RESUMO
In this issue of Developmental Cell, Schülle et al. address the mechanism that lays down the mammalian body plan and show that the first step in this process is associated with the activity of two T-box transcription factors whose interactions outline territories that will be further elaborated during gastrulation.
Assuntos
Gastrulação , Mamíferos , Animais , Fatores de Transcrição/genéticaRESUMO
A human embryo's legal definition and its entitlement to protection vary greatly worldwide. Recently, human pluripotent stem cells have been used to form in vitro models of early embryos that have challenged legal definitions and raised questions regarding their usage. In this light, we propose a refined legal definition of an embryo, suggest "tipping points" for when human embryo models could eventually be afforded similar protection to that of embryos, and then revisit basic ethical principles that might help to draft a roadmap for the gradual, justified usage of embryo models in a manner that aims to maximize benefits to society.
Assuntos
Pesquisas com Embriões , Embrião de Mamíferos , Humanos , Células-Tronco Pluripotentes , Pesquisas com Embriões/éticaRESUMO
The segmented body plan of vertebrates is established during somitogenesis, a well-studied process in model organisms; however, the details of this process in humans remain largely unknown owing to ethical and technical limitations. Despite recent advances with pluripotent stem cell-based approaches1-5, models that robustly recapitulate human somitogenesis in both space and time remain scarce. Here we introduce a pluripotent stem cell-derived mesoderm-based 3D model of human segmentation and somitogenesis-which we termed 'axioloid'-that captures accurately the oscillatory dynamics of the segmentation clock and the morphological and molecular characteristics of sequential somite formation in vitro. Axioloids show proper rostrocaudal patterning of forming segments and robust anterior-posterior FGF-WNT signalling gradients and retinoic acid signalling components. We identify an unexpected critical role of retinoic acid signalling in the stabilization of forming segments, indicating distinct, but also synergistic effects of retinoic acid and extracellular matrix on the formation and epithelialization of somites. Comparative analysis demonstrates marked similarities of axioloids to the human embryo, further validated by the presence of a Hox code in axioloids. Finally, we demonstrate the utility of axioloids for studying the pathogenesis of human congenital spine diseases using induced pluripotent stem cells with mutations in HES7 and MESP2. Our results indicate that axioloids represent a promising platform for the study of axial development and disease in humans.
Assuntos
Padronização Corporal , Técnicas de Cultura de Células em Três Dimensões , Somitos , Humanos , Padronização Corporal/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Técnicas In Vitro , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Mutação , Somitos/citologia , Somitos/efeitos dos fármacos , Somitos/embriologia , Somitos/metabolismo , Doenças da Coluna Vertebral/patologia , Tretinoína/metabolismo , Tretinoína/farmacologia , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Shaping the animal body plan is a complex process that involves the spatial organization and patterning of the different germ layers. Recent advances in live imaging have started to unravel the cellular choreography underlying this process in mammals, however, the sequence of events transforming an unpatterned cell ensemble into structured territories is largely unknown. Here, using gastruloids -3D aggregates of mouse embryonic stem cells- we study the formation of one of the three germ layers, the endoderm. We show that the endoderm is generated from an epiblast-like homogeneous state by a three-step mechanism: (i) a loss of E-cadherin mediated contacts in parts of the aggregate leading to the appearance of islands of E-cadherin expressing cells surrounded by cells devoid of E-cadherin, (ii) a separation of these two populations with islands of E-cadherin expressing cells flowing toward the aggregate tip, and (iii) their differentiation into an endoderm population. During the flow, the islands of E-cadherin expressing cells are surrounded by cells expressing T-Brachyury, reminiscent of the process occurring at the primitive streak. Consistent with recent in vivo observations, the endoderm formation in the gastruloids does not require an epithelial-to-mesenchymal transition, but rather a maintenance of an epithelial state for a subset of cells coupled with fragmentation of E-cadherin contacts in the vicinity, and a sorting process. Our data emphasize the role of signaling and tissue flows in the establishment of the body plan.
Assuntos
Endoderma , Camadas Germinativas , Animais , Caderinas , Diferenciação Celular , Movimento Celular , Gastrulação , Mamíferos , CamundongosRESUMO
The primitive streak, a transient embryonic structure, marks bilateral symmetry in mammalian and avian embryos and helps confer anterior-posterior and dorsal-ventral spatial information to early differentiating cells during gastrulation. Its recapitulation in vitro may facilitate derivation of tissues and organs with in vivolike complexity. Proper understanding of the primitive streak and what it entails in human development is key to achieving such research objectives. Here we provide an overview of the primitive streak and conclude that this structure is neither conserved nor necessary for gastrulation or early lineage diversification. We offer a model in which the primitive streak is viewed as part of a morphologically diverse yet molecularly conserved process of spatial coordinate acquisition. We predict that recapitulation of the primitive streak is dispensable for development in vitro.
