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.

Time-aligned hourglass gastrulation models in rabbit and mouse.

The hourglass model describes the convergence of species within the same phylum to a similar body plan during development; however, the molecular mechanisms underlying this phenomenon in mammals remain poorly described. Here, we compare rabbit and mouse time-resolved differentiation trajectories to revisit this model at single-cell resolution. We modeled gastrulation dynamics using hundreds of embryos sampled between gestation days 6.0 and 8.5 and compared the species using a framework for time-resolved single-cell differentiation-flows analysis. We find convergence toward similar cell-state compositions at E7.5, supported by the quantitatively conserved expression of 76 transcription factors, despite divergence in surrounding trophoblast and hypoblast signaling. However, we observed noticeable changes in specification timing of some lineages and divergence of primordial germ cell programs, which in the rabbit do not activate mesoderm genes. Comparative analysis of temporal differentiation models provides a basis for studying the evolution of gastrulation dynamics across mammals.

Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease.

Intestinal mesenchymal cells play essential roles in epithelial homeostasis, matrix remodeling, immunity, and inflammation. But the extent of heterogeneity within the colonic mesenchyme in these processes remains unknown. Using unbiased single-cell profiling of over 16,500 colonic mesenchymal cells, we reveal four subsets of fibroblasts expressing divergent transcriptional regulators and functional pathways, in addition to pericytes and myofibroblasts. We identified a niche population located in proximity to epithelial crypts expressing SOX6, F3 (CD142), and WNT genes essential for colonic epithelial stem cell function. In colitis, we observed dysregulation of this niche and emergence of an activated mesenchymal population. This subset expressed TNF superfamily member 14 (TNFSF14), fibroblastic reticular cell-associated genes, IL-33, and Lysyl oxidases. Further, it induced factors that impaired epithelial proliferation and maturation and contributed to oxidative stress and disease severity in vivo. Our work defines how the colonic mesenchyme remodels to fuel inflammation and barrier dysfunction in IBD.

Mesenchyme governs hair follicle induction.

Tissue interactions are essential for guiding organ development and regeneration. Hair follicle formation relies on inductive signalling between two tissues, the embryonic surface epithelium and the adjacent mesenchyme. Although previous research has highlighted the hair-inducing potential of the mesenchymal component of the hair follicle - the dermal papilla and its precursor, the dermal condensate - the source and nature of the primary inductive signal before dermal condensate formation have remained elusive. Here, we performed epithelial-mesenchymal tissue recombination experiments using hair-forming back skin and glabrous plantar skin from mouse embryos to unveil that the back skin mesenchyme is inductive even before dermal condensate formation. Moreover, the naïve, unpatterned mesenchyme was sufficient to trigger hair follicle formation even in the oral epithelium. Building on previous knowledge, we explored the hair-inductive ability of the Wnt agonist R-spondin 1 and a Bmp receptor inhibitor in embryonic skin explants. Although R-spondin 1 instigated precocious placode-specific transcriptional responses, it was insufficient for hair follicle induction, either alone or in combination with Bmp receptor inhibition. Our findings pave the way for identifying the hair follicle-inducing cue.

The ins and outs of the epithelial to mesenchymal transition in health and disease.

The epithelial to mesenchymal transition (EMT) converts epithelial cells into migratory and invasive cells and is a fundamental event in morphogenesis. Although its relevance in the progression of cancer and organ fibrosis had been debated until recently, the EMT is now established as an important step in the metastatic cascade of epithelial tumors. The similarities between pathological and developmental EMTs validate the embryo as the best model to understand the molecular and cellular mechanisms involved in this process, identifying those that are hijacked during the progression of cancer and organ degeneration. Our ever-increasing understanding of how transcription factors regulate the EMT has revealed complex regulatory loops coupled to posttranscriptional and epigenetic regulatory programs. The EMT is now integrated into the systemic activities of whole organisms, establishing links with cell survival, stemness, inflammation, and immunity. In addition, the EMT now constitutes a promising target for the treatment of cancer and organ-degenerative diseases.

Id genes are essential for early heart formation.

