Somite Patterning Research Articles

The people behind the papers - Julie Klepstad and Luciano Marcon.

The patterning of somites is coordinated by presomitic mesoderm cells through synchronised oscillations of Notch signalling, creating sequential waves of gene expression that propagate from the posterior to the anterior end of the tissue. In a new study, Klepstad and Marcon propose a new theoretical framework that recapitulates the dynamics of mouse somitogenesis observed in vivo and in vitro. To learn more about the story behind the paper, we caught up with first author Julie Klepstad and corresponding author Luciano Marcon, Principal Investigator at the Andalusian Center for Developmental Biology.

Notochord segmentation in zebrafish controlled by iterative mechanical signaling

In bony fishes, patterning of the vertebral column, or spine, is guided by a metameric blueprint established in the notochord sheath. Notochord segmentation begins days after somitogenesis concludes and can occur in its absence. However, somite patterning defects lead to imprecise notochord segmentation, suggesting thatthese processes are linked. Here, we identify that interactions between the notochord and the axial musculature ensure precise spatiotemporal segmentation of the zebrafish spine. We demonstrate that myoseptum-notochord linkages drive notochord segment initiation by locally deforming the notochord extracellular matrix and recruiting focal adhesion machinery at these contact points. Irregular somite patterning alters this mechanical signaling, causing non-sequential and dysmorphic notochord segmentation, leading to altered spine development. Using a model that captures myoseptum-notochord interactions, we find that a fixed spatial interval is critical for driving sequential segment initiation. Thus, mechanical coupling of axial tissues facilitates spatiotemporal spine patterning.

Ripply suppresses Tbx6 to induce dynamic-to-static conversion in somite segmentation

The metameric pattern of somites is created based on oscillatory expression of clock genes in presomitic mesoderm. However, the mechanism for converting the dynamic oscillation to a static pattern of somites is still unclear. Here, we provide evidence that Ripply/Tbx6 machinery is a key regulator of this conversion. Ripply1/Ripply2-mediated removal of Tbx6 protein defines somite boundary and also leads to cessation of clock gene expression in zebrafish embryos. On the other hand, activation of ripply1/ripply2 mRNA and protein expression is periodically regulated by clock oscillation in conjunction with an Erk signaling gradient. Whereas Ripply protein decreases rapidly in embryos, Ripply-triggered Tbx6 suppression persists long enough to complete somite boundary formation. Mathematical modeling shows that a molecular network based on results of this study can reproduce dynamic-to-static conversion in somitogenesis. Furthermore, simulations with this model suggest that sustained suppression of Tbx6 caused by Ripply is crucial in this conversion.

Deconstructing body axis morphogenesis in zebrafish embryos using robot-assisted tissue micromanipulation

Classic microsurgical techniques, such as those used in the early 1900s by Mangold and Spemann, have been instrumental in advancing our understanding of embryonic development. However, these techniques are highly specialized, leading to issues of inter-operator variability. Here we introduce a user-friendly robotic microsurgery platform that allows precise mechanical manipulation of soft tissues in zebrafish embryos. Using our platform, we reproducibly targeted precise regions of tail explants, and quantified the response in real-time by following notochord and presomitic mesoderm (PSM) morphogenesis and segmentation clock dynamics during vertebrate anteroposterior axis elongation. We find an extension force generated through the posterior notochord that is strong enough to buckle the structure. Our data suggest that this force generates a unidirectional notochord extension towards the tailbud because PSM tissue around the posterior notochord does not let it slide anteriorly. These results complement existing biomechanical models of axis elongation, revealing a critical coupling between the posterior notochord, the tailbud, and the PSM, and show that somite patterning is robust against structural perturbations.

Developmental toxicity of remdesivir, an anti-COVID-19 drug, is implicated by in vitro assays using morphogenetic embryoid bodies of mouse and human pluripotent stem cells.

