Search

Rhythmic and sequential segmentation of the growing vertebrate body relies on the segmentation clock, a multi-cellular oscillating genetic network. The clock is visible as tissue-level kinematic waves of gene expression that travel through the presomitic mesoderm (PSM) and arrest at the position of each forming segment. Here, we test how this hallmark wave pattern is driven by culturing single maturing PSM cells. We compare their cell-autonomous oscillatory and arrest dynamics to those we observe in the embryo at cellular resolution, finding similarity in the relative slowing of oscillations and arrest in concert with differentiation. This shows that cell-extrinsic signals are not required by the cells to instruct the developmental program underlying the wave pattern. We show that a cell-autonomous timing activity initiates during cell exit from the tailbud, then runs down in the anterior-ward cell flow in the PSM, thereby using elapsed time to provide positional information to the clock. Exogenous FGF lengthens the duration of the cell-intrinsic timer, indicating extrinsic factors in the embryo may regulate the segmentation clock via the timer. In sum, our work suggests that a noisy cell-autonomous, intrinsic timer drives the slowing and arrest of oscillations underlying the wave pattern, while extrinsic factors in the embryo tune this timer’s duration and precision. This is a new insight into the balance of cell-intrinsic and -extrinsic mechanisms driving tissue patterning in development.

During embryonic development, the timing of events at the cellular level must be coordinated across multiple length scales to ensure the formation of a well-proportioned body plan. This is clear during somitogenesis, where progenitors must be allocated to the axis over time whilst maintaining a progenitor population for continued elaboration of the body plan. However, the relative importance of intrinsic and extrinsic signals in timing progenitor addition at the single-cell level is not yet understood. Heterochronic grafts from older to younger embryos have suggested a level of intrinsic timing whereby later staged cells contribute to more posterior portions of the axis. To determine the precise step at which cells are delayed, we performed single-cell transcriptomic analysis on heterochronic grafts of somite progenitors in the chicken embryo. This revealed a previously undescribed cell state within which heterochronic grafted cells are stalled. The delayed exit of older cells from this state correlates with expression of posterior Hox genes. Using grafting and explant culture, we find that both Hox gene expression and the migratory capabilities of progenitor populations are intrinsically regulated at the population level. However, by grafting varied sizes of tissue, we find that small heterochronic grafts disperse more readily and contribute to more anterior portions of the body axis while still maintaining Hox gene expression. This enhanced dispersion is not replicated in explant culture, suggesting that it is a consequence of interaction between host and donor tissue and thus extrinsic to the donor tissue. Therefore, we demonstrate that the timing of cell dispersion and resulting axis contribution is impacted by a combination of both intrinsic and extrinsic cues.

Somitogenesis is a hallmark feature of all vertebrates and some invertebrate species that involves the periodic formation of block-like structures called somites. Somites are transient embryonic segments that eventually establish the entire vertebral column. A highly conserved molecular oscillator called the segmentation clock underlies this periodic event and the pace of this clock regulates the pace of somite formation. Although conserved signaling pathways govern the clock in most vertebrates, the mechanisms underlying the species-specific divergence in various clock characteristics remain elusive. For example, the segmentation clock in classical model species such as zebrafish, chick, and mouse embryos tick with a periodicity of ∼30, ∼90, and ∼120 min respectively. This enables them to form the species-specific number of vertebrae during their overall timespan of somitogenesis. Here, we perform a systematic review of the species-specific features of the segmentation clock with a keen focus on mouse embryos. We perform this review using three different perspectives: Notch-responsive clock genes, ligand-receptor dynamics, and synchronization between neighboring oscillators. We further review reports that use non-classical model organisms and in vitro model systems that complement our current understanding of the segmentation clock. Our review highlights the importance of comparative developmental biology to further our understanding of this essential developmental process.

Living systems exhibit an unmatched complexity, due to countless, entangled interactions across scales. Here, we aim to understand a complex system, that is, segmentation timing in mouse embryos, without a reference to these detailed interactions. To this end, we develop a coarse-grained approach, in which theory guides the experimental identification of the segmentation clock entrainment responses. We demonstrate period- and phase-locking of the segmentation clock across a wide range of entrainment parameters, including higher-order coupling. These quantifications allow to derive the phase response curve (PRC) and Arnold tongues of the segmentation clock, revealing its essential dynamical properties. Our results indicate that the somite segmentation clock has characteristics reminiscent of a highly non-linear oscillator close to an infinite period bifurcation and suggests the presence of long-term feedbacks. Combined, this coarse-grained theoretical-experimental approach reveals how we can derive simple, essential features of a highly complex dynamical system, providing precise experimental control over the pace and rhythm of the somite segmentation clock.

