The elongation rate of RNA polymerase II in zebrafish and its significance in the somite segmentation clock

Abstract

SUMMARYA 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. RESEARCH ARTICLE Gene length and the somite clockThis formula is derived for the idealised case of a single 'her1/7' autoinhibitory gene or, equivalently, of a pair of genes, her1 and her7, that have the same delays, lifetimes and regulation. Computer modelling shows that if her1 and her7 have somewhat different delays and lifetimes but are co-regulated, oscillations will occur with a period that is a compromise between that for a pure her1 oscillator and that for a pure her7 oscillator. Mutants in which her1 remains intact but her7 is functionally null, or vice versa, have recently become available, and in this paper we use them to test this prediction. Because the her1 and her7 genes are very different in length ( Fig. 2A), we anticipated that they should have different transcriptional delays, leading to different periods of oscillation. To our surprise, we found that the difference of period is actually very small. To resolve this paradox, we measured the elongation rate of RNA polymerase II (RNA Pol II), for the first time in vivo in a vertebrate. The value, as measured in the PSM cells of the zebrafish, is 4.8 kb/minute at 28.5°C. This unexpectedly high rate means that the time taken to transcribe the two genes is so short as to be insignificant in comparison with other sources of delay, such as the time required for splicing. These findings reconcile our theory with the experimental observations and remove an important objection to the proposition that her1 and her7 are pacemakers of the seg...

Spatial gradients of protein-level time delays set the pace of the traveling segmentation clock waves

“…We and others have also reported that traveling wave-like expression patterns are significantly disturbed in her7 −/− mutants; there is also a large variation from embryo to embryo from the same mutant clutch (Choorapoikayil et al, 2012;Hanisch et al, 2013;Schroter et al, 2012). Our model nicely recapitulates this phenotype.…”

“…We and others have recently reported that her1 −/− embryos halt their oscillations earlier in the PSM and manifest only one or two expression waves at mid-somitogenesis stages; this results in the formation of one less stripe in the anterior PSM in her1 −/− mutants (Choorapoikayil et al, 2012;Hanisch et al, 2013) compared with wild-type, hes6 −/− and her7 −/− ;hes6 −/− embryos (Schroter et al, 2012). Our model reproduced this observation successfully (Fig.…”

“…Quantitative data have become more available in the somitogenesis field in recent years. Molecular-level models coupled with experimentation have provided insight into how the dynamics of the segmentation clock in the posterior PSM cells can be affected in various mutant conditions (Ay et al, 2013;Hanisch et al, 2013;Özbudak and Lewis, 2008;Schroter et al, 2012). Although we have a good understanding of how the oscillator functions in the posterior PSM, the underlying mechanism that generates the spatiotemporal traveling waves in the whole PSM has remained elusive.…”

“…Although these studies provided insight into the system, these models were based on regulatory assumptions that have been corrected by recent experimental results (Hanisch et al, 2013;Schroter et al, 2012;Trofka et al, 2012). Furthermore, these models focused on simulating the system only in a wild-type background and were therefore largely unconstrained.…”

Modeling the Zebrafish Segmentation Clock’s Gene Regulatory Network Constrained by Expression Data Suggests Evolutionary Transitions Between Oscillating and Nonoscillating Transcription

“…The her/hes genes form both hetero-and homodimers and negatively regulate their own transcription (Hirata et al 2002;Oates and Ho 2002;Bessho et al 2003;Giudicelli et al 2007;Brend and Holley 2009a;Schröter et al 2012;Trofka et al 2012;Hanisch et al 2013). Due to a short half-life, this negative feedback results in repeated cycles of expression and repression of the her/hes genes within the presomitic mesoderm (PSM), the frequency of which correlates with the rate of somite formation (Lewis 2003;Monk 2003;Hirata et al 2004;Ishii et al 2008;Schröter and Oates 2010;Takashima et al 2011;Delaune et al 2012;Ay et al 2013;Harima et al 2013).…”