A single-cell time-lapse of mouse prenatal development from gastrula to birth.

The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans1,2. Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing3 to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data4-8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb.

Reconstruction and deconstruction of human somitogenesis in vitro.

The vertebrate body displays a segmental organization that is most conspicuous in the periodic organization of the vertebral column and peripheral nerves. This metameric organization is first implemented when somites, which contain the precursors of skeletal muscles and vertebrae, are rhythmically generated from the presomitic mesoderm. Somites then become subdivided into anterior and posterior compartments that are essential for vertebral formation and segmental patterning of the peripheral nervous system1-4. How this key somitic subdivision is established remains poorly understood. Here we introduce three-dimensional culture systems of human pluripotent stem cells called somitoids and segmentoids, which recapitulate the formation of somite-like structures with anteroposterior identity. We identify a key function of the segmentation clock in converting temporal rhythmicity into the spatial regularity of anterior and posterior somitic compartments. We show that an initial 'salt and pepper' expression of the segmentation gene MESP2 in the newly formed segment is transformed into compartments of anterior and posterior identity through an active cell-sorting mechanism. Our research demonstrates that the major patterning modules that are involved in somitogenesis, including the clock and wavefront, anteroposterior polarity patterning and somite epithelialization, can be dissociated and operate independently in our in vitro systems. Together, we define a framework for the symmetry-breaking process that initiates somite polarity patterning. Our work provides a platform for decoding general principles of somitogenesis and advancing knowledge of human development.

Reconstituting human somitogenesis in vitro.

The segmented body plan of vertebrates is established during somitogenesis, a well-studied process in model organisms; however, the details of this process in humans remain largely unknown owing to ethical and technical limitations. Despite recent advances with pluripotent stem cell-based approaches1-5, models that robustly recapitulate human somitogenesis in both space and time remain scarce. Here we introduce a pluripotent stem cell-derived mesoderm-based 3D model of human segmentation and somitogenesis-which we termed 'axioloid'-that captures accurately the oscillatory dynamics of the segmentation clock and the morphological and molecular characteristics of sequential somite formation in vitro. Axioloids show proper rostrocaudal patterning of forming segments and robust anterior-posterior FGF-WNT signalling gradients and retinoic acid signalling components. We identify an unexpected critical role of retinoic acid signalling in the stabilization of forming segments, indicating distinct, but also synergistic effects of retinoic acid and extracellular matrix on the formation and epithelialization of somites. Comparative analysis demonstrates marked similarities of axioloids to the human embryo, further validated by the presence of a Hox code in axioloids. Finally, we demonstrate the utility of axioloids for studying the pathogenesis of human congenital spine diseases using induced pluripotent stem cells with mutations in HES7 and MESP2. Our results indicate that axioloids represent a promising platform for the study of axial development and disease in humans.

In vitro characterization of the human segmentation clock.

The segmental organization of the vertebral column is established early in embryogenesis, when pairs of somites are rhythmically produced by the presomitic mesoderm (PSM). The tempo of somite formation is controlled by a molecular oscillator known as the segmentation clock1,2. Although this oscillator has been well-characterized in model organisms1,2, whether a similar oscillator exists in humans remains unknown. Genetic analyses of patients with severe spine segmentation defects have implicated several human orthologues of cyclic genes that are associated with the mouse segmentation clock, suggesting that this oscillator might be conserved in humans3. Here we show that human PSM cells derived in vitro-as well as those of the mouse4-recapitulate the oscillations of the segmentation clock. Human PSM cells oscillate with a period two times longer than that of mouse cells (5 h versus 2.5 h), but are similarly regulated by FGF, WNT, Notch and YAP signalling5. Single-cell RNA sequencing reveals that mouse and human PSM cells in vitro follow a developmental trajectory similar to that of mouse PSM in vivo. Furthermore, we demonstrate that FGF signalling controls the phase and period of oscillations, expanding the role of this pathway beyond its classical interpretation in 'clock and wavefront' models1. Our work identifying the human segmentation clock represents an important milestone in understanding human developmental biology.

