RESUMO
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.
Assuntos
Cóclea , Células-Tronco Neurais , Camundongos , Animais , Células-Tronco Neurais/metabolismo , Neurônios , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
OBJECTIVE: The cellular diversity of the inner ear has presented a technical challenge in obtaining molecular insight into its development and function. The application of technological advancements in cell type-specific expression enable clinicians and researchers to leap forward from classic genetics to obtaining mechanistic understanding of congenital and acquired hearing loss. This understanding is essential for development of therapeutics to prevent and reverse diseases of the inner ear, including hearing loss. The objective of this study is to describe and compare the available tools for cell type-specific analysis of the ear, as a means to support decision making in study design. STUDY DESIGN: Three major approaches for cell type-specific analysis of the ear including fluorescence-activated cell sorting (FACS), ribosomal and RNA pulldown techniques, and single cell RNA-seq (scRNA-seq) are compared and contrasted using both published and original data. RESULTS: We demonstrate the strength and weaknesses of these approaches leading to the inevitable conclusion that to maximize the utility of these approaches, it is important to match the experimental approach with the tissue of origin, cell type of interest, and the biological question. Often, a combined approach (eg, cell sorting and scRNA-seq or expression analysis using 2 separate approaches) is required. Finally, new tools for visualization and analysis of complex expression data, such as the gEAR platform (umgear.org), collate cell type-specific gene expression from the ear field and provide unprecedented access to both clinicians and researchers. LEVEL OF EVIDENCE: N/A Laryngoscope, 131:S1-S16, 2021.
Assuntos
Orelha Interna/citologia , Citometria de Fluxo/métodos , Perfilação da Expressão Gênica , RNA/isolamento & purificação , Análise de Sequência de RNA/métodos , Animais , Tomada de Decisões , Corantes Fluorescentes , Expressão Gênica , Perda Auditiva/congênito , Perda Auditiva/genética , Humanos , Camundongos , Camundongos Transgênicos , Órgão Espiral/citologia , Compostos de Piridínio , Compostos de Amônio Quaternário , Ribossomos/metabolismo , Análise de Célula Única/métodos , Junções ÍntimasRESUMO
Despite the known importance of the transcription factors ATOH1, POU4F3 and GFI1 in hair cell development and regeneration, their downstream transcriptional cascades in the inner ear remain largely unknown. Here, we have used Gfi1cre;RiboTag mice to evaluate changes to the hair cell translatome in the absence of GFI1. We identify a systematic downregulation of hair cell differentiation genes, concomitant with robust upregulation of neuronal genes in the GFI1-deficient hair cells. This includes increased expression of neuronal-associated transcription factors (e.g. Pou4f1) as well as transcription factors that serve dual roles in hair cell and neuronal development (e.g. Neurod1, Atoh1 and Insm1). We further show that the upregulated genes are consistent with the NEUROD1 regulon and are normally expressed in hair cells prior to GFI1 onset. Additionally, minimal overlap of differentially expressed genes in auditory and vestibular hair cells suggests that GFI1 serves different roles in these systems. From these data, we propose a dual mechanism for GFI1 in promoting hair cell development, consisting of repression of neuronal-associated genes as well as activation of hair cell-specific genes required for normal functional maturation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Células Ciliadas Auditivas Internas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação a DNA/genética , Células Ciliadas Auditivas Internas/citologia , Camundongos , Camundongos Transgênicos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fator de Transcrição Brn-3A/genética , Fator de Transcrição Brn-3A/metabolismo , Fatores de Transcrição/genéticaRESUMO
Alström syndrome (OMIM #203800) is an autosomal recessive obesity ciliopathy caused by loss-of-function mutations in the ALMS1 gene. In addition to multi-organ dysfunction, such as cardiomyopathy, retinal degeneration and renal dysfunction, the disorder is characterized by high rates of obesity, insulin resistance and early-onset type 2 diabetes mellitus (T2DM). To investigate the underlying mechanisms of T2DM phenotypes, we generated a loss-of-function deletion of alms1 in the zebrafish. We demonstrate conservation of hallmark clinical characteristics alongside metabolic syndrome phenotypes, including a propensity for obesity and fatty livers, hyperinsulinemia and glucose response defects. Gene expression changes in ß-cells isolated from alms1-/- mutants revealed changes consistent with insulin hypersecretion and glucose sensing failure, which were corroborated in cultured murine ß-cells lacking Alms1. We also found evidence of defects in peripheral glucose uptake and concomitant hyperinsulinemia in the alms1-/- animals. We propose a model in which hyperinsulinemia is the primary and causative defect underlying generation of T2DM associated with alms1 deficiency. These observations support the alms1 loss-of-function zebrafish mutant as a monogenic model for mechanistic interrogation of T2DM phenotypes.
