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 近四分之三的美国人在80岁时经历了嗅觉功能的衰退， 危害营养状况、人身安全，并对生活质量产生负面影响。不幸的是 目前没有有效的治疗方法来逆转年龄相关的嗅觉减退。这种感觉缺陷可以归因于 老年人嗅上皮（OE）内的病理恶化区域， 感觉神经元非神经元区域的存在是因为水平基底细胞（HBCs），OE的储备居民， 能够产生所有OE细胞类型干细胞群体仍然处于令人沮丧的休眠状态。激活 因此，在老年人OE中耐受的多能HBC代表了一种潜在的强大方法， 反向气味损失。我们的实验室先前已经证明了Notch的条件性敲除（cKO）， HBC中的Notch 1受体导致转录因子（TF）和主转录因子的HBC表达减少。 HBC状态的调节剂p63，并沉淀自发的HBC活化。然而，为了巧妙地操纵 目前已知的唯一信号通路调节HBCs的分子和功能状态，我们需要一个 深入了解Notch信号传导及其在指定HBC状态中的作用。这种需求是由于固有的 Notch信号通路的复杂性。这种复杂性的一个方面包括单个陷波的能力， 受体类型与多种配体相互作用，每种配体都可以不同地影响细胞分化。在 由HBCs表达的OE，Notch 1可以潜在地与几种Notch配体相互作用，包括Dll 1， Jagged 1也在HBCs中表达，Jagged 1定位于胶质样支持细胞。我的数据显示， HBc表达的Dll 1和Notch 1确实相互作用，Aim 1将确定Dll 1对HBc功能和免疫功能的影响。 通过（i）定量Hes 1，一种Notch信号传导- 在原代小鼠HBC培养物中Dll 1 cKO后的TF依赖性和p63表达。（二）利用 荧光激活细胞分选以定量HBC cKO后Hes 1和p63的体内HBC表达， Dll 1.为了探索Dll 1在HBCs中的长期功能作用，我将（iii）定量Dll 1在HBCs中的数量和身份。 Dll 1的HBC cKO后的HBC衍生后代。因为Notch配体-受体的功能输出 相互作用可以在组织形态发生过程中动态调节，目的2将阐明 Dll 1在由直接OE引起的HBC介导的OE再生模型中对活化HBC（aHBC）施加作用 损伤为此，我将（i）进行流式细胞术，以细胞分辨率将以下关系关联起来： HBC活性状态和Dll 1表达。我还将（ii）询问Dll 1的功能和分子影响， 在OE后立即通过病毒转导在HBCs中过表达Dll 1对再生有影响 损伤根据NIDCD的使命，该项目及其多学科方法将有助于设计医疗 治疗性激活HBCs并可控地再生OE以逆转年龄相关的 嗅觉减退因此，我将通过这项奖学金做好充分的准备，开始作为一名医生科学家的职业生涯。