Search

Sequential segmentation creates modular body plans of diverse metazoan embryos(1-4). Somitogenesis establishes segmental pattern of vertebrate body axis. A molecular segmentation clock in the presomitic mesoderm (PSM) sets the pace of somite formation(4). However, how cells are primed to form a segment boundary at a specific location remained unknown. Here we developed precise reporters for the clock and double phosphorylated ERK (ppERK) gradient in zebrafish. We discovered that the Her1/7 oscillator drives segmental commitment by periodically lowering ppERK, thus projecting its oscillation on the ppERK gradient. Pulsatile inhibition of the ppERK gradient can fully substitute the role of the clock and kinematic clock waves are dispensable for sequential segmentation. The clock functions upstream of the ppERK, which in turn enables neighboring cells to discretely establish somite boundaries in zebrafish(5). Molecularly divergent clocks and morphogen gradients were identified in sequentially segmenting species(3,4,6-8). Our findings imply versatile clocks may establish sequential segmentation in diverse species as long as they inhibit gradients.

Coordinated spatiotemporal expression of large sets of genes is required for the development and homeostasis of organisms. To achieve this goal, organisms use myriad strategies where they form operons, utilize bidirectional promoters, cluster genes, share enhancers among genes by DNA looping, and form topologically associated domains and transcriptional condensates. Coexpression achieved by these different strategies is hypothesized to have functional importance in minimizing gene expression variability, establishing dosage balance to ensure stoichiometry of protein complexes, and minimizing accumulation of toxic intermediate metabolites. By combining gene-editing tools with computational modeling, recent studies tested the advantages of adjacent genes located in pairs and clusters. We propose that with the advancement of gene editing, single-cell sequencing, and imaging tools, one could readily test the functional importance of different coexpression strategies in a variety of biological processes.

Gene expression is an inherently stochastic process1,2; however, organismal development and homeostasis require cells to coordinate the spatiotemporal expression of large sets of genes. In metazoans, pairs of co-expressed genes often reside in the same chromosomal neighbourhood, with gene pairs representing 10 to 50% of all genes, depending on the species3–6. Because shared upstream regulators can ensure correlated gene expression, the selective advantage of maintaining adjacent gene pairs remains unknown6. Here, using two linked zebrafish segmentation clock genes, her1 and her7, and combining single-cell transcript counting, genetic engineering, real-time imaging and computational modelling, we show that gene pairing boosts correlated transcription and provides phenotypic robustness for the formation of developmental patterns. Our results demonstrate that the prevention of gene pairing disrupts oscillations and segmentation, and the linkage of her1 and her7 is essential for the development of the body axis in zebrafish embryos. We predict that gene pairing may be similarly advantageous in other organisms, and our findings could lead to the engineering of precise synthetic clocks in embryos and organoids. The pairing of genes is essential for robust expression of segmentation clock genes during development in zebrafish embryos.

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from the presomitic mesoderm (PSM) as the tail end of the embryo elongates posteriorly. Somites form with regular periodicity and scale in size. Zebrafish is a popular model organism as it is genetically tractable and has transparent embryos that allow for live imaging. Nevertheless, during somitogenesis, fish embryos are wrapped around a large, rounding yolk. This geometry limits live imaging of PSM tissue in zebrafish embryos, particularly at higher resolutions that require a close objective working distance. Here, we present a flattened 3-D tissue culture method for live imaging of zebrafish tail explants. Tail explants mimic intact embryos by displaying a proportional slowdown of axis elongation and shortening of rostrocaudal somite lengths. We are further able to stall axis elongation speed through explant culture. This, for the first time, enables us to untangle the chemical input of signaling gradients from the mechanistic input of axial elongation. In future studies, this method can be combined with a microfluidic setup to allow time-controlled pharmaceutical perturbations or screening of vertebrate segmentation without any drug penetration concerns.

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., Graphical Abstract, In Brief 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.

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 development of multicellular organisms is controlled by transcriptional networks. Understanding the role of these networks requires a full understanding of transcriptome regulation during embryogenesis. Several microarray studies have characterized the temporal evolution of the transcriptome during development in different organisms [Wang QT, et al. (2004) Dev Cell 6:133–144; Furlong EE, Andersen EC, Null B, White KP, Scott MP (2001) Science 293:1629–1633; Mitiku N, Baker JC (2007) Dev Cell 13:897–907]. In all cases, however, experiments were performed on whole embryos, thus averaging gene expression among many different tissues. Here, we took advantage of the local synchrony of the differentiation process in the paraxial mesoderm. This approach provides a unique opportunity to study the systems-level properties of muscle differentiation. Using high-resolution, spatiotemporal profiling of the early stages of muscle development in the zebrafish embryo, we identified a major reorganization of the transcriptome taking place in the presomitic mesoderm. We further show that the differentiation process is associated with a striking modular compartmentalization of the transcription of essential components of cellular physiological programs. Particularly, we identify a tight segregation of cell cycle/DNA metabolic processes and translation/oxidative metabolism at the tissue level, highly reminiscent of the yeast metabolic cycle. These results should expand more investigations into the developmental control of metabolism.

