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Developmental Cell Jun 2024The anterior visceral endoderm (AVE) differs from the surrounding visceral endoderm (VE) in its migratory behavior and ability to restrict primitive streak formation to...
The anterior visceral endoderm (AVE) differs from the surrounding visceral endoderm (VE) in its migratory behavior and ability to restrict primitive streak formation to the opposite side of the mouse embryo. To characterize the molecular bases for the unique properties of the AVE, we combined single-cell RNA sequencing of the VE prior to and during AVE migration with phosphoproteomics, high-resolution live-imaging, and short-term lineage labeling and intervention. This identified the transient nature of the AVE with attenuation of "anteriorizing" gene expression as cells migrate and the emergence of heterogeneities in transcriptional states relative to the AVE's position. Using cell communication analysis, we identified the requirement of semaphorin signaling for normal AVE migration. Lattice light-sheet microscopy showed that Sema6D mutants have abnormalities in basal projections and migration speed. These findings point to a tight coupling between transcriptional state and position of the AVE and identify molecular controllers of AVE migration.
PubMed: 38843837
DOI: 10.1016/j.devcel.2024.05.014 -
ELife May 2024Large-scale cell flow characterizes gastrulation in animal development. In amniote gastrulation, particularly in avian gastrula, a bilateral vortex-like counter-rotating...
Large-scale cell flow characterizes gastrulation in animal development. In amniote gastrulation, particularly in avian gastrula, a bilateral vortex-like counter-rotating cell flow, called 'polonaise movements', appears along the midline. Here, through experimental manipulations, we addressed relationships between the polonaise movements and morphogenesis of the primitive streak, the earliest midline structure in amniotes. Suppression of the Wnt/planar cell polarity (PCP) signaling pathway maintains the polonaise movements along a deformed primitive streak. Mitotic arrest leads to diminished extension and development of the primitive streak and maintains the early phase of the polonaise movements. Ectopically induced Vg1, an axis-inducing morphogen, generates the polonaise movements, aligned to the induced midline, but disturbs the stereotypical cell flow pattern at the authentic midline. Despite the altered cell flow, induction and extension of the primitive streak are preserved along both authentic and induced midlines. Finally, we show that ectopic axis-inducing morphogen, Vg1, is capable of initiating the polonaise movements without concomitant PS extension under mitotic arrest conditions. These results are consistent with a model wherein primitive streak morphogenesis is required for the maintenance of the polonaise movements, but the polonaise movements are not necessarily responsible for primitive streak morphogenesis. Our data describe a previously undefined relationship between the large-scale cell flow and midline morphogenesis in gastrulation.
Topics: Animals; Gastrulation; Morphogenesis; Cell Movement; Primitive Streak; Cell Polarity; Gastrula; Chick Embryo
PubMed: 38727576
DOI: 10.7554/eLife.89948 -
BioRxiv : the Preprint Server For... Apr 2024A bilateral body plan is predominant throughout the animal kingdom. Bilaterality of amniote embryos becomes recognizable as midline morphogenesis begins at gastrulation,...
A bilateral body plan is predominant throughout the animal kingdom. Bilaterality of amniote embryos becomes recognizable as midline morphogenesis begins at gastrulation, bisecting an embryonic field into the left and right sides. Soon after, left-right asymmetry also starts. While a series of laterality genes expressed after the left-right compartmentalization has been extensively studied, the laterality patterning prior to and during midline morphogenesis has remained unclear. Here, through a biophysical quantification in a high spatial and temporal resolution, applied to a chick model system, we show that a large-scale bilateral counter-rotating cell flow, termed as 'polonaise movements', display left-right asymmetries in early gastrulation. This cell movement starts prior to the formation of the primitive streak, which is the earliest midline structure, and earlier than expression of laterality genes. The cell flow speed and vorticity unravel the location and timing of the left-right asymmetries. The bilateral cell flow exhibited a Left side asymmetry at the beginning, but a transition towards Right dominance. Mitotic arrest that diminishes primitive streak formation resulted in changes in the bilateral flow pattern, but the Right dominance persisted. Our data indicate that the left-right asymmetry in amniote gastrula becomes detectable prior to the point when the asymmetric regulation of the laterality signals at the node leads to the left-right patterning. More broadly, our results suggest that physical processes can play an unexpected but significant role in influencing left-right laterality during embryonic development.
PubMed: 38712212
DOI: 10.1101/2024.04.21.590437 -
PloS One 2024During vertebrate embryo development, the body is progressively segmented along the anterior-posterior (A-P) axis early in development. The rate of somite formation is...