Deciphering the fundamental mechanisms controlling cardiac specification is critical for our understanding of how heart formation is initiated during embryonic development and for applying stem cell biology to regenerative medicine and disease modeling. Using systematic and unbiased functional screening approaches, we discovered that the Id family of helix-loop-helix proteins is both necessary and sufficient to direct cardiac mesoderm formation in frog embryos and human embryonic stem cells. Mechanistically, Id proteins specify cardiac cell fate by repressing two inhibitors of cardiogenic mesoderm formation-Tcf3 and Foxa2-and activating inducers Evx1, Grrp1, and Mesp1. Most importantly, CRISPR/Cas9-mediated ablation of the entire Id (Id1-4) family in mouse embryos leads to failure of anterior cardiac progenitor specification and the development of heartless embryos. Thus, Id proteins play a central and evolutionarily conserved role during heart formation and provide a novel means to efficiently produce cardiovascular progenitors for regenerative medicine and drug discovery applications.

osr1 couples intermediate mesoderm cell fate with temporal dynamics of vessel progenitor cell differentiation.

Transcriptional regulatory networks refine gene expression boundaries to define the dimensions of organ progenitor territories. Kidney progenitors originate within the intermediate mesoderm (IM), but the pathways that establish the boundary between the IM and neighboring vessel progenitors are poorly understood. Here, we delineate roles for the zinc-finger transcription factor Osr1 in kidney and vessel progenitor development. Zebrafish osr1 mutants display decreased IM formation and premature emergence of lateral vessel progenitors (LVPs). These phenotypes contrast with the increased IM and absent LVPs observed with loss of the bHLH transcription factor Hand2, and loss of hand2 partially suppresses osr1 mutant phenotypes. hand2 and osr1 are expressed together in the posterior mesoderm, but osr1 expression decreases dramatically prior to LVP emergence. Overexpressing osr1 during this timeframe inhibits LVP development while enhancing IM formation, and can rescue the osr1 mutant phenotype. Together, our data demonstrate that osr1 modulates the extent of IM formation and the temporal dynamics of LVP development, suggesting that a balance between levels of osr1 and hand2 expression is essential to demarcate the kidney and vessel progenitor territories.

Capillarity and active cell movement at mesendoderm translocation in the Xenopus gastrula.

During Xenopus gastrulation, leading edge mesendoderm (LEM) advances animally as a wedge-shaped cell mass over the vegetally moving blastocoel roof (BCR). We show that close contact across the BCR-LEM interface correlates with attenuated net advance of the LEM, which is pulled forward by tip cells while the remaining LEM frequently separates from the BCR. Nevertheless, lamellipodia persist on the detached LEM surface. They attach to adjacent LEM cells and depend on PDGF-A, cell-surface fibronectin and cadherin. We argue that active cell motility on the LEM surface prevents adverse capillary effects in the liquid LEM tissue as it moves by being pulled. It counters tissue surface-tension effects with oriented cell movement and bulges the LEM surface out to keep it close to the curved BCR without attaching to it. Proximity to the BCR is necessary, in turn, for the maintenance and orientation of lamellipodia that permit mass cell movement with minimal substratum contact. Together with a similar process in epithelial invagination, vertical telescoping, the cell movement at the LEM surface defines a novel type of cell rearrangement: vertical shearing.

Mechanics of the cellular microenvironment as probed by cells in vivo during zebrafish presomitic mesoderm differentiation.

Tissue morphogenesis, homoeostasis and repair require cells to constantly monitor their three-dimensional microenvironment and adapt their behaviours in response to local biochemical and mechanical cues. Yet the mechanical parameters of the cellular microenvironment probed by cells in vivo remain unclear. Here, we report the mechanics of the cellular microenvironment that cells probe in vivo and in situ during zebrafish presomitic mesoderm differentiation. By quantifying both endogenous cell-generated strains and tissue mechanics, we show that individual cells probe the stiffness associated with deformations of the supracellular, foam-like tissue architecture. Stress relaxation leads to a perceived microenvironment stiffness that decreases over time, with cells probing the softest regime. We find that most mechanical parameters, including those probed by cells, vary along the anteroposterior axis as mesodermal progenitors differentiate. These findings expand our understanding of in vivo mechanosensation and might aid the design of advanced scaffolds for tissue engineering applications.

Ectoderm to mesoderm transition by down-regulation of actomyosin contractility.