Remdesivir is an antiviral drug approved for the treatment of COVID-19, whose developmental toxicity remains unclear. More information about the safety of remdesivir is urgently needed for people of childbearing potential, who are affected by the ongoing pandemic. Morphogenetic embryoid bodies (MEBs) are three-dimensional (3D) aggregates of pluripotent stem cells that recapitulate embryonic body patterning in vitro, and have been used as effective embryo models to detect the developmental toxicity of chemical exposures specifically and sensitively. MEBs were generated from mouse P19C5 and human H9 pluripotent stem cells, and used to examine the effects of remdesivir. The morphological effects were assessed by analyzing the morphometric parameters of MEBs after exposure to varying concentrations of remdesivir. The molecular impact of remdesivir was evaluated by measuring the transcript levels of developmental regulator genes. The mouse MEB morphogenesis was impaired by remdesivir at 1-8μM. Remdesivir affected MEBs in a manner dependent on metabolic conversion, and its potency was higher than GS-441524 and GS-621763, presumptive anti-COVID-19 drugs that act similarly to remdesivir. The expressions of developmental regulator genes, particularly those involved in axial and somite patterning, were dysregulated by remdesivir. The early stage of MEB development was more vulnerable to remdesivir exposure than the later stage. The morphogenesis and gene expression profiles of human MEBs were also impaired by remdesivir at 1-8μM. Remdesivir impaired mouse and human MEBs at concentrations that are comparable to the therapeutic plasma levels in humans, urging further investigation into the potential impact of remdesivir on developing embryos.

Hybrid reaction-diffusion and clock-and-wavefront model for the arrest of oscillations in the somitogenesis segmentation clock.

The clock and wavefront paradigm is arguably the most widely accepted model for explaining the embryonic process of somitogenesis. According to this model, somitogenesis is based upon the interaction between a genetic oscillator, known as segmentation clock, and a differentiation wavefront, which provides the positional information indicating where each pair of somites is formed. Shortly after the clock and wavefront paradigm was introduced, Meinhardt presented a conceptually different mathematical model for morphogenesis in general, and somitogenesis in particular. Recently, Cotterell et al. [A local, self-organizing reaction-diffusion model can explain somite patterning in embryos, Cell Syst. 1, 257-269 (2015)] rediscovered an equivalent model by systematically enumerating and studying small networks performing segmentation. Cotterell et al. called it a progressive oscillatory reaction-diffusion (PORD) model. In the Meinhardt-PORD model, somitogenesis is driven by short-range interactions and the posterior movement of the front is a local, emergent phenomenon, which is not controlled by global positional information. With this model, it is possible to explain some experimental observations that are incompatible with the clock and wavefront model. However, the Meinhardt-PORD model has some important disadvantages of its own. Namely, it is quite sensitive to fluctuations and depends on very specific initial conditions (which are not biologically realistic). In this work, we propose an equivalent Meinhardt-PORD model and then amend it to couple it with a wavefront consisting of a receding morphogen gradient. By doing so, we get a hybrid model between the Meinhardt-PORD and the clock-and-wavefront ones, which overcomes most of the deficiencies of the two originating models.

Somite Compartments in Amphioxus and Its Implications on the Evolution of the Vertebrate Skeletal Tissues.

Mineralized skeletal tissues of vertebrates are an evolutionary novelty within the chordate lineage. While the progenitor cells that contribute to vertebrate skeletal tissues are known to have two embryonic origins, the mesoderm and neural crest, the evolutionary origin of their developmental process remains unclear. Using cephalochordate amphioxus as our model, we found that cells at the lateral wall of the amphioxus somite express SPARC (a crucial gene for tissue mineralization) and various collagen genes. During development, some of these cells expand medially to surround the axial structures, including the neural tube, notochord and gut, while others expand laterally and ventrally to underlie the epidermis. Eventually these cell populations are found closely associated with the collagenous matrix around the neural tube, notochord, and dorsal aorta, and also with the dense collagen sheets underneath the epidermis. Using known genetic markers for distinct vertebrate somite compartments, we showed that the lateral wall of amphioxus somite likely corresponds to the vertebrate dermomyotome and lateral plate mesoderm. Furthermore, we demonstrated a conserved role for BMP signaling pathway in somite patterning of both amphioxus and vertebrates. These results suggest that compartmentalized somites and their contribution to primitive skeletal tissues are ancient traits that date back to the chordate common ancestor. The finding of SPARC-expressing skeletal scaffold in amphioxus further supports previous hypothesis regarding SPARC gene family expansion in the elaboration of the vertebrate mineralized skeleton.

