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

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

Setting the Tempo in Development: An Investigation of the Zebrafish Somite Clock Mechanism

The somites of the vertebrate embryo are clocked out sequentially from the presomitic mesoderm (PSM) at the tail end of the embryo. Formation of each somite corresponds to one cycle of oscillation of the somite segmentation clock—a system of genes whose expression switches on and off periodically in the cells of the PSM. We have previously proposed a simple mathematical model explaining how the oscillations, in zebrafish at least, may be generated by a delayed negative feedback loop in which the products of two Notch target genes, her1 and her7, directly inhibit their own transcription, as well as that of the gene for the Notch ligand DeltaC; Notch signalling via DeltaC keeps the oscillations of neighbouring cells in synchrony. Here we subject the model to quantitative tests. We show how to read temporal information from the spatial pattern of stripes of gene expression in the anterior PSM and in this way obtain values for the biosynthetic delays and molecular lifetimes on which the model critically depends. Using transgenic lines of zebrafish expressing her1 or her7 under heat-shock control, we confirm the regulatory relationships postulated by the model. From the timing of somite segmentation disturbances following a pulse of her7 misexpression, we deduce that although her7 continues to oscillate in the anterior half of the PSM, it governs the future somite segmentation behaviour of the cells only while they are in the posterior half. In general, the findings strongly support the mathematical model of how the somite clock works, but they do not exclude the possibility that other oscillator mechanisms may operate upstream from the her7/her1 oscillator or in parallel with it

Spatial Fold Change of FGF Signaling Encodes Positional Information for Segmental Determination in Zebrafish

Summary: Signal gradients encode instructive information for numerous decision-making processes during embryonic development. A striking example of precise, scalable tissue-level patterning is the segmentation of somites—the precursors of the vertebral column—during which the fibroblast growth factor (FGF), Wnt, and retinoic acid (RA) pathways establish spatial gradients. Despite decades of studies proposing roles for all three pathways, the dynamic feature of these gradients that encodes instructive information determining segment sizes remained elusive. We developed a non-elongating tail explant system, integrated quantitative measurements with computational modeling, and tested alternative models to show that positional information is encoded solely by spatial fold change (SFC) in FGF signal output. Neighboring cells measure SFC to accurately position the determination front and thus determine segment size. The SFC model successfully recapitulates results of spatiotemporal perturbation experiments on both explants and intact embryos, and it shows that Wnt signaling acts permissively upstream of FGF signaling and that RA gradient is dispensable. : Simsek et al. use an elongation-arrested 3D explant system, integrated with quantitative measurements and computational modeling, to show that positional information for segmentation is encoded solely by spatial fold change (SFC) in FGF signal output. Neighboring cells measure SFC to accurately determine somite segment sizes. Wnt signaling acts permissively upstream of FGF signaling

Patterning principles of morphogen gradients

Metazoan embryos develop from a single cell into three-dimensional structured organisms while groups of genetically identical cells attain specialized identities. Cells of the developing embryo both create and accurately interpret morphogen gradients to determine their positions and make specific decisions in response. Here, we first cover intellectual roots of morphogen and positional information concepts. Focusing on animal embryos, we then provide a review of current understanding on how morphogen gradients are established and how their spans are controlled. Lastly, we cover how gradients evolve in time and space during development, and how they encode information to control patterning. In sum, we provide a list of patterning principles for morphogen gradients and review recent advances in quantitative methodologies elucidating information provided by morphogens

Estimation of Transcriptional Delay from In Situ Hybridisation Pattern

<p>The spatial offset between the anterior margin of the band of nuclear dots and the anterior margin of the band of cytoplasmic signal in specimens stained as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050150#pbio-0050150-g003" target="_blank">Figure 3</a> gives a measure of the transcriptional delay. To measure this offset, we used Photoshop to generate from the image of each specimen a pair of pictures, one (left-hand panel) showing only the ISH signal that was nuclear (i.e., co-localized with DNA staining), the other (middle panel) showing only the signal that was cytoplasmic (i.e., co-localized with an absence of DNA staining); note, however, that because of the non-zero thickness of the optical section, the “nuclear” signal includes a sizeable contribution from cytoplasmic mRNA where the latter is plentiful. For each of these two images, we computed the smoothed mean intensity of staining as a function of distance <i>x</i> along the anteroposterior axis (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050150#s4" target="_blank">Materials and Methods</a>), and plotted the results together on the same graph (right-hand panel). The delay from the beginning of the rise in nuclear signal to the beginning of the rise in cytoplasmic signal corresponds to the spatial offset <i>δx</i> between the minima of the red and blue curves. We converted this offset to a time interval using Equation 3 and the local values of <i>x, L, S</i>(<i>x</i>), and <i>S₀</i> measured from the same specimen.</p

