The role of Eya1 in otic neurogenesis in Xenopus laevis
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Cranial placodes are specialized areas of thickening of the early embryonic ectoderm at the head and give rise to sensory organs and ganglia. In vertebrates, the inner ear is derived from one of these placodes, the otic placode. All cranial placodes arise from a common region of origin, the preplacodal ectoderm (PPE), determined by the expression of Eya1 and Six1 genes. Eya1 and Six1 were previously shown to play a crucial role both for the maintenance of proliferating progenitors and for neuronal differentiation in cranial placodes including the otic placode, but the mechanisms are still obscure. The main aim of this study is to elucidate the role of Eya1 during neurogenesis in the developing inner ear (otic vesicle) of Xenopus laevis with a particular focus on the role of Eya1 for cell proliferation, the formation of progenitors and differentiating neurons as well as for the distribution of cell polarity proteins during otic vesicle development. Since otic neurogenesis in Xenopus has not yet been studied in any detail, the first part of this study uses immunostaining and confocal microscopy to provide a detailed description of otic neurogenesis in Xenopus. It is shown that the otic vesicle of Xenopus 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 27 and 39. Delaminated cells congregate to form the vestibulocochlear ganglion, whose peripheral cells continue to proliferate (incorporate EdU), while central cells differentiate into Islet1/2-immunopositive neurons (stage 29) and send out neurites (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). Since only the expression of Eya1 mRNA but not of Eya1 protein has been previously analysed, the second part of the study then provides a detailed analysis of the subcellular distribution of Eya1 protein during development of the otic vesicle and its distribution in relation to markers of proliferation, progenitors and differentiating neurons using a Xenopus-specific Eya1 antibody, double-immunostaining with other antibodies and confocal microscopy. Eya1 protein localizes to both nuclei and cytoplasm in the otic epithelium, with levels of nuclear Eya1 declining in differentiating (Islet1/2+) ganglion neurons and in the developing sensory areas. The distribution of Eya1 in other cranial placodes throughout embryonic development is also characterized. Finally, in the third part of the study Eya1 and Six1 gain and loss of function experiments demonstrated that Eya1 and Six1 are essential for cell proliferation, progenitor maintenance and neuronal differentiation in the epithelium of the developing otic vesicle. Eya1 is also required to establish a proper apical distribution of cell polarity proteins and of N-cadherin in the otic epithelium. This suggests that Eya1 plays an important role for maintenance of epithelial cells with apicobasal polarity during otic neurogenesis. Further studies are needed to elucidate whether and how this role is linked to Eya1’s function in progenitor proliferation and neuronal differentiation.