Periodic inhibition of Erk activity drives sequential somite segmentation.

Sequential segmentation creates modular body plans of diverse metazoan embryos1-4. Somitogenesis establishes the segmental pattern of the vertebrate body axis. A molecular segmentation clock in the presomitic mesoderm sets the pace of somite formation4. However, how cells are primed to form a segment boundary at a specific location remains unclear. Here we developed precise reporters for the clock and double-phosphorylated Erk (ppErk) gradient in zebrafish. We show that the Her1-Her7 oscillator drives segmental commitment by periodically lowering ppErk, therefore projecting its oscillation onto the ppErk gradient. Pulsatile inhibition of the ppErk gradient can fully substitute for the role of the clock, and kinematic clock waves are dispensable for sequential segmentation. The clock functions upstream of ppErk, which in turn enables neighbouring cells to discretely establish somite boundaries in zebrafish5. Molecularly divergent clocks and morphogen gradients were identified in sequentially segmenting species3,4,6-8. Our findings imply that versatile clocks may establish sequential segmentation in diverse species provided that they inhibit gradients.

Oscillatory control of embryonic development.

Proper embryonic development depends on the timely progression of a genetic program. One of the key mechanisms for achieving precise control of developmental timing is to use gene expression oscillations. In this Review, we examine how gene expression oscillations encode temporal information during vertebrate embryonic development by discussing the gene expression oscillations occurring during somitogenesis, neurogenesis, myogenesis and pancreas development. These oscillations play important but varied physiological functions in different contexts. Oscillations control the period of somite formation during somitogenesis, whereas they regulate the proliferation-to-differentiation switch of stem cells and progenitor cells during neurogenesis, myogenesis and pancreas development. We describe the similarities and differences of the expression pattern in space (i.e. whether oscillations are synchronous or asynchronous across neighboring cells) and in time (i.e. different time scales) of mammalian Hes/zebrafish Her genes and their targets in different tissues. We further summarize experimental evidence for the functional role of their oscillations. Finally, we discuss the outstanding questions for future research.

Pairing of segmentation clock genes drives robust pattern formation.

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.

Regulatory mechanisms ensuring coordinated expression of functionally related genes.

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.

Zebrafish as a model to investigate a biallelic gain-of-function variant in MSGN1, associated with a novel skeletal dysplasia syndrome.

BACKGROUND/OBJECTIVES: Rare genetic disorders causing specific congenital developmental abnormalities often manifest in single families. Investigation of disease-causing molecular features are most times lacking, although these investigations may open novel therapeutic options for patients. In this study, we aimed to identify the genetic cause in an Iranian patient with severe skeletal dysplasia and to model its molecular function in zebrafish embryos. RESULTS: The proband displays short stature and multiple skeletal abnormalities, including mesomelic dysplasia of the arms with complete humero-radio-ulna synostosis, arched clavicles, pelvic dysplasia, short and thin fibulae, proportionally short vertebrae, hyperlordosis and mild kyphosis. Exome sequencing of the patient revealed a novel homozygous c.374G > T, p.(Arg125Leu) missense variant in MSGN1 (NM_001105569). MSGN1, a basic-Helix-Loop-Helix transcription factor, plays a crucial role in formation of presomitic mesoderm progenitor cells/mesodermal stem cells during early developmental processes in vertebrates. Initial in vitro experiments show protein stability and correct intracellular localization of the novel variant in the nucleus and imply retained transcription factor function. To test the pathogenicity of the detected variant, we overexpressed wild-type and mutant msgn1 mRNA in zebrafish embryos and analyzed tbxta (T/brachyury/ntl). Overexpression of wild-type or mutant msgn1 mRNA significantly reduces tbxta expression in the tailbud compared to control embryos. Mutant msgn1 mRNA injected embryos depict a more severe effect, implying a gain-of-function mechanism. In vivo analysis on embryonic development was performed by clonal msgn1 overexpression in zebrafish embryos further demonstrated altered cell compartments in the presomitic mesoderm, notochord and pectoral fin buds. Detection of ectopic tbx6 and bmp2 expression in these embryos hint to affected downstream signals due to Msgn1 gain-of-function. CONCLUSION: In contrast to loss-of-function effects described in animal knockdown models, gain-of-function of MSGN1 explains the only mildly affected axial skeleton of the proband and rather normal vertebrae. In this context we observed notochord bending and potentially disruption of pectoral fin buds/upper extremity after overexpression of msgn1 in zebrafish embryos. The latter might result from Msgn1 function on mesenchymal stem cells or on chondrogenesis in these regions. In addition, we detected ectopic tbx6 and bmp2a expression after gain of Msgn1 function in zebrafish, which are interconnected to short stature, congenital scoliosis, limb shortening and prominent skeletal malformations in patients. Our findings highlight a rare, so far undescribed skeletal dysplasia syndrome associated with a gain-of-function mutation in MSGN1 and hint to its molecular downstream effectors.

