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Mesoderm specification and diversification: from single cells to emergent tissues
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ScienceDirect Mesoderm specification and diversification: from single cells to emergent tissues Elisabetta Ferretti1 and Anna-Katerina Hadjantonakis2 The three germ layers — mesoderm, endoderm and ectoderm — constituting the cellular blueprint for the tissues and organs that will form during embryonic development, are specified at gastrulation. Cells of mesodermal origin are the most abundant in the human body, representing a great variety of cell types, including the musculoskeletal system (bone, cartilage and muscle), cardiovascular system (heart, blood and blood vessels), as well as the connective tissues found throughout our bodies. A long-standing question pertains how this panoply of mesodermal cell types arises in a stereotypical fashion in time and space. This review discusses the events associated with mesoderm specification, highlighting the reconstruction of putative developmental trajectories facilitated by recent single-cell ‘omic’ data. We will also discuss the potential of emergent organoid systems to emulate and interrogate the dynamics of lineage specification at cellular resolution. Addresses 1 The Novo Nordisk Foundation Center for Stem Cell Biology, University of Copenhagen, DK-2200 Copenhagen, Denmark 2 Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, USA Corresponding authors: Ferretti, Elisabetta ([email protected]), Hadjantonakis, Anna-Katerina ([email protected])
Current Opinion in Cell Biology 2019, 61:110–116 This review comes from a themed issue on Differentiation and disease
(cell-based) models have been made possible by employing more complex cellular models of organogenesis called organoids [5,6]. Organoids provide a cohort of scalable deconstructed systems for mimicking in vivo events and facilitating analyses ranging from biochemical to imaging of cells at high spatial and temporal resolutions. Such systems overcome the limitation of real time observation of mammalian development and permit the assessment of how cells dynamically acquire their final states. Several degenerative diseases affect mesoderm-derived organs; these include muscular dystrophy, as well as chronic end-stage cardiac and renal diseases. Accordingly, stem cell-based therapy has become an important area of interest in regenerative medicine, with promise for in vitro generation of cells and tissues for the treatment and cure of chronic diseases and for the repair of damaged organs [7]. Single-cell data are pinpointing the sequential cell states associated with cellular differentiation, offering critical insights to guide protocols for stem cell-based therapies. The knowledge gained through the identification of rare progenitors, key decision (or bifurcation) points, signature markers, and intrinsic molecular networks, coupled with an understanding of a cell’s lineage history, serves as the blueprint for the establishment of novel approaches for tissue regeneration and repair.
Edited by Sara Wickstro¨m and Yingzi Yang
Mesoderm is a recent evolutionary invention https://doi.org/10.1016/j.ceb.2019.07.012 0955-0674/ã 2019 Elsevier Ltd. All rights reserved.
Introduction Hierarchical interactions and regulatory networks driving cellular differentiation are being investigated at continually greater spatial and temporal resolution [1,2,3,4]. The implementation of new technologies has provided unprecedented insights into key open questions; from assessing cell-to-cell heterogeneities within populations, to the fine-tuning of distinct lineage identities and their eventual segregation. Complementing and bridging established in vivo (animal) and in vitro Current Opinion in Cell Biology 2019, 61:110–116
Although mesoderm-derived cells are the highly represented within the vertebrate body, the mesoderm is the youngest germ layer in evolutionary terms [8]. Three germ layers are a hallmark feature of metazoans; from flatworms to humans. The appearance of the third (mesodermal) germ layer facilitated the development of sophisticated strategies for cell movement, reproduction, as well as nutrient and gas exchange. It is associated with vivipary adaptation, and the growth of embryos inside their mother’s body. It also promoted development of circulatory systems, and the formation of features which drive cellular locomotion (for example an epithelialto-mesenchymal transition or EMT [9]), and represents the main supportive tissue of many internal organs. Furthermore, the appearance of mesoderm coincided with the evolution of excretory and reproductive systems, allowing adaptation to different environments. In this way, the advent of mesoderm pioneered the emergence of distinct cell types having unique functions. www.sciencedirect.com
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Generation of mesoderm in the embryo The development of key mesodermal organs such as kidneys, heart and axial skeleton requires maturation of mesodermal progenitors that are specified during the initial phases of gastrulation into distinct mesodermal lineages (extraembryonic, lateral, intermediate, paraxial and chorda mesoderm) [10–12] (Figure1). In the mouse, starting at embryonic (E) day E6.25, mesoderm arises from a pool of pluripotent epiblast cells that continuously emerge from a transient and dynamic zone called the primitive streak (PS). Cells lose pluripotency and acquire distinct mesodermal lineage identities as they progressively ingress through the PS, concomitantly undergoing EMT while spreading over the embryo in the space between the visceral (extra-embryonic) endoderm and epiblast (Figure1). The future fate of epiblast cells is determined based on their position relative to the PS, which in turn dictates their time of ingression (Figure1). However, predominantly cell identities are specified based on proximity to instructive signals in the PS. Fate mapping studies have demonstrated that the time and position of cell ingression along the anterior-posterior (AP) length of the PS are critical determinants for the acquisition of distinct mesodermal identities [12,13]. In mice, the first progenitors specified arise at the posterior PS and generate the extra-embryonic mesoderm. Concomitantly more anterior progenitors give rise to the cardiac and
cranial lineages, followed by intermediate, paraxial and midline mesoderm [12,14]. After this initial induction, progenitors differentiate and migrate towards their final destination while new progenitors emerge. As consequence different cell types arise from different regions of the PS at different time points.
