbr2 in the specification and maintenance of non-image forming RGCs.
Methods :
Tbr2CreER and Tbr2fl/+ that also harbor a tdTomato reporter are used to remove Tbr2 from adult retina while labeling the dendrites, soma, and axons of mutant cells. Retinas and brains were harvested 1 week after control and Tbr2 KO adult mice were fed tamoxifen by oral gavage. Melanopsin expression was examined by immunofluorescence on whole mount retinas. Brains were sectioned to visualize the axonal projections to retinorecipient nuclei.
Ectopic Tbr2 expression in the retina was performed through intravitreal injection of AAV-Tbr2 in the adult eye. A virus that expresses only a fluorescent reporter was used as a control. We quantified the population of cells infected with Tbr2 and control virus that coexpressed melanopsin and other RGC markers in whole mount retinas 2 weeks post infection, and also visualized the axonal connections of the infected RGCs in brain sections.
Results :
Removing Tbr2 from the retina in adult mice leads to a dramatic loss of melanopsin-positive RGCs when compared to the control (n=5 retinas for mutant and controls). There is a drastic loss of axonal inputs to the vLGN, OPN, and NOT in the mutants while SCN inputs remain (n=5 for mutant and controls). Ectopic Tbr2 expression in a random population of RGCs leads to melanopsin expression in image forming RGCs (n=5 retinas). Additionally, axonal inputs from image forming RGCs ectopically expressing Tbr2 were observed in the vLGN, OPN, and NOT (n=3).
Conclusions :
Our results are consistent with the hypothesis that Tbr2 is involved in maintaining RGC circuitry. Knocking out Tbr2 perturbs the pretectal connections known to be responsible for the pupillary light reflex, but not the connections to the SCN, responsible for circadian rhythms. Our results also show that Tbr2 is necessary and sufficient for melanopsin expression, and that Tbr2 expression in image forming RGCs is sufficient for rewiring axonal connections to specific non-image forming nuclei. Together, our evidence indicates that Tbr2 is necessary and sufficient for maintaining a non-image forming RGC fate.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.