RESUMO
The cellular complexity and scale of the early liver have constrained analyses examining its emergence during organogenesis. To circumvent these issues, we analyzed 45,334 single-cell transcriptomes from embryonic day (E)7.5, when endoderm progenitors are specified, to E10.5 liver, when liver parenchymal and non-parenchymal cell lineages emerge. Our data detail divergence of vascular and sinusoidal endothelia, including a distinct transcriptional profile for sinusoidal endothelial specification by E8.75. We characterize two distinct mesothelial cell types as well as early hepatic stellate cells and reveal distinct spatiotemporal distributions for these populations. We capture transcriptional profiles for hepatoblast specification and migration, including the emergence of a hepatomesenchymal cell type and evidence for hepatoblast collective cell migration. Further, we identify cell-cell interactions during the organization of the primitive sinusoid. This study provides a comprehensive atlas of liver lineage establishment from the endoderm and mesoderm through to the organization of the primitive sinusoid at single-cell resolution.
Assuntos
Linhagem da Célula/genética , Fígado/citologia , Fígado/metabolismo , Análise de Célula Única , Transcriptoma/genética , Animais , Movimento Celular , Embrião de Mamíferos/citologia , Endotélio/citologia , Mesoderma/citologia , Camundongos , Transdução de Sinais , Células-Tronco/citologiaRESUMO
Here we delineate the ontogeny of the mammalian endoderm by generating 112,217 single-cell transcriptomes, which represent all endoderm populations within the mouse embryo until midgestation. We use graph-based approaches to model differentiating cells, which provides a spatio-temporal characterization of developmental trajectories and defines the transcriptional architecture that accompanies the emergence of the first (primitive or extra-embryonic) endodermal population and its sister pluripotent (embryonic) epiblast lineage. We uncover a relationship between descendants of these two lineages, in which epiblast cells differentiate into endoderm at two distinct time points-before and during gastrulation. Trajectories of endoderm cells were mapped as they acquired embryonic versus extra-embryonic fates and as they spatially converged within the nascent gut endoderm, which revealed these cells to be globally similar but retain aspects of their lineage history. We observed the regionalized identity of cells along the anterior-posterior axis of the emergent gut tube, which reflects their embryonic or extra-embryonic origin, and the coordinated patterning of these cells into organ-specific territories.
Assuntos
Endoderma/citologia , Endoderma/embriologia , Intestinos/citologia , Intestinos/embriologia , Análise de Célula Única , Animais , Blastocisto/citologia , Padronização Corporal , Diferenciação Celular , Linhagem da Célula , Feminino , Gastrulação , Masculino , CamundongosRESUMO
Gene regulatory networks controlling functional activities of spatially and temporally distinct endodermal cell populations in the early mouse embryo remain ill defined. The T-box transcription factor Eomes, acting downstream from Nodal/Smad signals, directly activates the LIM domain homeobox transcription factor Lhx1 in the visceral endoderm. Here we demonstrate Smad4/Eomes-dependent Lhx1 expression in the epiblast marks the entire definitive endoderm lineage, the anterior mesendoderm, and midline progenitors. Conditional inactivation of Lhx1 disrupts anterior definitive endoderm development and impedes node and midline morphogenesis in part due to severe disturbances in visceral endoderm displacement. Transcriptional profiling and ChIP-seq (chromatin immunoprecipitation [ChIP] followed by high-throughput sequencing) experiments identified Lhx1 target genes, including numerous anterior definitive endoderm markers and components of the Wnt signaling pathway. Interestingly, Lhx1-binding sites were enriched at enhancers, including the Nodal-proximal epiblast enhancer element and enhancer regions controlling Otx2 and Foxa2 expression. Moreover, in proteomic experiments, we characterized a complex comprised of Lhx1, Otx2, and Foxa2 as well as the chromatin-looping protein Ldb1. These partnerships cooperatively regulate development of the anterior mesendoderm, node, and midline cell populations responsible for establishment of the left-right body axis and head formation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/embriologia , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos , Elementos Facilitadores Genéticos/fisiologia , Deleção de Genes , Perfilação da Expressão Gênica , Camadas Germinativas/metabolismo , Fator 3-beta Nuclear de Hepatócito/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fatores de Transcrição Otx/metabolismo , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Via de Sinalização WntRESUMO
The endoderm is a progenitor tissue that, in humans, gives rise to the majority of internal organs. Over the past few decades, genetic studies have identified many of the upstream signals specifying endoderm identity in different model systems, revealing them to be divergent from invertebrates to vertebrates. However, more recent studies of the cell behaviours driving endodermal morphogenesis have revealed a surprising number of shared features, including cells undergoing epithelial-to-mesenchymal transitions (EMTs), collective cell migration, and mesenchymal-to-epithelial transitions (METs). In this Review, we highlight how cross-organismal studies of endoderm morphogenesis provide a useful perspective that can move our understanding of this fascinating tissue forward.
