title: The elongation rate of RNA polymerase II in zebrafish and its significance in the somite segmentation clock.
abstract: A gene expression oscillator called the segmentation clock controls somite segmentation in the vertebrate embryo. In zebrafish, the oscillatory transcriptional repressor genes her1 and her7 are crucial for genesis of the oscillations, which are thought to arise from negative autoregulation of these genes. The period of oscillation is predicted to depend on delays in the negative-feedback loop, including, most importantly, the transcriptional delay - the time taken to make each molecule of her1 or her7 mRNA. her1 and her7 operate in parallel. Loss of both gene functions, or mutation of her1 combined with knockdown of Hes6, which we show to be a binding partner of Her7, disrupts segmentation drastically. However, mutants in which only her1 or her7 is functional show only mild segmentation defects and their oscillations have almost identical periods. This is unexpected because the her1 and her7 genes differ greatly in length. We use transgenic zebrafish to measure the RNA polymerase II elongation rate, for the first time, in the intact embryo. This rate is unexpectedly rapid, at 4.8 kb/minute at 28.5°C, implying that, for both genes, the time taken for transcript elongation is insignificant compared with other sources of delay, explaining why the mutants have similar clock periods. Our computational model shows how loss of her1 or her7 can allow oscillations to continue with unchanged period but with reduced amplitude and impaired synchrony, as manifested in the in situ hybridisation patterns of the single mutants.
authors.affiliation: Vertebrate Development Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK.
authors.forename: Anja
authors.initials: A
authors.lastname: Hanisch
authors.forename: Maxine V
authors.initials: MV
authors.lastname: Holder
authors.forename: Suma
authors.initials: S
authors.lastname: Choorapoikayil
authors.forename: Martin
authors.initials: M
authors.lastname: Gajewski
authors.forename: Ertugrul M
authors.initials: EM
authors.lastname: Özbudak
authors.forename: Julian
authors.initials: J
authors.lastname: Lewis
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D008954:Models, Biological; D008962:Models, Theoretical; D009154:Mutation; D009991:Oscillometry / Q000379:methods; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D019170:Somites / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000378:metabolism
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D005452:Fluoresceins; D012319:RNA Polymerase II; D014157:Transcription Factors; D029961:Zebrafish Proteins; C460099:HER7 protein, zebrafish; C491619:her1 protein, zebrafish; C007740:fluorexon