RESUMO
Retrospective lineage reconstruction of humans predicts that dramatic clonal imbalances in the body can be traced to the 2-cell stage embryo. However, whether and how such clonal asymmetries arise in the embryo is unclear. Here, we performed prospective lineage tracing of human embryos using live imaging, non-invasive cell labeling, and computational predictions to determine the contribution of each 2-cell stage blastomere to the epiblast (body), hypoblast (yolk sac), and trophectoderm (placenta). We show that the majority of epiblast cells originate from only one blastomere of the 2-cell stage embryo. We observe that only one to three cells become internalized at the 8-to-16-cell stage transition. Moreover, these internalized cells are more frequently derived from the first cell to divide at the 2-cell stage. We propose that cell division dynamics and a cell internalization bottleneck in the early embryo establish asymmetry in the clonal composition of the future human body.
Assuntos
Blastômeros , Linhagem da Célula , Embrião de Mamíferos , Feminino , Humanos , Blastômeros/citologia , Blastômeros/metabolismo , Divisão Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Masculino , Animais , CamundongosRESUMO
The three-dimensional (3D) organization of cells determines tissue function and integrity, and changes markedly in development and disease. Cell-based simulations have long been used to define the underlying mechanical principles. However, high computational costs have so far limited simulations to either simplified cell geometries or small tissue patches. Here, we present SimuCell3D, an efficient open-source program to simulate large tissues in three dimensions with subcellular resolution, growth, proliferation, extracellular matrix, fluid cavities, nuclei and non-uniform mechanical properties, as found in polarized epithelia. Spheroids, vesicles, sheets, tubes and other tissue geometries can readily be imported from microscopy images and simulated to infer biomechanical parameters. Doing so, we show that 3D cell shapes in layered and pseudostratified epithelia are largely governed by a competition between surface tension and intercellular adhesion. SimuCell3D enables the large-scale in silico study of 3D tissue organization in development and disease at a great level of detail.
Assuntos
Polaridade Celular , Simulação por Computador , Modelos Biológicos , Fenômenos Biomecânicos/fisiologia , Adesão Celular/fisiologia , Polaridade Celular/fisiologia , Forma Celular/fisiologia , Células Epiteliais/fisiologia , Células Epiteliais/citologia , Matriz Extracelular/fisiologia , Matriz Extracelular/química , Imageamento Tridimensional/métodos , SoftwareRESUMO
During kidney development, reciprocal signaling between the epithelium and the mesenchyme coordinates nephrogenesis with branching morphogenesis of the collecting ducts. The mechanism that positions the renal vesicles, and thus the nephrons, relative to the branching ureteric buds has remained elusive. By combining computational modeling and experiments, we show that geometric effects concentrate the key regulator, WNT9b, at the junctions between parent and daughter branches where renal vesicles emerge, even when uniformly expressed in the ureteric epithelium. This curvature effect might be a general paradigm to create non-uniform signaling in development.
Assuntos
Néfrons , Ureter , Rim , Transdução de Sinais , Epitélio , Morfogênese , MesodermaRESUMO
Robust embryonic development requires pattern formation with high spatial accuracy. In epithelial tissues that are patterned by morphogen gradients, the emerging patterns achieve levels of precision that have recently been explained by a simple one-dimensional reaction-diffusion model with kinetic noise. Here, we show that patterning precision is even greater if transverse diffusion effects are at play in such tissues. The positional error, a measure for spatial patterning accuracy, decreases in wider tissues but then saturates beyond a width of about ten cells. This demonstrates that the precision of gradient-based patterning in two- or higher-dimensional systems can be even greater than predicted by 1D models, and further attests to the potential of noisy morphogen gradients for high-precision tissue patterning.
