The central nervous system (CNS) consists of complex circuits that are established during development and fine-tuned through both activity-dependent and apoptotic mechanisms. However, how neurons form these complex circuits—and, more specifically, how cell surface molecules promote correct neuronal migration and synapse formation—is not fully resolved. Cell-adhesion molecules (CAMs) have been shown to be key mediators of CNS lamination and synaptogenesis. Work from ours and others focused on using the inner retina of the mouse as a developmental system to understand mechanisms regulating these developmental questions (Graham and Duan, 2021; Lefebvre et al., 2015; Sun et al., 2013; Matsuoka et al., 2011; Hoon et al., 2014). Specifically, our past studies showed that type II cadherin (Cdhs), in combination, plays key roles in establishing appropriate synapses between retinal ganglion cells (RGCs) and BCs, as well as RGCs with amacrine cells (ACs) (Duan et al., 2014; Duan et al., 2018). Among the molecular machinery that composes the adherens junction (AJ) complex, β-catenin has been shown to be important in neuronal laminar organization, leading to embryonic deficits and major retinal neuron loss (Fu et al., 2006).
While we assume that all intracellular components of cell-surface adhesion complexes are critical for retinal neuron survival and patterning, not all components of the AJ complex equally regulate the same aspects of these developmental programs. Afadin is a cytosolic adaptor protein that links Nectin, a Ca2+-independent immunoglobulin-like CAM, to F-actin microfilaments in the cytoskeleton (Mandai et al., 1997; Takai and Nakanishi, 2003; Takahashi et al., 1999; Takai et al., 2008). Afadin recruits cadherins to AJs mediated by Nectin, p120-catenin, and α-catenin (Takai and Nakanishi, 2003; Takahashi et al., 1999). While Afadin has multiple direct interactions with AJ proteins, it is not a core part of the cadherin–catenin complex (Sawyer et al., 2009).
Past genetic studies in the mouse CNS showed that loss of Afadin in both the hippocampal and cortical regions of the mouse brain leads to a decrease in dendritic spine density and number of synapses, with variable effects on dendritic arborization (Beaudoin et al., 2012). Additionally, Afadin plays an important role in cortical lamination: deletion of Afadin in the mouse telencephalon leads to cellular mislocalization and a resultant double-cortex (Yamamoto et al., 2015; Gil-Sanz et al., 2014). Notably, the drosophila homologue of Afadin is called Canoe (Yu and Zallen, 2020; Mandai et al., 2013), where the mutant phenotypes in the ommatidial eye were likely closely tied to the disruption of cellular junctions or synaptic complex, though given the broad role of Afadin (Canoe), they may also be due to other cell-surface signaling pathways (Matsuo et al., 1999).
The mouse neural retina offers a laminarly organized structure and well-characterized cellular composition across three cellular layers. During development, retinal progenitor cells span the retinal neuroepithelium via basal and apical processes, proliferate via asymmetric and symmetric divisions at the ventricular surface, and differentiate into six neuronal types via both transcriptional regulatory networks and environmental cues—these include rod and cone photoreceptors (PRs), horizontal cells (HCs), BCs, ACs, RGCs, and one glial cell type, Müller glia (MGs) (Turner and Cepko, 1987; Cepko, 2014; Livesey and Cepko, 2001; Yan et al., 2020). Thus, the distinct locations and temporal order offer a clear system to examine the roles of multifaceted molecules in every step of development, such as that for Afadin. By restricting the roles of Afadin into restricted RGC subsets or AC subsets, our recent study linked Afadin to the combinatorial Cdh complex that enables the selective RGC-AC synaptic choice (Duan et al., 2018). Yet it is unknown what role Afadin plays in neuronal migration, neuronal layer sorting, and brain target selection. Here, we utilized a developmental neural retina-specific Cre driver (Six3Cre) (Oliver et al., 1995; Liu and Cvekl, 2017; Diacou et al., 2018) to generate a conditional Afadin mutant (Six3Cre; AfadinF/F). This conditional mutant allows us to characterize the role of Afadin in early development. Here, we report that the Afadin mutant significantly alters retinal neuronal migration and neuronal layer sorting, though it has little effect on cellular differentiation within the inner retina.