Lineage-specific differentiation initiates in both temporal and spatially sequence Extra-embryonic mesoderm will form the mesodermal component of the visceral yolk sac; it will generate the amnion, chorion and allantois, and establish the respiration and nutritional support via the umbilical connection between the mother and fetus [15]. Extra-embryonic mesoderm generates the primitive vasculature and red blood cells. Primitive erythroblasts arise in two consecutives waves of progenitors: the first at E7.5 generates primitive erythroid cells, megakaryocytes and macrophages, while at E8.25 a second generates hemangioblast progenitors that will contribute to the definitive embryonic hematopoietic system [16]. during cardiovascular development, Similarly, progenitors arise in a spatiotemporally regulated manner [17]. Cardiac and cranial progenitors are among the first mesodermal cell populations to emerge from the PS between E6.5 and E7.5. The first wave of cardiac progenitors originates in close proximity to cranial progenitors. These populations coordinately migrate to
Figure 1
Epithelial- to-mesenchymal transition (EMT)
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Organization of the mouse gastrula stage embryo and regional specification of distinct cell types at the primitive streak. www.sciencedirect.com
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the opposite (anterior) side of the embryo where the cardiac progenitors will form a linear heart tube located caudal to the cranial region. Thereafter, cells of the socalled second heart field (SHF) are specified and added to the developing heart tube. Genetic fate mapping using the inducible lineage marker Mesp1, revealed that the first wave of progenitors predominantly contributes to the left ventricle of the heart [18], while the SHF gives rise to right ventricle and outflow tract (OFT), and the progenitors of the head muscles and bones [19]. The intermediate mesoderm, which arises in-between the nascent paraxial and lateral plate mesoderm, generates the urogenital system including the kidneys, gonads, and adrenal cortex. During subsequent stages of axis extension, mesoderm is generated from so-called axial progenitors. The chordoneural hinge (CNH) region at the anterior PS is gives rise to all axial cell types. Paraxial mesoderm originates from the axially located bi-potential neuromesodermal progenitors, which also contribute to the spinal cord of the thoracic tract [20–24]. Paraxial mesoderm is segmented into sequentially ordered blocks of cells referred to as somites, which differentiate into the sclerotome and dermomyotome, giving rise to axial skeleton and muscles respectively [25]. Posterior Hox genes modulate the rate of somite formation reducing mesoderm ingression and axial elongation [26]. Finally, the notochord (embryonic midline) also arises from the CNH region [22,27,28], acts as a key signalling center, and forms a transient rod-like structure around which sclerotome cells condense to form the vertebrae [29].
migrating away. Thus, a dynamic signalling landscape underlies the acquisition of distinct mesodermal identities in precise locations and at different times. In addition to signal transduction, cells evaluate their position by other mechanisms, including mechanical cues, density of extracellular matrix, cell–cell contacts and cell movements [31]. The architecture, assembly and dynamics of the extracellular matrix (ECM) regulate cell movement and migration. These factors also impact cell identity by modulating the diffusion of morphogens and signalling cues. How can this complex scenario be interpreted and converted into a clear and robust readout of cellular identity? The recent implementation of sc-RNAseq for the study of embryonic development shed light onto this conundrum by providing new insights on signalling requirement and early lineage segregation (Figure 2).
Interrogating the emergent mesodermal landscape at the level of single-cells
What sets the tempo and the order of mesodermal progenitor ingression into the PS and their subsequent migration? How is this process terminated? How do the cells sense their position within the embryo and how they choose the differentiation programs to execute?