Reduced dosage of ß-catenin provides significant rescue of cardiac outflow tract anomalies in a Tbx1 conditional null mouse model of 22q11.2 deletion syndrome.

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/ß-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 ß-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 ß-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, ß-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.

Spatial gradients of protein-level time delays set the pace of the traveling segmentation clock waves.

The vertebrate segmentation clock is a gene expression oscillator controlling rhythmic segmentation of the vertebral column during embryonic development. The period of oscillations becomes longer as cells are displaced along the posterior to anterior axis, which results in traveling waves of clock gene expression sweeping in the unsegmented tissue. Although various hypotheses necessitating the inclusion of additional regulatory genes into the core clock network at different spatial locations have been proposed, the mechanism underlying traveling waves has remained elusive. Here, we combined molecular-level computational modeling and quantitative experimentation to solve this puzzle. Our model predicts the existence of an increasing gradient of gene expression time delays along the posterior to anterior direction to recapitulate spatiotemporal profiles of the traveling segmentation clock waves in different genetic backgrounds in zebrafish. We validated this prediction by measuring an increased time delay of oscillatory Her1 protein production along the unsegmented tissue. Our results refuted the need for spatial expansion of the core feedback loop to explain the occurrence of traveling waves. Spatial regulation of gene expression time delays is a novel way of creating dynamic patterns; this is the first report demonstrating such a control mechanism in any tissue and future investigations will explore the presence of analogous examples in other biological systems.

The elongation rate of RNA polymerase II in zebrafish and its significance in the somite segmentation clock.

A gene expression oscillator called the segmentation clock controls somite segmentation in the vertebrate embryo. In zebrafish, the oscillatory transcriptional repressor genes her1 and her7 are crucial for genesis of the oscillations, which are thought to arise from negative autoregulation of these genes. The period of oscillation is predicted to depend on delays in the negative-feedback loop, including, most importantly, the transcriptional delay - the time taken to make each molecule of her1 or her7 mRNA. her1 and her7 operate in parallel. Loss of both gene functions, or mutation of her1 combined with knockdown of Hes6, which we show to be a binding partner of Her7, disrupts segmentation drastically. However, mutants in which only her1 or her7 is functional show only mild segmentation defects and their oscillations have almost identical periods. This is unexpected because the her1 and her7 genes differ greatly in length. We use transgenic zebrafish to measure the RNA polymerase II elongation rate, for the first time, in the intact embryo. This rate is unexpectedly rapid, at 4.8 kb/minute at 28.5°C, implying that, for both genes, the time taken for transcript elongation is insignificant compared with other sources of delay, explaining why the mutants have similar clock periods. Our computational model shows how loss of her1 or her7 can allow oscillations to continue with unchanged period but with reduced amplitude and impaired synchrony, as manifested in the in situ hybridisation patterns of the single mutants.

Short-lived Her proteins drive robust synchronized oscillations in the zebrafish segmentation clock.

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.

Control of segment number in vertebrate embryos.

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' 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.

Spatiotemporal control of pattern formation during somitogenesis.

Spatiotemporal patterns widely occur in biological, chemical, and physical systems. Particularly, embryonic development displays a diverse gamut of repetitive patterns established in many tissues and organs. Branching treelike structures in lungs, kidneys, livers, pancreases, and mammary glands as well as digits and bones in appendages, teeth, and palates are just a few examples. A fascinating instance of repetitive patterning is the sequential segmentation of the primary body axis, which is conserved in all vertebrates and many arthropods and annelids. In these species, the body axis elongates at the posterior end of the embryo containing an unsegmented tissue. Meanwhile, segments sequentially bud off from the anterior end of the unsegmented tissue, laying down an exquisite repetitive pattern and creating a segmented body plan. In vertebrates, the paraxial mesoderm is sequentially divided into somites. In this review, we will discuss the most prominent models, the most puzzling experimental data, and outstanding questions in vertebrate somite segmentation.

Spatiotemporal compartmentalization of key physiological processes during muscle precursor differentiation.

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.

Regulation of noise in the expression of a single gene.

