In Vivo Human Somitogenesis Guides Somite Development from hPSCs.
ABSTRACT: Somites form during embryonic development and give rise to unique cell and tissue types, such as skeletal muscles and bones and cartilage of the vertebrae. Using somitogenesis-stage human embryos, we performed transcriptomic profiling of human presomitic mesoderm as well as nascent and developed somites. In addition to conserved pathways such as WNT-?-catenin, we also identified BMP and transforming growth factor ? (TGF-?) signaling as major regulators unique to human somitogenesis. This information enabled us to develop an efficient protocol to derive somite cells in vitro from human pluripotent stem cells (hPSCs). Importantly, the in-vitro-differentiating cells progressively expressed markers of the distinct developmental stages that are known to occur during in vivo somitogenesis. Furthermore, when subjected to lineage-specific differentiation conditions, the hPSC-derived somite cells were multipotent in generating somite derivatives, including skeletal myocytes, osteocytes, and chondrocytes. This work improves our understanding of human somitogenesis and may enhance our ability to treat diseases affecting somite derivatives.
Project description:Bipotent axial stem cells residing in the caudal epiblast during late gastrulation generate neuroectodermal and presomitic mesodermal progeny that coordinate somitogenesis with neural tube formation, but the mechanism that controls these two fates is not fully understood. Retinoic acid (RA) restricts the anterior extent of caudal fibroblast growth factor 8 (Fgf8) expression in both mesoderm and neural plate to control somitogenesis and neurogenesis, however it remains unclear where RA acts to control the spatial expression of caudal Fgf8. Here, we found that mouse Raldh2-/- embryos, lacking RA synthesis and displaying a consistent small somite defect, exhibited abnormal expression of key markers of axial stem cell progeny, with decreased Sox2+ and Sox1+ neuroectodermal progeny and increased Tbx6+ presomitic mesodermal progeny. The Raldh2-/- small somite defect was rescued by treatment with an FGF receptor antagonist. Rdh10 mutants, with a less severe RA synthesis defect, were found to exhibit a small somite defect and anterior expansion of caudal Fgf8 expression only for somites 1-6, with normal somite size and Fgf8 expression thereafter. Rdh10 mutants were found to lack RA activity during the early phase when somites are small, but at the 6-somite stage RA activity was detected in neural plate although not in presomitic mesoderm. Expression of a dominant-negative RA receptor in mesoderm eliminated RA activity in presomitic mesoderm but did not affect somitogenesis. Thus, RA activity in the neural plate is sufficient to prevent anterior expansion of caudal Fgf8 expression associated with a small somite defect. Our studies provide evidence that RA restriction of Fgf8 expression in undifferentiated neural progenitors stimulates neurogenesis while also restricting the anterior extent of the mesodermal Fgf8 mRNA gradient that controls somite size, providing new insight into the mechanism that coordinates somitogenesis with neurogenesis.
Project description:<h4>Background</h4>Somitogenesis is the earliest sign of segmentation in the developing vertebrate embryo. This process starts very early, soon after gastrulation has initiated and proceeds in an anterior-to-posterior direction during body axis elongation. It is widely accepted that somitogenesis is controlled by a molecular oscillator with the same periodicity as somite formation. This periodic mechanism is repeated a specific number of times until the embryo acquires a defined specie-specific final number of somites at the end of the process of axis elongation. This final number of somites varies widely between vertebrate species. How termination of the process of somitogenesis is determined is still unknown.<h4>Results</h4>Here we show that during development there is an imbalance between the speed of somite formation and growth of the presomitic mesoderm (PSM)/tail bud. This decrease in the PSM size of the chick embryo is not due to an acceleration of the speed of somite formation because it remains constant until the last stages of somitogenesis, when it slows down. When the chick embryo reaches its final number of somites at stage HH 24-25 there is still some remaining unsegmented PSM in which expression of components of the somitogenesis oscillator is no longer dynamic. Finally, we identify a change in expression of retinoic acid regulating factors in the tail bud at late stages of somitogenesis, such that in the chick embryo there is a pronounced onset of Raldh2 expression while in the mouse embryo the expression of the RA inhibitor Cyp26A1 is downregulated.<h4>Conclusions</h4>Our results show that the chick somitogenesis oscillator is arrested before all paraxial mesoderm is segmented into somites. In addition, endogenous retinoic acid is probably also involved in the termination of the process of segmentation, and in tail growth in general.
Project description:During somitogenesis, Fgf8 maintains the predifferentiation stage of presomitic mesoderm (PSM) cells and its retraction gives a cue for somite formation. Delta/Notch initiates the expression of oscillation genes in the tail bud and subsequently contributes to somite formation in a periodic way. Whether there exists a critical factor coordinating Fgf8 and Notch signaling pathways is largely unknown. Here, we demonstrate that the loss of function of geminin gave rise to narrower somites as a result of derepressed Fgf8 gradient in the PSM and tail bud. Furthermore, in geminin morphants, the somite boundary could not form properly but the oscillation of cyclic genes was normal, displaying the blurry somitic boundary and disturbed somite polarity along the AP axis. In mechanism, these manifestations were mediated by the disrupted association of the geminin/Brg1 complex with intron 3 of mib1. The latter interaction was found to positively regulate mib1 transcription, Notch activity, and sequential somite segmentation during somitogenesis. In addition, geminin was also shown to regulate the expression of deltaD in mib1-independent way. Collectively, our data for the first time demonstrate that geminin regulates Fgf8 and Notch signaling to regulate somite segmentation during somitogenesis.