A comprehensive scRNA-seq analysis of developing mouse embryos ranging from E6.5 to E8.5 spanning the onset to cessation of gastrulation was recently conducted, providing predictive lineage trajectories [1,2,3]. This detailed temporal analysis defined sequential steps driving hemato-endothelial lineage emergence from extraembryonic mesoderm [1,2,3]. Pseudo-temporal analyses revealed two putative trajectories: at E7.5, a first wave of progenitors generating only embryonic primitive erythroids, and then at E8.25, a second wave of progenitors yielding endothelial and erythroid progenitors for definitive erythropoiesis1. Deeper analysis of these lineages led to the identification of rare megakaryocyte and myeloid progenitors. The latter carried signatures of microglial macrophages, suggesting the existence of early progenitors of brain microglia in the late gastrula embryos. If confirmed by lineage tracing in embryos, this observation will demonstrate that early myeloid progenitors can give rise to brain microglia and exist in the embryo before the onset of organogenesis.
In vertebrate embryos, PS formation is a readout of AP axis establishment [12,13]. Transplantation experiments revealed that mesodermal progenitors are plastic and commit upon reaching their final destinations [30]. Differentiation potential becomes restricted over time and concomitantly with patterning events leading to the stereotypical positioning and emergence of organs along the AP axis. Cell fate is progressively restricted through the integration of signalling cues (BMP, TGFb and WNT) that converge across the PS, and confer the spatial information critical for acquisition of specific identities at distinct positions and times [11]. However, the situation is complicated by the fact that the embryo grows during gastrulation, the PS elongates and regresses, and mesodermal progenitors are continuously being specified and
Mesp1 is a mesoderm-specific basic HLH transcription factor which is expressed through the PS and is critical for the development of cardiac mesoderm and epithelialization of paraxial mesoderm [32]. An inducible Mesp1 reporter allele was used to sort Mesp1-positive cardiac progenitor cells from E6.25 to E7.25 embryos [3]. scRNA-seq of these cells highlighted the presence of four different populations with distinct cardiac signatures localized within the PS: cardiomyocytes, endothelial, endocardial and SHF progenitors. These findings emphasized that cardiac progenitors have already segregated at the time of their exit from the PS. If this observation is confirmed by lineage tracing in embryos, it will corroborate the existence of early progenitors at the onset of organogenesis. As in other vertebrates such as the
Ordering time and space for the sequential acquisition of distinct mesodermal identities
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Figure 2
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Interconnecting embryos, single-cell RNAseq data, in silico (predictive) trajectories reconstructed from the single-cell RNAseq data, which in turn can be validated experimentally and spatially mapped back onto an embryo.
zebrafish, cell identity relationships based on scRNA-seq data have recently been coupled with lineage tracing using CRISPR-CAS9 barcode editing in mouse embryos [33,34,35,36,37]. One can envisage the use of such methods to obtain a complete lineage map of distinct and specialized mesoderm progenitors. Single cell ‘omics’ (sc-Omics) approaches have also been used to gain insight into the process of somitogenesis whereby the paraxial mesoderm becomes segmented into paired somites, and specifically the genes whose oscillatory expression establishes periodicity across the AP axis. Using Fgf8 expression as a positional landmark of posterior presomitic mesoderm, E8.25 mouse tail bud cells were ordered along a pseudo-space axis allowing the identification of genes with wave-like expression patterns [2]. This list included several well-known regulators of somitogenesis including Hes5, Lfn, Dll1, but also identified new oscillating genes such as Cited1. The latter has previously been described as a negative regulator of epithelial differentiation in the kidney [38], highlighting a possible role for this transcriptional coactivator in the maintenance of the mesenchymal nature of the interior of somites. A current limitation of sc-Omics approaches is the absence of spatial information. To spatially resolve transcriptomic data in vertebrate embryos, dissociated tissues were sequenced by scRNA-seq and then the cells were retrospectively mapped back to their respective anatomical location [34]. Similarly, cells dissected from defined www.sciencedirect.com