RESUMO
Tissue patterning during embryonic development is remarkably precise. Here, we numerically determine the impact of the cell diameter, gradient length and the morphogen source on the variability of morphogen gradients. We show that the positional error increases with the gradient length relative to the size of the morphogen source, and with the square root of the cell diameter and the readout position. We provide theoretical explanations for these relationships, and show that they enable high patterning precision over developmental time for readouts that scale with expanding tissue domains, as observed in the Drosophila wing disc. Our analysis suggests that epithelial tissues generally achieve higher patterning precision with small cross-sectional cell areas. An extensive survey of measured apical cell areas shows that they are indeed small in developing tissues that are patterned by morphogen gradients. Enhanced precision may thus have led to the emergence of pseudostratification in epithelia, a phenomenon for which the evolutionary benefit had so far remained elusive.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Estudos Transversais , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Desenvolvimento Embrionário , Tamanho Celular , Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Modelos Biológicos , Drosophila melanogaster/genéticaRESUMO
Morphogen gradients can instruct cells about their position in a patterned tissue. Non-linear morphogen decay has been suggested to increase gradient precision by reducing the sensitivity to variability in the morphogen source. Here, we use cell-based simulations to quantitatively compare the positional error of gradients for linear and non-linear morphogen decay. While we confirm that non-linear decay reduces the positional error close to the source, the reduction is very small for physiological noise levels. Far from the source, the positional error is much larger for non-linear decay in tissues that pose a flux barrier to the morphogen at the boundary. In light of this new data, a physiological role of morphogen decay dynamics in patterning precision appears unlikely.
Assuntos
Padronização Corporal , Modelos Biológicos , Padronização Corporal/fisiologiaRESUMO
Here, we present a protocol for collecting high-spatiotemporal-resolution datasets of undisturbed mouse embryonic epithelial rudiments using light-sheet fluorescence microscopy. We describe steps for rudiment dissection, clearing, and embedding for cleared and live imaging. We then detail procedures for light-sheet imaging followed by image processing and morphometric analysis. We provide protocol variations for imaging both growing and optically cleared lung explants to encourage the quantitative exploration of three-dimensional cell shapes, cell organization, and complex cell-cell dynamics. For complete details on the use and execution of this protocol, please refer to Gómez et al. (2021).1.
RESUMO
The cerebral cortex contains billions of neurons, and their disorganization or misspecification leads to neurodevelopmental disorders. Understanding how the plethora of projection neuron subtypes are generated by cortical neural stem cells (NSCs) is a major challenge. Here, we focused on elucidating the transcriptional landscape of murine embryonic NSCs, basal progenitors (BPs), and newborn neurons (NBNs) throughout cortical development. We uncover dynamic shifts in transcriptional space over time and heterogeneity within each progenitor population. We identified signature hallmarks of NSC, BP, and NBN clusters and predict active transcriptional nodes and networks that contribute to neural fate specification. We find that the expression of receptors, ligands, and downstream pathway components is highly dynamic over time and throughout the lineage implying differential responsiveness to signals. Thus, we provide an expansive compendium of gene expression during cortical development that will be an invaluable resource for studying neural developmental processes and neurodevelopmental disorders.
Assuntos
Células-Tronco Neurais , Neurônios , Animais , Camundongos , Diferenciação Celular , Linhagem da Célula/genética , Córtex Cerebral , Células-Tronco Embrionárias , Neurogênese/genética , Neurônios/metabolismoRESUMO
Kidneys develop via iterative branching of the ureteric epithelial tree and subsequent nephrogenesis at the branch points. Nephrons form in the cap mesenchyme as the metanephric mesenchyme (MM) condenses around the epithelial ureteric buds (UBs). Previous work has demonstrated that FGF8 is important for the survival of nephron progenitor cells (NPCs), and early deletion of Fgf8 leads to the cessation of nephron formation, which results in post-natal lethality. We now reveal a previously unreported function of FGF8. By combining transgenic mouse models, quantitative imaging assays and data-driven computational modelling, we show that FGF8 has a strong chemokinetic effect and that this chemokinetic effect is important for the condensation of NPCs to the UB. The computational model shows that the motility must be lower close to the UB to achieve NPC attachment. We conclude that the FGF8 signalling pathway is crucial for the coordination of NPC condensation at the UB. Chemokinetic effects have also been described for other FGFs and may be generally important for the formation of mesenchymal condensates.
