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Fbxo2/Fbx2 expression in whole embryos at E12.5. Whole mount immunohistochemistry was performed with the antibodies indicated and embryos were imaged with confocal microscopy. Fbx2 expression is shown in red, with Sox2 in green, and Tuj1 in white. (A–A″) Merged montages of maximum intensity z projections of confocal image stacks of a whole E12.5 embryo. (B–B″) show a projected stack of images through the otocyst region of the embryo shown in (A). Sox2 expression is seen in two bright vestibular prosensory patches in the anterior region of the vestibule, while Sox2 expression in other prosensory domains (such as posterior vestibule or cochlea areas) are not visible in this projection due to lower intensity signal and attenuation from optical density of the tissue. (C–C″) Single optical sections through the anterior vestibular prosensory patches showing Fbx2-positive epithelia contain prosensory domains expressing bright Sox2 and containing Tuj1-labeled neurites.
Source publication
Vertebrate embryogenesis gives rise to all cell types of an organism through the development of many unique lineages derived from the three primordial germ layers. The otic sensory lineage arises from the otic vesicle, a structure formed through invagination of placodal non-neural ectoderm. This developmental lineage possesses unique differentiatio...
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... we used an antibody against mouse Fbx2 to assess expression in the developing mouse, with whole mount preparations as well as tissue sections. We labeled whole embryos at early stages of otic development with anti-Fbx2 and anti-Sox2, optically cleared samples with the Scale method (Hama et al., 2011), and generated confocal z-stacks to assess expression in toto (Figures 4, 5). At E10.5, Fbx2 expression was robust throughout the OV including the Sox2+ prosensory and neurogenic domains and was undetectable in nearly all non-otic tissues (Figures 4A-C ′′ ). ...
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... E11 otic vesicle stained for Fbx2 and Sox10 shows strong Fbx2 expression in the OV and lower levels of expres- sion in delaminating cochleovestibular neuroblasts as well as the ninth cranial nerve ganglia (Figures 4D-E ′′ ). At E12.5, Fbx2 was detected only in the developing inner ear, where it was expressed throughout the membranous labyrinth (Figures 5A,B ′′ ), includ- ing prosensory epithelial regions that express Sox2 and contain Tuj1-labeled neurites (Figures 5C-C ′′ ). We stained for Fbx2 in tissue sections of embryos at E16.5 and E18.5 and co-labeled with antibodies to the prosensory/sensory domain markers Sox2 and Jag1 (Figure 6). ...
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... E11 otic vesicle stained for Fbx2 and Sox10 shows strong Fbx2 expression in the OV and lower levels of expres- sion in delaminating cochleovestibular neuroblasts as well as the ninth cranial nerve ganglia (Figures 4D-E ′′ ). At E12.5, Fbx2 was detected only in the developing inner ear, where it was expressed throughout the membranous labyrinth (Figures 5A,B ′′ ), includ- ing prosensory epithelial regions that express Sox2 and contain Tuj1-labeled neurites (Figures 5C-C ′′ ). We stained for Fbx2 in tissue sections of embryos at E16.5 and E18.5 and co-labeled with antibodies to the prosensory/sensory domain markers Sox2 and Jag1 (Figure 6). ...
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The stochastic resonance (SR) is a phenomenon of nonlinear systems in which the addition of an intermediate level of noise improves the response of such system. Although SR has been studied in isolated hair cells and in the bullfrog sacculus, the occurrence of this phenomenon in the vestibular system in development is unknown. The purpose of the pr...
Citations
... Based on differential gene expression ( Figure 2C), sub-clusters were annotated as sensory SCLCs and non-sensory epithelial cells (NSECs). 46 For example, the majority of SCLCs expressed markers such as Sox2, Jag1, and Lfng ( Figures 2C, 2D, and S1E), 33,47,48 reminiscent of SCs in the sensory epithelia of the inner ear, whereas cells of the smaller cluster expressed genes such as Oc90, Otol1, and Otoa ( Figures 2C-2E and S1E), [49][50][51] resembling the cell types lining the inner ear fluid spaces outside the sensory epithelia. During embryonic development of the cochlea, these cell types constitute the cochlear roof, which will later give rise to structures such as stria vascularis and Reissner's membrane. ...
