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Summary Gene regulatory networks govern pattern formation and differentiation during embryonic development. Segmentation of somites, precursors of the vertebral column among other tissues, is jointly controlled by temporal signals from the segmentation clock and spatial signals from morphogen gradients. To explore how these temporal and spatial signals are integrated, we combined time-controlled genetic perturbation experiments with computational modeling to reconstruct the core segmentation network in zebrafish. We found that Mesp family transcription factors link the temporal information of the segmentation clock with the spatial action of the fibroblast growth factor signaling gradient to establish rostrocaudal (head to tail) polarity of segmented somites. We further showed that cells gradually commit to patterning by the action of different genes at different spatiotemporal positions. Our study provides a blueprint of the zebrafish segmentation network, which includes evolutionarily conserved genes that are associated with the birth defect congenital scoliosis in humans., Graphical Abstract, Highlights • A core network establishes rostrocaudal polarity of segmented somites in zebrafish • mesp genes link the segmentation clock with the FGF signaling gradient • Gradual patterning is done by the action of different genes at different positions, Biological Sciences; Developmental Biology; Embryology; Mathematical Biosciences

The 22q11.2 deletion syndrome (22q11.2DS; velo-cardio-facial syndrome; DiGeorge syndrome) is a congenital anomaly disorder in which haploinsufficiency of TBX1, encoding a T-box transcription factor, is the major candidate for cardiac outflow tract (OFT) malformations. Inactivation of Tbx1 in the anterior heart field (AHF) mesoderm in the mouse results in premature expression of pro-differentiation genes and a persistent truncus arteriosus (PTA) in which septation does not form between the aorta and pulmonary trunk. Canonical Wnt/β-catenin has major roles in cardiac OFT development that may act upstream of Tbx1. Consistent with an antagonistic relationship, we found the opposite gene expression changes occurred in the AHF in β-catenin loss of function embryos compared to Tbx1 loss of function embryos, providing an opportunity to test for genetic rescue. When both alleles of Tbx1 and one allele of β-catenin were inactivated in the Mef2c-AHF-Cre domain, 61% of them (n = 34) showed partial or complete rescue of the PTA defect. Upregulated genes that were oppositely changed in expression in individual mutant embryos were normalized in significantly rescued embryos. Further, β-catenin was increased in expression when Tbx1 was inactivated, suggesting that there may be a negative feedback loop between canonical Wnt and Tbx1 in the AHF to allow the formation of the OFT. We suggest that alteration of this balance may contribute to variable expressivity in 22q11.2DS., Author summary To understand the genetic relationship between Tbx1 and canonical Wnt/β-catenin, we performed gene expression profiling and genetic rescue experiments. We found that Tbx1 and β-catenin may provide a negative feedback loop to restrict premature differentiation in the anterior heart field. This is relevant to understanding the basis of variable expressivity of 22q11.2DS, caused by haploinsufficiency of TBX1.

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.

The number of vertebrae, and hence the number of segments or 'somites' in the body, is highly variable among different vertebrate species. For instance, frogs have 10 vertebrae, while many snakes have over 300. But what controls vertebra number in a given species and why does it vary so much between species? Gomez et al. propose that the number depends on a balance struck early in embryogenesis between the division of the body into somites and the overall rate of development. They establish this by showing snakes have a much greater segmentation clock speed, relative to embryo development as a whole, than lizards and other vertebrates with fewer somites. The vertebrate body axis is divided into a number of segments or 'somites', such as the number of vertebrae. But what controls vertebra number, and its variation between species? This paper postulates that the number depends on a balance between the division of the body into somites (the segmentation clock rate) and the overall rate of development, as established by showing that the large number of vertebrae in snakes comes from a much greater segmentation clock speed in snakes, relative to development as a whole, than in other vertebrates. The vertebrate body axis is subdivided into repeated segments, best exemplified by the vertebrae that derive from embryonic somites. The number of somites is precisely defined for any given species but varies widely from one species to another. To determine the mechanism controlling somite number, we have compared somitogenesis in zebrafish, chicken, mouse and corn snake embryos. Here we present evidence that in all of these species a similar ‘clock-and-wavefront’1,2,3 mechanism operates to control somitogenesis; in all of them, somitogenesis is brought to an end through a process in which the presomitic mesoderm, having first increased in size, gradually shrinks until it is exhausted, terminating somite formation. In snake embryos, however, the segmentation clock rate is much faster relative to developmental rate than in other amniotes, leading to a greatly increased number of smaller-sized somites.

Oscillations are prevalent in natural systems. A gene expression oscillator, called the segmentation clock, controls segmentation of precursors of the vertebral column. Genes belonging to the Hes/her family encode the only conserved oscillating genes in all analyzed vertebrate species. Hes/Her proteins form dimers and negatively autoregulate their own transcription. Here, we developed a stochastic two-dimensional multicellular computational model to elucidate how the dynamics, i.e. period, amplitude and synchronization, of the segmentation clock are regulated. We performed parameter searches to demonstrate that autoregulatory negative-feedback loops of the redundant repressor Her dimers can generate synchronized gene expression oscillations in wild-type embryos and reproduce the dynamics of the segmentation oscillator in different mutant conditions. Our model also predicts that synchronized oscillations can be robustly generated as long as the half-lives of the repressor dimers are shorter than 6 minutes. We validated this prediction by measuring, for the first time, the half-life of Her7 protein as 3.5 minutes. These results demonstrate the importance of building biologically realistic stochastic models to test biological models more stringently and make predictions for future experimental studies.

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., Author Summary 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.

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., Author Summary Somites—the embryonic segments of the vertebrate body—are formed sequentially, with a spacing determined by a gene expression oscillator, the segmentation clock, operating in the cells at the tail end of the embryo. This system provides a rare opportunity to analyse how the timing of at least one set of developmental events is controlled. We previously proposed a mathematical model, showing how the oscillations could be generated by a delayed negative feedback loop, in which the products of two genes, her1 and her7, act as inhibitors of their own expression, and how Notch signalling between adjacent cells keeps their individual oscillations synchronised. Here we test and find support for this model in two ways. First, we show how to use the spatial pattern of gene expression to measure some of the temporal delays and molecular lifetimes that are critical for the occurrence of synchronised oscillations. Second, we use transgenic fish in which expression of her1 or her7 can be switched on at will by heat shock to probe the dynamics of the system and to analyse the logic of the control circuitry., The vertebrate somites are laid out in a regular pattern determined by the segmentation clock. Here the authors verify a mathematical model of the clock model and show that it quantitatively fits experimental details.

SummaryCellular polarization is often a response to distinct extracellular or intracellular cues, such as nutrient gradients or cortical landmarks. However, in the absence of such cues, some cells can still select a polarization axis at random. Positive feedback loops promoting localized activation of the GTPase Cdc42p are central to this process in budding yeast. Here, we explore spontaneous polarization during bud site selection in mutant yeast cells that lack functional landmarks. We find that these cells do not select a single random polarization axis, but continuously change this axis during the G1 phase of the cell cycle. This is reflected in traveling waves of activated Cdc42p which randomly explore the cell periphery. Our integrated computational and in vivo analyses of these waves reveal a negative feedback loop that competes with the aforementioned positive feedback loops to regulate Cdc42p activity and confer dynamic responsiveness on the robust initiation of cell polarization.