Molecular Analysis of Tweety Family Genes in Development and Tissue Homeostasis

在动物、植物甚至简单的真核生物中都发现了twety基因家族，该基因家族被认为编码门控基因。 氯通道在维持细胞体积中起重要作用。在脊椎动物中，twety基因家族高度 保守，由三个成员ttyh1、ttyh2和ttyh3组成，所有这些成员都有非常不同的 发育中胚胎的表达模式。Ttyh1和ttyh3几乎是互斥表达 发育中神经系统的模式，ttyh1在增殖区表达，ttyh3存在 几乎只存在于有丝分裂后区域。Ttyh2在眼睛和脑神经中表达。此外，两者 Ttyh1和ttyh3在RNA-Seq筛选中差异表达，以寻找调节胚胎能力的基因 在遗传和生理扰动后产生深刻的补偿性或再生反应。 尽管在脊椎动物谱系中有很强的保守性，并与广泛的人类 疾病，特别是侵袭性癌症，这个基因家族得到的关注相对较少。vt.给出 Ttyh1和ttyh3基因在胚胎发生、成体病理和再生中的动态表达 回应，这项建议的首要目标是阐明它们在发育中的胚胎和在 发育可塑性和动态平衡。具体目标1将侧重于确定ttyh1和ttyh1的作用 用ttyh3在正常胚胎发育过程中的功能获得和功能丧失实验。实验将会 利用非洲爪哇，一个经典的模型系统来剖析发育基因和 新兴的人类疾病模型，执行标准的、经过良好审查的异位表达方法 注射信使核糖核酸和CRISPR造成功能丧失。胚胎将在关键的发育阶段进行检测 利用一系列标记基因的整装原位杂交技术研究主要发育阶段 途径和细胞类型以及单细胞RNA-Seq，以获得单细胞水平的全球基因表达 目标是确定受功能增减实验影响的精确细胞类型和网络。 具体目标#2将集中于确定ttyh1和ttyh3在早期胚胎神经可塑性中的作用， 具体地说，它们在物理(前后轴旋转)后的恢复中的作用 遗传(Notch信号通路的过表达)扰动。基因和身体上的扰动将是 在通过CRISPR方法产生的ttyh1或ttyh3突变背景中进行。Ttyh1和Ttyh1的作用 Ttyh3基因敲除对恢复反应的分析将使用完整的补充方法 原位杂交和单细胞RNA-Sequ.综上所述，拟议的实验将进行 热心的本科生干部-无论是基于课程的研究还是指导的实验室研究-在 努力阐明twety基因家族的作用并解决基本的，但研究较少的问题 对再生医学有更广泛影响的发育生物学。

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.