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Semaphorin6A (Sema6A) is a repulsive guidance molecule that plays many roles in central nervous system, heart and bone development, as well as immune system responses and cell signaling in cancer. Loss of Sema6A or its receptor PlexinA2 in zebrafish leads to smaller eyes and improper retinal patterning. Here, we investigate a potential role for the Sema6A intracellular domain in zebrafish eye development and dissect which phenotypes rely on forward signaling and which rely on reverse signaling. We performed rescue experiments on zebrafish Sema6A morphants with either full-length Sema6A (Sema6A-FL) or Sema6A lacking its intracellular domain (Sema6A-ΔC). We identified that the intracellular domain is not required for eye size and retinal patterning, however it is required for retinal integrity, the number and end feet strength of Müller glia and protecting against retinal cell death. This novel function for the intracellular domain suggests a role for Sema6A reverse signaling in zebrafish eye development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Electrical synapses are neuronal gap junction (GJ) channels associated with a macromolecular complex called the electrical synapse density (ESD), which regulates development and dynamically modifies electrical transmission. However, the proteomic makeup and molecular mechanisms utilized by the ESD that direct electrical synapse formation are not well understood. Using the Mauthner cell of zebrafish as a model, we previously found that the intracellular scaffolding protein ZO1b is a member of the ESD, localizing postsynaptically, where it is required for GJ channel localization, electrical communication, neural network function, and behavior. Here, we show that the complexity of the ESD is further diversified by the genomic structure of the ZO1b gene locus. The ZO1b 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 and ZO1b-Gamma are broadly expressed throughout the nervous system and localize to electrical synapses. By contrast, ZO1b-Alpha is expressed mainly non-neuronally and is not found at synapses. We generate mutants in all individual isoforms, as well as double mutant combinations in cis on individual chromosomes, and find that ZO1b-Beta is necessary and sufficient for robust GJ channel localization. ZO1b-Gamma, despite its localization to the synapse, plays an auxiliary role in channel localization. This study expands the notion of molecular complexity at the ESD, revealing that an individual genomic locus can contribute distinct isoforms to the macromolecular complex at electrical synapses. Further, independent scaffold isoforms have differential contributions to developmental assembly of the interneuronal GJ channels. We propose that ESD molecular complexity arises both from the diversity of unique genes and from distinct isoforms encoded by single genes. Overall, ESD proteomic diversity is expected to have critical impacts on the development, structure, function, and plasticity of electrical transmission., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Michel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

To investigate electrical synapse formation in vivo we used forward genetics to disrupt genes affecting Mauthner cell electrical synapses in larval zebrafish. We identify the disconnect2 ( dis2 ) mutation for its failure to localize neural gap junction channels at electrical synapses. We mapped this mutation to chromosome 25 and identified a splice-altering mutation in the tjp1b gene. We demonstrated that the dis2 mutation disrupts tjp1b function using complementation analysis with CRISPR generated mutants. We conclude that the dis2 mutation disrupts the tjp1b gene that is required for electrical synapse formation., (Copyright: © 2022 by the authors.)

Electrical synaptic transmission relies on neuronal gap junctions containing channels constructed by Connexins. While at chemical synapses neurotransmitter-gated ion channels are critically supported by scaffolding proteins, it is unknown if channels at electrical synapses require similar scaffold support. Here, we investigated the functional relationship between neuronal Connexins and Zonula Occludens 1 (ZO1), an intracellular scaffolding protein localized to electrical synapses. Using model electrical synapses in zebrafish Mauthner cells, we demonstrated that ZO1 is required for robust synaptic Connexin localization, but Connexins are dispensable for ZO1 localization. Disrupting this hierarchical ZO1/Connexin relationship abolishes electrical transmission and disrupts Mauthner cell-initiated escape responses. We found that ZO1 is asymmetrically localized exclusively postsynaptically at neuronal contacts where it functions to assemble intercellular channels. Thus, forming functional neuronal gap junctions requires a postsynaptic scaffolding protein. The critical function of a scaffolding molecule reveals an unanticipated complexity of molecular and functional organization at electrical synapses., Competing Interests: AL, FE, SI, JM, EM, AM, ET, KM, AP, AM No competing interests declared, (© 2021, Lasseigne et al.)

In this review article, we will describe the recent advances made towards understanding the molecular and cell biological mechanisms of electrical synapse formation. New evidence indicates that electrical synapses, which are gap junctions between neurons, can have complex molecular compositions including protein asymmetries across joined cells, diverse morphological arrangements, and overlooked similarities with other junctions, all of which indicate new potential roles in neurodevelopmental disease. Aquatic organisms, and in particular the vertebrate zebrafish, have proven to be excellent models for elucidating the molecular mechanisms of electrical synapse formation. Zebrafish will serve as our main exemplar throughout this review and will be compared with other model organisms. We highlight the known cell biological processes that build neuronal gap junctions and compare these with the assemblies of adherens junctions, tight junctions, non-neuronal gap junctions, and chemical synapses to explore the unknown frontiers remaining in our understanding of the critical and ubiquitous electrical synapse., (Copyright © 2020 Martin, Lasseigne and Miller.)