Assuntos
Linhagem da Célula/fisiologia , Endoderma/embriologia , Endoderma/fisiologia , Morfogênese/fisiologia , Animais , Evolução Biológica , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Endoderma/citologia , Transição Epitelial-Mesenquimal/fisiologia , Humanos , Transdução de Sinais , Vertebrados/embriologiaRESUMO
Reciprocal inductive interactions between the embryonic and extraembryonic tissues establish the anterior-posterior (AP) axis of the early mouse embryo. The anterior visceral endoderm (AVE) signaling center emerges at the distal tip of the embryo at embryonic day 5.5 and translocates to the prospective anterior side of the embryo. The process of AVE induction and migration are poorly understood. Here we demonstrate that the T-box gene Eomesodermin (Eomes) plays an essential role in AVE recruitment, in part by directly activating the homeobox transcription factor Lhx1. Thus, Eomes function in the visceral endoderm (VE) initiates an instructive transcriptional program controlling AP identity.
Assuntos
Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Domínio T/metabolismo , Animais , Padronização Corporal/genética , Linhagem Celular , Embrião de Mamíferos , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Mutação , Proteínas com Domínio T/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Mouse embryonic stem cells (mESCs) are clonal populations derived from preimplantation mouse embryos that can be propagated in vitro and, when placed into blastocysts, contribute to all tissues of the embryo and integrate into the normal morphogenetic processes, i.e. they are pluripotent. However, although they can be steered to differentiate in vitro into all cell types of the organism, they cannot organise themselves into structures that resemble embryos. When aggregated into embryoid bodies they develop disorganised masses of different cell types with little spatial coherence. An exception to this rule is the emergence of retinas and anterior cortex-like structures under minimal culture conditions. These structures emerge from the cultures without any axial organisation. Here, we report that small aggregates of mESCs, of about 300 cells, self-organise into polarised structures that exhibit collective behaviours reminiscent of those that cells exhibit in early mouse embryos, including symmetry breaking, axial organisation, germ layer specification and cell behaviour, as well as axis elongation. The responses are signal specific and uncouple processes that in the embryo are tightly associated, such as specification of the anteroposterior axis and anterior neural development, or endoderm specification and axial elongation. We discuss the meaning and implications of these observations and the potential uses of these structures which, because of their behaviour, we suggest to call 'gastruloids'.
Assuntos
Padronização Corporal/fisiologia , Células-Tronco Embrionárias/fisiologia , Camadas Germinativas/embriologia , Sistema Nervoso/embriologia , Animais , Agregação Celular/fisiologia , Linhagem Celular , Polaridade Celular/fisiologia , Citometria de Fluxo , Camundongos , Microscopia de FluorescênciaRESUMO
The notochord is a structure common to all chordates, and the feature that the phylum Chordata has been named after. It is a rod-like mesodermal structure that runs the anterior-posterior length of the embryo, adjacent to the ventral neural tube. The notochord plays a critical role in embryonic tissue patterning, for example the dorsal-ventral patterning of the neural tube. The cells that will come to form the notochord are specified at gastrulation. Axial mesodermal cells arising at the anterior primitive streak migrate anteriorly as the precursors of the notochord and populate the notochordal plate. Yet, even though a lot of interest has centered on investigating the functional and structural roles of the notochord, we still have a very rudimentary understanding of notochord morphogenesis. The events driving the formation of the notochord are rapid, taking place over the period of approximately a day in mice. In this commentary, we provide an overview of our current understanding of mouse notochord morphogenesis, from the initial specification of axial mesendodermal cells at the primitive streak, the emergence of these cells at the midline on the surface of the embryo, to their submergence and organization of the stereotypically positioned notochord. We will also discuss some key open questions. Developmental Dynamics 245:547-557, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Morfogênese , Notocorda/embriologia , Animais , Mesoderma/citologia , Camundongos , Linha PrimitivaRESUMO
