Investigating the Roles of Epithelial Splice Regulatory Proteins in Inner Ear Development

研究上皮剪接调节蛋白在内耳发育中的作用

• 批准号: 9026485
• 负责人: ALEX ROHACEK
• 金额: $ 4.36万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9026485
• 关键词: Address; Adult; Affect; Alternative Splicing; Apical; Auditory; Auditory Brainstem Responses; Cadherins; Cell Cycle; Cell Differentiation process; Cell Maturation; Cells; Cleft Palate; Cochlea; Cochlear duct; Congenital Abnormality; Coupled; Defect; Development; Developmental Delay Disorders; Duct (organ) structure; Embryo; Epithelial; Epithelium; Event; Exhibits; Exons; Family; Fibroblast Growth Factor Receptors; General Population; Genes; Goals; Hair Cells; Head; Hearing; Hearing Impaired Persons; Human; Incidence; Individual; Infant; Kinetics; Knock-out; Knockout Mice; Labyrinth; Labyrinth Supporting Cells; Length; Life; Link; Maintenance; Mammals; Measures; Messenger RNA; Morphology; Mus; Mutant Strains Mice; Mutation; Myosin ATPase; Nature; Neonatal; Organ; Organ of Corti; Otic Vesicle; Paint; Pediatric Hospitals; Phase; Phenotype; Philadelphia; Process; Protein Isoforms; Proteins; RNA; RNA Splicing; RNA-Binding Proteins; Role; Sensorineural Hearing Loss; Sensory; Sensory Hair; Series; Space Perception; Specific qualifier value; Specificity; Structure; Supporting Cell; Temporal bone structure; Testing; Time; Tissues; Transcript; Transducers; Tube; United States; Vestibule; cell fate specification; cell type; cranium; deafness; ear development; epithelial to mesenchymal transition; exome sequencing; gene function; genetic regulatory protein; hearing impairment; improved; interest; loss of function mutation; mutant; mutant mouse model; next generation sequencing; novel; precursor cell; progenitor; public health relevance; recombinase; research study; sound; spatiotemporal; transcriptome sequencing

 DESCRIPTION (provided by applicant): The mammalian inner ear is a dual function organ comprised of the vestibular apparatus that senses spatial orientation and the cochlear duct which functions in sound transduction. The development of the cochlea is of particular interest due to the high incidence of hearing loss that occurs in the general population. The cochlear duct arises from a ventral outgrowth of the inner ear anlage, the otic vesicle. During its development the cochlea extends into a coiled tube that will contain the sensory hair and support cells of the Organ of Corti that are the transducers of sound. The function of the Organ of Corti is tightly coupled to the proper timing of events that occur during its formation. Sensory cell precursors must be specified from the otic epithelium, undergo cell-cycle exit and then differentiate into hair and support cells. The precise timing of these events is crucial for the proper development of the Organ of Corti, the cochlea as a whole, and, subsequently, the sense of hearing. While many of the genes responsible for each of these developmental events have been discovered the factors controlling their timing have yet to be identified. Recently, our collaborator, Dr. Ian Krantz (Children's Hospital of Philadelphia) identified mutations in the Epithelial Splicing Regulatory Protein 1 gene in several individuals with profound sensorineural deafness. In an effort to functionally link the deafness phenotype with mutations in Esrp1 we have begun to analyze mouse mutants in Esrp1 and/or Esrp2 for inner ear phenotypes. My preliminary results indicate that Esrp1 mutants exhibit a truncated cochlear duct and a significant delay in the development of auditory hair and support cells. The goal of this proposal is to determine the mechanism by which Esrp1 regulates the timing of sensory development in the inner ear. To accomplish this objective, I will systematically analyze the inner ears of Esrp mutants for alterations in the specification and cell-cycle exit of sensory progenitors, as well as the differentiation and maturation of cochlear hair and support cells (Aim1a). I will also perform RNAseq experiments on cochlear ducts from Esrp1-/- and control embryos to identify aberrantly spliced mRNA transcripts and potential targets of Esrp1 (Aim 1b). Finally, given the neonatal lethality of Esrp1-/- mutants from a cleft palate defect, I will generate a conditional knockout of Esrp1 in the inner ear to interrogate its requirement for hearing in adult mice (Aim 2). These experiments will uncover unique roles of Esrp genes in inner ear development and highlight the mechanism by which pathogenic mutations in Esrp1 result in deafness.

 描述（由申请人提供）：哺乳动物内耳是一种双重功能器官，由感知空间方向的前庭器和起声音传导作用的耳蜗管组成。由于普通人群中听力损失的发生率很高，因此耳蜗的发育受到特别关注。耳蜗管源自内耳原基（耳囊）的腹侧生长物。在其发育过程中，耳蜗延伸成一个盘绕管，其中包含感觉毛和柯蒂氏器的支持细胞，它们是声音的传感器。柯蒂氏器的功能与其形成过程中发生的事件的正确时间紧密相关。感官 细胞前体必须从耳上皮中分化出来，经历细胞周期退出，然后分化为毛发和支持细胞。这些事件的精确发生时间对于柯蒂氏器、整个耳蜗以及随后的听觉的正常发育至关重要。虽然许多负责这些发育事件的基因已被发现，但控制其发生时间的因素尚未确定。最近，我们的合作者 Ian Krantz 博士（费城儿童医院）在几名患有严重感音神经性耳聋的患者中发现了上皮剪接调节蛋白 1 基因的突变。为了在功能上将耳聋表型与 Esrp1 突变联系起来，我们已经开始分析小鼠 Esrp1 和/或 Esrp2 突变体的内耳表型。我的初步结果表明，Esrp1 突变体表现出耳蜗管截短以及听毛和支持细胞发育显着延迟。该提案的目标是确定 Esrp1 调节内耳感觉发育时间的机制。为了实现这一目标，我将系统地分析 Esrp 突变体的内耳，以了解感觉祖细胞的规格和细胞周期退出的变化，以及 耳蜗毛和支持细胞 (Aim1a) 的分化和成熟。我还将对 Esrp1-/- 和对照胚胎的耳蜗导管进行 RNAseq 实验，以识别异常剪接的 mRNA 转录本和 Esrp1 的潜在靶标（目标 1b）。最后，考虑到腭裂缺陷引起的 Esrp1-/- 突变体对新生儿的致死率，我将进行条件性敲除 内耳中的 Esrp1 询问其对成年小鼠听力的要求（目标 2）。这些实验将揭示 Esrp 基因在内耳发育中的独特作用，并强调 Esrp1 致病性突变导致耳聋的机制。