During vertebrate embryo development, the body is progressively segmented along the anterior-posterior (A-P) axis early in development. The rate of somite formation is controlled by the somitogenesis embryo clock (EC), which was first described as gene expression oscillations of hairy1 (hes4) in the presomitic mesoderm of chick embryos with 15-20 somites. Here, the EC displays the same periodicity as somite formation, 90 min, whereas the posterior-most somites (44-52) only arise every 150 minutes, matched by a corresponding slower pace of the EC. Evidence suggests that the rostral-most somites are formed faster, however, their periodicity and the EC expression dynamics in these early stages are unknown. In this study, we used time-lapse imaging of chicken embryos from primitive streak to somitogenesis stages with high temporal resolution (3-minute intervals). We measured the length between the anterior-most and the last formed somitic clefts in each captured frame and developed a simple algorithm to automatically infer both the length and time of formation of each somite. We found that the occipital somites (up to somite 5) form at an average rate of 75 minutes, while somites 6 onwards are formed approximately every 90 minutes. We also assessed the expression dynamics of hairy1 using half-embryo explants cultured for different periods of time. This showed that EC hairy1 expression is highly dynamic prior to somitogenesis and assumes a clear oscillatory behaviour as the first somites are formed. Importantly, using ex ovo culture and live-imaging techniques, we showed that the hairy1 expression pattern recapitulates with the formation of each new pair of somites, indicating that somite segmentation is coupled with EC oscillations since the onset of somitogenesis.
Topics: Animals; Chick Embryo; Somites; Chickens; Basic Helix-Loop-Helix Transcription Factors; Avian Proteins; Mesoderm
PubMed: 38635504
DOI: 10.1371/journal.pone.0297853 -
BioRxiv : the Preprint Server For... Mar 2024Micropatterned human pluripotent stem cells (hPSCs) treated with BMP4 (2D gastruloids) are among the most widely used stem cell models for human gastrulation. Due to its...
Micropatterned human pluripotent stem cells (hPSCs) treated with BMP4 (2D gastruloids) are among the most widely used stem cell models for human gastrulation. Due to its simplicity and reproducibility, this system is ideal for high throughput quantitative studies of tissue patterning and has led to many insights into the mechanisms of mammalian gastrulation. However, 2D gastruloids have only been studied up to 48h. Here we extended this system to 96h. We discovered a phase of highly reproducible morphogenesis during which directed migration from the primitive streak-like region gives rise to a mesodermal layer beneath an epiblast-like layer. Multiple types of mesoderm arise with striking spatial organization including lateral mesoderm-like cells on the colony border and paraxial mesoderm-like further inside the colony. Single cell transcriptomics showed strong similarity of these cells to mesoderm in human and non-human primate embryos. However, our data suggest that the annotation of the reference human embryo may need to be revised. This illustrates that extended culture of 2D gastruloids provides a powerful model for human mesoderm differentiation and morphogenesis.
PubMed: 38585971
DOI: 10.1101/2024.03.21.585753 -
Developmental Cell May 2024The blueprint of the mammalian body plan is laid out during gastrulation, when a trilaminar embryo is formed. This process entails a burst of proliferation, the...
The blueprint of the mammalian body plan is laid out during gastrulation, when a trilaminar embryo is formed. This process entails a burst of proliferation, the ingression of embryonic epiblast cells at the primitive streak, and their priming toward primitive streak fates. How these different events are coordinated remains unknown. Here, we developed and characterized a 3D culture of self-renewing mouse embryonic cells that captures the main transcriptional and architectural features of the early gastrulating mouse epiblast. Using this system in combination with microfabrication and in vivo experiments, we found that proliferation-induced crowding triggers delamination of cells that express high levels of the apical polarity protein aPKC. Upon delamination, cells become more sensitive to Wnt signaling and upregulate the expression of primitive streak markers such as Brachyury. This mechanistic coupling between ingression and differentiation ensures that the right cell types become specified at the right place during embryonic development.
Topics: Animals; Mice; Gastrulation; Cell Differentiation; Germ Layers; T-Box Domain Proteins; Pluripotent Stem Cells; Primitive Streak; Fetal Proteins; Wnt Signaling Pathway; Cell Proliferation; Gene Expression Regulation, Developmental; Embryo, Mammalian
PubMed: 38579720
DOI: 10.1016/j.devcel.2024.03.008 -
Developmental Cell Jun 2024Basement membranes (BMs) are sheet-like structures of extracellular matrix (ECM) that provide structural support for many tissues and play a central role in signaling....
