RESUMO
A gene expression atlas is an essential resource to quantify and understand the multiscale processes of embryogenesis in time and space. The automated reconstruction of a prototypic 4D atlas for vertebrate early embryos, using multicolor fluorescence in situ hybridization with nuclear counterstain, requires dedicated computational strategies. To this goal, we designed an original methodological framework implemented in a software tool called Match-IT. With only minimal human supervision, our system is able to gather gene expression patterns observed in different analyzed embryos with phenotypic variability and map them onto a series of common 3D templates over time, creating a 4D atlas. This framework was used to construct an atlas composed of 6 gene expression templates from a cohort of zebrafish early embryos spanning 6 developmental stages from 4 to 6.3 hpf (hours post fertilization). They included 53 specimens, 181,415 detected cell nuclei and the segmentation of 98 gene expression patterns observed in 3D for 9 different genes. In addition, an interactive visualization software, Atlas-IT, was developed to inspect, supervise and analyze the atlas. Match-IT and Atlas-IT, including user manuals, representative datasets and video tutorials, are publicly and freely available online. We also propose computational methods and tools for the quantitative assessment of the gene expression templates at the cellular scale, with the identification, visualization and analysis of coexpression patterns, synexpression groups and their dynamics through developmental stages.
Assuntos
Biologia Computacional/métodos , Embrião não Mamífero/citologia , Transcriptoma/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais , Bases de Dados Factuais , Embrião não Mamífero/metabolismo , Perfilação da Expressão GênicaRESUMO
MOTIVATION: Multi-view microscopy techniques such as Light-Sheet Fluorescence Microscopy (LSFM) are powerful tools for 3D + time studies of live embryos in developmental biology. The sample is imaged from several points of view, acquiring a set of 3D views that are then combined or fused in order to overcome their individual limitations. Views fusion is still an open problem despite recent contributions in the field. RESULTS: We developed a wavelet-based multi-view fusion method that, due to wavelet decomposition properties, is able to combine the complementary directional information from all available views into a single volume. Our method is demonstrated on LSFM acquisitions from live sea urchin and zebrafish embryos. The fusion results show improved overall contrast and details when compared with any of the acquired volumes. The proposed method does not need knowledge of the system's point spread function (PSF) and performs better than other existing PSF independent fusion methods. AVAILABILITY AND IMPLEMENTATION: The described method was implemented in Matlab (The Mathworks, Inc., USA) and a graphic user interface was developed in Java. The software, together with two sample datasets, is available at http://www.die.upm.es/im/software/SPIMFusionGUI.zip A public release, free of charge for non-commercial use, is planned after the publication of this article.
Assuntos
Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Ouriços-do-Mar/embriologia , Software , Peixe-Zebra/embriologia , Animais , HumanosRESUMO
We conducted a quantitative comparison of developing sea urchin embryos based on the analysis of five digital specimens obtained by automatic processing of in toto 3D+ time image data. These measurements served the reconstruction of a prototypical cell lineage tree able to predict the spatiotemporal cellular organisation of a normal sea urchin blastula. The reconstruction was achieved by designing and tuning a multi-level probabilistic model that reproduced embryo-level dynamics from a small number of statistical parameters characterising cell proliferation, cell surface area and cell volume evolution along the cell lineage. Our resulting artificial prototype was embedded in 3D space by biomechanical agent-based modelling and simulation, which allowed a systematic exploration and optimisation of free parameters to fit the experimental data and test biological hypotheses. The spherical monolayered blastula and the spatial arrangement of its different cell types appeared tightly constrained by cell stiffness, cell-adhesion parameters and blastocoel turgor pressure.
Assuntos
Blástula/citologia , Linhagem da Célula/fisiologia , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Modelos Estatísticos , Ouriços-do-Mar/embriologia , Animais , Fenômenos Biomecânicos , Blástula/fisiologia , Proliferação de Células , Tamanho Celular , Simulação por Computador , Imageamento Tridimensional , Ouriços-do-Mar/citologia , Ouriços-do-Mar/fisiologiaRESUMO
The quantitative and systematic analysis of embryonic cell dynamics from in vivo 3D+time image data sets is a major challenge at the forefront of developmental biology. Despite recent breakthroughs in the microscopy imaging of living systems, producing an accurate cell lineage tree for any developing organism remains a difficult task. We present here the BioEmergences workflow integrating all reconstruction steps from image acquisition and processing to the interactive visualization of reconstructed data. Original mathematical methods and algorithms underlie image filtering, nucleus centre detection, nucleus and membrane segmentation, and cell tracking. They are demonstrated on zebrafish, ascidian and sea urchin embryos with stained nuclei and membranes. Subsequent validation and annotations are carried out using Mov-IT, a custom-made graphical interface. Compared with eight other software tools, our workflow achieved the best lineage score. Delivered in standalone or web service mode, BioEmergences and Mov-IT offer a unique set of tools for in silico experimental embryology.