positions in the early mouse embryos were transcriptionally profiled in small defined cell population via RNA-seq [39]. In the future, a similar approach coupled with scRNA-seq could provide positional and temporal information to individual cells. Furthermore, the implementation of algorithms, statistical models and machine learning approaches will allow the accurate and automated reconstruction of cell lineage trajectories that elucidate upon the finely tuned molecular mechanisms guiding mesodermal progenitor segregation. Another limitation of scRNA-seq is the inability to identify the factors that direct cells towards particular identities. For example, differentiated cells, such as cardiomyocytes, acquire their identity through transitions between sequential intermediate states. Information on chromatin accessibility and enhancer usage can help identify cisregulatory regions driving these fate decisions. To this end, scATAC-seq was employed to identify transcription factor (TF) motifs that become accessible during cardiac differentiation. Isl1 is an homobox containing gene, primarily expressed in the cardiac progenitor cells of the SHF, which represent a reservoir of multipotent progenitors during cardiogenesis. Enhancer usage of the differentiating cardiac progenitors was assessed collecting Isl1-positive cells from E7.5 to E9.5 embryos and subjecting them to scATAC-seq [40]. Pseudo-temporal ordering of TF motifs revealed a specific usage and combination. Correlation with RNA expression provided putative matches, highlighting Hox and Gata protein cooperation within the cardiac lineage. Both Hox and Current Opinion in Cell Biology 2019, 61:110–116
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Gata proteins are important for heart development. Hox are evolutionarily highly conserved homeobox genes, which play a major role in the establishment of the body plan, providing positional information along the AP axis. Anterior Hox genes are specifically expressed in the SHF and contribute in the patterning of the outflow tract (OFT) [41]. GATA proteins are zinc finger TF that are present in the developing heart and regulate expression of several cardiac-specific genes [42]. Although it is still challenging to combine scRNA-seq and scATAC-seq data, exploiting information on transcriptomes with chromatin accessibility at the single-cell level will be critical for understanding the gene regulation of cell fate. Advances in a suite of sc-Omics approaches, including scATAC [43], scBisulfite [44], and scChIP [45], will reveal details of cellular responses to signalling cues, by elucidating the dynamic changes in the epigenetic landscape during differentiation, and how this impacts transcription.
Using single-cell approaches to dissect gene function Loss-of-function analyses are integral to the study of critical developmental processes, including cell fate specification, differentiation and patterning. Tal1 is a bHLH TF expressed in the posterior mesoderm and involved in the development of blood cells [46,47]. scRNA-seq combined with mutant embryo analyses have been used to highlight the unique role of Tal1 in blood lineage determination, by demonstrating that Tal1 is necessary not only for the establishment of a hematopoietic fate in both extra-embryonic and embryonic mesodermal tissues, but also for blocking alternative mesodermal fates [1,4]. Similarly, scRNA-seq analysis of Mesp1 mutant cardiac progenitors showed that Mesp1 promotes EMT, migration, and the acquisition of a cardiac fate [3].
Deconstructing mesoderm specification using organoid models While sc-Omics approaches will provide predictive lineage and developmental trajectories underlying the acquisition of distinct cell identities, tissue patterning represents the blueprint for organogenesis. The recent implementation of pluripotent stem cell-based organoid assays has helped deconstruct and interrogate the exit from pluripotency, and the orderly emergence of lineage identities in space and time. Mouse and human pluripotent embryonic stem cells (ESCs) plated on micropatterned discs self-organize, undergo EMT, and can be induced to differentiate into distinct lineages in a radial fashion [5,48,49,50,51]. Depending on the growth factors perceived (BMP, TGFb and/or WNT) distinct fates are adopted. While cells plated on micropatterns have radial symmetry and lack an AP axis [50,52,53], 3D self-organizing ESC-derived cellular bodies called gastruloids possess an AP axis and exhibit axial extension [53]. These different synthetic embryolike systems represent promising and complementary models to interrogate molecular mechanisms and cellular Current Opinion in Cell Biology 2019, 61:110–116
interactions. Thus, these approaches disentangle the relationships between signalling gradients, tissue growth, mechanical forces and cell fate. Furthermore, comparative sc-Omics approaches on embryos and organoids will establish whether the mechanisms guiding organoid development faithfully recapitulate aspects of embryonic development.
Concluding remarks and future directions Since early patterning events are inaccessible in human embryos, comparative analysis of human with mouse organoid systems, and mouse organoids with mouse embryos, will facilitate extrapolation to events taking place within the human embryo, identifying commonalities and distinctions. The coupling of multiple scOmic approaches with cell culture technologies will enable tissue regeneration, targeted differentiation and organ bio-engineering at an unprecedented scale, proving invaluable strategies to cure genetic and degenerative diseases.
Conflict of interest statement Nothing declared.
Acknowledgements We thank Josh Brickman, Niels Menezes and Clayton Schwarz for comments on this review. Work in the Ferretti lab is supported by Novo Nordisk Foundation Center for Stem Cell Biology (NNF17CC0027852), work in the Hadjantonakis lab is supported by grants from the National Institutes of Health (R01HD094868, R01DK084391 and P30CA00874).
