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The cochlear sensory epithelium derives from Wnt responsive cells in the dorsomedial otic cup.

ABSTRACT: Wnt1 and Wnt3a secreted from the dorsal neural tube were previously shown to regulate a gene expression program in the dorsal otic vesicle that is necessary for vestibular morphogenesis (Riccomagno et al., 2005. Genes Dev. 19, 1612-1623). Unexpectedly, Wnt1(-/-); Wnt3a(-/-) embryos also displayed a pronounced defect in the outgrowth of the ventrally derived cochlear duct. To determine how Wnt signaling in the dorsal otocyst contributes to cochlear development we performed a series of genetic fate mapping experiments using two independent Wnt responsive driver strains (TopCreER and Gbx2(CreER)) that when crossed to inducible responder lines (Rosa(lacZ) or Rosa(zsGreen)) permanently labeled dorsomedial otic progenitors and their derivatives. Tamoxifen time course experiments revealed that most vestibular structures showed some degree of labeling when recombination was induced between E7.75 and E12.5, consistent with continuous Wnt signaling activity in this tissue. Remarkably, a population of Wnt responsive cells in the dorsal otocyst was also found to contribute to the sensory epithelium of the cochlear duct, including auditory hair and support cells. Similar results were observed with both TopCreER and Gbx2(CreER) strains. The ventral displacement of Wnt responsive cells followed a spatiotemporal sequence that initiated in the anterior otic cup at, or immediately prior to, the 17-somite stage (E9) and then spread progressively to the posterior pole of the otic vesicle by the 25-somite stage (E9.5). These lineage-tracing experiments identify the earliest known origin of auditory sensory progenitors within a population of Wnt responsive cells in the dorsomedial otic cup.

SUBMITTER: Brown AS

PROVIDER: S-EPMC4339344 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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The cochlear sensory epithelium derives from Wnt responsive cells in the dorsomedial otic cup.

Brown Alexander S AS Rakowiecki Staci M SM Li James Y H JYH Epstein Douglas J DJ

Developmental biology 20150112 1

Wnt1 and Wnt3a secreted from the dorsal neural tube were previously shown to regulate a gene expression program in the dorsal otic vesicle that is necessary for vestibular morphogenesis (Riccomagno et al., 2005. Genes Dev. 19, 1612-1623). Unexpectedly, Wnt1(-/-); Wnt3a(-/-) embryos also displayed a pronounced defect in the outgrowth of the ventrally derived cochlear duct. To determine how Wnt signaling in the dorsal otocyst contributes to cochlear development we performed a series of genetic fat ...[more]

PMID: 25592224

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Project description:Nonmammalian vertebrates regenerate lost sensory hair cells by means of asymmetric division of supporting cells. Inner ear or lateral line supporting cells in birds, amphibians, and fish consequently serve as bona fide stem cells resulting in high regenerative capacity of hair cell-bearing organs. Hair cell regeneration does not happen in the mammalian cochlea, but cells with proliferative capacity can be isolated from the neonatal cochlea. These cells have the ability to form clonal floating colonies, so-called spheres, when cultured in nonadherent conditions. We noticed that the sphere population derived from mouse cochlear sensory epithelium cells was heterogeneous, consisting of morphologically distinct sphere types, hereby classified as solid, transitional, and hollow. Cochlear sensory epithelium-derived stem/progenitor cells initially give rise to small solid spheres, which subsequently transition into hollow spheres, a change that is accompanied by epithelial differentiation of the majority of sphere cells. Only solid spheres, and to a lesser extent, transitional spheres, appeared to harbor self-renewing stem cells, whereas hollow spheres could not be consistently propagated. Solid spheres contained significantly more rapidly cycling Pax-2-expressing presumptive otic progenitor cells than hollow spheres. Islet-1, which becomes upregulated in nascent sensory patches, was also more abundant in solid than in hollow spheres. Likewise, hair cell-like cells, characterized by the expression of multiple hair cell markers, differentiated in significantly higher numbers in cell populations derived from solid spheres. We conclude that cochlear sensory epithelium cell populations initially give rise to small solid spheres that have self-renewing capacity before they subsequently convert into hollow spheres, a process that is accompanied by loss of stemness and reduced ability to spontaneously give rise to hair cell-like cells. Solid spheres might, therefore, represent the most suitable sphere type for cell-based assays or animal model transplantation studies aimed at development of cell replacement therapies.

Project description:The peripheral hearing process taking place in the cochlea mainly depends on two distinct sensory cell types: the mechanosensitive hair cells and the spiral ganglion neurons (SGNs). The first respond to the mechanical stimulation exerted by sound pressure waves on their hair bundles by releasing neurotransmitters and thereby activating the latter. Loss of these sensorineural cells is associated with permanent hearing loss. Stem cell-based approaches aiming at cell replacement or in vitro drug testing to identify potential ototoxic, otoprotective, or regenerative compounds have lately gained attention as putative therapeutic strategies for hearing loss. Nevertheless, they rely on efficient and reliable protocols for the in vitro generation of cochlear sensory cells for their implementation. To this end, we have developed a differentiation protocol based on organoid culture systems, which mimics the most important steps of in vivo otic development, robustly guiding mouse embryonic stem cells (mESCs) toward otic sensory neurons (OSNs). The stepwise differentiation of mESCs toward ectoderm was initiated using a quick aggregation method in presence of Matrigel in serum-free conditions. Non-neural ectoderm was induced via activation of bone morphogenetic protein (BMP) signaling and concomitant inhibition of transforming growth factor beta (TGF?) signaling to prevent mesendoderm induction. Preplacodal and otic placode ectoderm was further induced by inhibition of BMP signaling and addition of fibroblast growth factor 2 (FGF2). Delamination and differentiation of SGNs was initiated by plating of the organoids on a 2D Matrigel-coated substrate. Supplementation with brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) was used for further maturation until 15 days of in vitro differentiation. A large population of neurons with a clear bipolar morphology and functional excitability was derived from these cultures. Immunostaining and gene expression analysis performed at different time points confirmed the transition trough the otic lineage and final expression of the key OSN markers. Moreover, the stem cell-derived OSNs exhibited functional electrophysiological properties of native SGNs. Our established in vitro model of OSNs development can be used for basic developmental studies, for drug screening or for the exploration of their regenerative potential.

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The cochlear sensory epithelium derives from Wnt responsive cells in the dorsomedial otic cup.

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The cochlear sensory epithelium derives from Wnt responsive cells in the dorsomedial otic cup.

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