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Analysis of Gene Expression in the Chicken Otocyst

ABSTRACT: The inner ear arises from multipotent placodal precursors that are gradually committed to the otic fate and further differentiate into all inner ear cell types, with the exception of a few neural crest-derived cells. The otocyst has a pivotal role during inner ear development: otic progenitor cells sub-compartmentalize into non-sensory regions and regions giving rise to prosensory domains where subsequently also hair cells differentiate. The genes and pathways underlying this progressive subdivision and differentiation process are not entirely known. The goal of this study was to identify a comprehensive set of genes expressed in the chicken otocyst using the serial analysis of gene expression (SAGE) method. Our analysis revealed several hundred transcriptional regulators, potential signaling proteins, and receptors. We identified a substantial collection of genes that were previously known in the context of inner ear development, but we also found many new candidate genes, such as Sox4, Sox5, Sox7, Sox8, Sox11, and Sox18, which previously were not known to be expressed in the developing inner ear. Despite its obvious limitation of not being all-inclusive, the generated otocyst SAGE library is a practical bioinformatics tool to study otocyst gene expression and to identify candidate genes for developmental studies.

ORGANISM(S): Gallus gallus

PROVIDER: GSE27176 | GEO | 2011/03/01

SECONDARY ACCESSION(S): PRJNA137363

REPOSITORIES: GEO

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Analysis of Gene Expression in the Chicken Otocyst

Project description:The inner ear arises from multipotent placodal precursors that are gradually committed to the otic fate and further differentiate into all inner ear cell types, with the exception of a few neural crest-derived cells. The otocyst has a pivotal role during inner ear development: otic progenitor cells sub-compartmentalize into non-sensory regions and regions giving rise to prosensory domains where subsequently also hair cells differentiate. The genes and pathways underlying this progressive subdivision and differentiation process are not entirely known. The goal of this study was to identify a comprehensive set of genes expressed in the chicken otocyst using the serial analysis of gene expression (SAGE) method. Our analysis revealed several hundred transcriptional regulators, potential signaling proteins, and receptors. We identified a substantial collection of genes that were previously known in the context of inner ear development, but we also found many new candidate genes, such as Sox4, Sox5, Sox7, Sox8, Sox11, and Sox18, which previously were not known to be expressed in the developing inner ear. Despite its obvious limitation of not being all-inclusive, the generated otocyst SAGE library is a practical bioinformatics tool to study otocyst gene expression and to identify candidate genes for developmental studies. Otocysts were dissected from HH stage 18-19 chicken embryos, total RNA was extracted, and subjected to a commercial long-SAGE protocol, resulting in a library of concatemerized tags. 3,512 individual clones of SAGE concatemers were sequenced resulting in 39,326 17-bp tags with tag counts up to 718 for the most abundant tag; 3,292 tags that were represented between two and five times, whereas the majority of tags (11,717) were only found once. Overall, we identified 16,008 unique sequence tags.

2011-03-01 | E-GEOD-27176 | biostudies-arrayexpress

Critical role of FGF, RA, and WNT signaling for otic lineage specification from human PSCs in a 2D/3D-combined differentiation system

Project description:The otic placode, from which the inner ear develops, initially forms as a thickened ectodermal patch adjacent to the dorsolateral hindbrain. Induction of otic placodal cells from human pluripotent stem cells (hPSCs) provides a robust approach to investigation of otic development. However, attempts to recapitulate otic lineage specification from hPSCs by stepwise differentiation methods have had limited success. One possible reason is that the role of signaling pathways in otic cell differentiation is not fully understood. Here, we developed a novel differentiation system involving use of suspension culture (3D floating culture) in combination with signaling factors for generating otic placodal cells via stepwise differentiation of hPSCs. We demonstrate that hPSC-derived pre-placodal cells acquired the potential to differentiate into posterior placodal cells after fibroblast growth factor 2 (FGF2) and retinoic acid (RA) treatment. Subsequent activation of WNT signaling following posterior placode specification induced differentiation of SIX1+/PAX8+/SOX2+ otic placodal cells. qPCR analysis showed that WNT activation increased expression of otocyst markers, including PAX8, PAX2, DLX5 and FBXO2. Moreover, induced otic cells exhibited similar gene expression patterns to the ventral and medial portions of the otocyst that give rise to the sensory epithelium of the inner ear. Collectively, our results indicate a critical role for FGF2, RA, and WNT signaling for the in vitro differentiation of the otic cell lineage from hPSCs. Our new culture system paves the way for improved modeling of early human inner ear development and will be of value to studies on the further differentiation of sensory epithelial cells.

