A Combinatorial Maf Transcription Factor Code Establishes Synaptic Heterogeneity Critical for Normal Hearing Function

Abstract

Spiral ganglion neuron (SGN) synaptic, functional, and molecular diversity is critical for hearing function. Heterogeneous inner hair cell (IHC)-SGN synapses transduce complex sound information from the ear to the brain. Type I SGNs encode the frequency, timing, and intensity of the sounds we hear. Each Type I SGN creates one to two synapses onto a single IHC. Immunohistochemical analyses have found variations in the subunit composition and volume of glutamate receptor puncta among SGN postsynaptic terminals. Synapses develop during the first postnatal week in mice and acquire their mature heterogeneous properties by one month. Single-cell RNA sequencing has shown that Type I SGNs can be divided into three molecularly distinct subtypes (IA, IB and IC). However, the molecular drivers of SGN synaptic heterogeneity remain elusive. MAF family transcription factors stand out as excellent candidates due to their known roles in synapse development. To define the contributions of MAFB and CMAF to synapse development and heterogeneity, we examined their expression patterns and assessed conditional knock-out strains for changes in synaptic morphology, auditory response, and gene expression. We used 10X single-cell RNA sequencing (scRNAseq) to independently assess cMaf and Mafb RNA expression levels across Type 1 SGN subtypes and to detect changes to synaptic genes in knockout strains. We have found that single-knockouts of CMAF and MAFB have both opposing and additive synaptic and functional phenotypes, with particularly severe deficits in double knockout animals. CMAF and MAFB are expressed in complementary patterns across SGN molecular subtypes and transcriptional analyses confirm that CMAF and MAFB contribute to subtype-specific and overall synaptic gene expression in SGNs. Here, we show that these transcription factors create a combinatorial code across SGN subtypes to establish the synaptic heterogeneity that is critical for normal hearing function.