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ScienceDirect Mesoderm specification and diversification: from single cells to emergent tissues Elisabetta Ferretti1 and Anna-Katerina Hadjantonakis2 The three germ layers — mesoderm, endoderm and ectoderm — constituting the cellular blueprint for the tissues and organs that will form during embryonic development, are specified at gastrulation. Cells of mesodermal origin are the most abundant in the human body, representing a great variety of cell types, including the musculoskeletal system (bone, cartilage and muscle), cardiovascular system (heart, blood and blood vessels), as well as the connective tissues found throughout our bodies. A long-standing question pertains how this panoply of mesodermal cell types arises in a stereotypical fashion in time and space. This review discusses the events associated with mesoderm specification, highlighting the reconstruction of putative developmental trajectories facilitated by recent single-cell ‘omic’ data. We will also discuss the potential of emergent organoid systems to emulate and interrogate the dynamics of lineage specification at cellular resolution. Addresses 1 The Novo Nordisk Foundation Center for Stem Cell Biology, University of Copenhagen, DK-2200 Copenhagen, Denmark 2 Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, USA Corresponding authors: Ferretti, Elisabetta ([email protected]), Hadjantonakis, Anna-Katerina ([email protected])
Current Opinion in Cell Biology 2019, 61:110–116 This review comes from a themed issue on Differentiation and disease
(cell-based) models have been made possible by employing more complex cellular models of organogenesis called organoids [5,6]. Organoids provide a cohort of scalable deconstructed systems for mimicking in vivo events and facilitating analyses ranging from biochemical to imaging of cells at high spatial and temporal resolutions. Such systems overcome the limitation of real time observation of mammalian development and permit the assessment of how cells dynamically acquire their final states. Several degenerative diseases affect mesoderm-derived organs; these include muscular dystrophy, as well as chronic end-stage cardiac and renal diseases. Accordingly, stem cell-based therapy has become an important area of interest in regenerative medicine, with promise for in vitro generation of cells and tissues for the treatment and cure of chronic diseases and for the repair of damaged organs [7]. Single-cell data are pinpointing the sequential cell states associated with cellular differentiation, offering critical insights to guide protocols for stem cell-based therapies. The knowledge gained through the identification of rare progenitors, key decision (or bifurcation) points, signature markers, and intrinsic molecular networks, coupled with an understanding of a cell’s lineage history, serves as the blueprint for the establishment of novel approaches for tissue regeneration and repair.
Edited by Sara Wickstro¨m and Yingzi Yang
Mesoderm is a recent evolutionary invention https://doi.org/10.1016/j.ceb.2019.07.012 0955-0674/ã 2019 Elsevier Ltd. All rights reserved.
Introduction Hierarchical interactions and regulatory networks driving cellular differentiation are being investigated at continually greater spatial and temporal resolution [1,2,3,4]. The implementation of new technologies has provided unprecedented insights into key open questions; from assessing cell-to-cell heterogeneities within populations, to the fine-tuning of distinct lineage identities and their eventual segregation. Complementing and bridging established in vivo (animal) and in vitro Current Opinion in Cell Biology 2019, 61:110–116
Although mesoderm-derived cells are the highly represented within the vertebrate body, the mesoderm is the youngest germ layer in evolutionary terms [8]. Three germ layers are a hallmark feature of metazoans; from flatworms to humans. The appearance of the third (mesodermal) germ layer facilitated the development of sophisticated strategies for cell movement, reproduction, as well as nutrient and gas exchange. It is associated with vivipary adaptation, and the growth of embryos inside their mother’s body. It also promoted development of circulatory systems, and the formation of features which drive cellular locomotion (for example an epithelialto-mesenchymal transition or EMT [9]), and represents the main supportive tissue of many internal organs. Furthermore, the appearance of mesoderm coincided with the evolution of excretory and reproductive systems, allowing adaptation to different environments. In this way, the advent of mesoderm pioneered the emergence of distinct cell types having unique functions. www.sciencedirect.com
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Generation of mesoderm in the embryo The development of key mesodermal organs such as kidneys, heart and axial skeleton requires maturation of mesodermal progenitors that are specified during the initial phases of gastrulation into distinct mesodermal lineages (extraembryonic, lateral, intermediate, paraxial and chorda mesoderm) [10–12] (Figure1). In the mouse, starting at embryonic (E) day E6.25, mesoderm arises from a pool of pluripotent epiblast cells that continuously emerge from a transient and dynamic zone called the primitive streak (PS). Cells lose pluripotency and acquire distinct mesodermal lineage identities as they progressively ingress through the PS, concomitantly undergoing EMT while spreading over the embryo in the space between the visceral (extra-embryonic) endoderm and epiblast (Figure1). The future fate of epiblast cells is determined based on their position relative to the PS, which in turn dictates their time of ingression (Figure1). However, predominantly cell identities are specified based on proximity to instructive signals in the PS. Fate mapping studies have demonstrated that the time and position of cell ingression along the anterior-posterior (AP) length of the PS are critical determinants for the acquisition of distinct mesodermal identities [12,13]. In mice, the first progenitors specified arise at the posterior PS and generate the extra-embryonic mesoderm. Concomitantly more anterior progenitors give rise to the cardiac and
cranial lineages, followed by intermediate, paraxial and midline mesoderm [12,14]. After this initial induction, progenitors differentiate and migrate towards their final destination while new progenitors emerge. As consequence different cell types arise from different regions of the PS at different time points.
