# ﻿Cyclin D1 is the G1 cyclin expressed throughout most of retinal development (Sicinski et al

﻿Cyclin D1 is the G1 cyclin expressed throughout most of retinal development (Sicinski et al., 1995). by microarray, followed by expression validation, revealed that ipsilateral RGCs have a distinct set of Ispinesib (SB-715992) genes that govern neurogenesis, differentiation, and axon guidance compared with contralateral RGCs. Elucidating these gene programs contributes to our understanding of how decussating systemsin particular, the binocular circuitare established. This information is critical for directing the appropriate RGC subtype differentiation and axon regeneration for repair after injury. Introduction The vertebrate central nervous system is composed of a complex network of highly diverse neurons defined by distinct molecular signatures that confer their unique properties in morphology, Ispinesib (SB-715992) connectivity, and function. The vertebrate retina, with its three cellular layers and six neuronal classes, has been a useful model for studying general principles of neurogenesis and axon guidance. Each class of retinal cells can be further divided into morphologically and functionally distinct subtypes, and recent efforts have identified the molecular programs that establish these differences within neuronal classes, such as amacrine, bipolar, and retinal ganglion cell (RGC) subtypes (Kim et al., 2008; Badea et al., 2009; Kay et al., 2011a, 2011b; Watson et al., 2012; Jiang et al., 2013; Sajgo et al., 2014; Macosko et al., 2015; Osterhout et al., 2015; Sanes and Masland, 2015; Tang et al., 2015; Jin et al., 2015; Rousso Rabbit polyclonal to ACYP1 et al., 2016; Shekhar et al., 2016). RGCs, as the only projection neurons of the retina, can be additionally distinguished by the laterality of their axonal projection to targets in the thalamus and midbrain. It is this decussation of the retinogeniculate projection that underlies binocular vision. Two different guidance programs direct the growth of the ipsilateral and contralateral projections at the mouse optic chiasm: EphB1 and EphrinB2 interactions repel ipsilateral axons from the midline, and an NrCAM/PlexinA1 complex reverses an inhibitory Sema6D signal to promote contralateral axon growth through the midline (Williams et al., 2003, 2006b; Kuwajima et al., 2012). Of the known retinal guidance receptors potentially regulated by these transcriptional programs, knockout mouse models show only partial changes in laterality (Williams et al., 2003, 2006a; Erskine et al., 2011; Kuwajima et al., 2012). Moreover, the molecular interactions between transcription factors (e.g., Zic2 and Islet2), downstream effectors (e.g., EphB1, Neuropilin, NrCAM, and PlexinA1), and upstream patterning genes (e.g., Foxd1 and Foxg1) within this genetic network have confirmed difficult to identify, suggesting the presence of Ispinesib (SB-715992) yet-unknown intermediate players that bridge these gaps (Herrera et al., 2003, 2004; Pak et al., 2004; Pratt et al., 2004; Tian et al., 2008; Picker et al., 2009; Carreres et al., 2011; Fotaki et al., 2013; Hernandez-Bejarano et al., 2015). For example, in overexpression studies, Zic2 is more potent than EphB1 in switching RGC projection laterality (Petros et al., 2009b) and thus may regulate additional downstream factors in the uncrossed guidance program. Even less is known about the transcriptional regulators and adhesion molecules that mediate organization of eye-specific RGC axon cohorts in the optic tract and innervation of target regions. One approach to tackling these questions is to analyze the molecular signatures of ipsilateral and contralateral RGCs to identify genes specific to these two RGC subtypes. Such an approach has confirmed useful in recent studies of other neuronal subtypes, such as cortical projection neurons (Lodato and Arlotta, 2015), and has been particularly successful in uncovering transcriptional networks that regulate postmitotic cell fate acquisition. An unbiased screen allows for identification of new candidates not previously described in other systems and not ascribed to the retina or RGCs. A challenge to such studies is usually that ipsilateral RGCs constitute a very small population of cells within the retina [only 3C5% of the final RGC number and 10% at embryonic day 16.5 (E16.5)]. Thus, the ipsilateral RGC population is particularly sensitive to contamination by other cell types when using anatomical isolation approaches. Here we present a novel method for purifying embryonic ipsilateral Ispinesib (SB-715992) and contralateral RGCs using retrograde labeling of live tissue coupled with fluorescence-activated cell sorting (FACS). Through gene expression profiling of purified ipsilateral and contralateral RGCs during the critical period of axon outgrowth and midline decussation, we have uncovered distinct molecular signatures that define and distinguish these two RGC cohorts during.