A spatio-temporal model of Notch signalling in the zebrafish segmentation clock: conditions for synchronised oscillatory dynamics

In the vertebrate embryo, tissue blocks called somites are laid down in head-to-tail succession, a process known as somitogenesis. Research into somitogenesis has been both experimental and mathematical. For zebrafish, there is experimental evidence for oscillatory gene expression in cells in the presomitic mesoderm (PSM) as well as evidence that Notch signalling synchronises the oscillations in neighbouring PSM cells. A biological mechanism has previously been proposed to explain these phenomena. Here we have converted this mechanism into a mathematical model of partial differential equations in which the nuclear and cytoplasmic diffusion of protein and mRNA molecules is explicitly considered. By performing simulations, we have found ranges of values for the model parameters (such as diffusion and degradation rates) that yield oscillatory dynamics within PSM cells and that enable Notch signalling to synchronise the oscillations in two touching cells. Our model contains a Hill coefficient that measures the co-operativity between two proteins (Her1, Her7) and three genes (her1, her7, deltaC) which they inhibit. This coefficient appears to be bounded below by the requirement for oscillations in individual cells and bounded above by the requirement for synchronisation. Consistent with experimental data and a previous spatially non-explicit mathematical model, we have found that signalling can increase the average level of Her1 protein. Biological pattern formation would be impossible without a certain robustness to variety in cell shape and size; our results possess such robustness. Our spatially-explicit modelling approach, together with new imaging technologies that can measure intracellular protein diffusion rates, is likely to yield significant new insight into somitogenesis and other biological processes.     

DeltaC and DeltaD interact as Notch ligands in the zebrafish segmentation clock

We describe the production and characterisation of two monoclonal antibodies, zdc2 and zdd2, directed against the zebrafish Notch ligands DeltaC and DeltaD, respectively. We use our antibodies to show that these Delta proteins can bind to one another homo- and heterophilically, and to study the localisation of DeltaC and DeltaD in the zebrafish nervous system and presomitic mesoderm (PSM). Our findings in the nervous system largely confirm expectations from previous studies, but in the PSM we see an unexpected pattern in which the localisation of DeltaD varies according to the level of expression of DeltaC: in the anterior PSM, where DeltaC is plentiful, the two proteins are colocalised in intracellular puncta, but in the posterior PSM, where DeltaC is at a lower level, DeltaD is seen mainly on the cell surface. Forced overexpression of DeltaC reduces the amount of DeltaD on the cell surface in the posterior PSM; conversely, loss-of-function mutation of DeltaC increases the amount of DeltaD on the cell surface in the anterior PSM. These findings suggest an explanation for a long-standing puzzle regarding the functions of the two Delta proteins in the somite segmentation clock--an explanation that is based on the proposition that they associate heterophilically to activate Notch.     

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.     

Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function

Cell division, differentiation and morphogenesis are coordinated during embryonic development, and frequently are in disarray in pathologies such as cancer. Here, we present a zebrafish mutant that ceases mitosis at the beginning of gastrulation, but that undergoes axis elongation and develops blood, muscle and a beating heart. We identify the mutation as being in early mitotic inhibitor 1 (emi1), a negative regulator of the Anaphase Promoting Complex, and use the mutant to examine the role of the cell cycle in somitogenesis. The mutant phenotype indicates that axis elongation during the segmentation period is driven substantially by cell migration. We find that the segmentation clock, which regulates somitogenesis, functions normally in the absence of cell cycle progression, and observe that mitosis is a modest source of noise for the clock. Somite morphogenesis involves the epithelialization of the somite border cells around a core of mesenchyme. As in wild-type embryos, somite boundary cells are polarized along a Fibronectin matrix in emi1(-/-). The mutants also display evidence of segment polarity. However, in the absence of a normal cell cycle, somites appear to hyper-epithelialize, as the internal mesenchymal cells exit the core of the somite after initial boundary formation. Thus, cell cycle progression is not required during the segmentation period for segmentation clock function but is necessary for the normal segmental arrangement of epithelial borders and internal mesenchymal cells.     

