Restriction of the Zebrafish Dorsal Organizer by Integrator Complex Subunit 6

项目摘要/摘要 脊椎动物的体轴由一个称为背部组织者的胚胎信号中心指定。独一无二的 我们实验室分离到的斑马鱼功能丧失突变体ints6p18aHub有望提供新的见解 了解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，纯合子ints6p18aHub突变雌性(称为M-ints6p18aHub胚胎)的后代显示扩大 组织器和多个躯干轴。斑马鱼中没有其他功能丧失突变产生多个身体 AXES：因此，这个突变体可能会揭示一种新的背部组织抑制机制。突变的 基因，整合子复合体亚基6(Ints6)，编码整合子复合体的一个成员，一个剪接小体 以前没有在脊椎动物胚胎模式中涉及的核RNA加工复合体。 这里提出的实验将揭示Ints6抑制背部组织者的机制。 初步的转录数据支持这样一种假设，即新发现的整合子的功能，称为 转录减弱，抑制腹外侧部组织中背部基因的表达，从而抑制组织者。 First(Aim 1)、RNA-seq和Chip-seq将用于M-ints6p18aHub胚胎，以评估集成商的五个已知 突变胚胎中的生化活动，决定哪一个负责抑制组织者。 第二个(目标2)，整合者的其他13个亚基将在M-ints6p18aHub突变胚胎中被击倒，以 确定哪些敲除增强了背侧表型，调查了其他哪些亚基可能 参与Ints6对背部组织者的抑制。最后(目标3)，空间定位的遗传拯救实验 将用于M-ints6p18aHub胚胎，以确定Ints6在哪里抑制组织者。这将揭示 Ints6抑制背部组织因子的新机制如何与已知胚胎的时间线相适应 斑马鱼中的图案事件。总之，这里提出的实验将揭示Ints6，一个新的 被识别的背部组织者的抑制子，限制了组织者的轴指定潜力。这项建议 奖学金将在合作和支持的环境中进行，以发展申请者的科学 独立，为从事科学事业做准备。申请者将由一位支持性的论文导师进行指导 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.