Project description:Vertebrate embryo somite formation is temporally controlled by the cyclic expression of somitogenesis clock genes in the presomitic mesoderm (PSM). The somitogenesis clock is believed to be an intrinsic property of this tissue, operating independently of embryonic midline structures and the signaling molecules produced therein, namely Sonic hedgehog (Shh). This work revisits the notochord signaling contribution to temporal control of PSM segmentation by assessing the rate and number of somites formed and somitogenesis molecular clock gene expression oscillations upon notochord ablation. The absence of the notochord causes a delay in somite formation, accompanied by an increase in the period of molecular clock oscillations. Shh is the notochord-derived signal responsible for this effect, as these alterations are recapitulated by Shh signaling inhibitors and rescued by an external Shh supply. We have characterized chick smoothened expression pattern and have found that the PSM expresses both patched1 and smoothened Shh signal transducers. Upon notochord ablation, patched1, gli1, and fgf8 are down-regulated, whereas gli2 and gli3 are overexpressed. Strikingly, notochord-deprived PSM segmentation rate recovers over time, concomitant with raldh2 overexpression. Accordingly, exogenous RA supplement rescues notochord ablation effects on somite formation. A model is presented in which Shh and RA pathways converge to inhibit PSM Gli activity, ensuring timely somite formation. Altogether, our data provide evidence that a balance between different pathways ensures the robustness of timely somite formation and that notochord-derived Shh is a component of the molecular network regulating the pace of the somitogenesis clock.
Project description:Segment formation in vertebrate embryos is a stunning example of biological self-organization. Here, we present an idealized framework, in which we treat the presomitic mesoderm (PSM) as a one-dimensional line of oscillators. We use the framework to derive constraints that connect the size of somites, and the timing of their formation, to the growth of the PSM and the gradient of the somitogenesis clock period across the PSM. Our analysis recapitulates the observations made recently in ex vivo cultures of mouse PSM cells, and makes predictions for how perturbations, such as increased Wnt levels, would alter somite widths. Finally, our analysis makes testable predictions for the shape of the phase profile and somite widths at different stages of PSM growth. In particular, we show that the phase profile is robustly concave when the PSM length is steady and slightly convex in an important special case when it is decreasing exponentially. In both cases, the phase profile scales with the PSM length; in the latter case, it scales dynamically. This has important consequences for the velocity of the waves that traverse the PSM and trigger somite formation, as well as the effect of errors in phase measurement on somite widths.
Project description:The reproducibility of embryonic development is remarkable, although molecular processes are intrinsically stochastic at the single-cell level. How the multicellular system resists the inevitable noise to acquire developmental reproducibility constitutes a fundamental question in developmental biology. Toward this end, we focused on vertebrate somitogenesis as a representative system, because somites are repeatedly reproduced within a single embryo whereas such reproducibility is lost in segmentation clock gene-deficient embryos. However, the effect of noise on developmental reproducibility has not been fully investigated, because of the technical difficulty in manipulating the noise intensity in experiments. In this study, we developed a computational model of ERK-mediated somitogenesis, in which bistable ERK activity is regulated by an FGF gradient, cell-cell communication, and the segmentation clock, subject to the intrinsic noise. The model simulation generated our previous in vivo observation that the ERK activity was distributed in a step-like gradient in the presomitic mesoderm, and its boundary was posteriorly shifted by the clock in a stepwise manner, leading to regular somite formation. Here, we showed that this somite regularity was robustly maintained against the noise. Removing the clock from the model predicted that the stepwise shift of the ERK activity occurs at irregular timing with irregular distance owing to the noise, resulting in somite size variation. This model prediction was recently confirmed by live imaging of ERK activity in zebrafish embryos. Through theoretical analysis, we presented a mechanism by which the clock reduces the inherent somite irregularity observed in clock-deficient embryos. Therefore, this study indicates a novel role of the segmentation clock in noise-resistant developmental reproducibility.
