Somite Differentiation Research Articles

Core planar cell polarity genes VANGL1 and VANGL2 in predisposition to congenital vertebral malformations

Congenital scoliosis (CS), affecting approximately 0.5 to 1 in 1,000 live births, is commonly caused by congenital vertebral malformations (CVMs) arising from aberrant somitogenesis or somite differentiation. While Wnt/ß-catenin signaling has been implicated in somite development, the function of Wnt/planar cell polarity (Wnt/PCP) signaling in this process remains unclear. Here, we investigated the role of Vangl1 and Vangl2 in vertebral development and found that their deletion causes vertebral anomalies resembling human CVMs. Analysis of exome sequencing data from multiethnic CS patients revealed a number of rare and deleterious variants in VANGL1 and VANGL2, many of which exhibited loss-of-function and dominant-negative effects. Zebrafish models confirmed the pathogenicity of these variants. Furthermore, we found that Vangl1 knock-in (p.R258H) mice exhibited vertebral malformations in a Vangl gene dose- and environment-dependent manner. Our findings highlight critical roles for PCP signaling in vertebral development and predisposition to CVMs in CS patients, providing insights into the molecular mechanisms underlying this disorder.

Sall4 regulates posterior trunk mesoderm development by promoting mesodermal gene expression and repressing neural genes in the mesoderm.

The trunk axial skeleton develops from paraxial mesoderm cells. Our recent study demonstrated that conditional knockout of the stem cell factor Sall4 in mice by TCre caused tail truncation and a disorganized axial skeleton posterior to the lumbar level. Based on this phenotype, we hypothesized that, in addition to the previously reported role of Sall4 in neuromesodermal progenitors, Sall4 is involved in the development of the paraxial mesoderm tissue. Analysis of gene expression and SALL4 binding suggests that Sall4 directly or indirectly regulates genes involved in presomitic mesoderm differentiation, somite formation and somite differentiation. Furthermore, ATAC-seq in TCre; Sall4 mutant posterior trunk mesoderm shows that Sall4 knockout reduces chromatin accessibility. We found that Sall4-dependent open chromatin status drives activation and repression of WNT signaling activators and repressors, respectively, to promote WNT signaling. Moreover, footprinting analysis of ATAC-seq data suggests that Sall4-dependent chromatin accessibility facilitates CTCF binding, which contributes to the repression of neural genes within the mesoderm. This study unveils multiple mechanisms by which Sall4 regulates paraxial mesoderm development by directing activation of mesodermal genes and repression of neural genes.

A transcriptional and regulatory map of mouse somite maturation

The mammalian body plan is shaped by rhythmic segmentation of mesoderm into somites, which are transient embryonic structures that form down each side of the neural tube. We have analyzed the genome-wide transcriptional and chromatin dynamics occurring within nascent somites, from early inception of somitogenesis to the latest stages of body plan establishment. We created matched gene expression andopen chromatin maps for the three leading pairs of somites at six time points during mouse embryonic development. We show that the rate of somite differentiation accelerates as development progresses. We identified a conserved maturation program followed by all somites, but somites from more developed embryos concomitantly switch on differentiation programs from derivative cell lineages soon after segmentation. Integrated analysis of the somitic transcriptional and chromatin activities identified opposing regulatory modules controlling the onset of differentiation. Our results provide a powerful, high-resolution view of the molecular genetics underlying somitic development in mammals.

Controlling human organoid symmetry breaking reveals signaling gradients drive segmentation clock waves

Axial development of mammals involves coordinated morphogenetic events, including axial elongation, somitogenesis, and neural tube formation. To gain insight into the signals controlling the dynamics of human axial morphogenesis, we generated axially elongating organoids by inducing anteroposterior symmetry breaking of spatially coupled epithelial cysts derived from human pluripotent stem cells. Each organoid was composed of a neural tube flanked by presomitic mesoderm sequentially segmented into somites. Periodic activation of the somite differentiation gene MESP2 coincided in space and time with anteriorly traveling segmentation clock waves in the presomitic mesoderm of the organoids, recapitulating critical aspects of somitogenesis. Timed perturbations demonstrated that FGF and WNT signaling play distinct roles in axial elongation and somitogenesis, and that FGF signaling gradients drive segmentation clock waves. By generating and perturbing organoids that robustly recapitulate the architecture of multiple axial tissues in human embryos, this work offers a means to dissect mechanisms underlying human embryogenesis.