Local modulation of the Wnt/β-catenin and bone morphogenic protein (BMP) pathways recapitulates rib defects analogous to cerebro-costo-mandibular syndrome.

Ribs are seldom affected by developmental disorders, however, multiple defects in rib structure are observed in the spliceosomal disease cerebro-costo-mandibular syndrome (CCMS). These defects include rib gaps, found in the posterior part of the costal shaft in multiple ribs, as well as missing ribs, shortened ribs and abnormal costotransverse articulations, which result in inadequate ventilation at birth and high perinatal mortality. The genetic mechanism of CCMS is a loss-of-function mutation in SNRPB, a component of the major spliceosome, and knockdown of this gene in vitro affects the activity of the Wnt/β-catenin and bone morphogenic protein (BMP) pathways. The aim of the present study was to investigate whether altering these pathways in vivo can recapitulate rib gaps and other rib abnormalities in the model animal. Chick embryos were implanted with beads soaked in Wnt/β-catenin and BMP pathway modulators during somitogenesis, and incubated until the ribs were formed. Some embryos were harvested in the preceding days for analysis of the chondrogenic marker Sox9, to determine whether pathway modulation affected somite patterning or chondrogenesis. Wnt/β-catenin inhibition manifested characteristic rib phenotypes seen in CCMS, including rib gaps (P<0.05) and missing ribs (P<0.05). BMP pathway activation did not cause rib gaps but yielded missing rib (P<0.01) and shortened rib phenotypes (P<0.05). A strong association with vertebral phenotypes was also noted with BMP4 (P<0.001), including scoliosis (P<0.05), a feature associated with CCMS. Reduced expression of Sox9 was detected with Wnt/β-catenin inhibition, indicating that inhibition of chondrogenesis precipitated the rib defects in the presence of Wnt/β-catenin inhibitors. BMP pathway activators also reduced Sox9 expression, indicating an interruption of somite patterning in the manifestation of rib defects with BMP4. The present study demonstrates that local inhibition of the Wnt/β-catenin and activation of the BMP pathway can recapitulate rib defects, such as those observed in CCMS. The balance of Wnt/β-catenin and BMP in the somite is vital for correct rib morphogenesis, and alteration of the activity of these two pathways in CCMS may perturb this balance during somite patterning, leading to the observed rib defects.

Role of somite patterning in the formation of Weberian apparatus and pleural rib in zebrafish.

In the vertebrate body, a metameric structure is present along the anterior-posterior axis. Zebrafish tbx6-/- larvae, in which somite boundaries do not form during embryogenesis, were shown to exhibit abnormal skeletal morphology such as rib, neural arch and hemal arch. In this study, we investigated the role of somite patterning in the formation of anterior vertebrae and ribs in more detail. Using three-dimensional computed tomography scans, we found that anterior vertebrae including the Weberian apparatus were severely affected in tbx6-/- larvae. In addition, pleural ribs of tbx6 mutants exhibited severe defects in the initial ossification, extension of ossification, and formation of parapophyses. Two-colour staining revealed that bifurcation of ribs was caused by fusion or branching of ribs in tbx6-/- . The parapophyses in tbx6-/- juvenile fish showed irregular positioning to centra and abnormal attachment to ribs. Furthermore, we found that the ossification of the distal portion of ribs proceeded along myotome boundaries even in irregularly positioned myotome boundaries. These results provide evidence of the contribution of somite patterning to the formation of the Weberian apparatus and rib in zebrafish.

The role of chick Ebf genes in the mediolateral patterning of the somites.