Vertebrate embryo somitogenesis is the earliest morphological manifestation of the characteristic patterned structure of the adult axial skeleton. Pairs of somites flanking the neural tube are formed periodically during early development, and the molecular mechanisms in temporal control of this early patterning event have been thoroughly studied. The discovery of a molecular Embryo Clock (EC) underlying the periodicity of somite formation shed light on the importance of gene expression dynamics for pattern formation. The EC is now known to be present in all vertebrate organisms studied and this mechanism was also described in limb development and stem cell differentiation. An outstanding question, however, remains unanswered: what sets the different EC paces observed in different organisms and tissues? This review aims to summarize the available knowledge regarding the pace of the EC, its regulation and experimental manipulation and to expose new questions that might help shed light on what is still to unveil.

Somitogenesis is the process by means of which a tissue known as presomitic mesoderm (PSM) is segmented in blocks of cells, called somites, along the anterior-posterior axis of the developing embryo in segmented animals. In vertebrates, somites give rise to axial skeleton, cartilage, tendons, skeletal muscle, and dermis. Somite formation occurs periodically, and this periodicity is driven by a genetic oscillator that operates within PSM cells and is known as the segmentation clock. The correct synchronization of the segmentation clock among PSM cells is essential for somitogenesis to develop normally. When synchronization is disrupted, somites form irregularly and, in consequence, the tissues that originate from them show clear malformations. In this work, based in a model for zebrafish segmentation clock, we investigate by means of a mathematical modeling approach, how PSM-cell synchronization is affected by factors like: the size of PSM-cell networks, the amount of cell-to-cell interactions per PSM cell, the strength of these interactions, and the inherent variability among PSM cells. Interestingly we found that very small PSM-cell networks are unable to synchronize. Moreover, the effect of decreasing the strength of interactions among PSM cells is corrected by increasing the network connectivity-level, and a moderated level of variability among cells can have a positive effect on synchronization, specially in large networks.

Congenital scoliosis (CS) is a congenital disease caused by malformations of vertebrae. Recent studies demonstrated that DNA modification could contribute to the pathogenesis of disease. This study aims to identify epigenetic perturbations that may contribute to the pathogenesis of CS. Four CS patients with hemivertebra were enrolled and underwent spine correction operations. DNA was extracted from the hemivertebrae and spinal process collected from the specimen during the hemivertebra resection. Genome-wide DNA methylation profiling was examined at base-pair resolution using whole-genome bisulfite sequencing (WGBS). We identified 343 genes with hyper-differentially methylated regions (DMRs) and 222 genes with hypo-DMRs, respectively. These genes were enriched in the mitogen-activated protein kinase (MAPK) signaling pathway, calcium signaling pathway, and axon guidance in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and were enriched in positive regulation of cell morphogenesis involved in differentiation, regulation of cell morphogenesis involved in differentiation, and regulation of neuron projection development in Biological Process of Gene Ontology (GO-BP) terms. Hyper-DMR-related genes, including IGHG1, IGHM, IGHG3, RNF213, and GSE1, and hypo DMR-related genes, including SORCS2, COL5A1, GRID1, RGS3, and ROBO2, may contribute to the pathogenesis of hemivertebra. The aberrant DNA methylation may be associated with the formation of hemivertebra and congenital scoliosis.