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 datasets 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.
... Both cell types were indeed identified in the day 26 RNA-seq dataset. IEO-derived OE and neuroblasts express markers previously found in the developing murine otocysts (Durruthy-Durruthy et al., 2014;Hartman et al., 2015), and based on our histological characterization match the CS13 stage of development. ...
... The OE derived in vitro co-expressed CDH1 and CDH2, whereas the latter was absent in the EP clusters (Fig. 5F; Fig. S5A). OE further expressed high levels of OC90, FBXO2, LMX1A and TBX2, as previously identified in mouse studies(Durruthy-Durruthy et al., 2014;Hartman et al., 2015;Kaiser et al., 2021;Sun et al., 2022) (Fig. 5F,G; Fig. S5A,D). The placodal genes SIX1, DLX5 and PAX8 were still expressed; PAX2 and SOX10 became clearly upregulated (Fig. 5F; ...
Our molecular understanding of the early stages of human inner ear development has been limited by the difficulty in accessing fetal samples at early gestational stages. As an alternative, previous studies have shown that inner ear morphogenesis can be partially recapitulated using induced pluripotent stem cells (iPSCs) directed to differentiate into Inner Ear Organoids (IEOs). Once validated and benchmarked, these systems could represent unique tools to complement and refine our understanding of human otic differentiation and model developmental defects. Here, we provide the first direct comparisons of the early human embryonic otocyst and fetal sensory organs to human IEOs. We use multiplexed immunostaining, and single-cell RNA sequencing to characterize IEOs at three key developmental steps, providing a new and unique signature of in vitro derived otic -placode, -epithelium, -neuroblasts, and -sensory epithelia. In parallel, we evaluate the expression and localization of critical markers at these equivalent stages in human embryos. Together, our data indicate that the current state-of-the-art protocol enables the specification of bona fide otic tissue, supporting the further application of IEOs to inform inner ear biology and disease.
... The sensory epithelia of the cochlea and the vestibular systems are derived from the otic vesicle, as are the neurons that relay auditory and vestibular information to the brain. [12][13][14] At the otic vesicle stage, the progenitors of the future cochlear and vestibular neurons are born. They delaminate as neuroblasts and migrate toward the cochleovestibular ganglion (CVG), where neuronal cell differentiation happens ( Figure 1A). ...
... Cluster 5 contained 58 differentially enriched genes, including otic vesicle markers Oc90, Trpm3, and Lmx1a. 12,38 Combined with the observed expression of pan-otic markers Six1 and Foxg1, cluster 5 was identified as the otic vesicle cells. Interestingly, cluster 5, as well as cluster 6 (38 differentially enriched genes), expressed the pan-epithelial marker Epcam. ...
... 39 However, the enrichment of Epcam in cluster 6 did not reach our cutoff (LFC = 0.74), small subset of cells in cluster 6 also express Oc90, which we have previously reported as a marker of the dorsal-most region of the otic vesicle at E10.5. 12,38 Further investigation of the genes enriched in cluster 6 revealed epidermal markers such as Anxa2 and Krt7 as more broadly expressed throughout cluster 6, as well as a subset of cells expressing the highly specific epidermal marker gene Trp63. [40][41][42][43] We therefore define cluster 6 as surface ectoderm/epidermis. ...
An abundance of research has recently highlighted the susceptibility of cochleovestibular ganglion (CVG) neurons to noise damage and aging in the adult cochlea, resulting in hearing deficits. Furthering our understanding of the transcriptional cascades that contribute to CVG development may provide insight into how these cells can be regenerated to treat inner ear dysfunction. Here we perform a high-depth single-cell RNA sequencing analysis of the E10.5 otic vesicle and its surrounding tissues, including CVG precursor neuroblasts and emerging CVG neurons. Clustering and trajectory analysis of otic-lineage cells reveals otic markers and the changes in gene expression that occur from neuroblast delamination toward the development of the CVG. This dataset provides a valuable resource for further identifying the mechanisms associated with CVG development from neurosensory competent cells within the otic vesicle.