Assuntos
Embriologia/métodos , Imageamento Tridimensional/métodos , Microscopia , Fluxo de Trabalho , Animais , Linhagem da Célula , Proliferação de Células , Ouriços-do-Mar , Urocordados , Peixe-ZebraRESUMO
We propose to directly process 3D + t image sequences with mathematical morphology operators using a new classification of the 3D+t structuring elements. Several methods (filtering, tracking, segmentation) dedicated to the analysis of 3D + t datasets of zebrafish embryogenesis are introduced and validated through a synthetic dataset. Then, we illustrate the application of these methods to the analysis of datasets of zebrafish early development acquired with various microscopy techniques. This processing paradigm produces spatio-temporal coherent results as it benefits from the intrinsic redundancy of the temporal dimension and minimizes the needs for human intervention in semi-automatic algorithms.
Assuntos
Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário/fisiologia , Imageamento Tridimensional/métodos , Microscopia/métodos , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/embriologia , Algoritmos , Animais , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise Espaço-TemporalRESUMO
Investigating cell dynamics during early zebrafish embryogenesis requires specific image acquisition and analysis strategies. Multiharmonic microscopy, i.e., second- and third-harmonic generations, allows imaging cell divisions and cell membranes in unstained zebrafish embryos from 1- to 1000-cell stage. This paper presents the design and implementation of a dedicated image processing pipeline (tracking and segmentation) for the reconstruction of cell dynamics during these developmental stages. This methodology allows the reconstruction of the cell lineage tree including division timings, spatial coordinates, and cell shape until the 1000-cell stage with minute temporal accuracy and micrometer spatial resolution. Data analysis of the digital embryos provides an extensive quantitative description of early zebrafish embryogenesis.
Assuntos
Rastreamento de Células/métodos , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Reconhecimento Automatizado de Padrão/métodos , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/embriologia , Algoritmos , Animais , Aumento da Imagem/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Nonlinear microscopy can be used to probe the intrinsic optical properties of biological tissues. Using femtosecond pulses, third-harmonic generation (THG) and four-wave mixing (FWM) signals can be efficiently produced and detected simultaneously. Both signals probe a similar parameter, i.e. the real part of the third-order nonlinear susceptibility χ((3)). However THG and FWM images result from different phase matching conditions and provide complementary information. We analyze this complementarity using calculations, z-scan measurements on water and oils, and THG-FWM imaging of cell divisions in live zebrafish embryos. The two signals exhibit different sensitivity to sample size and clustering in the half-wavelength regime. Far from resonance, THG images reveal spatial variations |Δχ((3))(-3ω;ω,ω,ω)| with remarkable sensitivity while FWM directly reflects the distribution of χ((3))(-2ω(1) + ω(2);ω(1), -ω(2), ω(1)). We show that FWM images provide χ((3)) maps useful for proper interpretation of cellular THG signals, and that combined imaging carries additional structural information. Finally we present simultaneous imaging of intrinsic THG, FWM, second-harmonic (SHG) and two-photon-excited fluorescence (2PEF) signals in live Caenorhabditis elegans worms illustrating the information provided by multimodal nonlinear imaging of unstained tissue.
RESUMO
Quantifying cell behaviors in animal early embryogenesis remains a challenging issue requiring in toto imaging and automated image analysis. We designed a framework for imaging and reconstructing unstained whole zebrafish embryos for their first 10 cell division cycles and report measurements along the cell lineage with micrometer spatial resolution and minute temporal accuracy. Point-scanning multiphoton excitation optimized to preferentially probe the innermost regions of the embryo provided intrinsic signals highlighting all mitotic spindles and cell boundaries. Automated image analysis revealed the phenomenology of cell proliferation. Blastomeres continuously drift out of synchrony. After the 32-cell stage, the cell cycle lengthens according to cell radial position, leading to apparent division waves. Progressive amplification of this process is the rule, contrasting with classical descriptions of abrupt changes in the system dynamics.