Assuntos
Síndrome de Alstrom/genética , Diabetes Mellitus Tipo 2/genética , Resistência à Insulina/genética , Insuficiência Renal/genética , Degeneração Retiniana/genética , Peixe-Zebra/genética , Síndrome de Alstrom/fisiopatologia , Animais , Animais Geneticamente Modificados , Linhagem Celular , Modelos Animais de Doenças , Intolerância à Glucose , Hiperinsulinismo/genética , Células Secretoras de Insulina/metabolismo , Camundongos , Modelos Biológicos , Obesidade/genética , Fenótipo , Peixe-Zebra/embriologiaRESUMO
The sensory cells that are responsible for hearing include the cochlear inner hair cells (IHCs) and outer hair cells (OHCs), with the OHCs being necessary for sound sensitivity and tuning1. Both cell types are thought to arise from common progenitors; however, our understanding of the factors that control the fate of IHCs and OHCs remains limited. Here we identify Ikzf2 (which encodes Helios) as an essential transcription factor in mice that is required for OHC functional maturation and hearing. Helios is expressed in postnatal mouse OHCs, and in the cello mouse model a point mutation in Ikzf2 causes early-onset sensorineural hearing loss. Ikzf2cello/cello OHCs have greatly reduced prestin-dependent electromotile activity, a hallmark of OHC functional maturation, and show reduced levels of crucial OHC-expressed genes such as Slc26a5 (which encodes prestin) and Ocm. Moreover, we show that ectopic expression of Ikzf2 in IHCs: induces the expression of OHC-specific genes; reduces the expression of canonical IHC genes; and confers electromotility to IHCs, demonstrating that Ikzf2 can partially shift the IHC transcriptome towards an OHC-like identity.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Ciliadas Auditivas Externas/citologia , Células Ciliadas Auditivas Externas/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transcriptoma/genética , Animais , Sequência de Bases , Biomarcadores/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
[This corrects the article DOI: 10.3389/fcell.2018.00047.].
RESUMO
The zebrafish inner ear organs and lateral line neuromasts are comprised of a variety of cell types, including mechanosensitive hair cells. Zebrafish hair cells are evolutionarily homologous to mammalian hair cells, and have been particularly useful for studying normal hair cell development and function. However, the relative scarcity of hair cells within these complex organs, as well as the difficulty of fine dissection at early developmental time points, makes hair cell-specific gene expression profiling technically challenging. Cell sorting methods, as well as single-cell RNA-Seq, have proved to be very informative in studying hair cell-specific gene expression. However, these methods require that tissues are dissociated, the processing for which can lead to changes in gene expression prior to RNA extraction. To bypass this problem, we have developed a transgenic zebrafish model to evaluate the translatome of the inner ear and lateral line hair cells in their native tissue environment; the Tg(myo6b:RiboTag) zebrafish. This model expresses both GFP and a hemagglutinin (HA) tagged rpl10a gene under control of the myo6b promoter (myo6b:GFP-2A-rpl10a-3xHA), resulting in HA-tagged ribosomes expressed specifically in hair cells. Consequently, intact zebrafish larvae can be used to enrich for actively translated hair cell mRNA via an immunoprecipitation protocol using an antibody for the HA-tag (similar to the RiboTag mice). We demonstrate that this model can be used to reliably enrich for actively translated zebrafish hair cell mRNA. Additionally, we perform a global hair cell translatome analysis using RNA-Seq and show enrichment of known hair cell expressed transcripts and depletion of non-hair cell expressed transcripts in the immunoprecipitated material compared with mRNA extracted from whole fish (input). Our results show that our model can identify novel hair cell expressed genes in intact zebrafish, without inducing changes to gene expression that result from tissue dissociation and delays during cell sorting. Overall, we believe that this model will be highly useful for studying changes in zebrafish hair cell-specific gene expression in response to developmental progression, mutations, as well as hair cell damage by noise or ototoxic drug exposure.