The vertebrate left-right axis is specified during neurulation by events occurring in a transient ciliated epithelium termed left-right organizer (LRO), which is made up of two distinct cell types. In the axial midline, central LRO (cLRO) cells project motile monocilia and generate a leftward fluid flow, which represents the mechanism of symmetry breakage. This directional fluid flow is perceived by laterally positioned sensory LRO (sLRO) cells, which harbor non-motile cilia. In sLRO cells on the left side, flow-induced signaling triggers post-transcriptional repression of the multi-pathway antagonist dand5. Subsequently, the co-expressed Tgf-β growth factor Nodal1 is released from Dand5-mediated repression to induce left-sided gene expression. Interestingly, Xenopus sLRO cells have somitic fate, suggesting a connection between LR determination and somitogenesis. Here, we show that doublesex and mab3-related transcription factor 2 (Dmrt2), known to be involved in vertebrate somitogenesis, is required for LRO ciliogenesis and sLRO specification. In dmrt2 morphants, misexpression of the myogenic transcription factors tbx6 and myf5 at early gastrula stages preceded the misspecification of sLRO cells at neurula stages. myf5 morphant tadpoles also showed LR defects due to a failure of sLRO development. The gain of myf5 function reintroduced sLRO cells in dmrt2 morphants, demonstrating that paraxial patterning and somitogenesis are functionally linked to LR axis formation in Xenopus.

Abstract Background The yak is a species of livestock which is crucial for local communities of the Qinghai-Tibet Plateau and adjacent regions and naturally owns one more thoracic vertebra than cattle. Recently, a sub-population of yak termed as the Jinchuan yak has been identified with over half its members own a thoracolumbar vertebral formula of T15L5 instead of the natural T14L5 arrangement. The novel T15L5 positioning is a preferred genetic trait leading to enhanced meat and milk production. Selective breeding of this trait would have great agricultural value and exploration of the molecular mechanisms underlying this trait would both accelerate this process and provide us insight into the development and regulation of somitogenesis. Results Here we investigated the genetic background of the Jinchuan yak through resequencing fifteen individuals, comprising five T15L5 individuals and ten T14L5 individuals with an average sequencing depth of > 10X, whose thoracolumbar vertebral formulae were confirmed by anatomical observation. Principal component analysis, linkage disequilibrium analysis, phylogenetic analysis, and selective sweep analysis were carried out to explore Jinchuan yak’s genetic background. Three hundred and thirty candidate markers were identified as associated with the additional thoracic vertebrae and target sequencing was used to validate seven carefully selected markers in an additional 51 Jinchuan yaks. The accuracies of predicting 15 thoracic vertebrae and 20 thoracolumbar vertebrae with these 7 markers were 100.00 and 33.33% despite they both could only represent 20% of all possible genetic diversity. Two genes, PPP2R2B and TBLR1, were found to harbour the most candidate markers associated with the trait and likely contribute to the unique somitic number and identity according to their reported roles in the mechanism of somitogenesis. Conclusions Our findings provide a clear depiction of the Jinchuan yak’s genetic background and a solid foundation for marker-assistant selection. Further exploitation of this unique population and trait could be promoted with the aid of our genomic resource.

During the development of the vertebrate embryo, segmented structures called somites are periodically formed from the presomitic mesoderm (PSM) and give rise to the vertebral column. While somite formation has been studied in several animal models, it is less clear how well this process is conserved in humans. Recent progress has made it possible to study aspects of human paraxial mesoderm (PM) development such as the human segmentation clock in vitro using human pluripotent stem cells (hPSCs); however, somite formation has not been observed in these monolayer cultures. Here, we describe the generation of human PM organoids from hPSCs (termed Somitoids), which recapitulate the molecular, morphological, and functional features of PM development, including formation of somite-like structures in vitro. Using a quantitative image-based screen, we identify critical parameters such as initial cell number and signaling modulations that reproducibly yielded formation of somite-like structures in our organoid system. In addition, using single-cell RNA-sequencing and 3D imaging, we show that PM organoids both transcriptionally and morphologically resemble their in vivo counterparts and can be differentiated into somite derivatives. Our organoid system is reproducible and scalable, allowing for the systematic and quantitative analysis of human spine development and disease in vitro.

Somitogenesis refers to the segmentation of the paraxial mesoderm, a tissue located on the back of the embryo, into regularly spaced and sized pieces, i.e., the somites. This periodicity is important to assure, for example, the formation of a functional vertebral column. Prevailing models of somitogenesis are based on the existence of a gene regulatory network capable of generating a striped pattern of gene expression, which is subsequently translated into periodic tissue boundaries. An alternative view is that the pre-pattern that guides somitogenesis is not chemical, but of a mechanical origin. A striped pattern of mechanical strain can be formed in physically connected tissues expanding at different rates, as it occurs in the embryo. Here we argue that both molecular and mechanical cues could drive somite periodicity and suggest how they could be integrated.

