Elsevier

Current Opinion in Neurobiology

Volume 59, December 2019, Pages 213-220
Current Opinion in Neurobiology

The generation of a protocadherin cell-surface recognition code for neural circuit assembly

https://doi.org/10.1016/j.conb.2019.10.001Get rights and content

Highlights

  • In vertebrates, neural self-avoidance requires the clustered Pcdh proteins.

  • Self-avoidance requires stochastic and combinatorial expression of multiple Pcdh isoforms.

  • Stochastic Pcdhα promoter choice is mediated by DNA loop-extrusion by CTCF/Cohesin.

The assembly of functional neural circuits in vertebrate organisms requires complex mechanisms of self-recognition and self-avoidance. Neurites (axons and dendrites) from the same neuron recognize and avoid self, but engage in synaptic interactions with other neurons. Vertebrate neural self-avoidance requires the expression of distinct repertoires of clustered Protocadherin (Pcdh) cell-surface protein isoforms, which act as cell-surface molecular barcodes that mediate highly specific homophilic self-recognition, followed by repulsion. The generation of sufficiently diverse cell-surface barcodes is achieved by the stochastic and combinatorial activation of a subset of clustered Pcdh promoters in individual neurons. This remarkable mechanism leads to the generation of enormous molecular diversity at the cell surface. Here we review recent studies showing that stochastic expression of individual Pcdhα isoforms is accomplished through an extraordinary mechanism involving the activation of ‘antisense strand’ promoter within Pcdhα ‘variable’ exons, antisense transcription of a long non-coding RNA through the upstream ‘sense strand’ promoter, demethylation of this promoter, binding of the CTCF/cohesin complex and DNA looping to a distant enhancer through a mechanism of chromatin ‘extrusion’.

Introduction

The human brain is estimated to be compromised of over 80 billion neurons, each of which may have as many as 1000 neurites (dendrites and axons), which assemble into complex neural circuits required for accurate transmission of signals and effective processing of sensory motor, and cognitive information. This process is dependent, in part, upon the ability of neurites to project into distinct regions of the nervous system during development to form specific and highly complex neural networks [1,2]. Most importantly, this process requires that as many as 1000 neurites of individual neurons remain separated from each other (self-avoidance) in order to maximize the formation of functional synaptic connections. Neural self-avoidance in both vertebrates and invertebrates has been shown to require the expression of unique combinations of cell surface homophilic recognition molecules to generate a molecular recognition code, that is, a single cell surface identity [3,4,5••].

An important and initially counter-intuitive observation that shed light on the molecular mechanism of neural self-recognition was the observation that the homophilic engagement between certain cell-surface proteins displayed on opposing membranes of neurites (self-avoidance), results in repulsion, rather than adhesion [3]. In an extraordinary example of convergent evolution, the same cell-surface mechanism involving specific homophilic interactions followed by repulsion is used in both Drosophila and vertebrates. However, in Drosophila, the diversity code required for self-avoidance is mediated by an extraordinary example of stochastic alternative splicing of the Down syndrome cell adhesion molecule, Dscam1, pre-mRNA, a process that can generate up to 18 ,000 distinct extracellular protein isoforms [3]. By contrast, single cell-surface diversity in mammals is generated by stochastic promoter choice in the Protocadherin gene cluster (see Ref. [5••] for recent review). Each of the two alleles of the Pcdh gene cluster generates distinct sets of Pcdh isoforms, which are displayed on the surface of neurons [5••]. Thus, in both flies and vertebrates, self-avoidance is provided by stochastic expression of multiple protein isoforms. However, this is accomplished by distinct mechanisms: stochastic alternative splicing of Dscam1 pre-mRNA in flies, and stochastic promoter choice of Pcdh genes in vertebrates [4]. Here, we focus on recent progress in understanding the detailed molecular mechanisms involved in the generation of Pcdh cell surface diversity in individual mammalian neurons.

Section snippets

The genomic organization of the Pcdh gene cluster

The generation of a Pcdh cell surface recognition code is a consequence of the genomic organization of the Pcdh gene cluster and a remarkable mechanism of stochastic activation of transcription (promoter choice). Pcdh genes are organized into three closely linked gene clusters (designated as Pcdh α, β and γ), which together, span nearly 1 million base pairs (bp) of genomic DNA [6••] (Figure 1a). These genes (50 in humans and 60 in mice) are organized into variable and constant exons, and their

A neuron-type specific Pcdh cell-surface identity code

Our understanding of the mechanisms by which a Pcdh cell-surface identity code is generated is based in large measure on recent studies performed in Olfactory Sensory Neurons (OSNs) in mice. Mature OSNs express distinct repertoires of Pcdh isoforms from all three clusters as revealed in single cell RNA sequencing studies (RNAseq) [5••,12••] (Figure 1b). Deletion of all three gene clusters results in a self-avoidance phenotype, whereby terminal axons from a single OSN are unable to recognize

Enhancer-dependent stochastic expression of Pcdhα isoforms

The expression of Pcdhα alternate exons requires long-range DNA looping between individual Pcdhα alternate promoters and a transcriptional enhancer, called HS5-1 (hypersensitivity site 5-1), which is located downstream of the Pcdhα variable and c-type exons [19,20] (Figure 3a). Importantly, deletion of the HS5-1 enhancer sequence results in a decrease in expression of Pcdhα alternate exons but not Pcdhαc2 [19,20]. Thus, the expression of Pcdhαc2 does not require the HS5-1 enhancer (Figure 3a).

Concluding remarks

Studies of the Pcdh gene cluster has provided deep insights into general principles of eukaryotic gene expression mechanisms, from genomic organization, chromatin regulation, and enhancer/promoter choice to transcription. However, many mechanistic questions regarding Pcdh expression regulation still remain to be answered. For example, how is the expression of Pcdhαc2 selectively activated in serotonergic neurons, while the alternate exon’s promoters silenced? Upon stochastic activation of an

Conflict of interest statement

Nothing declared.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgement

This work was supported by the N.I.H. grants 4R00GM121815 (D.C.), and 1R01MH108579 and 5R01NS088476 (T.M).

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