Patterning embryos with oscillations: structure, function and dynamics of the vertebrate segmentation clock

The segmentation clock is an oscillating genetic network thought to govern the rhythmic and sequential subdivision of the elongating body axis of the vertebrate embryo into somites: the precursors of the segmented vertebral column. Understanding how the rhythmic signal arises, how it achieves precision and how it patterns the embryo remain challenging issues. Recent work has provided evidence of how the period of the segmentation clock is regulated and how this affects the anatomy of the embryo. The ongoing development of real-time clock reporters and mathematical models promise novel insight into the dynamic behavior of the clock.     

And the segmentation clock keeps ticking

The vertebrate body is organized in segments, easily visible in the consecutive vertebrae of the skeleton. These are first defined in the embryo by the formation of somites. Somites are generated at regular intervals from the presomitic mesoderm by a combination of oscillating signals, known as the segmentation clock, which establish the pace at which new somites are formed, and signaling gradients that set the location of new intersomitic borders. Using a microarray approach, Dequéant et al. have now shown that the segmentation clock is more complex than previously thought and includes oscillating expression of genes from at least three signaling pathways organized in coordinated networks.     

斑马鱼生物钟研究进展

斑马鱼是生物钟研究领域中一种新兴的脊椎动物模型。文章总结了斑马鱼生物钟研究的一些进展, 以及利用斑马鱼研究生物钟的特点及优势。由于光照和温度作为重要的外部信号在斑马鱼生物钟调节中发挥重要作用, 文章主要就近期光和温度对斑马鱼钟基因及调节通路的研究进行了概述, 最后对斑马鱼生物钟研究的未来提出了展望。     

Light reaches the very heart of the zebrafish clock

Zebrafish are typically used as a model system to study various aspects of developmental biology, largely as a consequence of their ex vivo development, high degree of transparency, and, of course, ability to perform forward genetic mutant screens. More recently, zebrafish have been developed as a model system with which to study circadian clocks. Cell lines generated from early-stage zebrafish embryos contain clocks that are directly light-responsive. We describe recent experiments using single-cell luminescent imaging approaches to study clock function in this novel cell line system. Furthermore, studies examining the process of entrainment to light pulses within this cell population are described in this review, as are experiments examining light-responsiveness of early-stage zebrafish embryos.     

Evolution of segmentation: rolling back the clock

Recent work has revealed striking similarities in the genetic mechanisms underpinning somitogenesis in zebrafish and segmentation in the spider. Could this mean that the bilaterian common ancestor was segmented after all?     

The vertebrate segmentation clock: the tip of the iceberg

The vertebrate segmentation clock was identified 10 years ago as a molecular oscillator associated with the rhythmic production of embryonic somites. Since then, three major signaling pathways--Notch, FGF, and Wnt--have been shown to be activated periodically during segmentation and proposed to constitute the clockwork of the system. However, recent results from zebrafish embryonic studies demonstrate that Notch signaling is involved in the coupling of oscillations among cells rather than in the pacemaker of the oscillator. Furthermore, genetic analyses in mouse indicate that Wnt and FGF play only a permissive role in the control of the oscillations. Therefore, the nature of the segmentation clock pacemaker still remains elusive.     

Dopaminergic and noradrenergic circuit development in zebrafish

Dopaminergic and noradrenergic neurons constitute some of the major far projecting systems in the vertebrate brain and spinal cord that modulate the activity of circuits controlling a broad range of behaviors. Degeneration or dysfunction of dopaminergic neurons has also been linked to a number of neurological and psychiatric disorders, including Parkinson's disease.Zebrafish (Danio rerio) have emerged over the past two decades into a major genetic vertebrate model system,and thus contributed to a better understanding of developmental mechanisms controlling dopaminergic neuron specification and axonogenesis. In this review, we want to focus on conserved and dynamic aspects of the different catecholaminergic systems, which may help to evaluate the zebrafish as a model for dopaminergic and noradrenergic cellular specification and circuit function as well as biomedical aspects of catecholamine systems.