Coupling delay controls synchronized oscillation in the segmentation clock.

Individual cellular activities fluctuate but are constantly coordinated at the population level via cell-cell coupling. A notable example is the somite segmentation clock, in which the expression of clock genes (such as Hes7) oscillates in synchrony between the cells that comprise the presomitic mesoderm (PSM)1,2. This synchronization depends on the Notch signalling pathway; inhibiting this pathway desynchronizes oscillations, leading to somite fusion3-7. However, how Notch signalling regulates the synchronicity of HES7 oscillations is unknown. Here we establish a live-imaging system using a new fluorescent reporter (Achilles), which we fuse with HES7 to monitor synchronous oscillations in HES7 expression in the mouse PSM at a single-cell resolution. Wild-type cells can rapidly correct for phase fluctuations in HES7 oscillations, whereas the absence of the Notch modulator gene lunatic fringe (Lfng) leads to a loss of synchrony between PSM cells. Furthermore, HES7 oscillations are severely dampened in individual cells of Lfng-null PSM. However, when Lfng-null PSM cells were completely dissociated, the amplitude and periodicity of HES7 oscillations were almost normal, which suggests that LFNG is involved mostly in cell-cell coupling. Mixed cultures of control and Lfng-null PSM cells, and an optogenetic Notch signalling reporter assay, revealed that LFNG delays the signal-sending process of intercellular Notch signalling transmission. These results-together with mathematical modelling-raised the possibility that Lfng-null PSM cells shorten the coupling delay, thereby approaching a condition known as the oscillation or amplitude death of coupled oscillators8. Indeed, a small compound that lengthens the coupling delay partially rescues the amplitude and synchrony of HES7 oscillations in Lfng-null PSM cells. Our study reveals a delay control mechanism of the oscillatory networks involved in somite segmentation, and indicates that intercellular coupling with the correct delay is essential for synchronized oscillation.

An in vitro model of early anteroposterior organization during human development.

The body plan of the mammalian embryo is shaped through the process of gastrulation, an early developmental event that transforms an isotropic group of cells into an ensemble of tissues that is ordered with reference to three orthogonal axes1. Although model organisms have provided much insight into this process, we know very little about gastrulation in humans, owing to the difficulty of obtaining embryos at such early stages of development and the ethical and technical restrictions that limit the feasibility of observing gastrulation ex vivo2. Here we show that human embryonic stem cells can be used to generate gastruloids-three-dimensional multicellular aggregates that differentiate to form derivatives of the three germ layers organized spatiotemporally, without additional extra-embryonic tissues. Human gastruloids undergo elongation along an anteroposterior axis, and we use spatial transcriptomics to show that they exhibit patterned gene expression. This includes a signature of somitogenesis that suggests that 72-h human gastruloids show some features of Carnegie-stage-9 embryos3. Our study represents an experimentally tractable model system to reveal and examine human-specific regulatory processes that occur during axial organization in early development.

Recapitulating the human segmentation clock with pluripotent stem cells.

Pluripotent stem cells are increasingly used to model different aspects of embryogenesis and organ formation1. Despite recent advances in in vitro induction of major mesodermal lineages and cell types2,3, experimental model systems that can recapitulate more complex features of human mesoderm development and patterning are largely missing. Here we used induced pluripotent stem cells for the stepwise in vitro induction of presomitic mesoderm and its derivatives to model distinct aspects of human somitogenesis. We focused initially on modelling the human segmentation clock, a major biological concept believed to underlie the rhythmic and controlled emergence of somites, which give rise to the segmental pattern of the vertebrate axial skeleton. We observed oscillatory expression of core segmentation clock genes, including HES7 and DKK1, determined the period of the human segmentation clock to be around five hours, and demonstrated the presence of dynamic travelling-wave-like gene expression in in vitro-induced human presomitic mesoderm. Furthermore, we identified and compared oscillatory genes in human and mouse presomitic mesoderm derived from pluripotent stem cells, which revealed species-specific and shared molecular components and pathways associated with the putative mouse and human segmentation clocks. Using CRISPR-Cas9-based genome editing technology, we then targeted genes for which mutations in patients with segmentation defects of the vertebrae, such as spondylocostal dysostosis, have been reported (HES7, LFNG, DLL3 and MESP2). Subsequent analysis of patient-like and patient-derived induced pluripotent stem cells revealed gene-specific alterations in oscillation, synchronization or differentiation properties. Our findings provide insights into the human segmentation clock as well as diseases associated with human axial skeletogenesis.

Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids.

Gastruloids are three-dimensional aggregates of embryonic stem cells that display key features of mammalian development after implantation, including germ-layer specification and axial organization1-3. To date, the expression pattern of only a small number of genes in gastruloids has been explored with microscopy, and the extent to which genome-wide expression patterns in gastruloids mimic those in embryos is unclear. Here we compare mouse gastruloids with mouse embryos using single-cell RNA sequencing and spatial transcriptomics. We identify various embryonic cell types that were not previously known to be present in gastruloids, and show that key regulators of somitogenesis are expressed similarly between embryos and gastruloids. Using live imaging, we show that the somitogenesis clock is active in gastruloids and has dynamics that resemble those in vivo. Because gastruloids can be grown in large quantities, we performed a small screen that revealed how reduced FGF signalling induces a short-tail phenotype in embryos. Finally, we demonstrate that embedding in Matrigel induces gastruloids to generate somites with the correct rostral-caudal patterning, which appear sequentially in an anterior-to-posterior direction over time. This study thus shows the power of gastruloids as a model system for exploring development and somitogenesis in vitro in a high-throughput manner.

Repurposing of the enhancer-promoter communication underlies the compensation of Mesp2 by Mesp1.

Organisms are inherently equipped with buffering systems against genetic perturbations. Genetic compensation, the compensatory response by upregulating another gene or genes, is one such buffering mechanism. Recently, a well-conserved compensatory mechanism was proposed: transcriptional adaptation of homologs under the nonsense-mediated mRNA decay pathways. However, this model cannot explain the onset of all compensatory events. We report a novel genetic compensation mechanism operating over the Mesp gene locus. Mesp1 and Mesp2 are paralogs located adjacently in the genome. Mesp2 loss is partially rescued by Mesp1 upregulation in the presomitic mesoderm (PSM). Using a cultured PSM induction system, we reproduced the compensatory response in vitro and found that the Mesp2-enhancer is required to promote Mesp1. We revealed that the Mesp2-enhancer directly interacts with the Mesp1 promoter, thereby upregulating Mesp1 expression upon the loss of Mesp2. Of note, this interaction is established by genomic arrangement upon PSM development independently of Mesp2 disruption. We propose that the repurposing of this established enhancer-promoter communication is the mechanism underlying this compensatory response for the upregulation of the adjacent gene.

Cardiopharyngeal mesoderm origins of musculoskeletal and connective tissues in the mammalian pharynx.

Cardiopharyngeal mesoderm (CPM) gives rise to muscles of the head and heart. Using genetic lineage analysis in mice, we show that CPM develops into a broad range of pharyngeal structures and cell types encompassing musculoskeletal and connective tissues. We demonstrate that CPM contributes to medial pharyngeal skeletal and connective tissues associated with both branchiomeric and somite-derived neck muscles. CPM and neural crest cells (NCC) make complementary mediolateral contributions to pharyngeal structures, in a distribution established in the early embryo. We further show that biallelic expression of the CPM regulatory gene Tbx1, haploinsufficient in 22q11.2 deletion syndrome patients, is required for the correct patterning of muscles with CPM-derived connective tissue. Our results suggest that CPM plays a patterning role during muscle development, similar to that of NCC during craniofacial myogenesis. The broad lineage contributions of CPM to pharyngeal structures provide new insights into congenital disorders and evolution of the mammalian pharynx.