Whether or not the process of somitogenesis and myogenesis is affected by excessive caffeine intake still remains ambiguous. In this study, we first showed that caffeine treatment results in chest wall deformities and simultaneously reduced mRNA expressions of genes involved in myogenesis in the developing chicken embryos. We then used embryo cultures to assess in further detail how caffeine exposure affects the earliest steps of myogenesis, and we demonstrated that the caffeine treatment suppressed somitogenesis of chicken embryos by interfering with the expressions of crucial genes modulating apoptosis, proliferation, and differentiation of myogenic progenitors in differentiating somites. These phenotypes were abrogated by a retinoic acid (RA) antagonist in embryo cultures, even at low caffeine doses in C2C12 cells, implying that excess RA levels are responsible for these phenotypes in cells and possibly in vivo. These findings highlight that excessive caffeine exposure is negatively involved in regulating the development of myogenic progenitors through interfering with RA signaling. The RA somitogenesis/myogenesis pathway might be directly impacted by caffeine signaling rather than reflecting an indirect effect of the toxicity of excess caffeine dosage.

Integrity of rhythmic spatial gene expression patterns in the vertebrate segmentation clock requires local synchronization between neighboring cells by Delta-Notch signaling and its inhibition causes defective segment boundaries. Whether deformation of the oscillating tissue complements local synchronization during patterning and segment formation is not understood. We combine theory and experiment to investigate this question in the zebrafish segmentation clock. We remove a Notch inhibitor, allowing resynchronization, and analyze embryonic segment recovery. We observe unexpected intermingling of normal and defective segments, and capture this with a new model combining coupled oscillators and tissue mechanics. Intermingled segments are explained in the theory by advection of persistent phase vortices of oscillators. Experimentally observed changes in recovery patterns are predicted in the theory by temporal changes in tissue length and cell advection pattern. Thus, segmental pattern recovery occurs at two length and time scales: rapid local synchronization between neighboring cells, and the slower transport of the resulting patterns across the tissue through morphogenesis.

Fibronectin is essential for somite formation in the vertebrate embryo. Fibronectin matrix assembly starts as cells emerge from the primitive streak and ingress in the unsegmented presomitic mesoderm (PSM). PSM cells undergo cyclic waves of segmentation clock gene expression, followed by Notch-dependent upregulation of meso1 in the rostral PSM which induces somite cleft formation. However, the relevance of the fibronectin matrix for these molecular processes remains unknown. Here, we assessed the role of the PSM fibronectin matrix in the spatio-temporal regulation of chick embryo somitogenesis by perturbing (1) extracellular fibronectin matrix assembly, (2) integrin–fibronectin binding, (3) Rho-associated protein kinase (ROCK) activity and (4) non-muscle myosin II (NM II) function. We found that integrin–fibronectin engagement and NM II activity are required for cell polarization in the nascent somite. All treatments resulted in defective somitic clefts and significantly perturbed meso1 and segmentation clock gene expression in the PSM. Importantly, inhibition of actomyosin-mediated contractility increased the period of hairy1/hes4 oscillations from 90 to 120 min. Together, our work strongly suggests that the fibronectin–integrin–ROCK–NM II axis regulates segmentation clock dynamics and dictates the spatio-temporal localization of somitic clefts.

During vertebrate development, the presomitic mesoderm (PSM) periodically segments into somites, which will form the segmented vertebral column and associated muscle, connective tissue, and dermis. The periodicity of somitogenesis is regulated by a segmentation clock of oscillating Notch activity. Here, we examined mouse mutants lacking only Fgf4 or Fgf8, which we previously demonstrated act redundantly to prevent PSM differentiation. Fgf8 is not required for somitogenesis, but Fgf4 mutants display a range of vertebral defects. We analyzed Fgf4 mutants by quantifying mRNAs fluorescently labeled by hybridization chain reaction within Imaris-based volumetric tissue subsets. These data indicate that FGF4 maintains Hes7 levels and normal oscillatory patterns. To support our hypothesis that FGF4 regulates somitogenesis through Hes7, we demonstrate genetic synergy between Hes7 and Fgf4, but not with Fgf8. Our data indicate that Fgf4 is potentially important in a spectrum of human Segmentation Defects of the Vertebrae caused by defective Notch oscillations.

