Restriction of the Zebrafish Dorsal Organizer by Integrator Complex Subunit 6

集成复合体亚基 6 对斑马鱼背侧组织器的限制

基本信息

批准号：
10568987
负责人：
William Douglas Jones
金额：
$ 4.68万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-02 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10568987
关键词：
Alleles Attenuated Biochemical Body Patterning Cells ChIP-seq Complex DNA Polymerase II DNA-Directed RNA Polymerase Data Development Dorsal ENG gene Embryo Embryonic Development Enhancers Equilibrium Event Exhibits Fellowship Female Gene Expression Gene Expression Regulation Genes Genetic Genetic Complementation Test Genetic Transcription Genotype Human Human Development Introns Laboratories Lateral Malignant Neoplasms Mediating Mentors Messenger RNA Molecular Mutate Mutation Pattern Phenotype Proteins RNA RNA Processing Regulation Repression Reproduction Role Science Side Signal Transduction Small Nuclear RNA Small RNA Specific qualifier value Spliceosomes Temperature Testing TimeLine Tissues To specify Transcript Venus Vertebrates Zebrafish attenuation blastocyst career collaborative environment developmental disease differential expression embryo stage 2 exosome experimental study gastrulation helicase human female insight knock-down loss of function loss of function mutation member mutant overexpression prevent promoter supportive environment transcriptome sequencing transcriptomics

项目摘要

Project Summary/Abstract The vertebrate body axis is specified by an embryonic signaling center called the dorsal organizer. A unique zebrafish loss-of-function mutant isolated in our laboratory, called ints6p18ahub, promises to provide new insights into how the organizer is repressed to specify the proper balance of dorsoventral tissues. In this maternal-effect mutant, the progeny of homozygous ints6p18ahub mutant females (called M-ints6p18ahub embryos) display expanded organizers and multiple body axes. No other loss-of-function mutation in the zebrafish produces multiple body axes: therefore, this mutant will likely reveal a new mechanism of dorsal organizer repression. The mutated gene, Integrator complex subunit 6 (ints6), encodes a member of the Integrator complex, a spliceosomal small nuclear RNA processing complex that has not previously been implicated in vertebrate embryonic patterning. The experiments proposed here will uncover the mechanism by which Ints6 represses the dorsal organizer. Preliminary transcriptomic data support the hypothesis that a newly discovered function of Integrator, called transcriptional attenuation, represses dorsal gene expression in ventrolateral tissues to repress the organizer. First (Aim 1), RNA-seq and ChIP-seq will be utilized in M-ints6p18ahub embryos to evaluate Integrator’s five known biochemical activities in mutant embryos, determining which one is responsible for repressing the organizer. Second (Aim 2), Integrator’s 13 other subunits will be knocked down in M-ints6p18ahub mutant embryos to determine which knockdowns enhance the dorsalized phenotype, investigating which other subunits may participate in dorsal organizer repression by Ints6. Finally (Aim 3), a spatially localized genetic rescue experiment will be used in M-ints6p18ahub embryos to determine where Ints6 acts to repress the organizer. This will reveal how the new mechanism of dorsal organizer repression by Ints6 fits into the timeline of known embryonic patterning events in the zebrafish. In summary, the experiments proposed here will reveal how Ints6, a newly identified repressor of the dorsal organizer, restricts the organizer’s axis-specifying potential. This proposed fellowship will be performed in a collaborative and supportive environment to develop the applicant’s scientific independence and prepare for a career in science. The applicant will be mentored by a supportive thesis mentor and expert thesis committee.

项目概要/摘要脊椎动物的身体轴由称为“背部组织器”的胚胎信号中心指定。我们实验室分离出的斑马鱼功能丧失突变体，称为 ints6p18ahub，有望提供新的见解在这种母体效应中，组织者如何被抑制以指定背腹组织的适当平衡。突变体，纯合 ints6p18ahub 突变雌性的后代（称为 M-ints6p18ahub 胚胎）显示扩大斑马鱼中没有其他功能丧失突变产生多个身体。轴：因此，这种突变体可能会揭示背侧组织者抑制的新机制。基因，整合复合体亚基 6 (ints6)，编码整合复合体的一个成员，一个剪接体小核RNA加工复合物，以前从未涉及脊椎动物胚胎模式。这里提出的实验将揭示 Ints6 抑制背部组织者的机制。初步转录组数据支持这一假设，即新发现的 Integrator 功能称为转录减弱，抑制腹外侧组织中的背侧基因表达，从而抑制组织者。首先（目标 1），RNA-seq 和 ChIP-seq 将用于 M-ints6p18ahub 胚胎，以评估 Integrator 的五个已知突变胚胎中的生化活动，确定哪一种负责抑制组织者。其次（目标 2），Integrator 的其他 13 个亚基将在 M-ints6p18ahub 突变体胚胎中被敲除，以确定哪些敲低增强了背侧化表型，调查哪些其他亚基可能最后（目标 3）参与 Ints6 的背侧组织者抑制，这是一项空间局部基因拯救实验。将用于 M-ints6p18ahub 胚胎以确定 Ints6 在何处抑制组织者。 Ints6抑制背侧组织者的新机制如何适应已知胚胎的时间线总之，这里提出的实验将揭示新的 Ints6 是如何形成的。已确定的背部组织者的阻遏物，限制了组织者的轴指定潜力。奖学金将在协作和支持的环境中进行，以发展申请人的科学能力独立并为科学事业做好准备申请人将得到支持性论文导师的指导。和专家论文委员会。