The nephric mesenchyme lineage of intermediate mesoderm is derived from Tbx6-expressing derivatives of neuro-mesodermal progenitors via BMP-dependent Osr1 function.

In vertebrate embryos, the kidney primordium metanephros is formed from two distinct cell lineages, Wolffian duct and metanephric mesenchyme, which were classically grouped as intermediate mesoderm. Whereas the reciprocal interactions between these two cell populations in kidney development have been studied extensively, the mechanisms generating them remain elusive. Here, we show that the mouse cell lineage that forms nephric mesenchyme develops as a subpopulation of Tbx6-expressing mesodermal precursor derivatives of neuro-mesodermal progenitors (NMPs) under the condition of bone morphogenetic protein (BMP)-signal-dependent Osr1 expression. The Osr1-expressing nephric mesenchyme precursors were confirmed as descendants of NMPs because they were labeled by Sox2 N1 enhancer-EGFP. In Tbx6 mutant embryos, nephric mesenchyme changed its fate into neural tissues, which reflected its NMP origin. In Osr1 mutant embryos, the specific region of the Tbx6-expressing mesoderm precursor, which normally expresses Osr1 and develops into the nephric mesenchyme, instead expressed the somite marker FoxC2. BMP signaling activated Osr1 expression in a region of TBX6-expressing mesoderm and elicited nephric mesenchyme development. This study suggested a new model of cell lineage segregation during gastrulation.

A somitic contribution to the apical ectodermal ridge is essential for fin formation.

The transition from fins to limbs was an important terrestrial adaptation, but how this crucial evolutionary shift arose developmentally is unknown. Current models focus on the distinct roles of the apical ectodermal ridge (AER) and the signaling molecules that it secretes during limb and fin outgrowth. In contrast to the limb AER, the AER of the fin rapidly transitions into the apical fold and in the process shuts off AER-derived signals that stimulate proliferation of the precursors of the appendicular skeleton. The differing fates of the AER during fish and tetrapod development have led to the speculation that fin-fold formation was one of the evolutionary hurdles to the AER-dependent expansion of the fin mesenchyme required to generate the increased appendicular structure evident within limbs. Consequently, a heterochronic shift in the AER-to-apical-fold transition has been postulated to be crucial for limb evolution. The ability to test this model has been hampered by a lack of understanding of the mechanisms controlling apical fold induction. Here we show that invasion by cells of a newly identified somite-derived lineage into the AER in zebrafish regulates apical fold induction. Ablation of these cells inhibits apical fold formation, prolongs AER activity and increases the amount of fin bud mesenchyme, suggesting that these cells could provide the timing mechanism proposed in Thorogood's clock model of the fin-to-limb transition. We further demonstrate that apical-fold inducing cells are progressively lost during gnathostome evolution;the absence of such cells within the tetrapod limb suggests that their loss may have been a necessary prelude to the attainment of limb-like structures in Devonian sarcopterygian fish.

What we can learn from embryos to understand the mesenchymal-to-epithelial transition in tumor progression.

Epithelial plasticity involved the terminal and transitional stages that occur during epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET), both are essential at different stages of early embryonic development that have been co-opted by cancer cells to undergo tumor metastasis. These processes are regulated at multiple instances, whereas the post-transcriptional regulation of key genes mediated by microRNAs is gaining major attention as a common and conserved pathway. In this review, we focus on discussing the latest findings of the cellular and molecular basis of the less characterized process of MET during embryonic development, with special attention to the role of microRNAs. Although we take in consideration the necessity of being cautious when extrapolating the obtained evidence, we propose some commonalities between early embryonic development and cancer progression that can shed light into our current understanding of this complex event and might aid in the design of specific therapeutic approaches.

