Dendritic arbors of many neurons are patterned by a process called self-avoidance, in which branches arising from a single neuron repel each other1-7. By minimizing gaps and overlaps within the arbor, self-avoidance facilitates complete coverage of a neuron’s territory by its neurites1-3. Remarkably, some neurons that display self-avoidance interact freely with other neurons of the same subtype, implying that they discriminate self from non-self. Here, we demonstrate roles for the clustered protocadherins (Pcdhs) in dendritic self-avoidance and self/non-self discrimination. The Pcdh locus encodes ~60 related cadherin-like transmembrane proteins, at least some of which exhibit isoform-specific homophilic adhesion in heterologous cells and are expressed stochastically and combinatorially in single neurons7-11. Deletion of all 22 Pcdhs in the mouse gamma subcluster (Pcdhgs) disrupts self-avoidance of dendrites in retinal starburst amacrine cells (SACs) and cerebellar Purkinje cells. Further genetic analysis of SACs showed that Pcdhgs act cell-autonomously during development, and that replacement of the 22 Pcdhgs with a single isoform restores self-avoidance. Moreover, expression of the same single isoform in all SACs decreases interactions among dendrites of neighboring SACs (heteroneuronal interactions). These results suggest that homophilic Pcdhg interactions between sibling neurites (isoneuronal interactions) generate a repulsive signal that leads to self-avoidance. In this model, heteroneuronal interactions are normally permitted because dendrites seldom encounter a matched set of Pcdhgs unless they emanate from the same soma. In many respects, our results mirror those reported for Dscam1 in Drosophila: this complex gene encodes thousands of recognition molecules that exhibit stochastic expression and isoform-specific interactions, and mediate both self-avoidance and self/non-self discrimination4-7,12-15. Thus, although insect Dscams and vertebrate Pcdhs share no sequence homology, they appear to underlie similar strategies for endowing neurons with distinct molecular identities and patterning their arbors.

Summary Individual mammalian neurons express distinct repertoires of protocadherin (Pcdh) -α, -β and -γ proteins that function in neural circuit assembly. Here we show that all three types of Pcdhs can engage in specific homophilic interactions, that cell surface delivery of alternate Pcdhα isoforms requires cis interactions with other Pcdh isoforms, and that the extracellular cadherin domain EC6 plays a critical role in this process. Analysis of specific combinations of up to five Pcdh isoforms showed that Pcdh homophilic recognition specificities strictly depend on the identity of all of the expressed isoforms, such that mismatched isoforms interfere with cell-cell interactions. We present a theoretical analysis showing that the assembly of Pcdh-α, –β and –γ isoforms into multimeric recognition units, and the observed tolerance for mismatched isoforms can generate the cell surface diversity necessary for single-cell identity. However, competing demands of non-self discrimination and self-recognition place limitations on the mechanisms by which recognition units can function.

The vertebrate clustered protocadherin (Pcdh) cell surface proteins are encoded by three closely linked gene clusters (Pcdhα, Pcdhβ, and Pcdhγ). Here, we show that all three gene clusters functionally cooperate to provide individual mouse olfactory sensory neurons (OSNs) with the cell surface diversity required for their assembly into distinct glomeruli in the olfactory bulb. Although deletion of individual Pcdh clusters had subtle phenotypic consequences, the loss of all three clusters (tricluster deletion) led to a severe axonal arborization defect and loss of self-avoidance. By contrast, when endogenous Pcdh diversity is overridden by the expression of a single–tricluster gene repertoire (α and β and γ), OSN axons fail to converge to form glomeruli, likely owing to contact-mediated repulsion between axons expressing identical combinations of Pcdh isoforms.

The majority of vertebrate protocadherin (Pcdh) genes are clustered in a single genomic locus, and this remarkable genomic organization is highly conserved from teleosts to humans. These clustered Pcdhs are differentially expressed in individual neurons, they engage in homophilic trans-interactions as multimers and they are required for diverse neurodevelopmental processes, including neurite self-avoidance. Here, we provide a concise overview of the molecular and cellular biology of clustered Pcdhs, highlighting how they generate single cell diversity in the vertebrate nervous system and how such diversity may be used in neural circuit assembly.Key words: CTCF, Cohesin, Promoter choice, Single cell diversity, Homophilic interaction, Self-avoidance Introduction Protocadherins (Pcdhs), which are predominantly expressed in the nervous system, constitute the largest subfamily of the cadherin superfamily of cell-adhesion molecules. The founding members of the Pcdh gene family were discovered by Suzuki and co-workers in an effort to isolate novel cadherin repeat-containing genes (Sano et al., 1993). Distinct Pcdh members were subsequently cloned, and these included a set of eight cDNAs encoding cadherin-related neuronal receptors (CNRs) (Kohmura et al., 1998). A striking characteristic of the corresponding CNR mRNAs is that their 5Ј ends are distinct, whereas their 3Ј ends are all identical, suggesting that they are generated by alternative pre-mRNA splicing. Characterization of the human genomic DNA encoding the CNR mRNAs revealed that they are encoded in a large Pcdh gene cluster designated α (Pcdha), which is located immediately upstream of two additional Pcdh gene clusters designated β (Pcdhb) and γ (Pcdhg) (Wu and Maniatis, 1999). Remarkably, the genomic organization of the Pcdh gene clusters resembles that of the immunoglobulin and T-cell receptor genes, both of which generate enormous diversity in the immune system through a mechanism that Development 140, 3297-3302 (2013) DEVELOPMENT 3298 involves somatic cell DNA rearrangement. Subsequent studies confirmed the possibility that the clustered Pcdhs serve as a source of molecular diversity in the nervous system, albeit through a different mechanism. The enormous cell surface diversity resulting from the combinatorial expression of Pcdh isoforms, together with the extraordinary specificity afforded by their homophilic interactions, have led to the speculation that clustered Pcdhs are functional counterparts of the Drosophila Dscam1 proteins, which play a central role in neural circuit assembly in the invertebrate nervous system (Zipursky and Sanes, 2010). Indeed, recent studies have demonstrated that the Pcdhg gene cluster is required for neurite self-avoidance in the mouse, in a manner similar to that of the Dscam1 gene in the fly (Lefebvre et al., 2012). Thus, it appears that clustered Pcdhs may function as molecular barcodes for selfrecognition by individual neurons in the vertebrate nervous system. Here, we briefly review studies that led to this hypothesis...

Serotonergic neurons project their axons pervasively throughout the brain and innervate various target fields in a space-filling manner, leading to tiled arrangements of their axon terminals to allow optimal allocation of serotonin among target neurons. Here we show that conditional deletion of the mouse protocadherin α (Pcdhα) gene cluster in serotonergic neurons disrupts local axonal tiling and global assembly of serotonergic circuitries and results in depression-like behaviors. Genetic dissection and expression profiling revealed that this role is specifically mediated by Pcdhαc2, which is the only Pcdhα isoform expressed in serotonergic neurons. We conclude that, in contrast to neurite self-avoidance, which requires single-cell identity mediated by Pcdh diversity, a single cell-type identity mediated by the common C-type Pcdh isoform is required for axonal tiling and assembly of serotonergic circuitries.