During development, cells gradually assume specialized fates via changes of transcriptional dynamics, sometimes even within the same developmental stage. For anterior-posterior (AP) patterning in metazoans, it has been suggested that the gradual transition from a dynamic genetic regime to a static one is encoded by different transcriptional modules. In that case, the static regime has an essential role in pattern formation in addition to its maintenance function. In this work, we introduce a geometric approach to study such transition. We exhibit two types of genetic regime transitions arising through local or global bifurcations, respectively. We find that the global bifurcation type is more generic, more robust, and better preserves dynamical information. This could parsimoniously explain common features of metazoan segmentation, such as changes of periods leading to waves of gene expressions, ‘speed/frequency-gradient’ dynamics, and changes of wave patterns. Geometric approaches appear as possible alternatives to gene regulatory networks to understand development.

In development and disease, cells move as they exchange signals. One example is found in vertebrate development, during which the timing of segment formation is set by a ‘segmentation clock’, in which oscillating gene expression is synchronized across a population of cells by Delta-Notch signaling. Delta-Notch signaling requires local cell-cell contact, but in the zebrafish embryonic tailbud, oscillating cells move rapidly, exchanging neighbors. Previous theoretical studies proposed that this relative movement or cell mixing might alter signaling and thereby enhance synchronization. However, it remains unclear whether the mixing timescale in the tissue is in the right range for this effect, because a framework to reliably measure the mixing timescale and compare it with signaling timescale is lacking. Here, we develop such a framework using a quantitative description of cell mixing without the need for an external reference frame and constructing a physical model of cell movement based on the data. Numerical simulations show that mixing with experimentally observed statistics enhances synchronization of coupled phase oscillators, suggesting that mixing in the tailbud is fast enough to affect the coherence of rhythmic gene expression. Our approach will find general application in analyzing the relative movements of communicating cells during development and disease.

Somitogenesis is a critical process during vertebrate development that establishes the segmented body plan and gives rise to the vertebra, skeletal muscles, and dermis. While segmentation clock and wave front mechanisms have been elucidated to control the size and time of somite formation, regulation of the segmentation process that physically separates somites is not understood in detail. Here, we identified a cytoskeletal player, Cdc42 effector protein 3 (Cdc42ep3, CEP3) that is required for somite segmentation in Xenopus embryos. CEP3 is specifically expressed in somite tissue during somite segmentation. Loss-of-function experiments showed that CEP3 is not required for the specification of paraxial mesoderm, nor the differentiation of muscle cells, but is required for the segmentation process. Live imaging analysis further revealed that CEP3 is required for cell shape changes and alignment during somitogenesis. When CEP3 was knocked down, somitic cells did not elongate efficiently along the mediolateral axis and failed to undertake the 90° rotation. As a result, cells remained in a continuous sheet without an apparent segmentation cleft. CEP3 likely interacts with Cdc42 during this process, and both increased and decreased Cdc42 activity led to defective somite segmentation. Segmentation defects caused by Cdc42 knockdown can be partially rescued by the overexpression of CEP3. Conversely, loss of CEP3 resulted in the maintenance of high levels of Cdc42 activity at the cell membrane, which is normally reduced during and after somite segmentation. These results suggest that there is a feedback regulation between Cdc42 and CEP3 during somite segmentation and the activity of Cdc42 needs to be fine-tuned to control the coordinated cell shape changes and movement required for somite segmentation.

The metameric structure in vertebrates is based on the periodic formation of somites from the anterior end of the presomitic mesoderm (PSM). The segmentation boundary is defined by the Tbx6 expression domain, whose anterior limit is determined by Tbx6 protein destabilization via Ripply2. However, the molecular mechanism of this process is poorly understood. Here, we show that Ripply2 directly binds to Tbx6 in cultured cells without changing the stability of Tbx6, indicating an unknown mechanism for Tbx6 degradation in vivo. We succeeded in reproducing in vivo events using a mouse ES induction system, in which Tbx6 degradation occurred via Ripply2. Mass spectrometry analysis of the PSM-fated ES cells revealed that proteasomes are major components of the Ripply2-binding complex, suggesting that recruitment of a protein-degradation-complex is a pivotal function of Ripply2. Finally, we identified a motif in the T-box, which is required for Tbx6 degradation independent of binding with Ripply2 in vivo.

