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Molecular Analysis of Tweety Family Genes in Development and Tissue Homeostasis

Tweety 家族基因在发育和组织稳态中的分子分析

基本信息

批准号：
10806487
负责人：
MARGARET S SAHA
金额：
$ 45.21万
依托单位：
COLLEGE OF WILLIAM AND MARY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-20 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10806487
关键词：
Address Adult Affect Alzheimer&apos s Disease Animals Anterior Attention Biological Assay Biological Markers Biological Models CRISPR/Cas technology Cell Maintenance Cell Volumes Cells Chloride Channels Clinical Clustered Regularly Interspaced Short Palindromic Repeats Cranial Nerves Data Analyses Development Developmental Biology Developmental Gene Disease Ectopic Expression Embryo Embryonic Development Eukaryota Exhibits Eye Family Future Gastrula Gene Expression Gene Family Genes Genetic Goals Homeostasis In Situ Hybridization Injections Knock-out Knowledge Malignant Neoplasms Mentors Messenger RNA Modeling Molecular Analysis Morphology Nervous System Neuronal Plasticity Notch Signaling Pathway Organism Parkinson Disease Pathologic Pathology Pathway interactions Pattern Phylogenetic Analysis Physiological Physiological Processes Plants Process Publishing Recovery Regenerative Medicine Regenerative response Research Role Rotation Swimming Tadpoles Therapeutic Tissues Turner&apos s Syndrome Vertebrates Xenopus laevis cell type developmental plasticity differential expression experience experimental study gain of function human disease immune function knockout gene loss of function malignant neurologic neoplasms member mutant nerve stem cell nervous system disorder neural patterning neurodevelopment overexpression postmitotic regenerative response single-cell RNA sequencing transcriptome transcriptome sequencing undergraduate research experience undergraduate student

项目摘要

Identified in animals, plants, and even simple eukaryotes, the tweety gene family is thought to encode gated chloride channels important in the maintenance of cell volume. In vertebrates, the tweety gene family is highly conserved and consists of three members, ttyh1, ttyh2, and ttyh3, all of which have strikingly different expression patterns in the developing embryos. ttyh1 and ttyh3, display almost mutually exclusive expression patterns in the developing nervous system, with ttyh1 expressed in the proliferative zone and ttyh3 present almost exclusively in post-mitotic regions. ttyh2 is expressed in the eye and cranial nerves. Additionally, both ttyh1 and ttyh3 are differentially expressed in RNA-Seq screens for genes that regulate the ability of embryos to mount a profound compensatory or regenerative response following genetic and physical perturbations. Despite the strong conservation in the vertebrate lineage, and being implicated in a wide range of human diseases, particularly aggressive cancers, this family of genes has received relatively minimal attention. Given the dynamic expression of ttyh1 and ttyh3 genes in embryogenesis, adult pathologies, and regenerative responses, the overarching goal of this proposal is to elucidate their role in the developing embryo and in developmental plasticity and homeostasis. Specific Aim #1 will focus on determining the roles of ttyh1 and ttyh3 in normal embryogenesis using gain-of-function and loss of function experiments. Experiments will employ Xenopus laevis, a classic model system for dissecting the role of developmental genes and an emerging model for human disease, to perform standard, well-vetted approaches of ectopic expression using mRNA injection and CRISPR to generate loss-of-function. Embryos will be assayed at key developmental stages using whole mount in situ hybridization with a range of marker genes for the major developmental pathways and cell types and single cell RNA-Seq to obtain global gene expression at a single cell level with the goal of identifying the precise cell types and networks affected by the gain- and loss-of-function experiments. Specific Aim #2 will focus on determining the role of ttyh1 and ttyh3 in early embryonic neural plasticity, specifically their role in the recovery that occurs following physical (rotation of the anterior-posterior axis) or genetic (overexpression of the Notch signaling pathway) perturbation. Genetic and physical perturbation will be performed in a ttyh1 or ttyh3 mutant background generated via a CRISPR approach. The effect of ttyh1 and ttyh3 knockouts on the recovery response will be assayed using the complementary approaches of whole mount in situ hybridization and single cell RNA-Seq. Taken together, the proposed experiments will engage an eager cadre of undergraduate students—both in course-based research and mentored lab research—in an effort to elucidate the role of the tweety gene family and address fundamental, yet poorly studied, problems in developmental biology that have broader implications for regenerative medicine.

在动物，植物，甚至简单的真核生物中鉴定，tweety基因家族被认为编码门控氯离子通道在维持细胞体积方面很重要。在脊椎动物中，tweety基因家族高度保守的，由三个成员组成，ttyh 1，ttyh 2和ttyh 3，所有这些都有惊人的不同在发育中的胚胎中的表达模式。ttyh 1和ttyh 3，显示几乎互斥的表达式在发育中的神经系统中的模式，ttyh 1在增殖区表达，ttyh 3存在几乎只在有丝分裂后的区域。TTYH 2在眼和颅神经中表达。此外，两者 ttyh 1和ttyh 3在调节胚胎发育能力的基因的RNA-Seq筛选中差异表达在遗传和身体受到干扰后，产生深刻的补偿或再生反应。尽管在脊椎动物谱系中具有很强的保守性，并且与广泛的人类疾病有关，然而，对于癌症，特别是侵袭性癌症，这一基因家族受到的关注相对较少。给定 ttyh 1和ttyh 3基因在胚胎发生、成体病理和再生中的动态表达响应，这项建议的总体目标是阐明它们在发育胚胎中的作用，发育可塑性和内稳态。具体目标#1将侧重于确定ttyh 1和 ttyh 3在正常胚胎发生中使用功能获得和功能丧失实验。实验将采用非洲爪蟾，一个经典的模型系统解剖发育基因的作用，新兴的人类疾病模型，执行标准的，经过严格审查的异位表达方法， mRNA注射和CRISPR以产生功能丧失。将在关键发育阶段对胚胎进行检测阶段使用全坐骑原位杂交与一系列标记基因的主要发育途径和细胞类型以及单细胞RNA-Seq，以获得单细胞水平的全局基因表达，目标是识别受功能获得和丧失实验影响的精确细胞类型和网络。具体目标#2将侧重于确定ttyh 1和ttyh 3在早期胚胎神经可塑性中的作用，特别是它们在物理（前后轴的旋转）或遗传（Notch信号通路的过表达）干扰。遗传和物理干扰将是在通过CRISPR方法产生的ttyh 1或ttyh 3突变体背景中进行。ttyh 1和 ttyh 3基因敲除对恢复应答的影响将使用整体分析的互补方法进行分析。进行原位杂交和单细胞RNA-Seq。总之，拟议的实验将涉及一个热切的本科生干部-无论是在基于课程的研究和指导实验室研究-在一个努力阐明tweety基因家族的作用，并解决基本的，但研究不足的问题，发育生物学对再生医学有更广泛的影响。