Animal development requires coordinated communication between cells. The Connexin family of proteins is a major contributor to intercellular communication in vertebrates by forming gap junction channels that facilitate the movement of ions, small molecules, and metabolites between cells. Additionally, individual hemichannels can provide a conduit to the extracellular space for paracrine and autocrine signaling. Connexin-mediated communication is widely used in epithelial, neural, and vascular development and homeostasis, and most tissues likely use this form of communication. In fact, Connexin disruptions are of major clinical significance contributing to disorders developing from all major germ layers. Despite the fact that Connexins serve as an essential mode of cellular communication, the temporal and cell-type specific expression patterns of connexin genes remain unknown in vertebrates. A major challenge is the large and complex connexin gene family. To overcome this barrier, we determined the expression of all connexins in zebrafish using single-cell RNA-sequencing of entire animals across several stages of organogenesis. Our analysis of expression patterns has revealed that few connexins are broadly expressed, but rather, most are expressed in tissue- or cell-type-specific patterns. Additionally, most tissues possess a unique combinatorial signature of connexin expression with dynamic temporal changes across the organism, tissue, and cell. Our analysis has identified new patterns for well-known connexins and assigned spatial and temporal expression to genes with no-existing information. We provide a field guide relating zebrafish and human connexin genes as a critical step towards understanding how Connexins contribute to cellular communication and development throughout vertebrate organogenesis.

Electrical synapses are neuronal gap junction channels associated with a macromolecular complex called the electrical synapse density (ESD), which regulates development and dynamically modifies electrical transmission. However, the molecular mechanisms of ESD formation are not well understood. Using the Mauthner cell of zebrafish as a model, we previously found that the intracellular scaffolding protein ZO1 is a member of the ESD, localizing postsynaptically, where it is required for channel localization, electrical communication, neural network function, and behavior (Lasseigne et al., 2021). Here, we show that the complexity of the ESD is further diversified by the genomic structure of the ZO1 gene locus. The ZO1 gene is alternatively initiated at three transcriptional start sites resulting in isoforms with unique N-termini that we call ZO1b-Alpha, -Beta, and -Gamma. We demonstrate that ZO1b-Beta is localized to electrical synapses where it is necessary and sufficient for robust channel localization. By contrast, ZO1b-Gamma is localized to synapses yet plays a minor role in electrical synapse formation. This study expands the notion of molecular complexity at the ESD, revealing that an individual genomic locus can contribute multiple independent isoforms that differentially contributing to electrical synaptogenesis. We propose that ESD complexity, at the level of the proteome, but also in the diversity of individual protein isoforms, will have critical impacts on the structure, function, and plasticity of electrical transmission.

Animal development requires coordinated communication between cells. The Connexin family of proteins is a major contributor to intercellular communication in vertebrates by forming gap junction channels that facilitate the movement of ions, small molecules, and metabolites between cells. Additionally, individual hemichannels can provide a conduit to the extracellular space for paracrine and autocrine signaling. Connexin-mediated communication is well appreciated in epithelial, neural, and vascular development and homeostasis, and most tissues likely use this form of communication. In fact, Connexin disruptions are of major clinical significance contributing to disorders developing from all major germ layers. Despite the fact that Connexins serve as an essential mode of cellular communication, the temporal and cell-type specific expression patterns of connexin genes remain unknown in vertebrates. A major challenge is the large and complex connexin gene family. To overcome this barrier, we probed the expression of all connexins in zebrafish using single-cell RNA-sequencing of entire animals across several stages of organogenesis. Our analysis of expression patterns has revealed that few connexins are broadly expressed, but rather, most are expressed in tissue- or cell-type-specific patterns. Additionally, most tissues possess a unique combinatorial signature of connexin expression with dynamic temporal changes across the organism, tissue, and cell. Our analysis has identified new patterns for well-known connexins and assigned spatial and temporal expression to genes with no-existing information. We provide a field guide relating zebrafish and human connexin genes as a critical step towards understanding how Connexins contribute to cellular communication and development throughout vertebrate organogenesis.