The Role of Dll1 in Specifying the Molecular and Functional Status of Horizontal Basal Cells

Dll1 在指定水平基底细胞的分子和功能状态中的作用

• 批准号: 10400899
• 负责人: Jonathan Daniel Louie
• 金额: $ 5.18万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-16 至 2024-05-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10400899
• 关键词: Age; American; Anosmia; Basal Cell; Binding; Clinical Treatment; Communication; Data; Development; Developmental Biology; Failure; Fellowship; Flow Cytometry; Fluorescence-Activated Cell Sorting; Foundations; Genetic Transcription; Grant; Hour; Human; In Vitro; Knowledge; Laboratories; Lesion; Ligands; Mediating; Medical; Mission; Modeling; Molecular; Morphogenesis; Mus; National Institute on Deafness and Other Communication Disorders; Natural regeneration; Notch Signaling Pathway; Nutritional status; Olfactory Epithelial Cell; Olfactory Epithelium; Olfactory dysfunction; Output; Pathologic; Pathway interactions; Physicians; Play; Principal Investigator; Quality of life; Resolution; Role; Safety; Scientist; Sensory; Signal Pathway; Signal Transduction; Smell Perception; Specific qualifier value; Testing; Therapeutic; Tissues; Training; Up-Regulation; Viral; age related; aged; aging demographic; attenuation; career; cell type; conditional knockout; design; effective therapy; epithelial injury; epithelium regeneration; exhaust; experience; functional decline; functional status; hyposmia; in vivo; interdisciplinary approach; knock-down; mouse model; notch protein; novel; olfactory sensory neurons; overexpression; premature; prevent; receptor; stem; stem cell biology; stem cell population; sustentacular cell; transcription factor

Project Summary A functional decline in smell, which is experienced by nearly three-quarters of Americans by age 80, can endanger nutritional status, physical safety, and negatively impact quality of life. Unfortunately, there are currently no effective treatments to reverse age-related hyposmia. This sensory deficit can be attributed to pathologically deteriorated regions within aged human olfactory epithelium (OE) that are void of olfactory sensory neurons. Aneuronal regions persist because horizontal basal cells (HBCs), the OE's reserve resident stem cell population that is capable of giving rise to all OE cell types, remain frustratingly dormant. Activating multipotent HBCs that endure within aged human OE, therefore, represents a potentially powerful approach to reverse smell loss. Our laboratory has previously demonstrated that conditional knock out (cKO) of the Notch receptor, Notch1, in HBCs leads to decreased HBC expression of the transcription factor (TF) and master regulator of HBC status, p63, and precipitates spontaneous HBC activation. However, to deftly manipulate the only signaling pathway currently known to regulate the molecular and functional status of HBCs, we require a deeper understanding of Notch signaling and its role in specifying HBC status. This need is due to the inherent complexities of the Notch signaling pathway. One aspect of this intricacy includes the ability for a single Notch receptor type to interact with multiple ligands, each of which can differentially impact cellular differentiation. In the OE, Notch1 expressed by HBCs can potentially interact with several Notch ligands, including Dll1 that is also expressed by HBCs and Jagged1 localized to glial-like Sustentacular cells. As my data demonstrates that HBC-expressed Dll1 and Notch1 do indeed interact, Aim 1 will establish Dll1's effect on the functional and molecular identity of dormant HBCs (dHBCs) within uninjured OE by (i) quantifying Hes1, a Notch signaling- dependent TF, and p63 expression following Dll1 cKO in primary mouse HBC cultures. I will also (ii) utilize fluorescence activated cell sorting to quantify in vivo HBC expression of Hes1 and p63 following HBC cKO of Dll1. To probe the longer-term functional role of Dll1 in HBCs, I will (iii) quantify the number and identity of HBC-derived progeny following HBC cKO of Dll1. Because the functional output of Notch ligand-receptor interactions can be dynamically regulated during tissue morphogenesis, Aim 2 will elucidate the influence that Dll1 exerts on activated HBCs (aHBCs) in a model of HBC-mediated OE regeneration elicited by direct OE injury. To do so, I will (i) perform flow cytometry to correlate, with cellular resolution, the relationship between HBC activity status and Dll1 expression. I will also (ii) interrogate the functional and molecular impact that Dll1 has on regeneration by overexpressing Dll1 in HBCs via viral transduction in the immediate aftermath of OE injury. In line with NIDCD's mission, this project and its multidisciplinary approach will aid the design of medical treatments that therapeutically activate HBCs and controllably regenerate the OE to reverse age-related hyposmia. As a result, I will be well prepared by this fellowship to embark on a career as a physician-scientist.

项目摘要 气味的功能下降，到80岁的美国人近四分之三的气味会经历 危害营养状况，身体安全性并对生活质量产生负面影响。不幸的是，有 目前尚无有效的治疗方法来逆转与年龄相关的低血症。这种感觉赤字可以归因于 在老年人嗅觉上皮（OE）内的病理恶化区域，这些区域无效 感觉神经元。动脉元区域持续存在，因为水平基底细胞（HBC），OE的储备居民 能够引起所有OE细胞类型的干细胞群体仍然令人沮丧。激活 因此 反向气味损失。我们的实验室以前已经证明了条件淘汰（CKO） HBC中的受体Notch1导致转录因子（TF）和Master的HBC表达降低 HBC状态，p63和沉淀的调节剂自发HBC激活。但是，巧妙地操纵 仅当前已知的信号通路来调节HBC的分子和功能状态，我们需要一个 对Notch信号传导及其在指定HBC状态中的作用的更深入了解。这种需求是由于固有的 Notch信号通路的复杂性。这个复杂的一个方面包括单个缺口的能力 受体类型与多种配体相互作用，每种配体都会差异地影响细胞分化。在 HBC表达的OE，Notch1可能与几种缺口配体相互作用，包括DLL1 也由HBCS和JAGGEND1局部局限于神经胶质样的静脉内细胞表达。当我的数据表明 HBC表达的DLL1和Notch1确实确实相互作用，AIM 1将建立DLL1对功能的影响，并且 （i）量化Hes1（notch信号传导），在未伤害的OE中休眠HBC（DHBC）的分子身份 原代小鼠HBC培养物中DLL1 CKO后的依赖性TF和p63表达。我还将（ii）使用 HBC CKO之后，荧光激活的细胞分选中HES1和p63的体内HBC表达 DLL1。为了探测DLL1在HBC中的长期功能作用，我将（iii）量化 HBC衍生的后代DLL1之后。因为Notch配体受体的功能输出 可以在组织形态发生过程中动态调节相互作用，AIM 2将阐明影响的影响 DLL1在直接OE引起的HBC介导的OE再生模型中施加活化的HBC（AHBC） 受伤。为此，我将（i）执行流式细胞术以与蜂窝分辨率相关， HBC活动状态和DLL1表达。我还将（ii）询问DLL1的功能和分子影响 通过在OE立即通过病毒转导过度表达HBC中的DLL1通过过度表达DLL1的再生 受伤。根据NIDCD的使命，该项目及其多学科方法将有助于医疗设计 治疗的治疗方法可以激活HBC并可控制地再生OE以逆转与年龄有关 低质量。结果，这项奖学金将为我做好准备，以开始从事医师科学家的职业。