Gene expression profiling of Six1-null developing inner ear.
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE193334 | GEO | 2023/04/20
REPOSITORIES: GEO
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Project description:Several transcription factors are known to be expressed in discrete regions of the otic vesicle and Dlx5 is one of those that is expressed highly in the presumptive dorsal vestibular region. Mice lacking Dlx5 have vestibular defects. Specifically, they fail to form the endolymphatic duct (a defect visible as early as E10) as well as the anterior and posterior semi-circular canals. The lateral canal does form but is smaller, whereas the saccule, the utricle and the cochlea appear relatively normal. The goal of this study was to use microarrays to identify differentially expressed genes between wild-type and Dlx5-null otic vesicles microdissected from E10 and 10.5 and identify downstream targets of Dlx5 by searching the immediate 3kb promoter regions of the differentially expressed genes for homeodomain binding sites followed by chromatin immunoprecipitation in an otic vesicle-derived cell line over-expressing Dlx5. Normal vestibular morphogenesis is compromised in mice lacking Dlx5, a member of the Distal-less family of homeobox transcription factors. We identified its direct downstream targets in the developing mouse inner ear by gene expression profiling wild-type and Dlx5 null otic vesicles from embryonic stages E10 and E10.5. Four hundred genes were differentially expressed in mutants when compared to wild-type in at least one of the two stages. To further constrain the list of likely direct targets of Dlx5, we examined the genomic DNA sequences in the 3kb promoter regions immediately proximal to the transcriptional start sites of these genes. We searched for (i) one or more previously described binding site for Dlx5, (ii) one or more novel 12bp-long motifs with a canonical homeodomain response element (HDRE) shared by promoters of two or more genes, and (iii) 100% conservation of the 12bp-long HDRE-containing motifs in promoter regions of human orthologs. Forty genes passed one or more of these filters, 12 of which are known to be expressed in the developing otic vesicle in domains that at least partially overlap with that of Dlx5 in one or both stages that we examined. Chromatin immunoprecipitation using a Dlx5 antibody confirmed direct binding of Dlx5 to promoter regions of seven of these genes (Atbf1, Bmper, Large, Lrrtm1, and Msx1, all of which were down-regulated in mutants, and Ebf1 and Lhx1, both of which were up-regulated in mutants) in an otic vesicle-derived cell line over-expressing Dlx5. Gene expression profiling of this cell line showed that Bmper and Lrrtm1 transcripts were up-regulated, further supporting their identification as direct targets of Dlx5 activity. Otic vesicles from wild-type and Dlx5-null embryos were microdissected from mouse developmental stages E10 and E10.5 and expression profiled in duplicate for each stage and genotype. Dlx5-transfected and empty vector-transfected 2B1 cell line samples were also profiled in duplicate (independent cell cultures).
2011-07-01 | E-GEOD-22381 | biostudies-arrayexpress
Project description:The molecular characterization of early stages of human inner ear development is limited by the difficulty in accessing samples at early gestational stages. Some aspects of inner ear morphogenesis can be recapitulated using pluripotent stem cell directed differentiation in inner ear organoids (IEOs). Once validated and benchmarked, these models could provide a unique tool to complement and refine our understanding of human otic differentiation and could be used to model developmental defects.Here we provide a first characterization of early human embryonic otocyst development and compare the primary tissue to the iPSC-derived inner ear cell types. Multiplex immunostaining and single cell RNA sequencing were used to characterize human iPSC-derived IEOs at 3 key developmental steps, providing a new and unique signature of in vitro derived otic- placode, epithelium, neuroblasts and sensory epithelia. The expression and localization of key markers were further evaluated in human embryos. We show that the otic placode derived in vitro (day 8-12) matches marker expression of Carnegie Stage (CS) 11 embryos, and subsequently (day 20-40) gives rise to otic epithelia and neuroblasts comparable to the CS13 embryonic stage. Differentiation of sensory epithelia, including supporting cells and hair cells, starts in vitro at day 50-60 of culture. The maturity of these cells is equivalent to vestibular sensory epithelia at week 10 or cochlear tissue at week 12 of development, prior to functional onset. Taken together these data indicates that the current state of the art protocol enables the specification of bona fide otic tissue, supporting further application of IEOs to model inner ear biology and disease