This study was conducted to check whether the three chick Early B-cell Factor (Ebf) genes, particularly cEbf1, would be targets for Shh and Bmp signals during somites mediolateral (ML) patterning. Tissue manipulations and gain and loss of function experiments for Shh and Bmp4 were performed and the results revealed that cEbf1 expression was initiated in the cranial presomitic mesoderm by low dose of Bmp4 from the lateral mesoderm and maintained in the ventromedial part of the epithelial somite and the medial sclerotome by Shh from the notochord; while cEbf2/3 expression was induced and maintained by Bmp4 and inhibited by high dose of Shh. To determine whether Ebf1 plays a role in somite patterning, transfection of a dominant-negative construct was carried out; this showed suppression of cPax1 expression in the medial sclerotome and upregulation and medial expansion of cEbf3 and cPax3 expression in sclerotome and dermomyotome, respectively, suggesting that Ebf1 is important for ML patterning. Thus, it is possible that low doses of Bmp4 set up Ebf1 expression which, together with Shh from the notochord, leads to establishment of the medial sclerotome and suppression of lateral identities. These data also conclude that Bmp4 is required in both the medial and lateral domain of the somitic mesoderm to keep the ML identity of the sclerotome through maintenance of cEbf gene expression. These striking findings are novel and give a new insight on the role of Bmp4 on mediolateral patterning of somites.

CONTRIBUTION OF SCLEROTOME TO THE EMBRYONIC HEMATOPOIETIC STEM CELL NICHE AND VASCULAR SMOOTH MUSCLE

Hematopoietic stem cells (HSCs) are a self-renewing population that sustains production of blood lineages throughout life. Vertebrate HSCs are first specified in the embryo, arising from hemogenic endothelium in the ventral wall of the dorsal aorta (DA). The cell types within this three-dimensional environment and the signals they provide to promote HSC development are not well defined. A better understanding of native embryonic HSC specification niche could inform efficient directed differentiation of true HSCs from induced pluripotent stem cells for clinical and research applications. We previously showed that wnt16 is required for development of zebrafish HSCs, as well as normal patterning of the ventral somite, the sclerotome. We hypothesized that normal sclerotome patterning is required for HSC specification by contributing to the cellular environment that presents inductive signals. To follow sclerotome development in living embryos, we developed transgenic reporter zebrafish where fluorophore expression is driven by regulatory elements from the sclerotome gene, pax1a. We show that sclerotome-derived cells migrate to and make contact with hemogenic endothelium of the DA shortly before initiation of the definitive hematopoietic program, demonstrating that they are present at the right time and place to present specification signals. By live imaging and profiling of sorted cells, we show these cells also give rise to arterial vascular smooth muscle cells. Loss-of-function analyses demonstrate a requirement for multiple sclerotome genes, for both patterning of sclerotome, and subsequent specification of HSCs and downstream hematopoietic lineages. Our data indicate that sclerotomal cells contribute to the HSC specification niche of the early embryo, and form a basis for defining the niche architecture and signals that drive definitive hematopoietic programming.

Regulatory Network of the Scoliosis-Associated Genes Establishes Rostrocaudal Patterning of Somites in Zebrafish

SummaryGene 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.

Entrainment as a means of controlling phase waves in populations of coupled oscillators

We explore waves and entrainment in a model of coupled oscillators, inspired from the cellular oscillators in the presomitic mesoderm (PSM) of mice. The internal clock in each cell is based on a negative feedback loop which couples to the clocks of neighboring cells through a Notch mechanism. We investigate how a morphogen gradient in the mesoderm, which affects the period of oscillating cells, gives rise to phase waves traveling from the posterior to the anterior part of the PSM. We show that the phase waves can be entrained by an external periodic variation in this morphogen and also observe that multiple oscillatory solutions can coexist in the cell population. Together, these provide a way to potentially control phase waves and thereby manipulate somite patterning in embryos, based on entrainment properties of coupled nonlinear oscillators.