SUMMARY Taming cell-to-cell variability in gene expression is critical for precise pattern formation during embryonic development. To investigate the source and buffering mechanism of expression variability, we studied a biological clock, the vertebrate segmentation clock, controlling the precise spatiotemporal patterning of the vertebral column. By counting single transcripts of segmentation clock genes in zebrafish, we show that clock genes have low RNA amplitudes and expression variability is primarily driven by gene extrinsic sources, which is suppressed by Notch signaling. We further show that expression noise surprisingly increases from the posterior progenitor zone to the anterior segmentation and differentiation zone. Our computational model reproduces the spatial noise profile by incorporating spatially increasing time delays in gene expression. Our results, suggesting that expression variability is controlled by the balance of time delays and cell signaling in a vertebrate tissue, will shed light on the accuracy of natural clocks in multi-cellular systems and inspire engineering of robust synthetic oscillators., In Brief Keskin et al. show that segmentation clock transcription levels display low amplitude and high heterogeneity. Clock gene expression variability is primarily driven by gene extrinsic sources, which is suppressed by Notch signaling. Gene expression noise increases along the posteroanterior axis. Spatial gradients of time delays contribute to the noise gradient along the axis.

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.

Stochastic mechanisms are ubiquitous in biological systems. Biochemical reactions that involve small numbers of molecules are intrinsically noisy, being dominated by large concentration fluctuations 1‐3 . This intrinsic noise has been implicated in the random lysis/lysogeny decision of bacteriophage-λ 4 , in the loss of synchrony of circadian clocks 5,6 and in the decrease of precision of cell signals7. We sought to quantitatively investigate the extent to which the occurrence of molecular fluctuations within single cells (biochemical noise) could explain the variation of gene expression levels between cells in a genetically identical population (phenotypic noise). We have isolated the biochemical contribution to phenotypic noise from that of other noise sources by carrying out a series of differential measurements. We varied independently the rates of transcription and translation of a single fluorescent reporter gene in the chromosome of Bacillus subtilis, and we quantitatively measured the resulting changes in the phenotypic noise characteristics. We report that of these two parameters, increased translational efficiency is the predominant source of increased phenotypic noise. This effect is consistent with a stochastic model of gene expression in which proteins are produced in random and sharp bursts. Our results thus provide the first direct experimental evidence of the biochemical origin of phenotypic noise, demonstrating that the level of phenotypic variation in an isogenic population can be regulated by genetic parameters. We selected as our reporter system a single-copy chromosomal gene with an inducible promoter. As an estimated 50‐80% of bacterial genes are transcriptionally regulated 8 , this system typifies the majority of naturally occurring genes, allowing our results to be extended to natural systems. We incorporated a single copy of our reporter, the green fluorescent protein gene (gfp), into the chromosome of B. subtilis. We chose to integrate gfpinto the chromosome itself, rather than in the form of plasmids, as variation in plasmid copy number 9,10 can act as an additional and unwanted source of noise. Transcriptional efficiency was regulated by using an isopropyl-β-D-thiogalactopyranoside (IPTG)‐inducible promoter, Pspac, upstream of gfp, and varying the concentration of IPTG in the growth medium. Translational

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.

Fluctuations in protein numbers (noise) due to inherent stochastic effects in single cells can have large effects on the dynamic behavior of gene regulatory networks. Although deterministic models can predict the average network behavior, they fail to incorporate the stochasticity characteristic of gene expression, thereby limiting their relevance when single cell behaviors deviate from the population average. Recently, stochastic models have been used to predict distributions of steady-state protein levels within a population but not to predict the dynamic, presteady-state distributions. In the present work, we experimentally examine a system whose dynamics are heavily influenced by stochastic effects. We measure population distributions of protein numbers as a function of time in the Escherichia coli lactose uptake network ( lac operon). We then introduce a dynamic stochastic model and show that prediction of dynamic distributions requires only a few noise parameters in addition to the rates that characterize a deterministic model. Whereas the deterministic model cannot fully capture the observed behavior, our stochastic model correctly predicts the experimental dynamics without any fit parameters. Our results provide a proof of principle for the possibility of faithfully predicting dynamic population distributions from deterministic models supplemented by a stochastic component that captures the major noise sources.

Multistability, the capacity to achieve multiple internal states in response to a single set of external inputs, is the defining characteristic of a switch. Biological switches are essential for the determination of cell fate in multicellular organisms1, the regulation of cell-cycle oscillations during mitosis2,3 and the maintenance of epigenetic traits in microbes4. The multistability of several natural1,2,3,4,5,6 and synthetic7,8,9 systems has been attributed to positive feedback loops in their regulatory networks10. However, feedback alone does not guarantee multistability. The phase diagram of a multistable system, a concise description of internal states as key parameters are varied, reveals the conditions required to produce a functional switch11,12. Here we present the phase diagram of the bistable lactose utilization network of Escherichia coli13. We use this phase diagram, coupled with a mathematical model of the network, to quantitatively investigate processes such as sugar uptake and transcriptional regulation in vivo. We then show how the hysteretic response of the wild-type system can be converted to an ultrasensitive graded response14,15. The phase diagram thus serves as a sensitive probe of molecular interactions and as a powerful tool for rational network design.

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.