Project description:BACKGROUND: The vertebrate adult axial skeleton, trunk and limb skeletal muscles and dermis of the back all arise from early embryonic structures called somites. Somites are symmetrically positioned flanking the embryo axial structures (neural tube and notochord) and are periodically formed in a anterior-posterior direction from the presomitic mesoderm. The time required to form a somite pair is constant and species-specific. This extraordinary periodicity is proposed to depend on an underlying somitogenesis molecular clock, firstly evidenced by the cyclic expression of the chick hairy1 gene in the unsegmented presomitic mesoderm with a 90 min periodicity, corresponding to the time required to form a somite pair in the chick embryo. The number of hairy1 oscillations at any given moment is proposed to provide the cell with both temporal and positional information along the embryo's anterior-posterior axis. Nevertheless, how this is accomplished and what biological processes are involved is still unknown. Aiming at understanding the molecular events triggered by the somitogenesis clock Hairy1 protein, we have employed the yeast two-hybrid system to identify Hairy1 interaction partners. RESULTS: Sap18, an adaptor molecule of the Sin3/HDAC transcriptional repressor complex, was found to interact with the C-terminal portion of the Hairy1 protein in a yeast two-hybrid assay and the Hairy1/Sap18 interaction was independently confirmed by co-immunoprecipitation experiments. We have characterized the expression patterns of both sap18 and sin3a genes during chick embryo development, using in situ hybridization experiments. We found that both sap18 and sin3a expression patterns co-localize in vivo with hairy1 expression domains in chick rostral presomitic mesoderm and caudal region of somites. CONCLUSION: Hairy1 belongs to the hairy-enhancer-of-split family of transcriptional repressor proteins. Our results indicate that during chick somitogenesis Hairy1 may mediate gene transcriptional repression by recruiting the Sin3/HDAC complex, through a direct interaction with the Sap18 adaptor molecule. Moreover, since sap18 and sin3a are not expressed in the PSM territory where hairy1 presents cyclic expression, our study strongly points to different roles for Hairy1 throughout the PSM and in the prospective somite and caudal region of already formed somites.
Project description:Somites form during embryonic development and give rise to unique cell and tissue types, such as skeletal muscles and bones and cartilages of the vertebrae. Using somitogenesis stage human embryos, we performed the first ever transcriptomic profiling of human presomitic mesoderm as well as nascent and developed somites. Using this approach we uncovered novel regulators unique duing human somitogensis, and efficiently guided human pluripotent stem cells to differentiate to somite cells and downstream progeny. This work improves our understanding of human somite development and may enhance our ability to model and treat diseases affecting somite derivatives. Overall design: Human embryos of 4.5-5 weeks of gestation were obtained from electively aborted fetuses following informed consent and de-identification. After procurement, tissues were immediately washed in sterile PBS, placed in PBS supplemented with 5% fetal bovine serum, 1% Penicillin-Streptomycin and 2.5 µg/mL Amphotericin B, and shipped on ice. Within 48 hours, targeted tissues were micro-dissected from the embryos. Developed somites (SM Dev) included the two somite pairs at the forelimb bud level, nascent somites (SM) included the two somite pairs just anterior to the most caudal segmentation border that was visible, and presomitic mesoderm (PSM) included the tail region mesoderm posterior to SM. Duplicates were used for each sample type for RNA-seq.
Project description:Somites are transient embryonic structures consisting of hundreds of cells that bud off from the anterior tip of the presomitic mesoderm (PSM) on each side of the neural tube. They give rise to the vertebrae and associated skeletal muscles, nerves, and blood vessels. To characterise the transcriptional changes that orchestrate mouse somitogenesis, we have generated coupled RNA-seq and ATAC-seq profiles of individual somites, across embryonic development. We collected the three most posterior pairs of somites, which correspond to those most recently segmented, from embryos containing 8, 18, 21, 25, 27 and 35 pairs of somites. The three somites are labelled I, II and III from the most posterior to the most anterior. From each pair, one somite was used for RNA-seq and the other one for ATAC-seq.
Project description:Somites are transient segments formed in a rostro-caudal progression during vertebrate development. In chick embryos, segmentation of a new pair of somites occurs every 90 minutes and involves a mesenchyme-to-epithelium transition of cells from the presomitic mesoderm. Little is known about the cellular rearrangements involved, and, although it is known that the fibronectin extracellular matrix is required, its actual role remains elusive. Using 3D and 4D imaging of somite formation we discovered that somitogenesis consists of a complex choreography of individual cell movements. Epithelialization starts medially with the formation of a transient epithelium of cuboidal cells, followed by cell elongation and reorganization into a pseudostratified epithelium of spindle-shaped epitheloid cells. Mesenchymal cells are then recruited to this medial epithelium through accretion, a phenomenon that spreads to all sides, except the lateral side of the forming somite, which epithelializes by cell elongation and intercalation. Surprisingly, an important contribution to the somite epithelium also comes from the continuous egression of mesenchymal cells from the core into the epithelium via its apical side. Inhibition of fibronectin matrix assembly first slows down the rate, and then halts somite formation, without affecting pseudopodial activity or cell body movements. Rather, cell elongation, centripetal alignment, N-cadherin polarization and egression are impaired, showing that the fibronectin matrix plays a role in polarizing and guiding the exploratory behavior of somitic cells. To our knowledge, this is the first 4D in vivo recording of a full mesenchyme-to-epithelium transition. This approach brought new insights into this event and highlighted the importance of the extracellular matrix as a guiding cue during morphogenesis.