Restriction of the Zebrafish Dorsal Organizer by Integrator Complex Subunit 6

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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.