Assuntos
Rim , Néfrons , Camundongos , Animais , Néfrons/metabolismo , Rim/metabolismo , Organogênese , Fatores de Crescimento de Fibroblastos/metabolismo , Células-Tronco/metabolismo , Camundongos Transgênicos , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismoRESUMO
Development of the pancreas is driven by an intrinsic program coordinated with signals from other cell types in the epithelial environment. These intercellular communications have been so far challenging to study because of the low concentration, localized production and diversity of the signals released. Here, we combined scRNAseq data with a computational interactomic approach to identify signals involved in the reciprocal interactions between the various cell types of the developing pancreas. This in silico approach yielded 40,607 potential ligand-target interactions between the different main pancreatic cell types. Among this vast network of interactions, we focused on three ligands potentially involved in communications between epithelial and endothelial cells. BMP7 and WNT7B, expressed by pancreatic epithelial cells and predicted to target endothelial cells, and SEMA6D, involved in the reverse interaction. In situ hybridization confirmed the localized expression of Bmp7 in the pancreatic epithelial tip cells and of Wnt7b in the trunk cells. On the contrary, Sema6d was enriched in endothelial cells. Functional experiments on ex vivo cultured pancreatic explants indicated that tip cell-produced BMP7 limited development of endothelial cells. This work identified ligands with a restricted tissular and cellular distribution and highlighted the role of BMP7 in the intercellular communications contributing to vessel development and organization during pancreas organogenesis.
Assuntos
Células Endoteliais , Organogênese , Diferenciação Celular/fisiologia , Células Endoteliais/metabolismo , Ligantes , Organogênese/fisiologia , Pâncreas/metabolismoRESUMO
The trachea is a long tube that enables air passage between the larynx and the bronchi. C-shaped cartilage rings on the ventral side stabilise the structure. On its esophagus-facing dorsal side, deformable smooth muscle facilitates the passage of food in the esophagus. While the symmetry break along the dorsal-ventral axis is well understood, the molecular mechanism that results in the periodic Sox9 expression pattern that translates into the cartilage rings has remained elusive. Here, we review the molecular regulatory interactions that have been elucidated, and discuss possible patterning mechanisms. Understanding the principles of self-organisation is important, both to define biomedical interventions and to enable tissue engineering.
RESUMO
Neurulation is the process in early vertebrate embryonic development during which the neural plate folds to form the neural tube. Spinal neural tube folding in the posterior neuropore changes over time, first showing a median hinge point, then both the median hinge point and dorsolateral hinge points, followed by dorsolateral hinge points only. The biomechanical mechanism of hinge point formation in the mammalian neural tube is poorly understood. Here we employ a mechanical finite element model to study neural tube formation. The computational model mimics the mammalian neural tube using microscopy data from mouse and human embryos. While intrinsic curvature at the neural plate midline has been hypothesized to drive neural tube folding, intrinsic curvature was not sufficient for tube closure in our simulations. We achieved neural tube closure with an alternative model combining mesoderm expansion, nonneural ectoderm expansion, and neural plate adhesion to the notochord. Dorsolateral hinge points emerged in simulations with low mesoderm expansion and zippering. We propose that zippering provides the biomechanical force for dorsolateral hinge point formation in settings where the neural plate lateral sides extend above the mesoderm. Together, these results provide a perspective on the biomechanical and molecular mechanism of mammalian spinal neurulation.
Assuntos
Tubo Neural , Neurulação , Animais , Ectoderma/embriologia , Humanos , Camundongos , Placa Neural/embriologia , Tubo Neural/embriologia , Neurulação/fisiologia , Notocorda/embriologiaRESUMO
Despite molecular noise and genetic differences between individuals, developmental outcomes are remarkably constant. Decades of research has focused on the underlying mechanisms that ensure this precision and robustness. Recent quantifications of chemical gradients and epithelial cell shapes provide novel insights into the basis of precise development. In this review, we argue that these two aspects may be linked in epithelial morphogenesis.