BACKGROUND: The GATA-binding factor 6 (Gata6) gene encodes a zinc finger transcription factor that often functions as a key regulator of lineage specification during development. It is the earliest known marker of the primitive endoderm lineage in the mammalian blastocyst. During gastrulation, GATA6 is expressed in early cardiac mesoderm and definitive endoderm progenitors, and is necessary for development of specific mesoderm and endoderm-derived organs including the heart, liver, and pancreas. Furthermore, reactivation or silencing of the Gata6 locus has been associated with certain types of cancer affecting endodermal organs. RESULTS: We have generated a Gata6(H2B-Venus) knock-in reporter mouse allele for the purpose of labeling GATA6-expressing cells with a bright nuclear-localized fluorescent marker that is suitable for live imaging at single-cell resolution. CONCLUSIONS: Expression of the Venus reporter was characterized starting from embryonic stem (ES) cells, through mouse embryos and adult animals. The Venus reporter was not expressed in ES cells, but was activated upon endoderm differentiation. Gata6(H2B-Venus/H2B-Venus) homozygous embryos did not express GATA6 protein and failed to specify the primitive endoderm in the blastocyst. However, null blastocysts continued to express high levels of Venus in the absence of GATA6 protein, suggesting that early Gata6 transcription is independent of GATA6 protein expression. At early post-implantation stages of embryonic development, there was a strong correlation of Venus with endogenous GATA6 protein in endoderm and mesoderm progenitors, then later in the heart, midgut, and hindgut. However, there were discrepancies in reporter versus endogenous protein expression in certain cells, such as the body wall and endocardium. During organogenesis, detection of Venus in specific organs recapitulated known sites of endogenous GATA6 expression, such as in the lung bud epithelium, liver, pancreas, gall bladder, stomach epithelium, and vascular endothelium. In adults, Venus was observed in the lungs, pancreas, liver, gall bladder, ovaries, uterus, bladder, skin, adrenal glands, small intestine and corpus region of the stomach. Overall, Venus fluorescent protein under regulatory control of the Gata6 locus was expressed at levels that were easily visualized directly and could endure live and time-lapse imaging techniques. Venus is co-expressed with endogenous GATA6 throughout development to adulthood, and should provide an invaluable tool for examining the status of the Gata6 locus during development, as well as its silencing or reactivation in cancer or other disease states.
Assuntos
Fator de Transcrição GATA6/genética , Técnicas Genéticas , Camundongos/genética , Análise de Célula Única , Animais , Embrião de Mamíferos/metabolismo , Fator de Transcrição GATA6/metabolismo , Genes Reporter , Camundongos/embriologia , Camundongos KnockoutRESUMO
Mixl1 is the only member of the Mix/Bix homeobox gene family identified in mammals. During mouse embryogenesis, Mixl1 is first expressed at embryonic day (E)5.5 in cells of the visceral endoderm (VE). At the time of gastrulation, Mixl1 expression is detected in the vicinity of the primitive streak. Mixl1 is expressed in cells located within the primitive streak, in nascent mesoderm cells exiting the primitive streak, and in posterior VE overlying the primitive streak. Genetic ablation of Mixl1 in mice has revealed its crucial role in mesoderm and endoderm cell specification and tissue morphogenesis during early embryonic development. However, the early lethality of the constitutive Mixl1(-/-) mutant precludes the study of its role at later stages of embryogenesis and in adult mice. To circumvent this limitation, we have generated a conditional Mixl1 allele (Mixl1(cKO) that permits temporal as well as spatial control of gene ablation. Animals homozygous for the Mixl1(cKO) conditional allele were viable and fertile. Mixl1(KO/KO) embryos generated by crossing of Mixl1(cKO/cKO) mice with Sox2-Cre or EIIa-Cre transgenic mice were embryonic lethal at early somite stages. By contrast to wild-type embryos, Mixl1(KO/KO) embryos contained no detectable Mixl1, validating the Mixl1(cKO) as a protein null after Cre-mediated excision. Mixl1(KO/KO) embryos resembled the previously reported Mixl1(-/-) mutant phenotype. Therefore, the Mixl1 cKO allele provides a tool for investigating the temporal and tissue-specific requirements for Mixl1 in the mouse.