2023-08-22 | GSE229148 | GEO
Project description:The inner ear in mammals is derived from a simple ectodermal thickening called the otic placode. Through a series of complex morphological changes, the placode forms the mature inner ear comprising of the auditory organ (cochlea) and the vestibular/balance organs (utricle, saccule, and three semi-circular canals). The vast majority of genes known to be involved during inner ear development have been found through mutational screens or by chance. To identify genes that can serve as novel candidates required for inner ear development, and also candidate genes for uncloned human deafnesses, inner ear tissues from mouse embryos from E9 to E15 were microdissected and expression-profiled at half-day intervals. Also profiled was the non-inner ear mesenchymal tissue surrounding the inner ear tissue. Various patterns of gene expression were identified, and significant biological pathways that these genes represented were identified. Also identified were mouse genes whose human orthologs are located within uncloned non-syndromic deafness intervals, thus serving as candidates for sequence analysis. Experiment Overall Design: Inner ear tissues from E9 to E15 were microdissected at half-day intervals. E9 is the earliest stage when the otic placode is clearly visible and able to be microdissected cleanly. E15 is the stage when all the organs of the inner ear have become established, as have the sensory hair and non-sensory support cells within those organs. For each of the stages from E9 to E10, whole inner ears were profiled. For each of the stages from E10.5 to E12, the primordial cochlear and vestibular organs were profiled separately. For each of the stages from E12.5 to E15, the cochlea and the saccule were profiled separately, whereas the utricle and the three ampullae were combined and profiled together. Any given tissue from any given stage was a collection of anywhere between 4 to 17 identical tissues, and was obtained in duplicate (i.e. from different litters). Hence, a total of 58 inner ear samples were obtained. Moreover, non-inner ear tissue found in the immediate vicinity of inner ear tissue was also obtained and profiled. Specifically, all non-inner ear tissue from E9 was profiled in duplicate. Non-inner ear tissue from E9.5 to E10.5 was pooled and profiled together (in duplicate), whereas that from E11 to E15 was pooled and profiled together (also in duplicate). Therefore, a total of 6 non-inner samples were obtained.
2008-06-15 | E-GEOD-7536 | biostudies-arrayexpress
Project description:The morphogenesis of the otic vesicle (OV) to form inner ear organs serves as an excellent model system to understand cell fate acquisition on a single cell level. Tbx2 and Tbx3 (Tbx2/3) encode closely related T-box transcription factors that are expressed widely in the mammalian otic vesicle (OV). Inactivation of both genes in the Pax2-Cre lineage (Tbx2/3cKO) results in failed morphogenesis of the OV into inner ear organs. To understand the basis of these defects, single cell RNA-sequencing (scRNA-seq) was performed on the OV lineage using Pax2-Cre, in controls versus Tbx2/3cKO embryos, at stage E10.5. We identified a multipotent population termed otic progenitors that are localized to the anterior ventrolateral region of the OV in controls and are marked by expression of Fgf18, Sox3, and Cxcl12. The otic progenitor population was increased three-fold in Tbx2/3cKO embryos, concomitant with dysregulation of genes in these cells as well as reduced progression to more differentiated states of prosensory and nonsensory cells. An ectopic neural population of cells was detected in the posterior OV of Tbx2/3cKO embryos due to increased expression of Neurog1 and NeuroD1 but with reduced maturation. As all three cell fates were affected in Tbx2/3cKO embryos, we suggest that Tbx2/3 promotes progression of multipotent otic progenitors to more differentiated cell types in the OV.
2022-12-12 | GSE185172 | GEO
Project description:The first morphological evidence of the developing ear is a thickened disk of ectoderm known as the otic placode. However, signals for otogenesis are present even before the otic placode is physically apparent. Several inductive signals have been identified through candidate gene approaches, but there are still many gaps in the signaling cascade of otogenesis. Presently the candidate gene approach has largely exhausted known candidates. This project compares the pre-otic domain with a control region that is competent, but not specified to form otic placode. The purpose of this work is to identify genes that are differentially expressed in the pre-otic domain in order to generate a list of novel candidate genes for otic placode induction.
