The mammalian Pcdhg gene cluster encodes a family of 22 cell adhesion molecules, the gamma-Protocadherins (γ-Pcdhs), critical for neuronal survival and neural circuit formation. The extent to which isoform diversity-a γ-Pcdh hallmark-is required for their functions remains unclear. We used a CRISPR/Cas9 approach to reduce isoform diversity, targeting each Pcdhg variable exon with pooled sgRNAs to generate an allelic series of 26 mouse lines with 1 to 21 isoforms disrupted via discrete indels at guide sites and/or larger deletions/ rearrangements. Analysis of 5 mutant lines indicates that postnatal viability and neuronal survival do not require isoform diversity. Surprisingly, given reports that it might not independently engage in trans-interactions, we find that γC4, encoded by Pcdhgc4, is the only critical isoform. Because the human orthologue is the only PCDHG gene constrained in humans, our results indicate a conserved γC4 function that likely involves distinct molecular mechanisms.

14The mammalian Pcdhg gene cluster encodes a family of 22 cell adhesion molecules, the gamma-15 Protocadherins (γ-Pcdhs), critical for neuronal survival and neural circuit formation. The extent to which 16 isoform diversity-a γ-Pcdh hallmark-is required for their functions remains unclear. We used a 17 CRISPR/Cas9 approach to reduce isoform diversity, targeting each Pcdhg variable exon with pooled 18 sgRNAs to generate an allelic series of 26 mouse lines with 1 to 21 isoforms disrupted via discrete indels 19 at guide sites and/or larger deletions/rearrangements. Analysis of 5 mutant lines indicates that 20 postnatal viability and neuronal survival do not require isoform diversity. Surprisingly, as it is the only γ-21Pcdh that cannot independently engage in homophilic trans-interactions, we find that γC4, encoded by 22Pcdhgc4, is the only critical isoform. Because the human orthologue is the only PCDHG gene constrained 23 in humans, our results indicate a conserved γC4 function that likely involves distinct molecular 24 mechanisms. 25 26 mammalian analogue to fly Dscam1, although their diversity is generated by differential isoform 38 expression via promoter choice rather than alternative splicing (Zipursky and Sanes, 2010). 39

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The establishment of a functional cerebral cortex depends on the proper execution of multiple developmental steps, culminating in dendritic and axonal outgrowth and the formation and maturation of synaptic connections. Dysregulation of these processes can result in improper neuronal connectivity, including that associated with various neurodevelopmental disorders. The γ-Protocadherins (γ-Pcdhs), a family of 22 distinct cell adhesion molecules (CAMs) that share a C-terminal cytoplasmic domain, are involved in multiple aspects of neurodevelopment including neuronal survival, dendrite arborization, and synapse development. The extent to which individual γ-Pcdh family members play uniquevs.common roles remains unclear. We demonstrated previously that the γ-Pcdh-C3 isoform (γC3),viaits unique “variable” cytoplasmic domain (VCD), interacts in cultured cells with Axin1, a Wnt-pathway scaffold protein that regulates the differentiation and morphology of neurons. Here, we confirm that γC3 and Axin1 interact in the cortexin vivoand show that both male and female mice specifically lacking γC3 exhibit disrupted Axin1 localization to synaptic fractions, without obvious changes in dendritic spine density or morphology. However, both male and female γC3 knockout mice exhibit severely decreased dendritic complexity of cortical pyramidal neurons that is not observed in mouse lines lacking several other γ-Pcdh isoforms. Combining knockout with rescue constructs in cultured cortical neurons pooled from both male and female mice, we show that γC3 promotes dendritic arborization through an Axin1-dependent mechanism mediated through its VCD. Together, these data identify a novel mechanism though which γC3 uniquely regulates the formation of cortical circuitry.SIGNIFICANCE STATEMENT:The complexity of a neuron’s dendritic arbor is critical for its function. We showed previously that the γ-Protocadherin (γ-Pcdh) family of 22 cell adhesion molecules promotes arborization during development; it remained unclear whether individual family members played unique roles. Here, we show that one γ-Pcdh isoform, γC3, interacts in the brain with Axin1, a scaffolding protein known to influence dendrite development. A CRISPR/Cas9-generated mutant mouse line lacking γC3 (but not lines lacking other γ-Pcdhs) exhibits severely reduced dendritic complexity of cerebral cortex neurons. Using cultured γC3 knockout neurons and a variety of rescue constructs, we confirm that the γC3 cytoplasmic domain promotes arborization through an Axin1-dependent mechanism. Thus, γ-Pcdh isoforms are not interchangeable, but rather can play unique neurodevelopmental roles.

Cortical function critically depends on inhibitory/excitatory balance. Cortical inhibitory interneurons (cINs) are born in the ventral forebrain and migrate into cortex, where their numbers are adjusted by programmed cell death. Previously, we showed that loss of clustered gamma protocadherins (Pcdhγ), but not of genes in the alpha or beta clusters, increased dramatically cIN BAX-dependent cell death in mice. Here we show that the sole deletion of the Pcdhγc4 isoform, but not of the other 21 isoforms in the Pcdhγ gene cluster, increased cIN cell death in mice during the normal period of programmed cell death. Viral expression of the Pcdhγc4 isoform rescued transplanted cINs lacking Pcdhγ from cell death. We conclude that Pcdhγ, specifically Pcdhγc4, plays a critical role in regulating the survival of cINs during their normal period of cell death. This demonstrates a novel specificity in the role of Pcdhγ isoforms in cortical development.