Assuntos
Padronização Corporal , Modelos Biológicos , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Morfogênese/genética , Transdução de Sinais/genéticaRESUMO
Morphogen gradients encode positional information during development. How high patterning precision is achieved despite natural variation in both the morphogen gradients and in the readout process, is still largely elusive. Here, we show that the positional error of gradients in the mouse neural tube has previously been overestimated, and that the reported accuracy of the central progenitor domain boundaries in the mouse neural tube can be achieved with a single gradient, rather than requiring the simultaneous readout of opposing gradients. Consistently and independently, numerical simulations based on measured molecular noise levels likewise result in lower gradient variabilities than reported. Finally, we show that the patterning mechanism yields progenitor cell numbers with even greater precision than boundary positions, as gradient amplitude changes do not affect interior progenitor domain sizes. We conclude that single gradients can yield the observed developmental precision, which provides prospects for tissue engineering.
Assuntos
Padronização Corporal , Tubo Neural , Animais , Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Modelos BiológicosRESUMO
Generating comprehensive image maps, while preserving spatial three-dimensional (3D) context, is essential in order to locate and assess quantitatively specific cellular features and cell-cell interactions during organ development. Despite recent advances in 3D imaging approaches, our current knowledge of the spatial organization of distinct cell types in the embryonic pancreatic tissue is still largely based on two-dimensional histological sections. Here, we present a light-sheet fluorescence microscopy approach to image the pancreas in three dimensions and map tissue interactions at key time points in the mouse embryo. We demonstrate the utility of the approach by providing volumetric data, 3D distribution of three main cellular components (epithelial, mesenchymal and endothelial cells) within the developing pancreas, and quantification of their relative cellular abundance within the tissue. Interestingly, our 3D images show that endocrine cells are constantly and increasingly in contact with endothelial cells forming small vessels, whereas the interactions with mesenchymal cells decrease over time. These findings suggest distinct cell-cell interaction requirements for early endocrine cell specification and late differentiation. Lastly, we combine our image data in an open-source online repository (referred to as the Pancreas Embryonic Cell Atlas).
Assuntos
Imageamento Tridimensional/métodos , Pâncreas/anatomia & histologia , Animais , Embrião de Mamíferos/anatomia & histologia , Desenvolvimento Embrionário , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Epitélio/anatomia & histologia , Proteína Homeobox Nkx-2.5/deficiência , Proteína Homeobox Nkx-2.5/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de FluorescênciaRESUMO
During morphogenesis, epithelial sheets remodel into complex geometries. How cells dynamically organise their contact with neighbouring cells in these tightly packed tissues is poorly understood. We have used light-sheet microscopy of growing mouse embryonic lung explants, three-dimensional cell segmentation, and physical theory to unravel the principles behind 3D cell organisation in growing pseudostratified epithelia. We find that cells have highly irregular 3D shapes and exhibit numerous neighbour intercalations along the apical-basal axis as well as over time. Despite the fluidic nature, the cell packing configurations follow fundamental relationships previously described for apical epithelial layers, that is, Euler's polyhedron formula, Lewis' law, and Aboav-Weaire's law, at all times and across the entire tissue thickness. This arrangement minimises the lateral cell-cell surface energy for a given cross-sectional area variability, generated primarily by the distribution and movement of nuclei. We conclude that the complex 3D cell organisation in growing epithelia emerges from simple physical principles.
Assuntos
Pulmão/embriologia , Animais , Células Epiteliais/citologia , Epitélio/embriologia , Camundongos , MorfogêneseRESUMO
A central problem in developmental biology is to understand how cells interpret their positional information to give rise to spatial patterns, such as the process of periodic segmentation of the vertebrate embryo into somites. For decades, somite formation has been interpreted according to the clock-and-wavefront model. In this conceptual framework, molecular oscillators set the frequency of somite formation while the positional information is encoded in signaling gradients. Recent experiments using ex vivo explants have challenged this interpretation, suggesting that positional information is encoded in the properties of the oscillators, independent of long-range modulations such as signaling gradients. Here, we propose that positional information is encoded in the difference in the levels of neighboring oscillators. The differences gradually increase because both the amplitude and the period of the oscillators increase with time. When this difference exceeds a certain threshold, the segmentation program starts. Using this framework, we quantitatively fit experimental data from in vivo and ex vivo mouse segmentation, and propose mechanisms of somite scaling. Our results suggest a novel mechanism of spatial pattern formation based on the local interactions between dynamic molecular oscillators.