Dand5 is involved in zebrafish tailbud cell movement.

During vertebrate development, symmetry breaking occurs in the left-right organizer (LRO). The transfer of asymmetric molecular information to the lateral plate mesoderm is essential for the precise patterning of asymmetric internal organs, such as the heart. However, at the same developmental time, it is crucial to maintain symmetry at the somite level for correct musculature and vertebrae specification. We demonstrate how left-right signals affect the behavior of zebrafish somite cell precursors by using live imaging and fate mapping studies in dand5 homozygous mutants compared to wildtype embryos. We describe a population of cells in the vicinity of the LRO, named Non-KV Sox17:GFP+ Tailbud Cells (NKSTCs), which migrate anteriorly and contribute to future somites. We show that NKSTCs originate in a cluster of cells aligned with the midline, posterior to the LRO, and leave that cluster in a left-right alternating manner, primarily from the left side. Fate mapping revealed that more NKSTCs integrated somites on the left side of the embryo. We then abolished the asymmetric cues from the LRO using dand5-/- mutant embryos and verified that NKSTCs no longer displayed asymmetric patterns. Cell exit from the posterior cluster became bilaterally synchronous in dand5-/- mutants. Our study revealed a new link between somite specification and Dand5 function. The gene dand5 is well known as the first asymmetric gene involved in vertebrate LR development. This study revealed a new link for Dand5 as a player in cell exit from the maturation zone into the presomitic mesoderm, affecting the expression patterns of myogenic factors and tail size.

Ontogenesis and embryogenesis of Megaleporinus elongatus larvae (Characiformes: Anostomidae) from the Jequitinhonha River Basin

Five females and five males of Megaleporinus elongatus captured in the Jequitinhonha River were transferred to hypophysation tanks with a constant water flow at 26°C at the Machado Mineiro Fish Farm. They were submitted to induced reproduction by the hypophysation method. Crude carp pituitary extract (Cyprinus carpio) was injected into the coelomic cavity in two doses (2.5 and 5.0 mg/kg) for males and 3 doses (2.5, 5.0, and 2.5 mg/kg) for females. The doses were applied with a 24-hr interval between the 1st and 2nd doses and a 12-hr interval between the 2nd and 3rd ones. Oocytes and semen were obtained by manual extrusion, and the dry method was used for fertilisation. The eggs were kept in funnel type 20-litre incubators with constant water flow at 23°C. Egg samples were collected every 10 min and photographed under a stereomicroscope for analysis of embryonic development until hatching. The main events of embryogenesis and their respective duration times were: cleavage (45 min), blastula (3 hr 14 min), gastrula (5 hr 25 min), closure of blastopore (6 hr 34 min), differentiation of somites (13 hr 20 min), tail release (17 hr 50 min), and hatching (24 hr 50 min). During the larval ontogenesis, the yolk sac gradually decreased until its complete reabsorption on the 7th day after hatching. The opening of the mouth and digestive tract occurred from the 5th to the 7th day, indicating that larvae could feed exogenously. These results provide information to improve the techniques of induced spawning and cultivation of M. elongatus.

Nitrogen excretion during marsupial development in the terrestrial isopod Armadillidium vulgare

Marsupial embryos of Armadillidium vulgare (Isopoda: Oniscidea) were collected at different stages of development and assayed for products of nitrogen excretion. Stages were classified as early stage one, late stage one (clear embryo and somite differentiation), early stage two (chorion shed, prior to blastokinesis), late stage two (following blastokinesis), and mancae (vitelline membrane shed; second embryonic molt). Stage one and stage two embryos were primarily ammonotelic. Mancae showed a significant increase in stored uric acid and decrease in ammonia production, in most cases to undetectable levels. The increased metabolic rate of mancae, and the fact that they imbibe marsupial fluid prior to exiting the marsupium, may have favored a switch from ammonotely to uricotely to avoid ammonia toxicity. Protein metabolism, estimated from ammonia production, accounted for 7% of the measured catabolic rate in Stage 2 embryos. Newly emerged juveniles showed a >2-fold increase in metabolism relative to mancae, accompanying the transition from aquatic to aerial respiration. Following 48 h post-emergence, juveniles resumed ammonia excretion, volatilizing the base (NH3) as in later instars. Elevated ammonia excretion in early juveniles may derive from the catabolism of remaining yolk protein. A sharp increase in whole-animal glutamine in juveniles is consistent with its role as an intermediary nitrogen store during periodic ammonia excretion. Total ammonia concentration in the marsupial fluid fluctuated but did not increase significantly over time and ammonia was not volatilized across the oostegites, indicating that embryo ammonia is transported into the maternal hemolymph for excretion.

