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Rebecca James, Matthew Brown, Alex Kolodkin; Semaphorin 6A (Sema6A) elaborates direction-selective (DS) circuits by an unexpected mechanism. Invest. Ophthalmol. Vis. Sci. 2017;58(8):114.
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© ARVO (1962-2015); The Authors (2016-present)
DS circuitry enables object motion detection, a critical task for survival. Sema6A, a repulsive guidance cue, is required for DS circuit elaboration both locally in the retina and also in the brain, but how Sema6A guides local DS circuit refinement is unclear since it is expressed by both presynaptic starburst amacrine cells (SACs) and postsynaptic DS retinal ganglion cells (RGCs). As part of a comprehensive study of Sema6A function in DS circuit wiring, we tested the hypothesis that Sema6A in SACs enables retinal DS circuit development.
We made a mouse N-terminal HA-tagged Sema6A conditional allele (HA-Sema6AF). Expression of HA-Sema6A in the retina and brain of postnatal day 4 (P4), P10, and juvenile (~4 weeks) mice was studied. The source of HA-Sema6A in retinorecipient nuclei was probed by enucleation at P12 (tissue collection at P21). To ask if Sema6A in SACs drives SAC dendrite lamination and radial morphology, roles previously ascribed to Sema6A, recombination of HA-Sema6AF was performed using the SAC-specific ChatCre driver. Controls were heterozygous or Cre negative littermates. Conditional knockout (CKO) was also performed using panretinal Six3Cre and germline Sox2Cre drivers. SAC lamination was assayed by anti-ChAT immunolabeling, and scored as the relative frequency (±SEM) of parallel ChAT+ S2/S4 bands in CKO tests compared to control. Statistical significance was calculated by one-way ANOVA and Tukey HSD test. SAC morphology in SAC-specific CKO retinas was assessed by intravitreal injection of dilute AAV2-LSL-eGFP.
HA-Sema6A is broadly expressed in the retina and the brain. Many more RGCs express HA-Sema6A than expected, and HA-Sema6A+ RGCs project to major image-forming retinorecipient nuclei. Contrary to our hypothesis, Sema6A is not required in SACs for SAC lamination and morphology. However, SAC lamination is greatly impaired by germline (0.15±0.01, p=0.001) and retinal (0.24±0.02, p=0.001) CKO compared to control (0.99±0.01).
Our unexpected results suggest that RGCs interact with SACs during development to regulate the elaboration of SAC dendritic arbors. Further study of Sema6A function in SACs is required to define how SAC-derived Sema6A contributes to wiring DS circuitry. Preliminary studies suggest that Sema6A in SACs specifically regulates synaptogenesis by a novel mechanism.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
Retinal expression of HA-Sema6A.
Conditional knockout of HA-Sema6AF.
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