Comparative Analysis of Gene Regulatory Network Components in the Auditory Hindbrain of Mice and Chicken

Abstract

The neurons in the mammalian and avian auditory hindbrain nuclei share a number of significant morphological and physiological properties for fast, secure and precise neurotransmission, such as giant synapses, voltage-gated K⁺ channels and fast AMPA receptors. Based on the independent evolution of the middle ear in these two vertebrate lineages, on different embryonic origins of the nuclei and on marked differences on the circuit level, these similarities are assumed to reflect convergent evolution. Independent acquisition of similar phenotypes can be produced by divergent evolution of genetic mechanisms or by similar molecular mechanisms. The distinction between these two possibilities requires knowledge of the gene regulatory networks (GRNs) that orchestrate the development of auditory hindbrain structures. We therefore compared the expression pattern of GRN components, both transcription factors (TFs) and noncoding RNA, during terminal differentiation of the auditory hindbrain structures in mouse and chicken when neurons acquire their final morphological and electrophysiological properties. In general, we observed broad expression of these genes in the mouse auditory cochlear nucleus complex and the superior olivary complex at both postnatal day 4 (P4) and at P25, and for the chicken at the equivalent developmental stages, i.e. embryonic day 13 (E13) and at P14-P17. Our data are in agreement with a model based on similar molecular mechanisms underlying terminal differentiation and maintenance of neuronal cell identity in the auditory hindbrain of different vertebrate lineages. This conservation might reflect developmental constraints arising from the tagmatic organization of rhombomeres and the evolutionarily highly conserved GRNs operating in these structures.

Expression pattern of cochlear microRNAs in the mammalian auditory hindbrain

“…Both miRNAs belong to the miRNA cluster miR-183, miR-96 and miR-182, which is transcribed as a polycistronic transcript. Indeed, miR-183 showed a similar expression pattern as miR-96 in the auditory hindbrain (Pawlik et al 2016). A mutation in miR-96 causes reduced cell size, altered electrophysiological properties, changes in gene expression and impaired maturation of a giant synapse in the auditory hindbrain (Schlüter et al Fig.…”

“…Specificity of the probes was indicated by their different expression patterns. Furthermore, more than 40 sense probes in previous studies using the same protocol provided negative results (Ehmann et al 2013;Pawlik et al 2016).…”

Differential expression of microRNAs in the developing avian auditory hindbrain

“…RNA probe generation was conducted similarly to previous studies (Krohs et al, 2020; Pawlik et al, 2016). Sequences corresponding to pre‐miRNAs flanked by additional 25–75 nucleotides were amplified by PCR from chicken genomic DNA with corresponding primer pairs (Table 1).…”

“…Given the importance of GRN components, we have begun to establish comparative expression maps of these factors. Analysis of selected transcription factors showed a highly similar expression pattern in chicken and mice auditory hindbrain nuclei (Pawlik et al, 2016). The highly dynamic nature during development and evolution make microRNAs (miRNAs) another interesting entry point to such comparative studies (Bartel, 2018; Kittelmann & McGregor, 2019; Kosik & Nowakowski, 2018).…”

Differences in molecular mechanisms of K+ clearance in the auditory sensory epithelium of birds and mammals

“…Cross-sections of 20 µm thickness were cut on a cryostat (HM 400, Microm, Waldorf, Germany or CM1950, Leica Biosystems) and sections were stored at −80°C until use. On-slide in situ hybridization was performed at 50-60°C overnight in hybridization buffer [50% formamide, 5× SSC, 2% blocker (Roche), 0.02% SDS, 0.1% N-lauryl sarcosine; Wilms et al, 2016;Pawlik et al, 2016]. Bound probes were detected with an antidigoxigenin antibody conjugated to alkaline phosphatase (Roche).…”