Restriction of the Zebrafish Dorsal Organizer by Integrator Complex Subunit 6

项目概要/摘要 脊椎动物的体轴由称为背侧组织器的胚胎信号中心指定。一个独特的 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 研究组织者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 ints6p18ahub 突变雌性的后代（称为 M-ints6p18ahub 胚胎）显示扩大 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，这种突变体可能会揭示一种新的背部组织抑制机制。变异者 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样的假设：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 参与 Ints6 的背部组织者抑制。最后（目标 3），空间局部基因拯救实验 将用于 M-ints6p18ahub 胚胎以确定 Ints6 在何处发挥作用来抑制组织者。这将揭示 Ints6抑制背侧组织者的新机制如何适应已知胚胎的时间线 斑马鱼的模式化事件。总之，这里提出的实验将揭示 Ints6（一种新的 已识别的背部组织者的阻遏物，限制了组织者的轴指定潜力。这个提议 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。

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.