Assuntos
Linhagem da Célula , Embrião não Mamífero/citologia , Microscopia/métodos , Peixe-Zebra/embriologia , Animais , Blástula/citologia , Ciclo Celular , Processamento de Imagem Assistida por ComputadorRESUMO
During development, cell migration plays an important role in morphogenetic processes. The construction of the skeleton of the sea urchin embryo by a small number of cells, the primary mesenchyme cells (PMCs), offers a remarkable model to study cell migration and its involvement in morphogenesis. During gastrulation, PMCs migrate and become positioned along the ectodermal wall following a stereotypical pattern that determines skeleton morphology. Previous studies have shown that interactions between ectoderm and PMCs regulate several aspects of skeletal morphogenesis, but little is known at the molecular level. Here we show that VEGF signaling between ectoderm and PMCs is crucial in this process. The VEGF receptor (VEGFR) is expressed exclusively in PMCs, whereas VEGF expression is restricted to two small areas of the ectoderm, in front of the positions where the ventrolateral PMC clusters that initiate skeletogenesis will form. Overexpression of VEGF leads to skeletal abnormalities, whereas inhibition of VEGF/VEGFR signaling results in incorrect positioning of the PMCs, downregulation of PMC-specific genes and loss of skeleton. We present evidence that localized VEGF acts as both a guidance cue and a differentiation signal, providing a crucial link between the positioning and differentiation of the migrating PMCs and leading to morphogenesis of the embryonic skeleton.
Assuntos
Ectoderma/fisiologia , Mesoderma/fisiologia , Ouriços-do-Mar/embriologia , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem da Célula , Ectoderma/citologia , Ectoderma/metabolismo , Embrião não Mamífero , Gástrula , Hibridização In Situ , Mesoderma/citologia , Mesoderma/metabolismo , Microinjeções , Modelos Biológicos , Morfogênese , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ouriços-do-Mar/citologiaRESUMO
Drosophila ELAV is the founding member of an evolutionarily conserved family of RNA-binding proteins considered as key inducers of neuronal differentiation. Although several ELAV-specific targets have been identified, little is known about the role of elav during neural development. Here, we report a detailed characterization of the elav mutant commissural phenotype. The reduced number of commissures in elav mutant embryos is not due to loss or misspecification of neural cells but results from defects in commissural axon projections across the midline. We establish a causal relationship between the elav mutant commissural phenotype and a reduction in the expression of commissureless, a key component of the Robo/Slit growth cone repulsive signalling pathway. In the nerve cord of elav mutant embryos, comm mRNA expression is strongly reduced in neurons, but not in midline glial cells. Furthermore, specific expression of an elav transgene in posterior neurons of each segment of an elav mutant nerve cord restores comm mRNA expression in these cells, as well as the formation of posterior commissures. Finally, forced expression of comm in specific commissural neuron subsets rescues the midline crossing defects of these neurons in elav mutant embryos, further indicating that elav acts cell autonomously on comm expression.
Assuntos
Axônios , Padronização Corporal/fisiologia , Drosophila/embriologia , Neurônios/metabolismo , Proteínas de Ligação a RNA/fisiologia , Animais , Morte Celular , Sistema Nervoso Central/citologia , Sistema Nervoso Central/embriologia , Imuno-Histoquímica , Neurônios/citologia , RNA Mensageiro/genéticaRESUMO
Wnt signaling pathways play key roles in numerous developmental processes both in vertebrates and invertebrates. Their signals are transduced by Frizzled proteins, the cognate receptors of the Wnt ligands. This study focuses on the role of a member of the Frizzled family, Fz5/8, during sea urchin embryogenesis. During development, Fz5/8 displays restricted expression, beginning at the 60-cell stage in the animal domain and then from mesenchyme blastula stage, in both the animal domain and a subset of secondary mesenchyme cells (SMCs). Loss-of-function analyses in whole embryos and chimeras reveal that Fz5/8 is not involved in the specification of the main embryonic territories. Rather, it appears to be required in SMCs for primary invagination of the archenteron, maintenance of endodermal marker expression and apical localization of Notch receptors in endodermal cells. Furthermore, among the three known Wnt pathways, Fz5/8 appears to signal via the planar cell polarity pathway. Taken together, the results suggest that Fz5/8 plays a crucial role specifically in SMCs to control primary invagination during sea urchin gastrulation.