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Opposing activity of SOX9 and Wnt in the developing otocyst ensures SOX10 control of endolymphatic development

Project description:Sensorineural deafness can occur in campomelic dysplasia (CD). In a mouse model of the SOX9Y440X/+ CD mutation, SOX10 downregulation was previously shown to be associated with impaired development of the endolymphatic system at mid-gestation. The underlying molecular causes are unknown. Here we found at E10.5 when the otocyst lacks overt inner ear structures, Sox10 was downregulated in the dorsal and medial aspects of the Sox9Y440X/+ otic epithelium, regions associated with specification of the endolymphatic sac and duct. Single-cell transcriptomic profiling revealed differential elevation of Wnt signaling pathway genes associated with the domain-specific Sox10 reduction. Otocyst-specific forced elevation of Wnt signaling by expression of stabilized β-catenin suppressed Sox10 expression. Similarly, WNT exerted an inhibitory effect on SOX10 expression in human inner ear organoids derived from pluripotent stem cells. We propose a conserved regulatory model of SOX9-WNT antagonism governing SOX10 action in the otocyst underlies the specification of endolymphatic cell fate, with important implications for understanding human deafness syndromes.

2024-11-07 | GSE271849 | GEO

Expression data from E10.5 mouse otocyst

Project description:We established a novel EGFP reporter mouse line (named Tg(ETAR-EGFP)14Imeg), which enables the placode-derived inner ear sensory cell lineage to be visualized and monitored. At E10.5, EGFP expression was detected in the ventral and dorsomedial region of the otocyst. Using this reporter line and FACS-array technology, we performed gene expression profiling focused on the regional specificity of the otocyst. We sorted E10.5 otocyst epithelial cells of heterozygous mice into EGFP-positive and EGFP-negative cells for RNA extraction and hybridization on Affymetrix microarrays. We profiled the expression of the genes in FACS-enriched cell population and conducted transcriptome analysis.

2011-05-23 | E-GEOD-19618 | biostudies-arrayexpress

Expression data from E10.5 mouse otocyst

2011-05-23 | GSE19618 | GEO

Human Pluripotent stem cells-derived Inner ear organoids recapitulate otic development in vitro

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

CHD7 and SOX2 Act in a Common Gene Regulatory Network During Mammalian Semicircular Canal and Cochlear Development (CUT&Tag)

Project description:Inner ear morphogenesis requires tightly regulated epigenetic and transcriptional control of gene expression. CHD7, an ATP-dependent chromodomain helicase DNA-binding protein, and SOX2, an SRY-related HMG box pioneer transcription factor, are known to contribute to vestibular and auditory system development, but their genetic interactions in the ear have not been explored. Here, we analyzed inner ear development and the transcriptional regulatory landscapes in mice with variable dosage of Chd7 and/or Sox2. We show that combined haploinsufficiency for Chd7 and Sox2 results in reduced otic cell proliferation, severe malformations of semicircular canals, and shortened cochlea with ectopic hair cells. Examination of mice with conditional, inducible Chd7 loss by Sox2CreER reveals a critical period (~E9.5) of susceptibility in the inner ear to combined Chd7 and Sox2 loss. Data from genome-wide RNA-sequencing and CUT&Tag studies in the otocyst show that CHD7 regulates Sox2 expression and acts early in a gene regulatory network to control expression of key otic patterning genes, including Pax2 and Otx2. CHD7 and SOX2 directly bind independently and cooperatively at transcription start sites and enhancers to regulate otic progenitor cell proliferation and function. Together, our findings reveal essential roles for Chd7 and Sox2 in the early inner ear and may be applicable for CHD7 and SOX2 related syndromic and other forms of hearing or balance disorders.

2024-03-01 | GSE239362 | GEO

CHD7 and SOX2 Act in a Common Gene Regulatory Network During Mammalian Semicircular Canal and Cochlear Development (RNA-Seq)

2024-03-01 | GSE239361 | GEO

"Time-resolved quantitative proteomic analysis of the developing otic vesicle reveals putative congenital hearing loss candidates and a novel role for Tgfbi

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.

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