Abstract
Purpose :
To ensure correct wiring, developing neurons must prevent connections with non-circuit partners. A common strategy is the use of repulsive recognition cues to prevent neighboring circuits from forming inappropriate connections; however, circuit-specific cues are unknown. Our preliminary data suggest FLRT2 and Unc5C are well-positioned to mediate circuit-specific repulsion in retina. Here we utilized the mouse retinal direction-selective (DS) circuit to test the hypothesis that FLRT2-Unc5C repulsion mediates DS circuit laminar specificity.
Methods :
We utilized a genetic loss-of-function approach to determine whether FLRT2 and/or Unc5C are required for the selective laminar targeting of DS cells (i.e. starburst amacrine cells and DS ganglion cells). To selectively ablate FLRT2 in the retina, floxed Flrt2 mice were crossed to the retina-specific Six3-Cre line. For starburst-specific removal, the ChAT-Cre line was used. Unc5C was completely removed using a constitutive Unc5c mutant. These lines were further crossed to Hb9-GFP transgenic mice to mark a subset of DS ganglion cells. Eyes were collected at postnatal day 6, then eye cups were cryosectioned and immunohistochemistry was performed to label starbursts and DS ganglion cells. Confocal microscopy was used to examine DS cell dendrite morphology and quantify the frequency of DS cell laminar targeting errors in littermate controls versus mutants. Two-tailed Student’s t-test was used for statistical analysis.
Results :
In Six3-Cre Flrt2 mutants (n=7), DS ganglion cells made significantly more laminar targeting errors than controls (n=6; p=0.008), while starburst projections were normal. In ChAT-Cre Flrt2 mutant retinas (n=2), both starbursts and DS ganglion cells made laminar targeting errors, suggesting that the laminar targeting of DS ganglion cells requires starburst-derived FLRT2. Unc5c-/- DS ganglion cells (n=3) exhibited more laminar targeting defects than controls (n=2), similar to the full-retina Flrt2 mutants.
Conclusions :
Our results are consistent with the hypothesis that DS circuit-specific FLRT2 expression mediates repulsion to maintain DS circuit integrity, but the selective requirement for FLRT2 in DS circuit cell types suggests additional levels of regulation. The effect of Unc5C removal suggests it acts non-cell autonomously to control the stratification of DS circuit cells. These mechanisms are now under investigation.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.