Assuntos
Endoderma/embriologia , Técnicas de Inativação de Genes/métodos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mesoderma/embriologia , Animais , Cruzamentos Genéticos , Endoderma/metabolismo , Gastrulação , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Genes Letais , Mesoderma/metabolismo , CamundongosRESUMO
Paraxial mesoderm is the tissue which gives rise to the skeletal muscles and vertebral column of the body. A gene regulatory network operating in the formation of paraxial mesoderm has been described. This network hinges on three key factors, Wnt3a, Msgn1 and Tbx6, each of which is critical for paraxial mesoderm formation, since absence of any one of these factors results in complete absence of posterior somites. In this study we determined and compared the spatial and temporal patterns of expression of Wnt3a, Msgn1 and Tbx6 at a time when paraxial mesoderm is being formed. Then, we performed a comparative characterization of mutants in Wnt3a, Msgn1 and Tbx6. To determine the epistatic relationship between these three genes, and begin to decipher the complex interplay between them, we analyzed double mutant embryos and compared their phenotypes to the single mutants. Through the analysis of molecular markers in mutants, our data support the bipotential nature of the progenitor cells for paraxial mesoderm and establish regulatory relationships between genes involved in the choice between neural and mesoderm fates.
Assuntos
Embrião de Mamíferos/metabolismo , Mesoderma/citologia , Animais , Diferenciação Celular , Transição Epitelial-Mesenquimal , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Camundongos , Mutação , Somitos/metabolismoRESUMO
BACKGROUND: Live imaging provides an essential methodology for understanding complex and dynamic cell behaviors and their underlying molecular mechanisms. Genetically-encoded reporter expressing mouse strains are an important tool for use in live imaging experiments. Such reporter strains can be engineered by placing cis-regulatory elements of interest to direct the expression of desired reporter genes. If these cis-regulatory elements are downstream targets, and thus activated as a consequence of signaling pathway activation, such reporters can provide read-outs of the signaling status of a cell. The Notch signaling pathway is an evolutionary conserved pathway operating in multiple developmental processes as well as being the basis for several congenital diseases. The transcription factor CBF1 is a central evolutionarily conserved component of the Notch signaling pathway. It binds the active form of the Notch receptor (NICD) and subsequently binds to cis-regulatory regions (CBF1 binding sites) in the promoters of Notch responsive genes. In this way, CBF1 binding sites represent a good target for the design of a Notch signaling reporter. RESULTS: To generate a single-cell resolution Notch signaling reporter, we used a CBF responsive element to direct the expression of a nuclear-localized fluorescent protein. To do this, we linked 4 copies of a consensus CBF1 binding site to the basal simian virus 40 (SV40) promoter, placed this cassette in front of a fluorescent protein fusion comprising human histone H2B linked to the yellow fluorescent protein (YFP) Venus, one of the brightest available YFPs. We used the CBF:H2B-Venus construct to generate both transgenic embryonic mouse stem (ES) cell lines and a strain of transgenic mice that would report Notch signaling activity. CONCLUSION: By using multiple CBF1 binding sites together with a subcellular-localized, genetically-encoded fluorescent protein, H2B-Venus, we have generated a transgenic strain of mice that faithfully recapitulates Notch signaling at single-cell resolution. This is the first mouse reporter strain in which individual cells transducing a Notch signal can be visualized. The improved resolution of this reporter makes it ideal for live imaging developmental processes regulated by the Notch signaling pathway as well as a short-term lineage tracer of Notch expressing cells due to the perdurance of the fluorescent reporter. Taken together, the CBF:H2B-Venus mouse strain is a unique tool to study and understand the morphogenetic events regulated by the Notch signaling pathway.