2010-06-21 | GSE16918 | GEO
Project description:Otic ectoderm gives rise to almost all cell types of the inner ear; however, the mechanisms that link transcription factors, chromatin, lineage commitment and differentiation capacity are largely unknown. Here we show that Brg1 chromatin-remodeling factor is required for specifying neurosensory lineage in the otocyst and for inducing hair and supporting cell fates in the cochlear sensory epithelium. Brg1 interacts with the critical neurosensory-specific transcription factors Eya1/Six1, both of which simultaneously interact with BAF60a or BAF60c. Chromatin immunoprecipitation-sequencing (ChIP-seq) and ChIP assays demonstrate Brg1 association with discrete regulatory elements at the Eya1 and Six1 loci. Brg1-deficiency leads to markedly decreased Brg1 binding at these elements and loss of Eya1 and Six1 expression. Furthermore, ChIP-seq reveals Brg1-bound promoter-proximal and distal regions near genes essential for inner ear morphogenesis and cochlear sensory epithelium development. These findings uncover essential functions for chromatin-remodeling in the activation of neurosensory fates during inner ear development.
2020-04-17 | GSE119545 | GEO
Project description:Mutations in the chromatin remodeling enzyme CHD7 cause CHARGE syndrome, which affects multiple organs including the inner ear. We investigated how CHD7 mutations affect otic development in human inner ear organoids. We found loss of CHD7 or its chromatin remodeling activity leads to complete absence of hair cells and supporting cells, which can be explained by dysregulation of key otic development-associated genes in mutant otic progenitors. Further analysis of the mutant otic progenitors suggested that CHD7 can regulate otic genes through a chromatin remodeling-independent mechanism. Results from transcriptome profiling of hair cells revealed disruption of deafness gene expression as a potential underlying mechanism of CHARGE-associated sensorineural hearing loss. Notably, co-differentiating CHD7 knockout and wild-type cells in chimeric organoids partially rescued mutant phenotypes by restoring otherwise severely dysregulated otic genes. Taken together, our results suggest that CHD7 plays a critical role in regulating human otic lineage differentiation and deafness gene expression.
2022-07-21 | GSE208585 | GEO
Project description:Inner ear organoids recapitulate development and are intended to generate cell types of the otic lineage for applications such as basic science research and cell replacement strategies. Here, we use single-cell sequencing to study the cellular heterogeneity of late-stage mouse inner ear organoid sensory epithelia, which we validated by comparison with data sets of the mouse cochlea and vestibular epithelia. We resolved supporting cell sub-types, cochlear like hair cells, and vestibular Type I and Type II like hair cells. While cochlear like hair cells aligned best with an outer hair cell trajectory, vestibular like hair cells followed developmental trajectories similar to in vivo programs branching into Type II and then Type I extrastriolar hair cells. These results highlight the transcriptional accuracy of the organoid developmental program but will also inform future strategies to improve synaptic connectivity and regional specification.
2024-02-13 | GSE242675 | GEO
Project description:The inner ear continues to grow and develop until the auditory and vestibular systems reach full maturity and all of the genes involved in this process have yet to be identified. Previous gene based analysis have primarily focused on the early developmental stages following induction and initial formation of the inner ear. The aim of this study is to identify new candidate genes for inner ear development. Microarrays were used to produce expression profiles from larval stages 56,57,58 of the Xenopus laevis inner ear. The data produced from this work represent an annotated resource that can be utilized by the Xenopus community to provide candidates for further functional analysis. Xenopus inner ears were isolated from larval animals for RNA extraction and hybridization to Affymetrix GeneChip microarrays.
2016-02-29 | E-GEOD-73829 | biostudies-arrayexpress
Project description:A large number of congenital hearing loss cases have an unknown genetic etiology. So far, transcriptomic approaches have successfully identified many candidate regulators of otic development, little is known about the abundance of their protein products during the development of the inner eat. Herein we used a multiplexed quantitative mass spectrometry-based proteomic approach to determine temporal trends in protein abundances during inner ear (otic) development in Xenopus. Wild type Xenopus embryos were cultured to larval stages and their otic tissues were manually dissected at five stages that represent that represent key transitions in otic morphology. The samples were processed using a bottom-up proteomic workflow and analyzed using LC-MS3.The analysis revealed dynamic expression of proteins related to cytoskeletal regulation, integrin signaling, and the extracellular matrix as inner ear structures developed. We correlated the dynamically regulated proteins in our dataset with previously published putative downstream targets of syndromic hearing loss genes SIX1 and CHD7 to identify novel candidate genes for congenital hearing loss.
2023-10-24 | PXD041991 | Pride