Collective migration of cohesive tissues is a fundamental process in morphogenesis and is particularly well illustrated during gastrulation by the rapid and massive internalization of the mesoderm, which contrasts with the much more modest movements of the ectoderm. In the Xenopus embryo, the differences in morphogenetic capabilities of ectoderm and mesoderm can be connected to the intrinsic motility of individual cells, very low for ectoderm, high for mesoderm. Surprisingly, we find that these seemingly deep differences can be accounted for simply by differences in Rho-kinases (Rock)-dependent actomyosin contractility. We show that Rock inhibition is sufficient to rapidly unleash motility in the ectoderm and confer it with mesoderm-like properties. In the mesoderm, this motility is dependent on two negative regulators of RhoA, the small GTPase Rnd1 and the RhoGAP Shirin/Dlc2/ArhGAP37. Both are absolutely essential for gastrulation. At the cellular and tissue level, the two regulators show overlapping yet distinct functions. They both contribute to decrease cortical tension and confer motility, but Shirin tends to increase tissue fluidity and stimulate dispersion, while Rnd1 tends to favor more compact collective migration. Thus, each is able to contribute to a specific property of the migratory behavior of the mesoderm. We propose that the "ectoderm to mesoderm transition" is a prototypic case of collective migration driven by a down-regulation of cellular tension, without the need for the complex changes traditionally associated with the epithelial-to-mesenchymal transition.

Specifying mouse embryonic germ cells.

Male germ cells are induced to form from the epiblast of the mouse embryo by a combination of WNT and bone morphogenetic protein signals. Ohinata et al. (2009) now clarify the steps of mouse germ cell formation and use this genetic insight to direct the specification and differentiation of germline progenitor cells in vitro.

A signaling principle for the specification of the germ cell lineage in mice.

Specification of the germ cell lineage is vital to development and heredity. In mice, the germ cell fate is induced in pluripotent epiblast cells by signaling molecules, yet the underlying mechanism remains unknown. Here we demonstrate that germ cell fate in the epiblast is a direct consequence of Bmp4 signaling from the extraembryonic ectoderm (ExE), which is antagonized by the anterior visceral endoderm (AVE). Strikingly, Bmp8b from the ExE restricts AVE development, thereby contributing to Bmp4 signaling. Furthermore, Wnt3 in the epiblast ensures its responsiveness to Bmp4. Serum-free, defined cultures revealed that, in response to Bmp4, competent epiblast cells uniformly expressed key transcriptional regulators Blimp1 and Prdm14 and acquired germ-cell properties, including genome-wide epigenetic reprogramming, in an orderly fashion. Notably, the induced cells contributed to both spermatogenesis and fertility of offspring. By identifying a signaling principle in germ cell specification, our study establishes a robust strategy for reconstituting the mammalian germ cell lineage in vitro.

Tissue stiffening coordinates morphogenesis by triggering collective cell migration in vivo.

Collective cell migration is essential for morphogenesis, tissue remodelling and cancer invasion. In vivo, groups of cells move in an orchestrated way through tissues. This movement involves mechanical as well as molecular interactions between cells and their environment. While the role of molecular signals in collective cell migration is comparatively well understood, how tissue mechanics influence collective cell migration in vivo remains unknown. Here we investigated the importance of mechanical cues in the collective migration of the Xenopus laevis neural crest cells, an embryonic cell population whose migratory behaviour has been likened to cancer invasion. We found that, during morphogenesis, the head mesoderm underlying the cephalic neural crest stiffens. This stiffening initiates an epithelial-to-mesenchymal transition in neural crest cells and triggers their collective migration. To detect changes in their mechanical environment, neural crest cells use mechanosensation mediated by the integrin-vinculin-talin complex. By performing mechanical and molecular manipulations, we show that mesoderm stiffening is necessary and sufficient to trigger neural crest migration. Finally, we demonstrate that convergent extension of the mesoderm, which starts during gastrulation, leads to increased mesoderm stiffness by increasing the cell density underneath the neural crest. These results show that convergent extension of the mesoderm has a role as a mechanical coordinator of morphogenesis, and reveal a link between two apparently unconnected processes-gastrulation and neural crest migration-via changes in tissue mechanics. Overall, we demonstrate that changes in substrate stiffness can trigger collective cell migration by promoting epithelial-to-mesenchymal transition in vivo. More broadly, our results raise the idea that tissue mechanics combines with molecular effectors to coordinate morphogenesis.

pmar1/phb homeobox genes and the evolution of the double-negative gate for endomesoderm specification in echinoderms.