Lineage-specific differentiation initiates in both temporal and spatially sequence Extra-embryonic mesoderm will form the mesodermal component of the visceral yolk sac; it will generate the amnion, chorion and allantois, and establish the respiration and nutritional support via the umbilical connection between the mother and fetus [15]. Extra-embryonic mesoderm generates the primitive vasculature and red blood cells. Primitive erythroblasts arise in two consecutives waves of progenitors: the first at E7.5 generates primitive erythroid cells, megakaryocytes and macrophages, while at E8.25 a second generates hemangioblast progenitors that will contribute to the definitive embryonic hematopoietic system [16]. during cardiovascular development, Similarly, progenitors arise in a spatiotemporally regulated manner [17]. Cardiac and cranial progenitors are among the first mesodermal cell populations to emerge from the PS between E6.5 and E7.5. The first wave of cardiac progenitors originates in close proximity to cranial progenitors. These populations coordinately migrate to
Figure 1
Epithelial- to-mesenchymal transition (EMT)
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itive S (PS treak ) Ca
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Organization of the mouse gastrula stage embryo and regional specification of distinct cell types at the primitive streak. www.sciencedirect.com
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the opposite (anterior) side of the embryo where the cardiac progenitors will form a linear heart tube located caudal to the cranial region. Thereafter, cells of the socalled second heart field (SHF) are specified and added to the developing heart tube. Genetic fate mapping using the inducible lineage marker Mesp1, revealed that the first wave of progenitors predominantly contributes to the left ventricle of the heart [18], while the SHF gives rise to right ventricle and outflow tract (OFT), and the progenitors of the head muscles and bones [19]. The intermediate mesoderm, which arises in-between the nascent paraxial and lateral plate mesoderm, generates the urogenital system including the kidneys, gonads, and adrenal cortex. During subsequent stages of axis extension, mesoderm is generated from so-called axial progenitors. The chordoneural hinge (CNH) region at the anterior PS is gives rise to all axial cell types. Paraxial mesoderm originates from the axially located bi-potential neuromesodermal progenitors, which also contribute to the spinal cord of the thoracic tract [20–24]. Paraxial mesoderm is segmented into sequentially ordered blocks of cells referred to as somites, which differentiate into the sclerotome and dermomyotome, giving rise to axial skeleton and muscles respectively [25]. Posterior Hox genes modulate the rate of somite formation reducing mesoderm ingression and axial elongation [26]. Finally, the notochord (embryonic midline) also arises from the CNH region [22,27,28], acts as a key signalling center, and forms a transient rod-like structure around which sclerotome cells condense to form the vertebrae [29].
migrating away. Thus, a dynamic signalling landscape underlies the acquisition of distinct mesodermal identities in precise locations and at different times. In addition to signal transduction, cells evaluate their position by other mechanisms, including mechanical cues, density of extracellular matrix, cell–cell contacts and cell movements [31]. The architecture, assembly and dynamics of the extracellular matrix (ECM) regulate cell movement and migration. These factors also impact cell identity by modulating the diffusion of morphogens and signalling cues. How can this complex scenario be interpreted and converted into a clear and robust readout of cellular identity? The recent implementation of sc-RNAseq for the study of embryonic development shed light onto this conundrum by providing new insights on signalling requirement and early lineage segregation (Figure 2).
Interrogating the emergent mesodermal landscape at the level of single-cells
What sets the tempo and the order of mesodermal progenitor ingression into the PS and their subsequent migration? How is this process terminated? How do the cells sense their position within the embryo and how they choose the differentiation programs to execute?