Assuntos
Genes Reporter , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Encéfalo/metabolismo , Gastrulação , Expressão Gênica , Histonas/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Rim/metabolismo , Camundongos , Regiões Promotoras GenéticasRESUMO
BACKGROUND: Velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) is caused by a 1.5-3 Mb microdeletion of chromosome 22q11.2, frequently referred to as 22q11.2 deletion syndrome (22q11DS). This region includes TBX1, a T-box transcription factor gene that contributes to the etiology of 22q11DS. The requirement for TBX1 in mammalian development is dosage-sensitive, such that loss-of-function (LOF) and gain-of-function (GOF) of TBX1 in both mice and humans results in disease relevant congenital malformations. RESULTS: To further gain insight into the role of Tbx1 in development, we have targeted the Rosa26 locus to generate a new GOF mouse model in which a Tbx1-GFP fusion protein is expressed conditionally using the Cre/LoxP system. Tbx1-GFP expression is driven by the endogenous Rosa26 promoter resulting in ectopic and persistent expression. Tbx1 is pivotal for proper ear and heart development; ectopic activation of Tbx1-GFP in the otic vesicle by Pax2-Cre and Foxg1-Cre represses neurogenesis and produces morphological defects of the inner ear. Overexpression of a single copy of Tbx1-GFP using Tbx1Cre/+ was viable, while overexpression of both copies resulted in neonatal lethality with cardiac outflow tract defects. We have partially rescued inner ear and heart anomalies in Tbx1Cre/- null embryos by expression of Tbx1-GFP. CONCLUSIONS: We have generated a new mouse model to conditionally overexpress a GFP-tagged Tbx1 protein in vivo. This provides a useful tool to investigate in vivo direct downstream targets and protein binding partners of Tbx1.
Assuntos
Orelha/embriologia , Coração/embriologia , Modelos Animais , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Animais , Orelha/patologia , Embrião de Mamíferos , Dosagem de Genes , Proteínas de Fluorescência Verde/metabolismo , Camundongos , RNA não Traduzido/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Primitive erythroid (EryP) progenitors are the first cell type specified from the mesoderm late in gastrulation. We used a transgenic reporter to image and purify the earliest blood progenitors and their descendants from developing mouse embryos. EryP progenitors exhibited remarkable proliferative capacity in the yolk sac immediately before the onset of circulation, when these cells comprise nearly half of all cells of the embryo. Global expression profiles generated at 24-hour intervals from embryonic day 7.5 through 2.5 revealed 2 abrupt changes in transcript diversity that coincided with the entry of EryPs into the circulation and with their late maturation and enucleation, respectively. These changes were paralleled by the expression of critical regulatory factors. Experiments designed to test predictions from these data demonstrated that the Wnt-signaling pathway is active in EryP progenitors, which display an aerobic glycolytic profile and the numbers of which are regulated by transforming growth factor-ß1 and hypoxia. This is the first transcriptome assembled for a single hematopoietic lineage of the embryo over the course of its differentiation.
Assuntos
Linhagem da Célula/genética , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/metabolismo , Eritropoese/genética , Redes Reguladoras de Genes , Animais , Sequência de Bases , Citocinas/genética , Primers do DNA/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Glicólise/genética , Proteínas de Fluorescência Verde/genética , Substâncias de Crescimento/genética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Oxigênio/metabolismo , Gravidez , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais/genética , Globinas épsilon/genéticaRESUMO
Two distinct fates, pluripotent epiblast (EPI) and primitive (extra-embryonic) endoderm (PrE), arise from common progenitor cells, the inner cell mass (ICM), in mammalian embryos. To study how these sister identities are forged, we leveraged embryonic (ES) and eXtraembryonic ENdoderm (XEN) stem cells - in vitro counterparts of the EPI and PrE. Bidirectional reprogramming between ES and XEN coupled with single-cell RNA and ATAC-seq analyses uncovered distinct rates, efficiencies and trajectories of state conversions, identifying drivers and roadblocks of reciprocal conversions. While GATA4-mediated ES-to-iXEN conversion was rapid and nearly deterministic, OCT4, KLF4 and SOX2-induced XEN-to-iPS reprogramming progressed with diminished efficiency and kinetics. The dominant PrE transcriptional program, safeguarded by Gata4, and globally elevated chromatin accessibility of EPI underscored the differential plasticities of the two states. Mapping in vitro trajectories to embryos revealed reprogramming in either direction tracked along, and toggled between, EPI and PrE in vivo states without transitioning through the ICM.