Assuntos
Embrião de Mamíferos/fisiologia , Embrião não Mamífero/fisiologia , Gastrulação , Linha Primitiva/fisiologia , Vertebrados/embriologia , Animais , Evolução Biológica , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/citologia , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/citologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Morfogênese , FilogeniaRESUMO
Over the last few years an intense activity in the areas of advanced microscopy and quantitative cell biology has put the focus on the morphogenetic events that shape embryos. The interest in these processes is taking place against the backdrop of genomic studies, particularly of global patterns of gene expression at the level of single cells, which cannot fully account for the way cells build tissues and organs. Here we discuss the need to integrate the activity of genes with that of cells and propose the need to develop a framework, based on cellular processes and cell interactions, that parallels that which has been created for gene activity in the form of Gene Regulatory Networks (GRNs). We begin to do this by suggesting elements for building Cell Regulatory Networks (CRNs). In the same manner that GRNs create schedules of gene expression that result in the emergence of cell fates over time, CRNs create tissues and organs i.e. space. We also suggest how GRNs and CRNs might interact in the building of embryos through feedback loops involving mechanics and tissue tectonics.
Assuntos
Fenômenos Fisiológicos Celulares , Redes Reguladoras de Genes , Redes Reguladoras de Genes/genética , Genômica , MorfogêneseRESUMO
In recent years, a diverse array of in vitro cell-derived models of mammalian development have been described that hold immense potential for exploring fundamental questions in developmental biology, particularly in the case of the human embryo where ethical and technical limitations restrict research. These models open up new avenues toward biomedical advances in in vitro fertilization, clinical research, and drug screening with potential to impact wider society across many diverse fields. These technologies raise challenging questions with profound ethical, regulatory, and social implications that deserve due consideration. Here, we discuss the potential impacts of embryo-like models, and their biomedical potential and current limitations.
Assuntos
Pesquisa Biomédica , Embrião de Mamíferos/fisiologia , Mamíferos/embriologia , Modelos Biológicos , Sociedades , Animais , Descoberta de Drogas , HumanosRESUMO
The connectivity patterns of neurons sustaining the functionality of spinal locomotor circuits rely on the specification of hundreds of motor neuron and interneuron subtypes precisely arrayed within the embryonic spinal cord. Knowledge acquired by developmental biologists on the molecular mechanisms underpinning this process in vivo has supported the development of 2D and 3D differentiation strategies to generate spinal neuronal diversity from mouse and human pluripotent stem cells (PSCs). Here, we review recent breakthroughs in this field and the perspectives opened up by models of in vitro embryogenesis to approach the mechanisms underlying neuronal diversification and the formation of functional mouse and human locomotor circuits. Beyond serving fundamental investigations, these new approaches should help engineering neuronal circuits differentially impacted in neuromuscular disorders, such as amyotrophic lateral sclerosis or spinal muscular atrophies, and thus open new avenues for disease modeling and drug screenings.
Assuntos
Esclerose Lateral Amiotrófica , Medula Espinal , Animais , Interneurônios , Mamíferos , Camundongos , Neurônios MotoresRESUMO
Floating spheroidal aggregates of mouse embryonic stem cells can develop into polarized/elongated organoids, namely gastruloids. We set up a high-performing assay to measure gastruloid formation efficiency (GFE), and found that GFE decreases as pluripotency progresses from naive (GFE ≥ 95%) to primed (GFE = 0) state. Specifically, we show that primed EpiSCs fail to generate proper cell aggregates, while early-primed EpiLCs aggregate but eventually fail to develop into elongated gastruloids. Moreover, we characterized proline-induced cells (PiCs), a LIF-dependent reversible early-primed state of pluripotency, and show that PiCs are able to generate gastruloids (GFE â¼ 50%) and are also competent to differentiate into primordial germ cell-like cells. Thus, we propose the GFE assay as a valuable functional tool to discriminate different states of the pluripotency continuum.
Assuntos
Desenvolvimento Embrionário , Fator de Crescimento Epidérmico/metabolismo , Camadas Germinativas/metabolismo , Glicoproteínas de Membrana/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas de Neoplasias/metabolismo , Organogênese , Organoides/embriologia , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular , Fator de Crescimento Epidérmico/genética , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genéticaRESUMO
Technical and ethical limitations create a challenge to study early human development, especially following the first 3 weeks of development after fertilization, when the fundamental aspects of the body plan are established through the process called gastrulation. As a consequence, our current understanding of human development is mostly based on the anatomical and histological studies on Carnegie Collection of human embryos, which were carried out more than half a century ago. Due to the 14-day rule on human embryo research, there have been no experimental studies beyond the fourteenth day of human development. Mutagenesis studies on animal models, mostly in mouse, are often extrapolated to human embryos to understand the transcriptional regulation of human development. However, due to the existence of significant differences in their morphological and molecular features as well as the time scale of their development, it is obvious that complete knowledge of human development can be achieved only by studying the human embryo. These studies require a cellular framework. Here we summarize the cellular, molecular, and temporal aspects associated with human gastrulation and discuss how they relate to existing human PSCs based models of early development.
