DNER and NFIA are expressed by developing and mature AII amacrine cells in the mouse retina

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Excerpt

The mammalian retina is a thin neural tissue that lines the back of the eye and is responsible for the transmission of incoming light signals to higher visual regions of the brain. Within the retina, this information is already beginning to be processed as separate parallel visual pathways. This is accomplished by many different cell types: current estimates suggest that there are approximately 85 unique neuronal populations in the retina (Masland, 2012; Macosko et al., 2015; Shekhar et al., 2016; Baden et al., 2016). One such cell type is the AII amacrine cell, a narrow‐field amacrine cell initially reported to have a unique radial orientation and apparent separation of its dendrites into pre‐ and post‐synaptic compartments (Famiglietti & Kolb, 1975; Strettoi, Raviola, & Dacheux, 1992). Subsequent anatomical and physiological studies have not only confirmed, but expounded on this compartmentalization, revealing a stunning complexity in how this cell is integrated into the retinal circuitry: the AII amacrine cell forms synapses with no less than 28 retinal cell types (Marc, Anderson, Jones, Sigulinsky, & Lauritzen, 2014), participating in multiple retinal circuits, with a particularly critical role in mediating scotopic vision (Demb & Singer, 2012). Little is known, however, about the developmental processes and genetic underpinnings that establish this neuronal population, create their complex dendritic arbors, and integrate these cells into the retinal circuitry.
Understanding the mechanisms underlying AII amacrine cell development requires knowledge of the molecular players expressed by these cells at different ages. Many proteins have been identified to be expressed by mature AII amacrine cells, including calretinin, parvalbumin, Dab1, and Prox1 (Wässle, Grünert, & Röhrenbeck, 1993; Massey & Mills, 1999; Rice & Curran, 2000; Dyer, Livesey, Cepko, & Oliver, 2003). Only a few proteins, however, have been shown to be important for developing AII amacrine cells, such as DSCAML1 and NeuroD2, both implicated in the growth of AII neurites (Fuerst et al., 2009; Cherry et al., 2011). The present study has sought to identify other novel proteins that are expressed by developing and mature AII amacrine cells, by taking advantage of recent advances in molecular profiling, particularly in single cell RNA‐sequencing, that have begun to differentiate the subtleties that exist between different cell types through their transcriptome‐wide molecular signatures (Kay, Chu, & Sanes, 2012; Siegert et al., 2012; Macosko et al., 2015; Shekhar et al., 2016). These gene expression profiles offer a comprehensive look into the proteins that must be critical for the development and function of specific cell types in the retina, including the AII amacrine cell.
In this study, we have used recently published expression profiles of retinal cell types to identify genes that may have roles critical for the development of AII amacrine cells. We found two genes, Delta/Notch‐like EGF repeat containing (Dner) and nuclear factor I/A (Nfia), that are highly expressed in these cells, both in development and adulthood. We subsequently validated the expression of their protein products using immunofluorescent techniques, and determined that probing for both proteins conjointly is a reliable method for identifying the population of AII amacrine cells. As both genes are known to play a role in neural development outside the retina, they are prime candidates for understanding the mechanisms that underlie the development of the AII amacrine cell.
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