Basement membranes (BMs) are sheet-like structures of extracellular matrix (ECM) that provide structural support for many tissues and play a central role in signaling. They are key regulators of cell behavior and tissue functions, and defects in their assembly or composition are involved in numerous human diseases. Due to the differences between human and animal embryogenesis, ethical concerns, legal constraints, the scarcity of human tissue material, and the inaccessibility of the in vivo condition, BM regulation during human embryo development has remained elusive. Using the post-implantation amniotic sac embryoid (PASE), we delineate BM assembly upon post-implantation development and BM disassembly during primitive streak (PS) cell dissemination. Further, we show that the transcription factor Oct4 regulates the expression of BM structural components and receptors and controls BM development by regulating Akt signaling and the small GTPase Rac1. These results represent a relevant step toward a more comprehensive understanding of early human development.
Topics: Humans; Basement Membrane; Embryonic Development; Octamer Transcription Factor-3; rac1 GTP-Binding Protein; Signal Transduction; Proto-Oncogene Proteins c-akt; Gene Expression Regulation, Developmental; Primitive Streak; Laminin; Extracellular Matrix
PubMed: 38579716
DOI: 10.1016/j.devcel.2024.03.007 -
Developmental Cell May 2024The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos...
The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to generate HSCs for 2.5 days (∼E8.5-E11) but subsequently cease, delimiting a narrow time frame for HSC formation in vivo. Guided by the arterial origins of blood, we efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10 days. hPSC-derived hematopoietic progenitors generate T, B, NK, erythroid, and myeloid cells in vitro and, critically, express hallmark HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly enriched HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of unwanted lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could avail both basic research and cellular therapies.
Topics: Animals; Humans; Mice; Cell Differentiation; Cell Lineage; Endothelial Cells; Hematopoiesis; Hematopoietic Stem Cells; Homeodomain Proteins; Pluripotent Stem Cells; Transcription Factors; Basic-Leucine Zipper Transcription Factors
PubMed: 38569552
DOI: 10.1016/j.devcel.2024.03.003 -
BioRxiv : the Preprint Server For... Mar 2024The Wnt pathway is essential for inducing the primitive streak, the precursor of the mesendoderm, as well as setting anterior-posterior coordinates. How Wnt coordinates...
The Wnt pathway is essential for inducing the primitive streak, the precursor of the mesendoderm, as well as setting anterior-posterior coordinates. How Wnt coordinates these diverse activities remains incompletely understood. Here, we show that in Wnt-treated human pluripotent cells, endogenous Nodal signaling is a crucial switch between posteriorizing and primitive streak-including activities. While treatment with Wnt posteriorizes cells in standard culture, in micropatterned colonies, higher levels of endogenously induced Nodal signaling combine with exogenous Wnt to drive endoderm differentiation. Inhibition of Nodal signaling restores dose-dependent posteriorization by Wnt. In the absence of Nodal inhibition, micropatterned colonies undergo spontaneous, elaborate morphogenesis concomitant with endoderm differentiation even in the absence of added extracellular matrix proteins like Matrigel. Our study shows how Wnt and Nodal combinatorially coordinate germ layer differentiation with AP patterning and establishes a system to study a natural self-organizing morphogenetic event in culture.
PubMed: 38559061
DOI: 10.1101/2024.03.13.584912 -
Nature Communications Feb 2024Many amniote vertebrate species including humans can form identical twins from a single embryo, but this only occurs rarely. It has been suggested that the...
Many amniote vertebrate species including humans can form identical twins from a single embryo, but this only occurs rarely. It has been suggested that the primitive-streak-forming embryonic region emits signals that inhibit streak formation elsewhere but the signals involved, how they are transmitted and how they act has not been elucidated. Here we show that short tracks of calcium firing activity propagate through extraembryonic tissue via gap junctions and prevent ectopic primitive streak formation in chick embryos. Cross-regulation of calcium activity and an inhibitor of primitive streak formation (Bone Morphogenetic Protein, BMP) via NF-κB and NFAT establishes a long-range BMP gradient spanning the embryo. This mechanism explains how embryos of widely different sizes can maintain positional information that determines embryo polarity. We provide evidence for similar mechanisms in two different human embryo models and in Drosophila, suggesting an ancient evolutionary origin.
Topics: Animals; Chick Embryo; Humans; Calcium; Bone Morphogenetic Proteins; Gastrulation; Primitive Streak; Reproduction
PubMed: 38368410
DOI: 10.1038/s41467-024-45772-4