4D imaging reveals stage dependent random and directed cell motion during somite morphogenesis

Somites are paired embryonic segments that form in a regular sequence from unsegmented mesoderm during vertebrate development. Although transient structures they are of fundamental importance as they generate cell lineages of the musculoskeletal system in the trunk such as cartilage, tendon, bone, endothelial cells and skeletal muscle. Surprisingly, very little is known about cellular dynamics underlying the morphological transitions during somite differentiation. Here, we address this by examining cellular rearrangements and morphogenesis in differentiating somites using live multi-photon imaging of transgenic chick embryos, where all cells express a membrane-bound GFP. We specifically focussed on the dynamic cellular changes in two principle regions within the somite, the medial and lateral domains, to investigate extensive morphological transformations. Furthermore, by using quantitative analysis and cell tracking, we capture for the first time a directed movement of dermomyotomal progenitor cells towards the rostro-medial domain of the dermomyotome, where skeletal muscle formation initiates.

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.

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay.

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

Paraxis is required for somite morphogenesis and differentiation in Xenopus laevis.

In most vertebrates, the segmentation of the paraxial mesoderm involves the formation of metameric units called somites through a mesenchymal-epithelial transition. However, this process is different in Xenopus laevis because it does not form an epithelial somite. Xenopus somitogenesis is characterized by a complex cells rearrangement that requires the coordinated regulation of cell shape, adhesion, and motility. The molecular mechanisms that control these cell behaviors underlying somite formation are little known. Although the Paraxis has been implicated in the epithelialization of somite in chick and mouse, its role in Xenopus somite morphogenesis has not been determined. Using a morpholino and hormone-inducible construction approaches, we showed that both gain and loss of function of paraxis affect somite elongation, rotation and alignment, causing a severe disorganization of somitic tissue. We further found that depletion or overexpression of paraxis in the somite led to the downregulation or upregulation, respectively, of cell adhesion expression markers. Finally, we demonstrated that paraxis is necessary for the proper expression of myotomal and sclerotomal differentiation markers. Our results demonstrate that paraxis regulates the cell rearrangements that take place during the somitogenesis of Xenopus by regulating cell adhesion. Furthermore, paraxis is also required for somite differentiation.

Sprouty2 mediated tuning of signalling is essential for somite myogenesis.

BackgroundNegative regulators of signal transduction cascades play critical roles in controlling different aspects of normal embryonic development. Sprouty2 (Spry2) negatively regulates receptor tyrosine kinases (RTK) and FGF signalling and is important in differentiation, cell migration and proliferation. In vertebrate embryos, Spry2 is expressed in paraxial mesoderm and in forming somites. Expression is maintained in the myotome until late stages of somite differentiation. However, its role and mode of action during somite myogenesis is still unclear.ResultsHere, we analysed chick Spry2 expression and showed that it overlaps with that of myogenic regulatory factors MyoD and Mgn. Targeted mis-expression of Spry2 led to inhibition of myogenesis, whilst its C-terminal domain led to an increased number of myogenic cells by stimulating cell proliferation.ConclusionsSpry2 is expressed in somite myotomes and its expression overlaps with myogenic regulatory factors. Overexpression and dominant-negative interference showed that Spry2 plays a crucial role in regulating chick myogenesis by fine tuning of FGF signaling through a negative feedback loop. We also propose that mir-23, mir-27 and mir-128 could be part of the negative feedback loop mechanism. Our analysis is the first to shed some light on in vivo Spry2 function during chick somite myogenesis.

Circadian genes, xBmal1 and xNocturnin, modulate the timing and differentiation of somites in Xenopus laevis.