Zebrafish embryos exposed to deltamethrin exhibit abnormalities despite induced expression of related genes (you, you-too, momo and u-boot)

Evaluation of the toxic effects of a widely used synthetic pyrethroid, deltamethrin (DM), was carried out in this study. This pesticide is preferred for pest control because of its low environmental persistence and toxicity. We investigated the expression pattern of four genes, namely, you ( you), yot ( you-too), momo ( mom) and ubo ( u-boot) during early development of zebrafish, that is, from 12 hpf to 48 hpf stages. These stages are selected as most of the important developmental aspects take place during this period. All four genes are known to play a vital role in development of notochord and somites. To understand the effect of DM on development, embryos of 4 hpf stage were exposed to two concentrations (100 and 200 µg/L) of DM, and observations were made at 12, 24 and 48 hpf stages. Our earlier studies have shown phenotypic abnormalities such as notochord bending, tail deformation, yolk sac and pericardial edema, lightening of body and eye pigmentation and interfered in somite patterning, during these stages of development. Understanding the relationship of phenotypic abnormalities with these four genes has been our primary objective. These four genes were analyzed by Reverse transcription (RT)-polymerase chain reaction and intensity of the bands has shown induction in their expression after exposure to the toxicant. In spite of the expression of genes, it was noticed that DM caused abnormalities. It can be said from the results that translational pathway could have been affected.

The ADAMTS5 Metzincin Regulates Zebrafish Somite Differentiation.

The ADAMTS5 metzincin, a secreted zinc-dependent metalloproteinase, modulates the extracellular matrix (ECM) during limb morphogenesis and other developmental processes. Here, the role of ADAMTS5 was investigated by knockdown of zebrafish adamts5 during embryogenesis. This revealed impaired Sonic Hedgehog (Shh) signaling during somite patterning and early myogenesis. Notably, synergistic regulation of myod expression by ADAMTS5 and Shh during somite differentiation was observed. These roles were not dependent upon the catalytic activity of ADAMTS5. These data identify a non-enzymatic function for ADAMTS5 in regulating an important cell signaling pathway that impacts on muscle development, with implications for musculoskeletal diseases in which ADAMTS5 and Shh have been associated.

Bifurcation Analysis About a Mathematical Model of Somitogenesis Based on the Runge–Kutta Method

In this paper, we investigated the modified two dimensional model which can explain somite patterning in embryos. It is suitable for exploring a design space of somitogenesis and can explain aspects of somitogenesis that previous models cannot. Here, we mainly studied the non-diffusing case. We have used the Hopf bifurcation theorem, the Center manifold theorem and Runge---Kutta method in our investigation. First, we investigate its dynamical behaviors and put forward a sufficient condition for the oscillation of the small network. Then, we give the mathematical simulation based on the Runge---Kutta method. In the process of solving ordinary differential equations, the four order Runge---Kutta method has the advantages of high accuracy, convergence and stability (under certain conditions), which can change the step size and do not need to calculate higher order derivatives. Therefore, it has become the most commonly used numerical solution. At the same time, we get the sufficient condition in which the bistable state of the system exists and give the numerical simulation. Because somitogenesis occupies an important position in the process of biological development, and as a pattern process can be used to study pattern formation and many aspects of embryogenesis. So our study have a great help for embryonic development, gene expression, cell differentiation. In addition, it is beneficial to study the clone animal variation problem of spinal bone number and is of great help to the treatment and prevention of defects of human spine disease.

Engrailed controls epaxial-hypaxial muscle innervation and the establishment of vertebrate three-dimensional mobility

Chordates are characterised by contractile muscle on either side of the body that promotes movement by side-to-side undulation. In the lineage leading to modern jawed vertebrates (crown group gnathostomes), this system was refined: body muscle became segregated into distinct dorsal (epaxial) and ventral (hypaxial) components that are separately innervated by the medial and hypaxial motors column, respectively, via the dorsal and ventral ramus of the spinal nerves. This allows full three-dimensional mobility, which in turn was a key factor in their evolutionary success. How the new gnathostome system is established during embryogenesis and how it may have evolved in the ancestors of modern vertebrates is not known.Vertebrate Engrailed genes have a peculiar expression pattern as they temporarily demarcate a central domain of the developing musculature at the epaxial-hypaxial boundary. Moreover, they are the only genes known with this particular expression pattern. The aim of this study was to investigate whether Engrailed genes control epaxial-hypaxial muscle development and innervation.Investigating chick, mouse and zebrafish as major gnathostome model organisms, we found that the Engrailed expression domain was associated with the establishment of the epaxial-hypaxial boundary of muscle in all three species. Moreover, the outgrowing epaxial and hypaxial nerves orientated themselves with respect to this Engrailed domain. In the chicken, loss and gain of Engrailed function changed epaxial-hypaxial somite patterning. Importantly, in all animals studied, loss and gain of Engrailed function severely disrupted the pathfinding of the spinal motor axons, suggesting that Engrailed plays an evolutionarily conserved role in the separate innervation of vertebrate epaxial-hypaxial muscle.