Assuntos
Embrião de Mamíferos/metabolismo , Gastrulação , Animais , Padronização Corporal , Embrião de Mamíferos/citologia , Humanos , Modelos AnimaisRESUMO
Gastrulation is an inherently complicated process involving a well-orchestrated series of collective cellular behaviours that lead to the emergence of the body plan of the organism. A convenient method to explore the mechanical and chemical interactions that underpin this process, is to isolate specific tissues and to allow them to develop in isolation or in a novel environment. These approaches are the essence of experimental embryology and have enabled an understanding of the underlying principles of embryogenesis, in a way that observation alone could not. The recent rise of 3D culture systems using Embryonic Stem Cells (ESCs) has enabled the extension of this approach to mammalian systems. Here, we argue that these ESC based methods are consistent with the program of experimental embryology, and discuss the insights that can be gained from this perspective, particularly focussing the process of gastrulation and the associated emergence of the body plan.
Assuntos
Desenvolvimento Embrionário , Gastrulação , Modelos Biológicos , Células-Tronco Pluripotentes/citologia , Animais , HumanosRESUMO
Pluripotent stem cells derived from the early mammalian embryo offer a convenient model system for studying cell fate decisions in embryogenesis. The last 10 years have seen a boom in the popularity of two-dimensional micropatterns and three-dimensional stem cell culture systems as a way to recreate the architecture and interactions of particular cell populations during development. These methods enable the controlled exploration of cellular organization and patterning during development, using cell lines instead of embryos. They have established a new class of in vitro model system for pre-implantation and peri-implantation embryogenesis, ranging from models of the blastocyst stage, through gastrulation and toward early organogenesis. This review aims to set these systems in context and to highlight the strengths and suitability of each approach in modelling early mammalian development.
Assuntos
Embrião de Mamíferos/citologia , Desenvolvimento Embrionário , Mamíferos/embriologia , Modelos Biológicos , Células-Tronco Pluripotentes/metabolismo , Animais , Humanos , Organoides/citologia , Organoides/embriologiaRESUMO
The body plan of the mammalian embryo is shaped through the process of gastrulation, an early developmental event that transforms an isotropic group of cells into an ensemble of tissues that is ordered with reference to three orthogonal axes1. Although model organisms have provided much insight into this process, we know very little about gastrulation in humans, owing to the difficulty of obtaining embryos at such early stages of development and the ethical and technical restrictions that limit the feasibility of observing gastrulation ex vivo2. Here we show that human embryonic stem cells can be used to generate gastruloids-three-dimensional multicellular aggregates that differentiate to form derivatives of the three germ layers organized spatiotemporally, without additional extra-embryonic tissues. Human gastruloids undergo elongation along an anteroposterior axis, and we use spatial transcriptomics to show that they exhibit patterned gene expression. This includes a signature of somitogenesis that suggests that 72-h human gastruloids show some features of Carnegie-stage-9 embryos3. Our study represents an experimentally tractable model system to reveal and examine human-specific regulatory processes that occur during axial organization in early development.
Assuntos
Padronização Corporal , Gástrula/citologia , Células-Tronco Embrionárias Humanas/citologia , Organoides/citologia , Organoides/embriologia , Somitos/citologia , Somitos/embriologia , Padronização Corporal/genética , Gástrula/embriologia , Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Técnicas In Vitro , Organoides/metabolismo , Transdução de Sinais , Somitos/metabolismo , TranscriptomaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Gastruloids are three-dimensional aggregates of embryonic stem cells that display key features of mammalian development after implantation, including germ-layer specification and axial organization1-3. To date, the expression pattern of only a small number of genes in gastruloids has been explored with microscopy, and the extent to which genome-wide expression patterns in gastruloids mimic those in embryos is unclear. Here we compare mouse gastruloids with mouse embryos using single-cell RNA sequencing and spatial transcriptomics. We identify various embryonic cell types that were not previously known to be present in gastruloids, and show that key regulators of somitogenesis are expressed similarly between embryos and gastruloids. Using live imaging, we show that the somitogenesis clock is active in gastruloids and has dynamics that resemble those in vivo. Because gastruloids can be grown in large quantities, we performed a small screen that revealed how reduced FGF signalling induces a short-tail phenotype in embryos. Finally, we demonstrate that embedding in Matrigel induces gastruloids to generate somites with the correct rostral-caudal patterning, which appear sequentially in an anterior-to-posterior direction over time. This study thus shows the power of gastruloids as a model system for exploring development and somitogenesis in vitro in a high-throughput manner.