... 3c, 4d). In addition, GSE analysis also revealed dysregulation of FGF and WNT signaling pathways, as well as enrichment of multiple gene sets closely related to the cellular functions of the otic progenitors, such as cell junction organization and extracellular matrix organization (Fig. 4d) identity is affected, we compared our dataset with a list of otic lineagespecific genes systematically identified by Hartman et al. 30 . While 57.7% of these otic-specific genes remained largely unaffected, 38.5% of them, including the highest ranked otic-specific genes FBXO2, COL9A2, and OC90, were significantly downregulated in CHD7 KO/KO otic progenitors, suggesting that the otic identity is partially impaired (Figs. ...
... Differential expression (DE) analysis was performed using the DESeq2 package 72 in conjunction with zingeR in R, and DE genes were visualized on volcano plots using the EnhancedVolcano R package (https://github.com/kevinblighe/EnhancedVolcano). To generate the bubble plot for differentially expressed otic-specific genes, the gene expression fold-change values of E10.5 otic vesicles versus non-otic tissues reported by Hartman et al. 30 were used to calculate the area of gene-correlated spots on the volcano plot. ...
Mutations in CHD7 cause CHARGE syndrome, affecting multiple organs including the inner ear in humans. We investigate how CHD7 mutations affect inner ear development using human pluripotent stem cell-derived organoids as a model system. We find that 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 suggests that CHD7 can regulate otic genes through a chromatin remodeling-independent mechanism. Results from transcriptome profiling of hair cells reveal 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 rescues 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 specification and hair cell differentiation.
... The otic markers, Oc90, Fbxo2, and Sox10, were seen in D10, which is consistent with findings in mouse E10.5. 44 In addition to the otic placode, there was evidence for induction of neurogenic placodes (clusters 6 and 9 on D8 and cluster 7 on D10) during inner ear organoid induction. The epibranchial placode is also derived from the pPPE 45 ; therefore, it is unsurprising that Phox2b + epibranchial placodal cells (cluster 6 on D8) were present in our cultures. ...
The inner ear is derived from the otic placode, one of numerous cranial sensory placodes that emerges from the pre-placodal ectoderm (PPE) along its anterior-posterior axis. However, the molecular dynamics underlying how the PPE is regionalized are poorly resolved. We used stem cell-derived organoids to investigate the effects of Wnt signaling on early PPE differentiation and found that modulating Wnt signaling significantly increased inner ear organoid induction efficiency and reproducibility. Alongside single-cell RNA sequencing, our data reveal that the canonical Wnt signaling pathway leads to PPE regionalization and, more specifically, medium Wnt levels during the early stage induce 1) expansion of the caudal neural plate border (NPB), which serves as a precursor for the posterior PPE, and 2) a caudal microenvironment that is required for otic specification. Our data further demonstrate Wnt-mediated induction of rostral and caudal cells in organoids and more broadly suggest that Wnt signaling is critical for anterior-posterior patterning in the PPE.
... Therefore, these neuroblast clusters (otocyst-derived neuroblasts-1e4) were seemingly the delaminated cell populations from the otic epithelial cells. Of note, cells in anteromedial otocyst-1e4, anteroventral otocyst-1e3 and otocystderived neuroblasts-1, 3, and 4 expressed FBXO2, a specific marker of otocysts in E10.5 mice [46,50]. ...
... Since the expression of known otocyst marker genes, such as DLX5, GBX2, PAX8, and PAX2, is spatially and temporally restricted in rodents [5], our scRNA-seq data may represent the presence of different regions of otocyst cells in each cluster. Importantly, in most otocyst cell clusters, we detected the expression of FBXO2, which is expressed specifically in the mouse otocyst at E10.5 and is highly expressed in the anteromedial portion of the otocyst [46,50]. Further optimization of the culture method may be required to decrease the heterogeneity of the otocyst lineage cell populations. ...
Introduction
Efficient induction of the otic placode, the developmental origin of the inner ear from human pluripotent stem cells (hPSCs), provides a robust platform for otic development and sensorineural hearing loss modelling. Nevertheless, there remains a limited capacity of otic lineage specification from hPSCs by stepwise differentiation methods, since the critical factors for successful otic cell differentiation have not been thoroughly investigated. In this study, we developed a novel differentiation system involving the use of a three-dimensional (3D) floating culture with signalling factors for generating otic cell lineages via stepwise differentiation of hPSCs.