Shaping the zebrafish myotome by intertissue friction and active stress.

Organ formation is an inherently biophysical process, requiring large-scale tissue deformations. Yet, understanding how complex organ shape emerges during development remains a major challenge. During zebrafish embryogenesis, large muscle segments, called myotomes, acquire a characteristic chevron morphology, which is believed to aid swimming. Myotome shape can be altered by perturbing muscle cell differentiation or the interaction between myotomes and surrounding tissues during morphogenesis. To disentangle the mechanisms contributing to shape formation of the myotome, we combine single-cell resolution live imaging with quantitative image analysis and theoretical modeling. We find that, soon after segmentation from the presomitic mesoderm, the future myotome spreads across the underlying tissues. The mechanical coupling between the future myotome and the surrounding tissues appears to spatially vary, effectively resulting in spatially heterogeneous friction. Using a vertex model combined with experimental validation, we show that the interplay of tissue spreading and friction is sufficient to drive the initial phase of chevron shape formation. However, local anisotropic stresses, generated during muscle cell differentiation, are necessary to reach the acute angle of the chevron in wild-type embryos. Finally, tissue plasticity is required for formation and maintenance of the chevron shape, which is mediated by orientated cellular rearrangements. Our work sheds light on how a spatiotemporal sequence of local cellular events can have a nonlocal and irreversible mechanical impact at the tissue scale, leading to robust organ shaping.

Neuromesodermal progenitors are a conserved source of spinal cord with divergent growth dynamics.

During gastrulation, embryonic cells become specified into distinct germ layers. In mouse, this continues throughout somitogenesis from a population of bipotent stem cells called neuromesodermal progenitors (NMps). However, the degree of self-renewal associated with NMps in the fast-developing zebrafish embryo is unclear. Using a genetic clone-tracing method, we labelled early embryonic progenitors and found a strong clonal similarity between spinal cord and mesoderm tissues. We followed individual cell lineages using light-sheet imaging, revealing a common neuromesodermal lineage contribution to a subset of spinal cord tissue across the anterior-posterior body axis. An initial population subdivides at mid-gastrula stages and is directly allocated to neural and mesodermal compartments during gastrulation. A second population in the tailbud undergoes delayed allocation to contribute to the neural and mesodermal compartment only at late somitogenesis. Cell tracking and retrospective cell fate assignment at late somitogenesis stages reveal these cells to be a collection of mono-fated progenitors. Our results suggest that NMps are a conserved population of bipotential progenitors, the lineage of which varies in a species-specific manner due to vastly different rates of differentiation and growth.

Corepressor SMRT is required to maintain Hox transcriptional memory during somitogenesis.

Nuclear hormone receptors (NRs), such as retinoic acid receptors (RARs), play critical roles in vertebrate development and homeostasis by regulating target gene transcription. Their activity is controlled by ligand-dependent release of corepressors and subsequent recruitment of coactivators, but how these individual receptor modes contribute to development are unknown. Here, we show that mice carrying targeted knockin mutations in the corepressor Silencing Mediator of Retinoid and Thyroid hormone receptor (SMRT) that specifically disable SMRT function in NR signaling (SMRTmRID), display defects in cranial neural crest cell-derived structures and posterior homeotic transformations of axial vertebrae. SMRTmRID embryos show enhanced transcription of RAR targets including Hox loci, resulting in respecification of vertebral identities. Up-regulated histone acetylation and decreased H3K27 methylation are evident in the Hox loci whose somitic expression boundaries are rostrally shifted. Furthermore, enhanced recruitment of super elongation complex is evident in rapidly induced non-Pol II-paused targets in SMRTmRID embryonic stem cells. These results demonstrate that SMRT-dependent repression of RAR is critical to establish and maintain the somitic Hox code and segmental identity during fetal development via epigenetic marking of target loci.

