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

Abstract

The clustered protocadherins (Pcdhs) comprise 58 cadherin-related proteins encoded by three tandemly arrayed gene clusters, <i>Pcdh</i>-α, <i>Pcdh</i>-β, and <i>Pcdh</i>-γ (<i>Pcdha</i>, <i>Pcdhb</i>, and <i>Pcdhg</i>, 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 <i>Pcdha</i> and <i>Pcdhg</i> clusters. To circumvent neonatal lethality associated with deletion of <i>Pcdhgs</i>, we used Crispr-Cas9 genome editing in mice to combine a constitutive <i>Pcdha</i> mutant allele with a conditional <i>Pcdhg</i> 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 <i>Pcdha</i> and <i>Pcdhg</i> clusters led to far more dramatic defects in survival and self-avoidance than <i>Pcdhg</i> 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.<b>SIGNIFICANCE STATEMENT</b> 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 <i>Pcdh</i>-α, <i>Pcdh</i>-β, and <i>Pcdh</i>-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 <i>Pcdh</i>-α and <i>Pcdh</i>-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.