Nascent and Mature Transcripts Visualized by In Situ Hybridisation

<p>Fluorescent staining by ISH for <i>her1</i> (top left)<i>, her7</i> (top middle)<i>,</i> and <i>deltaC</i> (top right)<i>,</i> using tyramide chemistry (green), reveals nuclear dots corresponding to nascent transcripts and cytoplasmic signal corresponding to mature mRNA. The bottom panel is a magnified detail of the top left panel. The images are confocal optical sections of flat-mounted specimens, counterstained for DNA with TOPRO3 (blue false colour). Red staining shows <i>myoD</i> expression by dual ISH.</p

Estimation of Clock Rate <i>ω</i>(<i>x</i>) and Period <i>T</i>(<i>x</i>) as a Function of Distance <i>x</i> along the PSM

<div><p>(A) Flat mount of a seven-somite embryo stained by ISH for <i>her1</i> (purple, NBT/BCIP detection chemistry) to show the oscillation pattern and for <i>myoD</i> (red, Fast Red detection chemistry) as a marker of formed somites. Spatial wavelength as a function of position, <i>S</i>(<i>x</i>), is measured as the interval between one <i>her1</i> peak and the next, or one trough and the next, where <i>x</i> is the midpoint of that interval. <i>S</i>₀ is the width of one formed somite. Position <i>x</i> = 0 corresponds to the tail end of the notochord, and position <i>x</i> = <i>L</i> corresponds to the anterior end of the PSM.</p> <p>(B and C) Clock frequency <i>ω</i>(<i>x</i>) and period <i>T</i>(<i>x</i>) as a function of distance <i>x</i> along the PSM. Each data point corresponds to one measurement of spatial wavelength <i>S</i>(<i>x</i>). The graph represents data from ten different, randomly chosen specimens of the type shown in (A) fixed at the 7–15-somite stages. The line (a hyperbola) is a least-squares fit to the data. To combine data from different specimens, distances are scaled relative to the length of the PSM. Clock rate and period are calculated for each data point according to the Equation 2 shown in the text. We use a simple linear approximation for <i>u</i>(<i>x</i>), the speed of forward movement of the cells along the anteroposterior axis relative to the tail end of the notochord: by definition, at <i>x</i> = 0, <i>u</i>(<i>x</i>) = 0, and at <i>x/L</i> = 1, <i>u</i>(<i>x</i>) = 1 (in somite lengths per somite cycle), so we assume <i>u</i>(<i>x</i>) = <i>x/L</i> at intermediate values of <i>x</i>.</p></div

Mapping of Temporal Oscillations into a Spatial Wave Pattern during Somite Segmentation, and a Model of the Oscillator Mechanism

<div><p>(A) The expression pattern of the oscillating gene <i>deltaC</i> in a zebrafish embryo at the ten-somite stage.</p> <p>(B) In each cell of the presomitic mesoderm, it is proposed that a <i>her1</i> or <i>her7</i> autoinhibition negative feedback loop generates oscillations.</p> <p>(C) Communication via the Delta-Notch pathway is proposed to keep oscillations in adjacent cells synchronized. The oscillations depend critically on the delays (<i>T</i>_m, <i>T</i>_p, <i>T</i>_md, and <i>T</i>_pd) in the feedback loops.</p> <p>(A–C) are slightly modified from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050150#pbio-0050150-b025" target="_blank">25</a>].</p></div

The Pattern of <i>deltaC</i> Transcripts Compared with the Pattern of DeltaC Protein in a Doubly Stained Specimen

<p>The left-hand panel shows the distribution of <i>deltaC</i> mRNA, as revealed by ISH; arrows indicate the three most recently formed somites. The middle panel shows the distribution of DeltaC protein in the same optical section, immunostained with the zdc2 monoclonal antibody. The right-hand panel shows the two patterns superimposed in the doubly stained specimen. The protein pattern is shifted by almost exactly one somite width relative to the mRNA pattern; one somite cycle time earlier, the cells currently displaying peak levels of protein would have been in the locations of the cells currently expressing the peak levels of mRNA and would have been expressing those levels of mRNA themselves. Thus there is a delay of approximately one somite cycle time (30 min) from the accumulation of the mRNA in the cell to the accumulation of the protein translated from it.</p

Delayed Somite Segmentation Defects following a Heat-Shock–Induced Pulse of HA-Her7

<p>The photographs show effects of a 60-min heat shock at 37 °C initiated at the five-somite stage, followed by 7 h of recovery at the normal incubation temperature, for (A) an <i>hsp70:HA-her7</i> transgenic and (B) a wild-type sibling control. In the transgenic, four somites (s6 to s9) have formed normally after the onset of heat shock, but a block of somites subsequent to that has been disrupted (arrowhead). Embryos are lightly fixed, but unstained, and viewed with dark-field illumination.</p