RARβ2 is required for vertebrate somitogenesis.

During vertebrate somitogenesis, retinoic acid is known to establish the position of the determination wavefront, controlling where new somites are permitted to form along the anteroposterior body axis. Less is understood about how RAR regulates somite patterning, rostral-caudal boundary setting, specialization of myotome subdivisions or the specific RAR subtype that is required for somite patterning. Characterizing the function of RARβ has been challenging due to the absence of embryonic phenotypes in murine loss-of-function studies. Using the Xenopus system, we show that RARβ2 plays a specific role in somite number and size, restriction of the presomitic mesoderm anterior border, somite chevron morphology and hypaxial myoblast migration. Rarβ2 is the RAR subtype whose expression is most upregulated in response to ligand and its localization in the trunk somites positions it at the right time and place to respond to embryonic retinoid levels during somitogenesis. RARβ2 positively regulates Tbx3 a marker of hypaxial muscle, and negatively regulates Tbx6 via Ripply2 to restrict the anterior boundaries of the presomitic mesoderm and caudal progenitor pool. These results demonstrate for the first time an early and essential role for RARβ2 in vertebrate somitogenesis.

Supt20 is required for development of the axial skeleton

Somitogenesis and subsequent axial skeletal development is regulated by the interaction of pathways that determine the periodicity of somite formation, rostrocaudal somite polarity and segment identity. Here we use a hypomorphic mutant mouse line to demonstrate that Supt20 (Suppressor of Ty20) is required for development of the axial skeleton. Supt20 hypomorphs display fusions of the ribs and vertebrae at lower thoracic levels along with anterior homeotic transformation of L1 to T14. These defects are preceded by reduction of the rostral somite and posterior shifts in Hox gene expression. While cycling of Notch target genes in the posterior presomitic mesoderm (PSM) appeared normal, expression of Lfng was reduced. In the anterior PSM, Mesp2 expression levels and cycling were unaffected; yet, expression of downstream targets such as Lfng, Ripply2, Mesp1 and Dll3 in the prospective rostral somite was reduced accompanied by expansion of caudal somite markers such as EphrinB2 and Hes7. Supt20 interacts with the Gcn5-containing SAGA histone acetylation complex. Gcn5 hypomorphic mutant embryos show similar defects in axial skeletal development preceded by posterior shift of Hoxc8 and Hoxc9 gene expression. We demonstrate that Gcn5 and Supt20 hypomorphs show similar defects in rostral-caudal somite patterning potentially suggesting shared mechanisms.

Quadruple zebrafish mutant reveals different roles of Mesp genes in somite segmentation between mouse and zebrafish.

The segmental pattern of somites is generated by sequential conversion of the temporal periodicity provided by the molecular clock. Whereas the basic structure of this clock is conserved among different species, diversity also exists, especially in terms of the molecular network. The temporal periodicity is subsequently converted into the spatial pattern of somites, and Mesp2 plays crucial roles in this conversion in the mouse. However, it remains unclear whether Mesp genes play similar roles in other vertebrates. In this study, we generated zebrafish mutants lacking all four zebrafish Mesp genes by using TALEN-mediated genome editing. Contrary to the situation in the mouse Mesp2 mutant, in the zebrafish Mesp quadruple mutant embryos the positions of somite boundaries were clearly determined and morphological boundaries were formed, although their formation was not completely normal. However, each somite was caudalized in a similar manner to the mouse Mesp2 mutant, and the superficial horizontal myoseptum and lateral line primordia were not properly formed in the quadruple mutants. These results clarify the conserved and species-specific roles of Mesp in the link between the molecular clock and somite morphogenesis.