Abstract
Purpose :
Structure and function are intimately connected within the nervous system. Neurons, in the brain and retina, have a diverse array of cellular morphologies that are unique and specific among the different functional populations. While much work has been done to uncover the exact physiological reasons a cell class has its shape, we are still in the dark on the genetic contribution to these structures. Using two closely related function cell types, rod and cone photoreceptors, that have similar but distinct morphologies, we sought to investigate those genes that could be playing a role in defining the morphological differences between the two.
Methods :
To accomplish our goal, we first quantitatively characterized rod and cone (and cone-like - having an intermediate structure between the two native types) synaptic terminal structures: terminal size, plexiform layer position, ribbon number, and presence/absence of telodendrites. The transcriptional landscape of developing rod and cone-like photoreceptors along with transcription factor binding profiles of key regulators of photoreceptor fate to identify a more workable list of candidates, 720 in total. From this, we selected and knocked down approximately 10% of these genes (selected based on expression profiles and G.O. analysis) through in vivo electroporation of shRNAs combined with a rod-specific fluorescent reporter and assayed individual cell terminals through quantitative analysis of the pre-synaptic terminal structure, specifically the depth within the plexiform layer and the size of the terminals.
Results :
We have currently found 18 genes (25%) whereby knockdown in rod photoreceptors resulted in differential morphological features that are more similar to the cone terminal than to the native rod, either in the size of the terminal, the position of it within the connective layer, or both. Confidence in these data is increased through knockdown of the same genes with different constructs as well as rescue of selected phenotypes.
Conclusions :
Our data show that we can identify some of the molecular players that lead to cell-specific morphology in at least a subset of neurons. Current work is focused on finding loss of function mice that complement the morphology knockdown screen to see how this morphology affects the synaptic transmission. This work provides an in-depth look at the formation of a cell's gross morphology and looks to link that structure with overall function.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.