International Journal of Molecular Sciences Review Development in the Mammalian Auditory System Depends on Transcription Factors Karen L. Elliott 1, Gabriela Pavlínková 2 , Victor V. Chizhikov 3, Ebenezer N. Yamoah 4 and Bernd Fritzsch 1,* 1 Department of Biology, University of Iowa, Iowa City, IA 52242, USA; [email protected] 2 Institute of Biotechnology of the Czech Academy of Sciences, 25250 Vestec, Czechia; [email protected] 3 Department of Anatomy and Neurobiology, The University of Tennessee Health Science Center, Memphis, TN 38163, USA; [email protected] 4 Department of Physiology and Cell Biology, School of Medicine, University of Nevada, Reno, NV 89557, USA; [email protected] * Correspondence: [email protected] Abstract: We review the molecular basis of several transcription factors (Eya1, Sox2), including the three related genes coding basic helix–loop–helix (bHLH; see abbreviations) proteins (Neurog1, Neurod1, Atoh1) during the development of spiral ganglia, cochlear nuclei, and cochlear hair cells. Neuronal development requires Neurog1, followed by its downstream target Neurod1, to cross- regulate Atoh1 expression. In contrast, hair cells and cochlear nuclei critically depend on Atoh1 and require Neurod1 expression for interactions with Atoh1. Upregulation of Atoh1 following Neurod1 loss changes some vestibular neurons’ fate into “hair cells”, highlighting the significant interplay between the bHLH genes. Further work showed that replacing Atoh1 by Neurog1 rescues some hair cells from complete absence observed in Atoh1 null mutants, suggesting that bHLH genes can partially replace Citation: Elliott, K.L.; Pavlínková, G.; one another. The inhibition of Atoh1 by Neurod1 is essential for proper neuronal cell fate, and in Chizhikov, V.V.; Yamoah, E.N.; the absence of Neurod1, Atoh1 is upregulated, resulting in the formation of “intraganglionic” HCs. Fritzsch, B. Development in the Additional genes, such as Eya1/Six1, Sox2, Pax2, Gata3, Fgfr2b, Foxg1, and Lmx1a/b, play a role in the Mammalian Auditory System Depends on Transcription Factors. auditory system. Finally, both Lmx1a and Lmx1b genes are essential for the cochlear organ of Corti, Int. J. Mol. Sci. 2021, 22, 4189. spiral ganglion neuron, and cochlear nuclei formation. We integrate the mammalian auditory system https://doi.org/10.3390/ development to provide comprehensive insights beyond the limited perception driven by singular ijms22084189 investigations of cochlear neurons, cochlear hair cells, and cochlear nuclei. A detailed analysis of gene expression is needed to understand better how upstream regulators facilitate gene interactions Academic Editor: Toshio Ohshima and mammalian auditory system development. Received: 13 February 2021 Keywords: transcription factors; neuronal differentiation; bHLH genes; spiral ganglion neurons; Accepted: 16 April 2021 cochlea hair cells; cochlear nuclei Published: 18 April 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- The mammalian auditory system evolved in tetrapods out of the vestibular system [1,2] iations. that diversifies into distinct hair cells with a unique innervation of spiral ganglion neurons (SGNs) which reach the cochlear nuclei for information processing [3,4]. The development of the auditory system depends on sequential activation of various genes. Once early development is completed, age-dependent loss progresses to reduce auditory hair cells Copyright: © 2021 by the authors. that cannot be replaced [5,6]. Loss of hair cells may result in a delayed loss of SGNs with Licensee MDPI, Basel, Switzerland. age [7,8], which will affect the cochlear nuclei [9]. We aim to provide an overview of This article is an open access article transcription factors to allow a framework to develop the auditory system’s earliest steps, distributed under the terms and from neurons to hair cells and cochlear nuclei. conditions of the Creative Commons The mammalian auditory system consists of SGNs, two types of cochlear hair cells Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ (inner and outer hair cells; IHC, OHC), and the cochlear nuclei subdivided into three 4.0/). nuclei: the anteroventral, posteroventral, and dorsal cochlear nucleus. Insights into these Int. J. Mol. Sci. 2021, 22, 4189. https://doi.org/10.3390/ijms22084189 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 21 The mammalian auditory system consists of SGNs, two types of cochlear hair cells Int. J. Mol. Sci. 2021, 22, 4189 2 of 21 (inner and outer hair cells; IHC, OHC), and the cochlear nuclei subdivided into three nu- clei: the anteroventral, posteroventral, and dorsal cochlear nucleus. Insights into these in- terlinked neurosensory components will be