RESUMO
Live imaging of genetically encoded fluorescent protein reporters is increasingly being used to investigate details of the cellular behaviors that underlie the large-scale tissue rearrangements that shape the embryo. However, the majority of mouse fluorescent reporter strains are based on the green fluorescent protein (GFP). Mouse reporter strains expressing fluorescent colors other than GFP are therefore valuable for co-visualization studies with GFP, where relative positioning and relationship between two different tissues or compartments within cells are being investigated. Here, we report the generation and characterization of a transgenic Afp::mCherry mouse strain in which cis-regulatory elements from the Alpha-fetoprotein (Afp) locus were used to drive expression of the monomeric Cherry red fluorescent protein. The Afp::mCherry transgene is based on and recapitulates reporter expression of a previously described Afp::GFP strain. However, we note that perdurance of mCherry protein is not as prolonged as GFP, making the Afp::mCherry line a more faithful reporter of endogenous Afp expression. Afp::mCherry transgenic mice expressed mCherry specifically in the visceral endoderm and its derivatives, including the visceral yolk sac, gut endoderm, fetal liver, and pancreas of the embryo. The Afp::mCherry reporter was also noted to be expressed in other documented sites of Afp expression including hepatocytes as well as in pancreas, digestive tract, and brain of postnatal mice.
Assuntos
Endoderma/citologia , Endoderma/metabolismo , Genes Reporter , Proteínas Luminescentes/genética , alfa-Fetoproteínas/genética , Animais , Linhagem Celular Tumoral , Embrião de Mamíferos/metabolismo , Proteínas de Fluorescência Verde/genética , Hepatócitos , Humanos , Camundongos , Camundongos Transgênicos , Sequências Reguladoras de Ácido Nucleico , Transgenes , Vísceras/citologia , Vísceras/metabolismo , Saco Vitelino/embriologia , Saco Vitelino/metabolismo , Proteína Vermelha FluorescenteRESUMO
BACKGROUND: To exploit the flood of data from advances in high throughput imaging of optically sectioned nuclei, image analysis methods need to correctly detect thousands of nuclei, ideally in real time. Variability in nuclear appearance and undersampled volumetric data make this a challenge. RESULTS: We present a novel 3D nuclear identification method, which subdivides the problem, first segmenting nuclear slices within each 2D image plane, then using a shape model to assemble these slices into 3D nuclei. This hybrid 2D/3D approach allows accurate accounting for nuclear shape but exploits the clear 2D nuclear boundaries that are present in sectional slices to avoid the computational burden of fitting a complex shape model to volume data. When tested over C. elegans, Drosophila, zebrafish and mouse data, our method yielded 0 to 3.7% error, up to six times more accurate as well as being 30 times faster than published performances. We demonstrate our method's potential by reconstructing the morphogenesis of the C. elegans pharynx. This is an important and much studied developmental process that could not previously be followed at this single cell level of detail. CONCLUSIONS: Because our approach is specialized for the characteristics of optically sectioned nuclear images, it can achieve superior accuracy in significantly less time than other approaches. Both of these characteristics are necessary for practical analysis of overwhelmingly large data sets where processing must be scalable to hundreds of thousands of cells and where the time cost of manual error correction makes it impossible to use data with high error rates. Our approach is fast, accurate, available as open source software and its learned shape model is easy to retrain. As our pharynx development example shows, these characteristics make single cell analysis relatively easy and will enable novel experimental methods utilizing complex data sets.
Assuntos
Núcleo Celular/ultraestrutura , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Animais , Caenorhabditis elegans/fisiologia , Processamento de Imagem Assistida por Computador , Camundongos , Análise de Célula ÚnicaRESUMO
Gastrulation is a central process in mammalian development in which a spatiotemporally coordinated series of events driven by cross-talk between adjacent embryonic and extra-embryonic tissues results in stereotypical morphogenetic cell behaviors, massive cell proliferation and the acquisition of distinct cell identities. Gastrulation provides the blueprint of the body plan of the embryo, as well as generating extra-embryonic cell types of the embryo to make a connection with its mother. Gastrulation involves the specification of mesoderm and definitive endoderm from pluripotent epiblast, concomitant with a highly ordered elongation of tissue along the anterior-posterior (AP) axis. Interestingly, cells with an endoderm identity arise twice during mouse development. Cells with a primitive endoderm identity are specified in the preimplantation blastocyst, and which at gastrulation intercalate with the emergent definitive endoderm to form a mosaic tissue, referred to as the gut endoderm. The gut endoderm gives rise to the gut tube, which will subsequently become patterned along its AP axis into domains possessing unique visceral organ identities, such as thyroid, lung, liver and pancreas. In this way, proper endoderm development is essential for vital organismal functions, including the absorption of nutrients, gas exchange, detoxification and glucose homeostasis.