A comprehensive scRNA-seq analysis of developing mouse embryos ranging from E6.5 to E8.5 spanning the onset to cessation of gastrulation was recently conducted, providing predictive lineage trajectories [1,2,3]. This detailed temporal analysis defined sequential steps driving hemato-endothelial lineage emergence from extraembryonic mesoderm [1,2,3]. Pseudo-temporal analyses revealed two putative trajectories: at E7.5, a first wave of progenitors generating only embryonic primitive erythroids, and then at E8.25, a second wave of progenitors yielding endothelial and erythroid progenitors for definitive erythropoiesis1. Deeper analysis of these lineages led to the identification of rare megakaryocyte and myeloid progenitors. The latter carried signatures of microglial macrophages, suggesting the existence of early progenitors of brain microglia in the late gastrula embryos. If confirmed by lineage tracing in embryos, this observation will demonstrate that early myeloid progenitors can give rise to brain microglia and exist in the embryo before the onset of organogenesis.
In vertebrate embryos, PS formation is a readout of AP axis establishment [12,13]. Transplantation experiments revealed that mesodermal progenitors are plastic and commit upon reaching their final destinations [30]. Differentiation potential becomes restricted over time and concomitantly with patterning events leading to the stereotypical positioning and emergence of organs along the AP axis. Cell fate is progressively restricted through the integration of signalling cues (BMP, TGFb and WNT) that converge across the PS, and confer the spatial information critical for acquisition of specific identities at distinct positions and times [11]. However, the situation is complicated by the fact that the embryo grows during gastrulation, the PS elongates and regresses, and mesodermal progenitors are continuously being specified and
Mesp1 is a mesoderm-specific basic HLH transcription factor which is expressed through the PS and is critical for the development of cardiac mesoderm and epithelialization of paraxial mesoderm [32]. An inducible Mesp1 reporter allele was used to sort Mesp1-positive cardiac progenitor cells from E6.25 to E7.25 embryos [3]. scRNA-seq of these cells highlighted the presence of four different populations with distinct cardiac signatures localized within the PS: cardiomyocytes, endothelial, endocardial and SHF progenitors. These findings emphasized that cardiac progenitors have already segregated at the time of their exit from the PS. If this observation is confirmed by lineage tracing in embryos, it will corroborate the existence of early progenitors at the onset of organogenesis. As in other vertebrates such as the
Ordering time and space for the sequential acquisition of distinct mesodermal identities
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Figure 2
embryo
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Interconnecting embryos, single-cell RNAseq data, in silico (predictive) trajectories reconstructed from the single-cell RNAseq data, which in turn can be validated experimentally and spatially mapped back onto an embryo.
zebrafish, cell identity relationships based on scRNA-seq data have recently been coupled with lineage tracing using CRISPR-CAS9 barcode editing in mouse embryos [33,34,35,36,37]. One can envisage the use of such methods to obtain a complete lineage map of distinct and specialized mesoderm progenitors. Single cell ‘omics’ (sc-Omics) approaches have also been used to gain insight into the process of somitogenesis whereby the paraxial mesoderm becomes segmented into paired somites, and specifically the genes whose oscillatory expression establishes periodicity across the AP axis. Using Fgf8 expression as a positional landmark of posterior presomitic mesoderm, E8.25 mouse tail bud cells were ordered along a pseudo-space axis allowing the identification of genes with wave-like expression patterns [2]. This list included several well-known regulators of somitogenesis including Hes5, Lfn, Dll1, but also identified new oscillating genes such as Cited1. The latter has previously been described as a negative regulator of epithelial differentiation in the kidney [38], highlighting a possible role for this transcriptional coactivator in the maintenance of the mesenchymal nature of the interior of somites. A current limitation of sc-Omics approaches is the absence of spatial information. To spatially resolve transcriptomic data in vertebrate embryos, dissociated tissues were sequenced by scRNA-seq and then the cells were retrospectively mapped back to their respective anatomical location [34]. Similarly, cells dissected from defined www.sciencedirect.com
