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

• 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
• 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

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.

项目总结/摘要 脊椎动物的身体轴是由一个称为背侧组织者的胚胎信号中心指定的。一个独特 在我们的实验室中分离出一种名为ints 6p 18 ahub的斑马鱼功能丧失突变体，有望提供新的见解 组织者是如何被抑制以指定背腹组织的适当平衡。在这种母性效应中， 突变体，纯合ints 6p 18 ahub突变体雌性的后代（称为M-ints 6p 18 ahub胚胎）显示扩增 组织者和多个身体轴。斑马鱼中没有其他功能丧失突变产生多体 轴：因此，该突变体将可能揭示背侧组织抑制的新机制。突变的 整合子复合体亚基6（ints 6）基因编码整合子复合体的一个成员，一个剪接体小 核RNA加工复合物，以前没有涉及脊椎动物胚胎图案。 这里提出的实验将揭示Ints 6抑制背侧组织者的机制。 初步的转录组学数据支持一个新发现的Integrator功能的假设， 转录衰减，抑制背侧基因表达的腹外侧组织抑制组织者。 首先（目的1），将在M-ints 6p 18 ahub胚胎中使用RNA-seq和ChIP-seq来评估Integrator的五个已知的 突变胚胎的生化活动，决定哪一个负责抑制组织者。 第二（目的2），整合子的13个其他亚基将在M-ints 6p 18 ahub突变胚胎中被敲低， 确定哪些敲除增强背侧化表型，研究哪些其他亚基可能 参与Ints 6对背侧组织者的抑制。最后（目标3），空间定位的遗传拯救实验 将在M-ints 6p 18 ahub胚胎中使用，以确定Ints 6在何处抑制组织者。这将揭示 Ints 6抑制背侧组织者的新机制如何符合已知胚胎发育的时间轴 在斑马鱼中形成模式总之，这里提出的实验将揭示Ints 6，一个新的 确定的阻遏物的背组织者，限制组织者的轴指定的潜力。这一拟议 奖学金将在一个合作和支持的环境中进行，以发展申请人的科学 独立并为科学事业做好准备。申请人将由支持论文导师指导 专家论文委员会。