In several model animals, the earliest phases of embryogenesis are regulated by lineage-specific genes, such as Drosophila bicoid Sea urchin (echinoid) embryogenesis is initiated by zygotic expression of pmar1, a paired-class homeobox gene that has been considered to be present only in the lineage of modern urchins (euechinoids). In euechinoids, Pmar1 promotes endomesoderm specification by repressing the hairy and enhancer of split C (hesC) gene. Here, we have identified the basal echinoid (cidaroid) pmar1 gene, which also promotes endomesoderm specification but not by repressing hesC A further search for related genes demonstrated that other echinoderms have pmar1-related genes named phb Functional analyses of starfish Phb proteins indicated that, similar to cidaroid Pmar1, they promote activation of endomesoderm regulatory gene orthologs via an unknown repressor that is not HesC. Based on these results, we propose that Pmar1 may have recapitulated the regulatory function of Phb during the early diversification of echinoids and that the additional repressor HesC was placed under the control of Pmar1 in the euechinoid lineage. This case provides an exceptional model for understanding how early developmental processes diverge.

Distinct populations of crypt-associated fibroblasts act as signaling hubs to control colon homeostasis.

Despite recent progress in recognizing the importance of mesenchymal cells for the homeostasis of the intestinal system, the current picture of how these cells communicate with the associated epithelial layer remains unclear. To describe the relevant cell populations in an unbiased manner, we carried out a single-cell transcriptome analysis of the adult murine colon, producing a high-quality atlas of matched colonic epithelium and mesenchyme. We identify two crypt-associated colonic fibroblast populations that are demarcated by different strengths of platelet-derived growth factor receptor A (Pdgfra) expression. Crypt-bottom fibroblasts (CBFs), close to the intestinal stem cells, express low levels of Pdgfra and secrete canonical Wnt ligands, Wnt potentiators, and bone morphogenetic protein (Bmp) inhibitors. Crypt-top fibroblasts (CTFs) exhibit high Pdgfra levels and secrete noncanonical Wnts and Bmp ligands. While the Pdgfralow cells maintain intestinal stem cell proliferation, the Pdgfrahigh cells induce differentiation of the epithelial cells. Our findings enhance our understanding of the crosstalk between various colonic epithelial cells and their associated mesenchymal signaling hubs along the crypt axis-placing differential Pdgfra expression levels in the spotlight of intestinal fibroblast identity.

Spatial mapping of tissue properties in vivo reveals a 3D stiffness gradient in the mouse limb bud.

Numerous hypotheses invoke tissue stiffness as a key parameter that regulates morphogenesis and disease progression. However, current methods are insufficient to test hypotheses that concern physical properties deep in living tissues. Here we introduce, validate, and apply a magnetic device that generates a uniform magnetic field gradient within a space that is sufficient to accommodate an organ-stage mouse embryo under live conditions. The method allows rapid, nontoxic measurement of the three-dimensional (3D) spatial distribution of viscoelastic properties within mesenchyme and epithelia. Using the device, we identify an anteriorly biased mesodermal stiffness gradient along which cells move to shape the early limb bud. The stiffness gradient corresponds to a Wnt5a-dependent domain of fibronectin expression, raising the possibility that durotaxis underlies cell movements. Three-dimensional stiffness mapping enables the generation of hypotheses and potentially the rigorous testing of mechanisms of development and disease.

Retinoic acid signaling restricts the size of the first heart field within the anterior lateral plate mesoderm.

Retinoic acid (RA) signaling is required to restrict heart size through limiting the posterior boundary of the vertebrate cardiac progenitor field within the anterior lateral plate mesoderm (ALPM). However, we still do not fully understand how different cardiac progenitor populations that contribute to the developing heart, including earlier-differentiating first heart field (FHF), later-differentiating second heart field (SHF), and neural crest-derived progenitors, are each affected in RA-deficient embryos. Here, we quantified the number of cardiac progenitors and differentiating cardiomyocytes (CMs) in RA-deficient zebrafish embryos. While Nkx2.5+ cells were increased overall in the nascent hearts of RA-deficient embryos, unexpectedly, we found that the major effect within this population was a significant expansion in the number of differentiating FHF CMs. In contrast to the expansion of the FHF, there was a progressive decrease in SHF progenitors at the arterial pole as the heart tube elongated. Temporal differentiation assays and immunostaining in RA-deficient embryos showed that the outflow tracts (OFTs) of the hearts were significantly smaller, containing fewer differentiated SHF-derived ventricular CMs and a complete absence of SHF-derived smooth muscle at later stages. At the venous pole of the heart, pacemaker cells of the sinoatrial node also failed to differentiate in RA-deficient embryos. Interestingly, genetic lineage tracing showed that the number of neural-crest derived CMs was not altered within the enlarged hearts of RA-deficient zebrafish embryos. Altogether, our data show that the enlarged hearts in RA-deficient zebrafish embryos are comprised of an expansion in earlier differentiating FHF-derived CMs coupled with a progressive depletion of the SHF, suggesting RA signaling determines the relative ratios of earlier- and later-differentiation cardiac progenitors within an expanded cardiac progenitor pool.

