Notch Signalling Synchronizes the Zebrafish Segmentation Clock but Is Not Needed To Create Somite Boundaries

Abstract

Somite segmentation depends on a gene expression oscillator or clock in the posterior presomitic mesoderm (PSM) and on read-out machinery in the anterior PSM to convert the pattern of clock phases into a somite pattern. Notch pathway mutations disrupt somitogenesis, and previous studies have suggested that Notch signalling is required both for the oscillations and for the read-out mechanism. By blocking or overactivating the Notch pathway abruptly at different times, we show that Notch signalling has no essential function in the anterior PSM and is required only in the posterior PSM, where it keeps the oscillations of neighbouring cells synchronized. Using a GFP reporter for the oscillator gene her1, we measure the influence of Notch signalling on her1 expression and show by mathematical modelling that this is sufficient for synchronization. Our model, in which intracellular oscillations are generated by delayed autoinhibition of her1 and her7 and synchronized by Notch signalling, explains the observations fully, showing that there are no grounds to invoke any additional role for the Notch pathway in the patterning of somite boundaries in zebrafish. The somites—the embryonic segments of the vertebrate body—form one after another from tissue at the tail end of the embryo. A gene expression oscillator, the somite segmentation clock, operating in this tail region, marks out a periodic spatial pattern and so controls the segmentation process. Evidence from mutants shows that the Notch cell-cell signalling pathway has a critical role in the clock mechanism. However, when we switch on a blockade of Notch signalling, by immersing zebrafish embryos in the chemical inhibitor DAPT, the next ∼12 somites form normally, and only after that do disrupted somites appear. We show that this is because Notch signalling is needed only to maintain synchrony between the clocks of individual cells. The cells take about seven cycles to drift out of synchrony when Notch-mediated communication is blocked, and then a further five cycles to pass from the site where the tissue receives its “time-stamp” to the site where overt segmentation begins. By mathematical modelling, backed up with measurements on transgenic embryos, we show how Notch signalling may act at a molecular level to synchronise the intracellular oscillators of adjacent individual cells.