We have been investigating whether xBmal1 and xNocturnin play a role in somitogenesis, a cyclic developmental process with an ultradian period. Previous work from our lab shows that circadian genes (xPeriod1, xPeriod2, xBmal1, and xNocturnin) are expressed in developing somites. Somites eventually form the vertebrae, muscles of the back, and dermis. In Xenopus, a pair of somites is formed about every 50 minutes from anterior to posterior. We were intrigued by the co-localization of circadian genes in an embryonic tissue known to be regulated by an ultradian clock. Cyclic expression of genes involved in Notch signaling has been implicated in the somite clock. Disruption of Notch signaling in humans has been linked to skeletal defects in the vertebral column. We found that both depletion (morpholino) and overexpression (mRNA) of xBMAL1 protein (bHLH transcription factor) or xNOCTURNIN protein (deadenylase) on one side of the developing embryo led to a significant decrease in somite number with respect to the untreated side (p<0.001). These manipulations also significantly affect expression of a somite clock component (xESR9; p<0.05). We observed opposing effects on somite size. Depletion of xBMAL1 or xNOCTURNIN caused a statistically significant decrease in somite area (quantified using NIH ImageJ; p<0.002), while overexpression of these proteins caused a significant dose dependent increase in somite area (p<0.02; p<0.001, respectively). We speculate that circadian genes may play two separate roles during somitogenesis. Depletion and overexpression of xBMAL1 and NOCTURNIN both decrease somite number and influence expression of a somite clock component, suggesting that these proteins may modulate the timing of the somite clock in the undifferentiated presomitic mesoderm. The dosage dependent effects on somite area suggest that xBMAL1 and xNOCTURNIN may also act during somite differentiation to promote myogenesis.

The avian embryo as a model system for skeletal myogenesis.

This review will focus on the use of the chicken and quail as model systems to analyze myogenesis and as such will emphasize the experimental approaches that are strongest in these systems-the amenability of the avian embryo to manipulation and in ovo observation. During somite differentiation, a wide spectrum of developmental processes occur such as cellular differentiation, migration, and fusion. Cell lineage studies combined with recent advancements in cell imaging allow these biological phenomena to be readily observed and hypotheses tested extremely rapidly-a strength that is restricted to the avian system. A clear weakness of the chicken in the past has been genetic approaches to modulate gene function. Recent advances in the electroporation of expression vectors, siRNA constructs, and use of tissue specific reporters have opened the door to increasingly sophisticated experiments that address questions of interest not only to the somite/muscle field in particular but also fundamental to biology in general. Importantly, an ever-growing body of evidence indicates that somite differentiation in birds is indistinguishable to that of mammals; therefore, these avian studies complement the complex genetic models of the mouse.

Haematopoietic stem cell induction by somite-derived endothelial cells controlled by meox1.

Haematopoietic stem cells (HSCs) are self-renewing stem cells capable of replenishing all blood lineages. In all vertebrate embryos that have been studied, definitive HSCs are generated initially within the dorsal aorta (DA) of the embryonic vasculature by a series of poorly understood inductive events. Previous studies have identified that signalling relayed from adjacent somites coordinates HSC induction, but the nature of this signal has remained elusive. Here we reveal that somite specification of HSCs occurs via the deployment of a specific endothelial precursor population, which arises within a sub-compartment of the zebrafish somite that we have defined as the endotome. Endothelial cells of the endotome are specified within the nascent somite by the activity of the homeobox gene meox1. Specified endotomal cells consequently migrate and colonize the DA, where they induce HSC formation through the deployment of chemokine signalling activated in these cells during endotome formation. Loss of meox1 activity expands the endotome at the expense of a second somitic cell type, the muscle precursors of the dermomyotomal equivalent in zebrafish, the external cell layer. The resulting increase in endotome-derived cells that migrate to colonize the DA generates a dramatic increase in chemokine-dependent HSC induction. This study reveals the molecular basis for a novel somite lineage restriction mechanism and defines a new paradigm in induction of definitive HSCs.

MyomiR-dependent switching of BAF60 variant incorporation into Brg1 chromatin remodeling complexes during embryo myogenesis.