positions in the early mouse embryos were transcriptionally profiled in small defined cell population via RNA-seq [39]. In the future, a similar approach coupled with scRNA-seq could provide positional and temporal information to individual cells. Furthermore, the implementation of algorithms, statistical models and machine learning approaches will allow the accurate and automated reconstruction of cell lineage trajectories that elucidate upon the finely tuned molecular mechanisms guiding mesodermal progenitor segregation. Another limitation of scRNA-seq is the inability to identify the factors that direct cells towards particular identities. For example, differentiated cells, such as cardiomyocytes, acquire their identity through transitions between sequential intermediate states. Information on chromatin accessibility and enhancer usage can help identify cisregulatory regions driving these fate decisions. To this end, scATAC-seq was employed to identify transcription factor (TF) motifs that become accessible during cardiac differentiation. Isl1 is an homobox containing gene, primarily expressed in the cardiac progenitor cells of the SHF, which represent a reservoir of multipotent progenitors during cardiogenesis. Enhancer usage of the differentiating cardiac progenitors was assessed collecting Isl1-positive cells from E7.5 to E9.5 embryos and subjecting them to scATAC-seq [40]. Pseudo-temporal ordering of TF motifs revealed a specific usage and combination. Correlation with RNA expression provided putative matches, highlighting Hox and Gata protein cooperation within the cardiac lineage. Both Hox and Current Opinion in Cell Biology 2019, 61:110–116
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Gata proteins are important for heart development. Hox are evolutionarily highly conserved homeobox genes, which play a major role in the establishment of the body plan, providing positional information along the AP axis. Anterior Hox genes are specifically expressed in the SHF and contribute in the patterning of the outflow tract (OFT) [41]. GATA proteins are zinc finger TF that are present in the developing heart and regulate expression of several cardiac-specific genes [42]. Although it is still challenging to combine scRNA-seq and scATAC-seq data, exploiting information on transcriptomes with chromatin accessibility at the single-cell level will be critical for understanding the gene regulation of cell fate. Advances in a suite of sc-Omics approaches, including scATAC [43], scBisulfite [44], and scChIP [45], will reveal details of cellular responses to signalling cues, by elucidating the dynamic changes in the epigenetic landscape during differentiation, and how this impacts transcription.
Using single-cell approaches to dissect gene function Loss-of-function analyses are integral to the study of critical developmental processes, including cell fate specification, differentiation and patterning. Tal1 is a bHLH TF expressed in the posterior mesoderm and involved in the development of blood cells [46,47]. scRNA-seq combined with mutant embryo analyses have been used to highlight the unique role of Tal1 in blood lineage determination, by demonstrating that Tal1 is necessary not only for the establishment of a hematopoietic fate in both extra-embryonic and embryonic mesodermal tissues, but also for blocking alternative mesodermal fates [1,4]. Similarly, scRNA-seq analysis of Mesp1 mutant cardiac progenitors showed that Mesp1 promotes EMT, migration, and the acquisition of a cardiac fate [3].
Deconstructing mesoderm specification using organoid models While sc-Omics approaches will provide predictive lineage and developmental trajectories underlying the acquisition of distinct cell identities, tissue patterning represents the blueprint for organogenesis. The recent implementation of pluripotent stem cell-based organoid assays has helped deconstruct and interrogate the exit from pluripotency, and the orderly emergence of lineage identities in space and time. Mouse and human pluripotent embryonic stem cells (ESCs) plated on micropatterned discs self-organize, undergo EMT, and can be induced to differentiate into distinct lineages in a radial fashion [5,48,49,50,51]. Depending on the growth factors perceived (BMP, TGFb and/or WNT) distinct fates are adopted. While cells plated on micropatterns have radial symmetry and lack an AP axis [50,52,53], 3D self-organizing ESC-derived cellular bodies called gastruloids possess an AP axis and exhibit axial extension [53]. These different synthetic embryolike systems represent promising and complementary models to interrogate molecular mechanisms and cellular Current Opinion in Cell Biology 2019, 61:110–116
interactions. Thus, these approaches disentangle the relationships between signalling gradients, tissue growth, mechanical forces and cell fate. Furthermore, comparative sc-Omics approaches on embryos and organoids will establish whether the mechanisms guiding organoid development faithfully recapitulate aspects of embryonic development.
Concluding remarks and future directions Since early patterning events are inaccessible in human embryos, comparative analysis of human with mouse organoid systems, and mouse organoids with mouse embryos, will facilitate extrapolation to events taking place within the human embryo, identifying commonalities and distinctions. The coupling of multiple scOmic approaches with cell culture technologies will enable tissue regeneration, targeted differentiation and organ bio-engineering at an unprecedented scale, proving invaluable strategies to cure genetic and degenerative diseases.
Conflict of interest statement Nothing declared.
Acknowledgements We thank Josh Brickman, Niels Menezes and Clayton Schwarz for comments on this review. Work in the Ferretti lab is supported by Novo Nordisk Foundation Center for Stem Cell Biology (NNF17CC0027852), work in the Hadjantonakis lab is supported by grants from the National Institutes of Health (R01HD094868, R01DK084391 and P30CA00874).
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