Combinatorial Effects of Alpha- and Gamma-Protocadherins on Neuronal Survival and Dendritic Self-Avoidance.

The clustered protocadherins (Pcdhs) comprise 58 cadherin-related proteins encoded by three tandemly arrayed gene clusters, Pcdh-α, Pcdh-ß, and Pcdh-γ (Pcdha, Pcdhb, and Pcdhg, respectively). Pcdh isoforms from different clusters are combinatorially expressed in neurons. They form multimers that interact homophilically and mediate a variety of developmental processes, including neuronal survival, synaptic maintenance, axonal tiling, and dendritic self-avoidance. Most studies have analyzed clusters individually. Here, we assessed functional interactions between Pcdha and Pcdhg clusters. To circumvent neonatal lethality associated with deletion of Pcdhgs, we used Crispr-Cas9 genome editing in mice to combine a constitutive Pcdha mutant allele with a conditional Pcdhg allele. We analyzed roles of Pcdhas and Pcdhgs in the retina and cerebellum from mice (both sexes) lacking one or both clusters. In retina, Pcdhgs are essential for survival of inner retinal neurons and dendritic self-avoidance of starburst amacrine cells, whereas Pcdhas are dispensable for both processes. Deletion of both Pcdha and Pcdhg clusters led to far more dramatic defects in survival and self-avoidance than Pcdhg deletion alone. Comparisons of an allelic series of mutants support the conclusion that Pcdhas and Pcdhgs function together in a dose-dependent and cell-type-specific manner to provide a critical threshold of Pcdh activity. In the cerebellum, Pcdhas and Pcdhgs also cooperate to mediate self-avoidance of Purkinje cell dendrites, with modest but significant defects in either single mutant and dramatic defects in the double mutant. Together, our results demonstrate complex patterns of redundancy between Pcdh clusters and the importance of Pcdh cluster diversity in postnatal CNS development.SIGNIFICANCE STATEMENT The formation of neural circuits requires diversification and combinatorial actions of cell surface proteins. Prominent among them are the clustered protocadherins (Pcdhs), a family of ∼60 neuronal recognition molecules. Pcdhs are encoded by three closely linked gene clusters called Pcdh-α, Pcdh-ß, and Pcdh-γ. The Pcdhs mediate a variety of developmental processes, including neuronal survival, synaptic maintenance, and spatial patterning of axons and dendrites. Most studies to date have been limited to single clusters. Here, we used genome editing to assess interactions between Pcdh-α and Pcdh-γ gene clusters. We examined two regions of the CNS, the retina and cerebellum and show that the 14 α-Pcdhs and 22 γ-Pcdhs act synergistically to mediate neuronal survival and dendrite patterning.

Gamma-protocadherins regulate dendrite self-recognition and dynamics to drive self-avoidance.

Neurons form cell-type-specific morphologies that are shaped by cell-surface molecules and their cellular events governing dendrite growth. One growth rule is dendrite self-avoidance, whereby dendrites distribute uniformly within a neuron's territory by avoiding sibling branches. In mammalian neurons, dendrite self-avoidance is regulated by a large family of cell-recognition molecules called the clustered protocadherins (cPcdhs). Genetic and molecular studies suggest that the cPcdhs mediate homophilic recognition and repulsion between self-dendrites. However, this model has not been tested through direct investigation of self-avoidance during development. Here, we performed live imaging and four-dimensional (4D) quantifications of dendrite morphogenesis to define the dynamics and cPcdh-dependent mechanisms of self-avoidance. We focused on the mouse retinal starburst amacrine cell (SAC), which requires the gamma-Pcdhs (Pcdhgs) and self/non-self-recognition to establish a stereotypic radial morphology while permitting dendritic interactions with neighboring SACs. Through morphogenesis, SACs extend dendritic protrusions that iteratively fill the growing arbor and contact and retract from nearby self-dendrites. Compared to non-self-contacting protrusions, self-contacting events have longer lifetimes, and a subset persists as loops. In the absence of the Pcdhgs, non-self-contacting dynamics are unaffected but self-contacting retractions are significantly diminished. Self-contacting bridges accumulate, leading to the bundling of dendritic processes and disruption to the arbor shape. By tracking dendrite self-avoidance in real time, our findings establish that the γ-Pcdhs mediate self-recognition and retraction between contacting sibling dendrites. Our results also illustrate how self-avoidance shapes stochastic and space-filling dendritic outgrowth for robust pattern formation in mammalian neurons.

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations.

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a powerful model system for the investigation of cell type-specific mechanisms of neuronal morphogenesis and connectivity. The organization and composition of retinal subtypes are well-defined, and genetic tools are available to access specific types during development. Many retinal cell types also constrain their dendrites and/or axons to narrow layers, which facilitates time-lapse imaging. Mouse retina explant cultures are well suited for live-cell imaging using confocal or multiphoton microscopy, but methods optimized for imaging dendrite dynamics with temporal and structural resolution are lacking. Presented here is a method to sparsely label and image the development of specific retinal populations marked by the Cre-Lox system. Commercially available adeno-associated viruses (AAVs) used here expressed membrane-targeted fluorescent proteins in a Cre-dependent manner. Intraocular delivery of AAVs in neonatal mice produces fluorescent labeling of targeted cell types by 4-5 days post-injection (dpi). The membrane fluorescent signals are detectable by confocal imaging and resolve fine branch structures and dynamics. High-quality videos spanning 2-4 h are acquired from imaging retinal flat-mounts perfused with oxygenated artificial cerebrospinal fluid (aCSF). Also provided is an image postprocessing pipeline for deconvolution and three-dimensional (3D) drift correction. This protocol can be used to capture several cellular behaviors in the intact retina and to identify novel factors controlling neurite morphogenesis. Many developmental strategies learned in the retina will be relevant for understanding the formation of neural circuits elsewhere in the central nervous system.