Methods
We differentiated hPSCs into preplacodal cells under a two-dimensional (2D) monolayer culture. Then, we transferred the induced preplacodal cells into a 3D floating culture under the control of the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), retinoic acid (RA) and WNT signalling pathways. We evaluated the characteristics of the induced cells using immunocytochemistry, quantitative PCR (qPCR), population averaging, and single-cell RNA-seq (RNA-seq) analysis. We further investigated the methods for differentiating otic progenitors towards hair cells by overexpression of defined transcription factors.
Results
We demonstrated that hPSC-derived preplacodal cells acquired the potential to differentiate into posterior placodal cells in 3D floating culture with FGF2 and RA. Subsequent activation of WNT signalling induced otic placodal cell formation. By single-cell RNA-seq (scRNA-seq) analysis, we identified multiple clusters of otic placode- and otocyst marker-positive cells in the induced spheres. Moreover, the induced otic cells showed the potential to generate hair cell-like cells by overexpression of the transcription factors ATOH1, POU4F3 and GFI1.
Conclusions
We demonstrated the critical role of FGF2, RA and WNT signalling in a 3D environment for the in vitro differentiation of otic lineage cells from hPSCs. The induced otic cells had the capacity to differentiate into inner ear hair cells with stereociliary bundles and tip link-like structures. The protocol will be useful for in vitro disease modelling of sensorineural hearing loss and human inner ear development and thus contribute to drug screening and stem cell-based regenerative medicine.
... Undifferentiated CVG cells originate from the otic vesicle (OV), which is the primordium of the inner ear and is formed adjacent to hindbrain rhombomeres 4-6 around embryonic day 9 (E9) in the mouse (Morsli et al., 1998). OV epithelium cells express several sensory markers, such as Sox2, Fbxo2, Tbx2, Foxg1, and Pax2 (Hartman et al., 2015;Kaiser et al., 2021;Kiernan et al., 2005;Ohyama and Groves, 2004;Pauley et al., 2006;Shirai et al., 2009;Xu et al., 2021). One of the early cellular events in the OV is the formation of the anterior-posterior polarity mediated by retinoic acid (Bok et al., 2011;Wu and Kelley, 2012). ...
... We next annotated cell identities in the clusters E9.5-5, E9.5-6, E9.5-8, and E9.5-12. First, cluster E9.5-12 includes OV cells expressing Fbxo2 and Tbx2 ( Figure 1G), two markers recognized to be highly expressed in OV cells (Hartman et al., 2015;Kaiser et al., 2021;Shirai et al., 2009). Accordingly, cluster E9.5-12 cells also express other known otic markers, such as Foxg1, Pax2, Sox2, Six1, Eya1, Lmx1a, and Tbx1 (Ahmed et al., 2012;Burton et al., 2004;Kiernan et al., 2005;Koo et al., 2009;Mann et al., 2017;Raft et al., 2004) (Figures S1A-S1B 00 ). ...
Inner ear vestibular and spiral ganglion neurons (VGNs and SGNs) are known to play pivotal roles in balance control and sound detection. However, the molecular mechanisms underlying otic neurogenesis at early embryonic ages have remained unclear. Here, we use single-cell RNA sequencing to reveal the transcriptomes of mouse otic tissues at three embryonic ages, embryonic day 9.5 (E9.5), E11.5, and E13.5, covering proliferating and undifferentiated otic neuroblasts and differentiating VGNs and SGNs. We validate the high quality of our studies by using multiple assays, including genetic fate mapping analysis, and we uncover several genes upregulated in neuroblasts or differentiating VGNs and SGNs, such as Shox2, Myt1, Casz1, and Sall3. Notably, our findings suggest a general cascaded differentiation trajectory during early otic neurogenesis. The comprehensive understanding of early otic neurogenesis provided by our study holds critical implications for both basic and translational research.