In vertebrate development, the sequential and rhythmic segmentation of the body axis is regulated by a “segmentation clock”. This clock is comprised of a population of coordinated oscillating cells that together produce rhythmic gene expression patterns in the embryo. Whether individual cells autonomously maintain oscillations, or whether oscillations depend on signals from neighboring cells is unknown. Using a transgenic zebrafish reporter line for the cyclic transcription factor Her1, we recorded single tailbud cells in vitro. We demonstrate that individual cells can behave as autonomous cellular oscillators. We described the observed variability in cell behavior using a theory of generic oscillators with correlated noise. Single cells have longer periods and lower precision than the tissue, highlighting the role of collective processes in the segmentation clock. Our work reveals a population of cells from the zebrafish segmentation clock that behave as self-sustained, autonomous oscillators with distinctive noisy dynamics.

Abstract Segmentation is a common feature of disparate clades of metazoans, and its evolution is a central problem of evolutionary developmental biology. We evolved in silico regulatory networks by a mutation/selection process that just rewards the number of segment boundaries. For segmentation controlled by a static gradient, as in long‐germ band insects, a cascade of adjacent repressors reminiscent of gap genes evolves. For sequential segmentation controlled by a moving gradient, similar to vertebrate somitogenesis, we invariably observe a very constrained evolutionary path or funnel. The evolved state is a cell autonomous ‘clock and wavefront’ model, with the new attribute of a separate bistable system driven by an autonomous clock. Early stages in the evolution of both modes of segmentation are functionally similar, and simulations suggest a possible path for their interconversion. Our computation illustrates how complex traits can evolve by the incremental addition of new functions on top of pre‐existing traits.

Although recently the properties of a single somite cell oscillator have been intensively investigated, the system-level nature of the segmentation clock remains largely unknown. To elaborate qualitatively this question, we examine the possibility to transform a well-known time delay somite cell oscillator to dynamical system of differential equations allowing qualitative analysis.

Abstract Background The Deepwater Horizon disaster was the largest marine oil spill in history, and total vertical exposure of oil to the water column suggests it could impact an enormous diversity of ecosystems. The most vulnerable organisms are those encountering these pollutants during their early life stages. Water-soluble components of crude oil and specific polycyclic aromatic hydrocarbons have been shown to cause defects in cardiovascular and craniofacial development in a variety of teleost species, but the developmental origins of these defects have yet to be determined. We have adopted zebrafish, Danio rerio, as a model to test whether water accumulated fractions (WAF) of the Deepwater Horizon oil could impact specific embryonic developmental processes. While not a native species to the Gulf waters, the developmental biology of zebrafish has been well characterized and makes it a powerful model system to reveal the cellular and molecular mechanisms behind Macondo crude toxicity. Results WAF of Macondo crude oil sampled during the oil spill was used to treat zebrafish throughout embryonic and larval development. Our results indicate that the Macondo crude oil causes a variety of significant defects in zebrafish embryogenesis, but these defects have specific developmental origins. WAF treatments caused defects in craniofacial development and circulatory function similar to previous reports, but we extend these results to show they are likely derived from an earlier defect in neural crest cell development. Moreover, we demonstrate that exposure to WAFs causes a variety of novel deformations in specific developmental processes, including programmed cell death, locomotor behavior, sensory and motor axon pathfinding, somitogenesis and muscle patterning. Interestingly, the severity of cell death and muscle phenotypes decreased over several months of repeated analysis, which was correlated with a rapid drop-off in the aromatic and alkane hydrocarbon components of the oil. Conclusions Whether these teratogenic effects are unique to the oil from the Deepwater Horizon oil spill or generalizable for most crude oil types remains to be determined. This work establishes a model for further investigation into the molecular mechanisms behind crude oil mediated deformations. In addition, due to the high conservation of genetic and cellular processes between zebrafish and other vertebrates, our work also provides a platform for more focused assessment of the impact that the Deepwater Horizon oil spill has had on the early life stages of native fish species in the Gulf of Mexico and the Atlantic Ocean.