Myogenesis involves the stable commitment of progenitor cells followed by the execution of myogenic differentiation, processes that are coordinated by myogenic regulatory factors, microRNAs and BAF chromatin remodeling complexes. BAF60a, BAF60b and BAF60c are structural subunits of the BAF complex that bind to the core ATPase Brg1 to provide functional specificity. BAF60c is essential for myogenesis; however, the mechanisms regulating the subunit composition of BAF/Brg1 complexes, in particular the incorporation of different BAF60 variants, are not understood. Here we reveal their dynamic expression during embryo myogenesis and uncover the concerted negative regulation of BAF60a and BAF60b by the muscle-specific microRNAs (myomiRs) miR-133 and miR-1/206 during somite differentiation. MicroRNA inhibition in chick embryos leads to increased BAF60a or BAF60b levels, a concomitant switch in BAF/Brg1 subunit composition and delayed myogenesis. The phenotypes are mimicked by sustained BAF60a or BAF60b expression and are rescued by morpholino knockdown of BAF60a or BAF60b. This suggests that myomiRs contribute to select BAF60c for incorporation into the Brg1 complex by specifically targeting the alternative variants BAF60a and BAF60b during embryo myogenesis, and reveals that interactions between tissue-specific non-coding RNAs and chromatin remodeling factors confer robustness to mesodermal lineage determination.

NANOG and CDX2 Pattern Distinct Subtypes of Human Mesoderm during Exit from Pluripotency

Mesoderm is induced at the primitive streak (PS) and patterns subsequently into mesodermal subtypes and organ precursors. It is unclear whether mesoderm induction generates a multipotent PS progenitor or several distinct ones with restricted subtype potentials. We induced mesoderm in human pluripotent stem cells with ACTIVIN and BMP or with GSK3-β inhibition. Both approaches induced BRACHYURY(+) mesoderm of distinct PS-like identities, which had differing patterning potential. ACTIVIN and BMP-induced mesoderm patterned into cardiac but not somitic subtypes. Conversely, PS precursors induced by GSK3-β inhibition did not generate lateralplate and cardiac mesoderm and favored instead somitic differentiation. The mechanism of thesecell fate decisions involved mutual repression ofNANOG and CDX2. Although NANOG was required for cardiac specification but blocked somitic subtypes, CDX2 was required for somitic mesoderm but blocked cardiac differentiation. In sum, rather than forming a common PS progenitor, separate induction mechanisms distinguish human mesoderm subtypes.

Active repression by RARγ signaling is required for vertebrate axial elongation.

Retinoic acid receptor gamma 2 (RARγ2) is the major RAR isoform expressed throughout the caudal axial progenitor domain in vertebrates. During a microarray screen to identify RAR targets, we identified a subset of genes that pattern caudal structures or promote axial elongation and are upregulated by increased RAR-mediated repression. Previous studies have suggested that RAR is present in the caudal domain, but is quiescent until its activation in late stage embryos terminates axial elongation. By contrast, we show here that RARγ2 is engaged in all stages of axial elongation, not solely as a terminator of axial growth. In the absence of RA, RARγ2 represses transcriptional activity in vivo and maintains the pool of caudal progenitor cells and presomitic mesoderm. In the presence of RA, RARγ2 serves as an activator, facilitating somite differentiation. Treatment with an RARγ-selective inverse agonist (NRX205099) or overexpression of dominant-negative RARγ increases the expression of posterior Hox genes and that of marker genes for presomitic mesoderm and the chordoneural hinge. Conversely, when RAR-mediated repression is reduced by overexpressing a dominant-negative co-repressor (c-SMRT), a constitutively active RAR (VP16-RARγ2), or by treatment with an RARγ-selective agonist (NRX204647), expression of caudal genes is diminished and extension of the body axis is prematurely terminated. Hence, gene repression mediated by the unliganded RARγ2-co-repressor complex constitutes a novel mechanism to regulate and facilitate the correct expression levels and spatial restriction of key genes that maintain the caudal progenitor pool during axial elongation in Xenopus embryos.

FGF negative regulation during early myogenesis

Negative regulators of signal transduction cascades play critical roles in controlling different aspects of normal embryonic development. Sprouty2 negatively regulates FGF signaling and Receptor Tyrosine Kinases (RTK) and is important in differentiation, cell migration and proliferation. In vertebrate embryos, Sprouty2 is expressed in pre-segmented mesoderm and in forming somites. Expression is maintained in the myotome until late stages of somite differentiation. However, its role and mode of action during somite myogenesis is still unclear. In the current study, we analysed chick Sprouty2 expression and showed that it overlaps with that of Myogenic Regulatory Factors (MRF) MyoD and Mgn. Targeted mis-expression of Sprouty2 led to inhibition of myogenesis, whilst its C-terminal domain interference led to an increased number of myogenic cells by stimulating cell proliferation. Our results show that Sprouty2 plays crucial role in regulating chick myogenesis by fine tuning of FGF signaling through a negative feedback loop.