... To model phenotype manifestation of CHARGE syndrome, we next created mono-and bi-allelic CHD7 KO hESC lines by targeting the first of the 38 coding exons of CHD7 with CRISPR. Frameshift indel formation at this early coding region leads to nonsense-mediated mRNA decay (NMD) 18 , or if the mutated transcript escapes the NMD mechanism, it results in early truncation of the CHD7 protein prior to any functional domain, therefore creating a null deletion. We chose two clonal lines with frameshift indels in one, or both alleles, and designated them as CHD7 KO/+ and CHD7 KO/KO hESC lines for further analysis ( Fig. 2a and Supplementary Fig. 3-4). ...
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.
... The mechanical cues from the ECM have been recently reported to promote the survival, expansion and differentiation of inner ear progenitors, partly through activation of WNT signaling [30]. Our HTS screen identified ECM-encoding genes that have been previously reported in the mouse inner ear, such collagen as (COL1A1, COL1A2, COL2A1, COL9A2) [31,32]. In addition, the HTS revealed the second group of unknown ECM components in the inner ear that included additional types of collagen (COL18A, COL26A1), glycoprotein (FN1), and proteoglycan decorin (DCN). ...
... In the inner ear, COL2A1 expression has been detected in both ectodermal and mesodermally derived structures in mammals [33] including connective tissue of neuronal cells, stria vascularis and organ of Corti [34]. The COL9A2 expression was observed in the otic vesicle at E10.5 and in organ of Corti and spiral ligament during mouse postnatal development [32]. Furthermore, HTS screen also revealed several ITG targets that were enriched in day 13 cultures. ...
... This pathway senses and transmits mechanical cues from ECM, and any deleterious change often results in syndromic HL [30]. ECM-encoding genes (COL2A1 and COL9A2) we identified in HTS and qPCR analyzes during hiPSC differentiation were also previously reported in rodents and human inner ear [32][33][34]. The immunostaining revealed a spatio-temporal expression of ECM (COL2A1, COL9A2 and ITGAV) in fetal human inner ear. ...
We analyzed transcriptomic data from otic sensory cells differentiated from human induced pluripotent stem cells (hiPSCs) by a previously described method to gain new insights into the early human otic neurosensory lineage. We identified genes and biological networks not previously described to occur in the human otic sensory developmental cell lineage. These analyses identified and ranked genes known to be part of the otic sensory lineage program (SIX1, EYA1, GATA3, etc.), in addition to a number of novel genes encoding extracellular matrix (ECM) (COL3A1, COL5A2, DCN, etc.) and integrin (ITG) receptors (ITGAV, ITGA4, ITGA) for ECM molecules. The results were confirmed by quantitative PCR analysis of a comprehensive panel of genes differentially expressed during the time course of hiPSC differentiation in vitro. Immunocytochemistry validated results for select otic and ECM/ITG gene markers in the in vivo human fetal inner ear. Our screen shows ECM and ITG gene expression changes coincident with hiPSC differentiation towards human otic neurosensory cells. Our findings suggest a critical role of ECM-ITG interactions with otic neurosensory lineage genes in early neurosensory development and cell fate determination in the human fetal inner ear.
... Conos analysis revealed that supporting cells are transcriptionally distinct from other peripheral glia yet express many glial genes. We identified cluster g8 as supporting cells based on expression of known markers Fbxo2 and Tecta (Hartman et al., 2015;Kim et al., 2019) (Figure 7A). Although there are several types of adult supporting cells (Wan et al., 2013), our analysis revealed a single cluster, possibly due to underrepresentation of P14 cells. ...
The peripheral nervous system responds to a wide variety of sensory stimuli, a process that requires great neuronal diversity. These diverse neurons are closely associated with glial cells originating from the neural crest. However, the molecular nature and diversity among peripheral glia are not understood. Here, we used single-cell RNA sequencing to profile developing and mature glia from somatosensory dorsal root ganglia and auditory spiral ganglia. We found that glial precursors (GPs) in these two systems differ in their transcriptional profiles. Despite their unique features, somatosensory and auditory GPs undergo convergent differentiation to generate molecularly uniform myelinating and non-myelinating Schwann cells. By contrast, somatosensory and auditory satellite glial cells retain system-specific features. Lastly, we identified a glial signature gene set, providing new insights into commonalities among glia across the nervous system. This survey of gene expression in peripheral glia constitutes a resource for understanding functions of glia across different sensory modalities.