Assuntos
Padronização Corporal , Desenvolvimento Embrionário/fisiologia , Receptores Frizzled/metabolismo , Gástrula/fisiologia , Ouriços-do-Mar , Animais , Polaridade Celular , Receptores Frizzled/classificação , Receptores Frizzled/genética , Gástrula/citologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Mesoderma/citologia , Mesoderma/metabolismo , Filogenia , Receptores Notch/metabolismo , Ouriços-do-Mar/anatomia & histologia , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/crescimento & desenvolvimento , Transdução de Sinais/fisiologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
The Wnt pathways are evolutionarily well-conserved signal transduction pathways that are known to play important roles in all Metazoans investigated to date. Here, we examine the Wnt pathway genes and target genes present in the genome of the echinoderm Strongylocentrotus purpuratus. Analysis of the Wnt genes revealed that eleven of the thirteen reported Wnt subfamilies are represented in sea urchin, with the intriguing identification of a Wnt-A ortholog thought to be absent in deuterostomes. A phylogenetic study of the Frizzled proteins, the Wnt receptors, performed throughout the animal kingdom showed that not all Frizzled subfamilies were present in the metazoan common ancestor, e.g. Fz3/6 emerged later during evolution. Using sequence analysis, orthologs of the vast majority of the cellular machinery involved in transducing the three types of Wnt pathways were found in the sea urchin genome. Furthermore, of about one hundred target genes identified in other organisms, more than half have clear echinoderm orthologs. Thus, these analyses produce new inputs in the evolutionary history of the Wnt genes in an animal occupying a position that offers great insights into the basal properties of deuterostomes.
Assuntos
Genoma , Ouriços-do-Mar/genética , Proteínas Wnt/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ouriços-do-Mar/classificação , Ouriços-do-Mar/embriologia , Homologia de Sequência de AminoácidosRESUMO
The Receptor Tyrosine kinase (RTK) and TGF-beta signaling pathways play essential roles during development in many organisms and regulate a plethora of cellular responses. From the genome sequence of Strongylocentrotus purpuratus, we have made an inventory of the genes encoding receptor tyrosine kinases and their ligands, and of the genes encoding cytokines of the TGF-beta superfamily and their downstream components. The sea urchin genome contains at least 20 genes coding for canonical receptor tyrosine kinases. Seventeen of the nineteen vertebrate RTK families are represented in the sea urchin. Fourteen of these RTK among which ALK, CCK4/PTK7, DDR, EGFR, EPH, LMR, MET/RON, MUSK, RET, ROR, ROS, RYK, TIE and TRK are present as single copy genes while pairs of related genes are present for VEGFR, FGFR and INSR. Similarly, nearly all the subfamilies of TGF-beta ligands identified in vertebrates are present in the sea urchin genome including the BMP, ADMP, GDF, Activin, Myostatin, Nodal and Lefty, as well as the TGF-beta sensu stricto that had not been characterized in invertebrates so far. Expression analysis indicates that the early expression of nodal, BMP2/4 and lefty is restricted to the oral ectoderm reflecting their role in providing positional information along the oral-aboral axis of the embryo. The coincidence between the emergence of TGF-beta-related factors such as Nodal and Lefty and the emergence of the deuterostome lineage strongly suggests that the ancestral function of Nodal could have been related to the secondary opening of the mouth which characterizes this clade, a hypothesis supported by functional data in the extant species. The sea urchin genome contains 6 genes encoding TGF-beta receptors and 4 genes encoding prototypical Smad proteins. Furthermore, most of the transcriptional activators and repressors shown to interact with Smads in vertebrates have orthologues in echinoderms. Finally, the sea urchin genome contains an almost complete repertoire of genes encoding extracellular modulators of BMP signaling including Chordin, Noggin, Sclerotin, SFRP, Gremlin, DAN and Twisted gastrulation. Taken together, these findings indicate that the sea urchin complement of genes of the RTK and TGF-beta signaling pathways is qualitatively very similar to the repertoire present in vertebrates, and that these genes are part of the common genetool kit for intercellular signaling of deuterostomes.