Assuntos
Gástrula , Células-Tronco Embrionárias Murinas/citologia , Organoides/citologia , Organoides/embriologia , Análise de Célula Única , Somitos/citologia , Somitos/embriologia , Transcriptoma , Animais , Colágeno , Combinação de Medicamentos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Feminino , Gástrula/citologia , Gástrula/embriologia , Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Laminina , Masculino , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Organoides/metabolismo , Proteoglicanas , RNA-Seq , Somitos/metabolismo , Fatores de TempoRESUMO
The EMBO-EMBL Symposium 'Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture' was held in Heidelberg, Germany, in March 2019, with 150 participants seeking to reverse-engineer embryogenesis, emphasizing quantitative simulation and the use of synthetic systems to test models. This highly dynamic, interdisciplinary mix of quantitative developmental genetics, bioengineering, synthetic biology and artificial life aimed to reveal how evolution exploits physical forces and genetics to implement the cell- and tissue-level decision-making required for complex morphogenesis.
Assuntos
Bioengenharia/organização & administração , Congressos como Assunto , Biologia do Desenvolvimento/métodos , Engenharia Genética , Animais , Bioengenharia/métodos , Bioengenharia/tendências , Congressos como Assunto/organização & administração , Congressos como Assunto/normas , Biologia do Desenvolvimento/organização & administração , Biologia do Desenvolvimento/tendências , Desenvolvimento Embrionário/fisiologia , Redes Reguladoras de Genes/fisiologia , Engenharia Genética/métodos , Engenharia Genética/tendências , Alemanha , Humanos , Modelos Biológicos , Morfogênese/fisiologia , Biologia Sintética/métodos , Biologia Sintética/organização & administração , Biologia Sintética/tendênciasRESUMO
The mammalian embryo's caudal lateral epiblast (CLE) harbours bipotent progenitors, called neural mesodermal progenitors (NMPs), that contribute to the spinal cord and the paraxial mesoderm throughout axial elongation. Here, we performed a single cell analysis of different in vitro NMP populations produced either from embryonic stem cells (ESCs) or epiblast stem cells (EpiSCs) and compared them with E8.25 CLE mouse embryos. In our analysis of this region, our findings challenge the notion that NMPs can be defined by the exclusive co-expression of Sox2 and T at mRNA level. We analyse the in vitro NMP-like populations using a purpose-built support vector machine (SVM) based on the embryo CLE and use it as a classification model to compare the in vivo and in vitro populations. Our results show that NMP differentiation from ESCs leads to heterogeneous progenitor populations with few NMP-like cells, as defined by the SVM algorithm, whereas starting with EpiSCs yields a high proportion of cells with the embryo NMP signature. We find that the population from which the Epi-NMPs are derived in culture contains a node-like population, which suggests that this population probably maintains the expression of T in vitro and thereby a source of NMPs. In conclusion, differentiation of EpiSCs into NMPs reproduces events in vivo and suggests a sequence of events for the emergence of the NMP population.
Assuntos
Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Camadas Germinativas/citologia , Mesoderma/embriologia , Células-Tronco Pluripotentes/citologia , Células-Tronco/citologia , Animais , Padronização Corporal/genética , Diferenciação Celular , Linhagem da Célula , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/metabolismo , Técnicas In Vitro , Masculino , Camundongos , Células-Tronco Neurais/citologia , Medula Espinal/embriologia , Máquina de Vetores de Suporte , TranscriptomaRESUMO
The caudal lateral epiblast of mammalian embryos harbours bipotent progenitors that contribute to the spinal cord and the paraxial mesoderm in concert with the body axis elongation. These progenitors, called neural mesodermal progenitors (NMPs), are identified as cells that co-express Sox2 and T/brachyury, a criterion used to derive NMP-like cells from embryonic stem cells in vitro However, unlike embryonic NMPs, these progenitors do not self-renew. Here, we find that the protocols that yield NMP-like cells in vitro initially produce a multipotent population that, in addition to NMPs, generates progenitors for the lateral plate and intermediate mesoderm. We show that epiblast stem cells (EpiSCs) are an effective source of these multipotent progenitors, which are further differentiated by a balance between BMP and Nodal signalling. Importantly, we show that NMP-like cells derived from EpiSCs exhibit limited self-renewal in vitro and a gene expression signature like their embryonic counterparts.