Abstract
Using immunostaining and confocal microscopy, we here provide the first detailed description of otic neurogenesis in Xenopus laevis. We show that the otic vesicle comprises a pseudostratified epithelium with apicobasal polarity (apical enrichment of Par3, aPKC, phosphorylated Myosin light chain, N-cadherin) and interkinetic nuclear migration (apical localization of mitotic, pH3-positive cells). A Sox3-immunopositive neurosensory area in the ventromedial otic vesicle gives rise to neuroblasts, which delaminate through breaches in the basal lamina between stages 26/27 and 39. Delaminated cells congregate to form the vestibulocochlear ganglion, whose peripheral cells continue to proliferate (as judged by EdU incorporation), while central cells differentiate into Islet1/2-immunopositive neurons from stage 29 on and send out neurites at stage 31. The central part of the neurosensory area retains Sox3 but stops proliferating from stage 33, forming the first sensory areas (utricular/saccular maculae). The phosphatase and transcriptional coactivator Eya1 has previously been shown to play a central role for otic neurogenesis but the underlying mechanism is poorly understood. Using an antibody specifically raised against Xenopus Eya1, we characterize the subcellular localization of Eya1 proteins, their levels of expression as well as their distribution in relation to progenitor and neuronal differentiation markers during otic neurogenesis. We show that Eya1 protein localizes to both nuclei and cytoplasm in the otic epithelium, with levels of nuclear Eya1 declining in differentiating (Islet1/2+) vestibulocochlear ganglion neurons and in the developing sensory areas. Morpholino-based knockdown of Eya1 leads to reduction of proliferating, Sox3- and Islet1/2-immunopositive cells, redistribution of cell polarity proteins and loss of N-cadherin suggesting that Eya1 is required for maintenance of epithelial cells with apicobasal polarity, progenitor proliferation and neuronal differentiation during otic neurogenesis.
Highlights
Time Course of Neuronal Delamination and Differentiation In Xenopus, the otic vesicle begins to invaginate from the posterior placodal area at stage 22/23 and has completely separated from the surface ectoderm by stage 28 (Schlosser and Northcutt, 2000; Schlosser and Ahrens, 2004)
Whereas a previous study has reported that neurons of the vestibulocochlear ganglion first differentiate at stage 31 (Quick and Serrano, 2005), a detailed schedule of neuronal delamination and differentiation has not yet been described
Our study provides the first detailed description of otic neurogenesis in Xenopus laevis
Summary
The otic placode of vertebrates contains a neurosensory domain with neurosensory progenitor pools that give rise to the sensory hair cells of the inner ear responding to vestibular and auditory stimuli as well as to the sensory neurons of the vestibulocochlear ganglion (reviewed in Alsina et al, 2009; Wu and Kelley, 2012; Maier et al, 2014; Alsina, 2020; Elliott et al, 2021; Schlosser, 2021).Otic Neurogenesis in XenopusBecause of its central importance for the vertebrate senses of balance and hearing, the generation of sensory hair cells and sensory neurons from the otic vesicle has been described in several vertebrate model organisms, viz. mouse, chick and zebrafish but not in Xenopus (Carney and Silver, 1983; Hemond and Morest, 1991; Haddon and Lewis, 1996; Alsina et al, 2004; Raft et al, 2004, 2007; Neves et al, 2007). Whereas Sox2/3 and Neurog or the related protein Neurog play important roles for neurogenesis in the inner ear and in the central nervous system (Schmidt et al, 2013; Schlosser, 2021), other transcriptional regulators such as the transcription factor Six and its coactivator Eya are required for the generation of sensory and neuronal cells from the otic vesicle and other placode-derived structures (Xu et al, 1999; Laclef et al, 2003; Zheng et al, 2003; Zou et al, 2004; Bricaud and Collazo, 2006; Schlosser et al, 2008; Ahmed et al, 2012a,b). Eya protein binds directly to other proteins and serves as a phosphatase in either the nucleus or the cytoplasm, but these functions are still poorly characterized (Fan et al, 2000; Li et al, 2003; Embry et al, 2004; Cook et al, 2009; Krishnan et al, 2009; Xiong et al, 2009; Ahmed et al, 2012a,b; Merk et al, 2020)
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