Assuntos
Embrião de Mamíferos/fisiologia , Endoderma/fisiologia , Trato Gastrointestinal/fisiologia , Gastrulação , Camadas Germinativas/fisiologia , Mesoderma/fisiologia , Morfogênese , Animais , Embrião de Mamíferos/citologia , Endoderma/citologia , Trato Gastrointestinal/citologia , Camadas Germinativas/citologia , Mesoderma/citologia , CamundongosRESUMO
To simultaneously follow multiple subcellular characteristics, for example, cell position and cell morphology, in living specimens requires multiple subcellular labels. Toward this goal, we generated dual-tagged mouse embryonic stem (ES) cells constitutively expressing differentially localized, spectrally distinct, genetically encoded fluorescent protein fusions. We have used human histone H2B fusions to fluorescent proteins to mark chromatin. This provides a descriptor of cell position, division, and death. An additional descriptor of cell morphology is achieved by combining this transgene with select lipid-modified fluorescent protein fusions that mark the plasma membrane. Using this strategy, wewere able to live image cellular dynamics in three dimensions over time both in cultured ES cells and in mouse embryos generated using dual-tagged ES cells. This study, therefore, presents the feasibility of applying multiple spectrally and subcellularly distinct fluorescent protein reporters for live imaging studies in ES cells and mouse embryos. Furthermore, the increasing availability of spectral variant fluorescent proteins along with the development of methods that permit improved spectral separation now facilitate multiplexing of fluorescent reporters to provide readouts of a variety of anatomical and physiological behaviors simultaneously in living specimens.
Assuntos
Membrana Celular/metabolismo , Cromatina/metabolismo , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/metabolismo , Transgenes/genética , Animais , Agregação Celular , Linhagem Celular , Quimera , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Estudos de Viabilidade , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia de Fluorescência/métodos , Plasmídeos/genética , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Transfecção/métodos , Proteína Vermelha FluorescenteRESUMO
Microscopy has always been an obligate tool in the field of developmental biology, a goal of which is to elucidate the essential cellular and molecular interactions that coordinate the specification of different cell types and the establishment of body plans. The 2008 Nobel Prize in chemistry was awarded 'for the discovery and development of the green fluorescent protein, GFP' in recognition that the discovery of genetically encoded fluorescent proteins (FPs) has spearheaded a revolution in applications for imaging of live cells. With the development of more-sophisticated imaging technology and availability of FPs with different spectral characteristics, dynamic processes can now be live-imaged at high resolution in situ in embryos. Here, we review some recent advances in this rapidly evolving field as applied to live-imaging capabilities in the mouse, the most genetically tractable mammalian model organism for embryologists.
Assuntos
Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Imunofluorescência/métodos , Proteínas Luminescentes/metabolismo , Camundongos/metabolismo , Microscopia de Fluorescência/métodos , Animais , Imunofluorescência/tendências , Microscopia de Fluorescência/tendênciasRESUMO
Tbx1, a T-box transcription factor, and an important gene for velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) in humans, causes outflow tract (OFT) heart defects when inactivated in the mouse. Tbx1 is expressed in the second heart field (SHF) and is required in this tissue for OFT development. To identify Tbx1 regulated genetic pathways in the SHF, we performed gene expression profiling of the caudal pharyngeal region in Tbx1(-/-) and wild type embryos. Isl1, a key marker for the SHF, as well as Hod and Nkx2-6, were downregulated in Tbx1(-/-) mutants, while genes required for cardiac morphogenesis, such as Raldh2, Gata4, and Tbx5, as well as a subset of muscle contractile genes, signifying myocardial differentiation, were ectopically expressed. Pan-mesodermal ablation of Tbx1 resulted in similar gene expression changes, suggesting cell-autonomous roles of Tbx1 in regulating these genes. Opposite expression changes concomitant with SHF-derived cardiac defects occurred in TBX1 gain-of-function mutants, indicating that appropriate levels of Tbx1 are required for heart development. When taken together, our studies show that Tbx1 acts upstream in a genetic network that positively regulates SHF cell proliferation and negatively regulates differentiation, cell-autonomously in the caudal pharyngeal region.