Lineage tracing of sclerotome cells in amphibian reveals that multipotent somitic cells originate from lateral somitic frontier.

The two somite compartments, dorso-lateral dermomyotome and medio-ventral sclerotome are major vertebrate novelties, but little is known about their evolutionary origin. We determined that sclerotome cells in Xenopus come from lateral somitic frontier (LSF) by lineage tracing, ablation experiments and histological analysis. We identified Twist1 as marker of migrating sclerotome progenitors in two amphibians, Xenopus and axolotl. From these results, three conclusions can be drawn. First, LSF is made up of multipotent somitic cells (MSCs) since LSF gives rise to sclerotome but also to dermomytome as already shown in Xenopus. Second, the basic scheme of somite compartmentalization is conserved from cephalochordates to anamniotes since in both cases, lateral cells envelop dorsally and ventrally the ancestral myotome, suggesting that lateral MSCs should already exist in cephalochordates. Third, the transition from anamniote to amniote vertebrates is characterized by extension of the MSCs domain to the entire somite at the expense of ancestral myotome since amniote somite is a naive tissue that subdivides into sclerotome and dermomyotome. Like neural crest pluripotent cells, MSCs are at the origin of major vertebrate novelties, namely hypaxial region of the somite, dermomyotome and sclerotome compartments. Hence, change in MSCs properties and location is involved in somite evolution.

Fine scale differences within the vagal neural crest for enteric nervous system formation.

The enteric nervous system is mostly derived from vagal neural crest (NC) cells adjacent to somites (s)1-7. We used in ovo focal fluorescent vital dyes and focal electroporation of fluorophore-encoding plasmids in quail embryos to investigate NC cell migration to the foregut initially and later throughout the entire gut. NC cells of different somite-level origins were largely separate until reaching the foregut at about QE2.5, when all routes converged. By QE3.5, NC cells of different somite-levels became mixed, although s1-s2 NC cells were mainly confined to rostral foregut. Mid-vagal NC-derived cells (s3 and s4 level) arrived earliest at the foregut, and occurred in greatest number. By QE6.5 ENS was present from foregut to hindgut. Mid-vagal NC-derived cells occurred in greatest numbers from foregut to distal hindgut. NC-derived cells of s2, s5, and s6 levels were fewer and were widely distributed but were never observed in the distal hindgut. Rostro-vagal (s1) and caudo-vagal (s7) levels were few and restricted to the foregut. Single somite levels of quail neural tube/NC from s1 to s8 were combined with chick aneural ChE4.5 midgut and hindgut and the ensemble was grown on the chorio-allantoic membrane for 6 days. This tests ENS-forming competence in the absence of intra-segmental competition between NC cells, of differential influences of segmental paraxial tissues, and of positional advantage. All vagal NC-levels, but not s8 level, furnished enteric plexuses in the recipient gut, but the density of both ENS cells in total and neurons was highest from mid-vagal level donors, as was the length colonised. We conclude that the fate and competence for ENS formation of vagal NC sub-levels is not uniform over the vagal level but is biased to favour mid-vagal levels. Overviewing this and prior studies suggests the vagal region is, as in its traditional sense, a natural unit but with complex sub-divisions.

Making muscle: skeletal myogenesis in vivo and in vitro.

Skeletal muscle is the largest tissue in the body and loss of its function or its regenerative properties results in debilitating musculoskeletal disorders. Understanding the mechanisms that drive skeletal muscle formation will not only help to unravel the molecular basis of skeletal muscle diseases, but also provide a roadmap for recapitulating skeletal myogenesis in vitro from pluripotent stem cells (PSCs). PSCs have become an important tool for probing developmental questions, while differentiated cell types allow the development of novel therapeutic strategies. In this Review, we provide a comprehensive overview of skeletal myogenesis from the earliest premyogenic progenitor stage to terminally differentiated myofibers, and discuss how this knowledge has been applied to differentiate PSCs into muscle fibers and their progenitors in vitro.