Trim-Away mediated knock down uncovers a new function for Lbh during gastrulation of Xenopus laevis.

We previously identified the protein Lbh as necessary for cranial neural crest (CNC) cell migration in Xenopus through the use of morpholinos. However, Lbh is a maternally deposited protein and morpholinos achieve knockdowns through prevention of translation. In order to investigate the role of Lbh in earlier embryonic events, we employed the new technique "Trim-Away" to degrade this maternally deposited protein. Trim-Away utilizes the E3 ubiquitin ligase trim21 to degrade proteins targeted with an antibody and was developed in mammalian systems. Our results show that Xenopus is amenable to the Trim-Away technique. We also show that early knockdown of Lbh in Xenopus results in defects in gastrulation that present with a decrease in fibronectin matrix assembly, an increased in mesodermal cell migration and decrease in endodermal cell cohesion. We further show that the technique is also effective on a second abundant maternal protein PACSIN2. We discuss potential advantages and limit of the technique in Xenopus embryos as well as the mechanism of gastrulation inhibition.

Netrin expressed by the ventral ectoderm lineage guides mesoderm migration in epibolic gastrulation of the leech.

Netrin is a remarkably conserved midline landmark, serving as a chemotactic factor that organizes the bilateral neural architecture in the post-gastrula bilaterian embryos. Netrin signal also guides cell migration in many other neural and non-neural organogenesis events in later developmental stages but has never been found to participate in gastrulation - the earliest cell migration in metazoan embryogenesis. Here, we found that the netrin signaling molecules and their receptors are expressed during gastrulation of the leech Helobdella. Intriguingly, Hau-netrin-1 was expressed in the N lineage, which gives rise in part to the ventral midline of ectoderm, at the onset of gastrulation. We demonstrated that the N lineage is required for the entrance of mesoderm into the germinal band and that misexpression of Hau-netrin-1 in early gastrulation prevented mesoderm from entering the germinal band. Together, these results suggested that Hau-netrin-1 secreted by the N lineage guides mesoderm migration during germinal band assembly. Furthermore, ectopic expression of Hau-netrin-1 after the completion of germinal band assembly disrupted the epibolic migration of the germinal bands in a later stage of gastrulation. Thus, Hau-netrin-1 is likely involved in two distinct events in sequential stages of leech gastrulation: the assembly of germinal bands in early gastrulation and their epibolic migration in mid-gastrulation. Given that the leech netrin is expressed in the precursor cells of the ventral midline during gastrulation, we propose that a heterochronic change from the midline netrin expression had taken place in the evolution of a novel mode of gastrulation in the directly developing leech embryos.

Neural differentiation, selection and transcriptomic profiling of human neuromesodermal progenitor-like cells in vitro.

Robust protocols for directed differentiation of human pluripotent cells are required to determine whether mechanisms operating in model organisms are relevant to our own development. Recent work in vertebrate embryos has identified neuromesodermal progenitors as a bipotent cell population that contributes to paraxial mesoderm and spinal cord. However, precise protocols for in vitro differentiation of human spinal cord progenitors are lacking. Informed by signalling in amniote embryos, we show here that transient dual-SMAD inhibition, together with retinoic acid (dSMADi-RA), provides rapid and reproducible induction of human spinal cord progenitors from neuromesodermal progenitor-like cells. Using CRISPR-Cas9 to engineer human embryonic stem cells with a GFP-reporter for neuromesodermal progenitor-associated gene Nkx1.2 we facilitate selection of this cell population. RNA-sequencing was then used to identify human and conserved neuromesodermal progenitor transcriptional signatures, to validate this differentiation protocol and to reveal new pathways/processes in human neural differentiation. This optimised protocol, novel reporter line and transcriptomic data are useful resources with which to dissect molecular mechanisms regulating human spinal cord generation and allow the scaling-up of distinct cell populations for global analyses, including proteomic, biochemical and chromatin interrogation.