detailed beyond the initial morphogenetic interlinkedearly steps neurosensory[9,10]. The proliferation components pattern will be within detailed each beyond auditory the system initial morphogenetic component af- earlyfects the steps temporal [9,10]. The and proliferation spatial differentiation pattern within and interconnects each auditory with system various component neurosensory affects thecells. temporal SGNs are and the spatial first cells differentiation of the auditory and interconnectssystem to exit with the cell various cycle, neurosensory progressing cells.from SGNsbase-to are-apex the from first ~E10.5 cells of–12.5, the auditoryfollowed systemby cochlear to exit HCs, the which cell cycle, exit the progressing cell cycle oppo- from base-to-apexsite direction, from from ~E10.5–12.5, apex-to-base followed between by ~E12.5 cochlear–14.5 HCs, [11, which12]. The exit temporal the cell cyclepattern opposite of pro- direction,liferation fromprogression apex-to-base in the betweencochlea raises ~E12.5–14.5 questions: [11,12 why]. The do temporal developing pattern neurons of prolifer- prolif- ationerate progressionfrom the apex in- theto-base cochlea, and raises the cochlear questions: HCs why proliferate do developing from base neurons-to-apex proliferate (Figure from1)? The the cochlear apex-to-base, nuclei and exit the the cochlear cell cycle HCs between proliferate E10.5 from–14.5 base-to-apex [13], partially (Figure overlapping1)? The cochlearwith spiral nuclei ganglion exit the neurons. cell cycle G betweenranular cells E10.5–14.5 exit the [13 cell], partially cycle between overlapping E12-18, with which spiral is gangliondelayed relativ neurons.e to Granularspiral ganglion cells exitproliferation the cell cycle progression between [14] E12-18,. We want which to is follow delayed the relativeformation to spiralof SGN ganglions, hair cells, proliferation and cochlear progression nuclei and [14 ].relate We wantthem to to follow the three the interlinked formation ofconnections. SGNs, hair cells, and cochlear nuclei and relate them to the three interlinked connections. SpiralSpiral ganglionganglion neurons: neurons: SGNs SGNs depend depend upon uponEya1 Eya1and andSox2, Sox2,followed followed by Neurog1 by Neurog[15]1 and[15] Neurod1and Neurod1[16]. [Neurog116]. Neurog1was shownwas shown to be to regulated be regulated by Neurod1 by Neurod1[17]. [ In17] contrast. In contrast to the to completethe complete loss loss of neurons of neurons in Neurog1 in Neurog1null micenull mice [15], [ a15] few, a SGNsfew SGNs remain remain following following the loss the oflossNeurod1 of Neurod1[16,18 [16,]. A18] degree. A degree of interaction of interaction and crosstalkand crosstalk between betweenNeurog1, Neurog1, Neurod1 Neurod1, and, Atoh1and Atoh1and variousand various additional additional transcription transcription factors factors remain remain incompletely incompletely understood understood and willand bewill discussed be discussed in this in review.this review. Figure 1. A composite set of development of the auditory system is revealed—Base-to-apex pro- Figure 1. A composite set of development of the auditory system is revealed—Base-to-apex progres- gression of α9-AChr expression in differentiating hair cells along the cochlea (A,B). The two differ- α sionent types of 9-AChr of spiral expression ganglion neurons in differentiating connect the hair cochlear cells along hair thecells cochlea with the (A cochlear,B). The nuclei two different (ante- typesroventral of spiral (AVCN ganglion), posteroventral neurons connect (PVCN the), cochlear dorsal (DCN hair cells)), maintaining with the cochlear basal to nuclei apical (anteroventral tonotopic (AVCN),representation posteroventral (C). The PVCN, (PVCN), located dorsal in (DCN)),rhombomere maintaining 4, expresses basal Hoxb1. to apical An tonotopicapex-to-base represen- prolif- tationeration (C (E12.5). The–14.5) PVCN, for located cochlear in hair rhombomere cells is followed 4, expresses by a base Hoxb1.-to-apex An differenti apex-to-baseation proliferation initiated by (E12.5–14.5)Atoh1 (E13.5for–P0). cochlear The organ hair of cells Corti is followed contains bytwo a different base-to-apex types differentiation of hair cells, inner initiated and byouter Atoh1 hair (E13.5–P0).cells (IHC, OHC) The organ arranged of Corti from contains high-frequency two different detection types at of the hair base cells, (thick inner basilar and outermembrane) hair cells to (IHC,low-frequency OHC) arranged detection from at the high-frequency apex (thinner detection basilar membrane). at the base Approximately (thick basilar membrane) 15 type I fibers to low- converge on a single IHC, whereas a single