Stochastic mechanisms are ubiquitous in biological systems. Biochemical reactions that involve small numbers of molecules are intrinsically noisy, being dominated by large concentration fluctuations. This intrinsic noise has been implicated in the random lysis/lysogeny decision of bacteriophage-lambda, in the loss of synchrony of circadian clocks and in the decrease of precision of cell signals. 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.

Human stem cell models unravel mechanisms of somite segmentation.

In vitro models to study human somitogenesis, the formation of the segmented body plan, have so far been limited.1 Two papers in Nature now report the creation of pluripotent stem cell (PSC)-derived 3D culture systems that recapitulate the formation of somite-like structures and help gain insights into this developmental process.2,3.

A design logic for sequential segmentation across organisms.

Multitudes of organisms display metameric compartmentalization of their body plan. Segmentation of these compartments happens sequentially in diverse phyla. In several sequentially segmenting species, periodically active molecular clocks and signaling gradients have been found. The clocks are proposed to control the timing of segmentation, while the gradients are proposed to instruct the positions of segment boundaries. However, the identity of the clock and gradient molecules differs across species. Furthermore, sequential segmentation of a basal chordate, Amphioxus, continues at late stages when the small tail bud cell population cannot establish long-range signaling gradients. Thus, it remains to be explained how a conserved morphological trait (i.e., sequential segmentation) is achieved by using different molecules or molecules with different spatial profiles. Here, we first focus on sequential segmentation of somites in vertebrate embryos and then draw parallels with other species. Thereafter, we propose a candidate design principle that has the potential to answer this puzzling question.

Using single-molecule fluorescence in situ hybridization and immunohistochemistry to count RNA molecules in single cells in zebrafish embryos.

Taming gene expression variability is critical for robust pattern formation during embryonic development. Here, we describe an optimized protocol for single-molecule fluorescence in situ hybridization and immunohistochemistry in zebrafish embryos. We detail how to count segmentation clock RNAs and calculate their variability among neighboring cells. This approach is easily adaptable to count RNA numbers of any gene and calculate transcriptional variability among neighboring cells in diverse biological settings. For complete details on the use and execution of this protocol, please refer to Keskin et al. (2018),1 Zinani et al. (2021),2 and Zinani et al. (2022).3.

The Role of Fibroblast Growth Factor Signaling in Somitogenesis.

Fibroblast growth factor (FGF) signaling is conserved from cnidaria to mammals (Ornitz and Itoh, 2022) and it regulates several critical processes such as differentiation, proliferation, apoptosis, cell migration, and embryonic development. One pivotal process FGF signaling controls is the division of vertebrate paraxial mesoderm into repeated segmented units called somites (i.e., somitogenesis). Somite segmentation occurs periodically and sequentially in a head-to-tail manner, and lays down the plan for compartmentalized development of the vertebrate body axis (Gomez et al., 2008). These somites later give rise to vertebrae, tendons, and skeletal muscle. Somite segments form sequentially from the anterior end of the presomitic mesoderm (PSM). The periodicity of somite segmentation is conferred by the segmentation clock, comprising oscillatory expression of Hairy and enhancer-of-split (Her/Hes) genes in the PSM. The positional information for somite boundaries is instructed by the double phosphorylated extracellular signal-regulated kinase (ppERK) gradient, which is the relevant readout of FGF signaling during somitogenesis (Sawada et al., 2001; Delfini et al., 2005; Simsek and Ozbudak, 2018; Simsek et al., 2023). In this review, we summarize the crosstalk between the segmentation clock and FGF/ppERK gradient and discuss how that leads to periodic somite boundary formation. We also draw attention to outstanding questions regarding the interconnected roles of the segmentation clock and ppERK gradient, and close with suggested future directions of study.

Stochastic gene expression and environmental stressors trigger variable somite segmentation phenotypes.

Mutations of several genes cause incomplete penetrance and variable expressivity of phenotypes, which are usually attributed to modifier genes or gene-environment interactions. Here, we show stochastic gene expression underlies the variability of somite segmentation defects in embryos mutant for segmentation clock genes her1 or her7. Phenotypic strength is further augmented by low temperature and hypoxia. By performing live imaging of the segmentation clock reporters, we further show that groups of cells with higher oscillation amplitudes successfully form somites while those with lower amplitudes fail to do so. In unfavorable environments, the number of cycles with high amplitude oscillations and the number of successful segmentations proportionally decrease. These results suggest that individual oscillation cycles stochastically fail to pass a threshold amplitude, resulting in segmentation defects in mutants. Our quantitative methodology is adaptable to investigate variable phenotypes of mutant genes in different tissues.

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.

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.