4.1Ba is necessary for glutamatergic synapse formation in the sensorimotor circuit of developing zebrafish.

Javier Fierro,Philip Washbourne,Dylan R Haynes

doi:10.1371/journal.pone.0205255

Abstract

During the process of synapse formation, thousands of proteins assemble at prospective sites of cell-cell communication. Although many of these proteins have been identified, the roles they play in generating functional connections during development remain unknown. 4.1 scaffolding proteins have been implicated in synapse formation and maturation in vitro, but in vivo studies for some family members have suggested these proteins are not important for this role. We examined the role of family member 4.1B because it has been implicated in glutamatergic synaptogenesis, but has not been described in vivo. We identified two 4.1B genes in zebrafish, 4.1Ba and 4.1Bb, by sequence comparisons and synteny analysis. In situ hybridization shows these genes are differentially expressed, with 4.1Ba expressed primarily in the nervous system and 4.1Bb expressed in the nervous system and muscle, but not the spinal cord. We focused our studies on 4.1Ba in the spinal cord. 4.1Ba knockdown reduced the number of glutamatergic synapses at caudal primary motor neurons and caused an increase in the duration of touch-evoked coiling. These results suggest 4.1Ba is important for the formation of functional glutamatergic synapses in the developing zebrafish spinal cord.

Highlights

The central nervous system (CNS) is composed of trillions of synapses, important cellular structures that together coordinate the multitude of functions of the brain
Given the sequence similarity between 4.1 family members, we performed an analysis of conserved synteny to ensure the identified genes are co-orthologs of mammalian 4.1B, i.e. duplicates of the single mammalian ortholog that arose from a genome duplication event [21]
As 4.1Ba is expressed throughout the spinal cord, i.e. in other neurons synapsing onto motor neurons, we examined whether the phenotype associated with 4.1Ba knockdown is due to a cell autonomous or non-cell autonomous effect

Summary

Introduction

The central nervous system (CNS) is composed of trillions of synapses, important cellular structures that together coordinate the multitude of functions of the brain. The circuit underlying touch-evoked coiling is well characterized and is reliant on glutamatergic synapses [20] that can be individually identified [18] These characteristics make the developing spinal cord of zebrafish an ideal model for studying the influence of 4.1B on glutamatergic synapses and their function. Knockdown studies of 4.1Ba showed a reduction in the number of glutamatergic synapses at motor neurons, and an overall increase in the duration of touch-evoked coiling at 26 hpf These results suggest 4.1Ba is important for the formation and maturation of glutamatergic synapses in the developing zebrafish spinal cord

Results

Discussion

Materials and methods