Spatio-temporal filtering with morphological operators for robust cell migration estimation in "in-vivo" images

The understanding of the embryogenesis in living systems requires reliable quantitative analysis of the cell migration throughout all the stages of development. This is a major challenge of the "in-toto" reconstruction based on different modalities of "in-vivo" imaging techniques -spatio-temporal resolution and image artifacts and noise. Several methods for cell tracking are available, but expensive manual interaction -time and human resources- is always required to enforce coherence. Because of this limitation it is necessary to restrict the experiments or assume an uncontrolled error rate. Is it possible to obtain automated reliable measurements of migration? can we provide a seed for biologists to complete cell lineages efficiently? We propose a filtering technique that considers trajectories as spatio-temporal connected structures that prunes out those that might introduce noise and false positives by using multi-dimensional morphological operators

An Automatic Quantification and Registration Strategy to Create a Gene Expression Atlas of Zebrafish Embryogenesis

In order to properly understand and model the gene regulatory networks in animals development, it is crucial to obtain detailed measurements, both in time and space, about their gene expression domains. In this paper, we propose a complete computational framework to fulfill this task and create a 3D Atlas of the early zebrafish embryogenesis annotated with both the cellular localizations and the level of expression of different genes at different developmental stages. The strategy to construct such an Atlas is described here with the expression pattern of 5 different genes at 6 hours of development post fertilization

3D + t Morphological Processing: Applications to Embryogenesis Image Analysis

We propose to directly process 3D + t image sequences with mathematical morphology operators, using a new classi?cation of the 3D+t structuring elements. Several methods (?ltering, tracking, segmentation) dedicated to the analysis of 3D + t datasets of zebra?sh embryogenesis are introduced and validated through a synthetic dataset. Then, we illustrate the application of these methods to the analysis of datasets of zebra?sh early development acquired with various microscopy techniques. This processing paradigm produces spatio-temporal coherent results as it bene?ts from the intrinsic redundancy of the temporal dimension, and minimizes the needs for human intervention in semi-automatic algorithms

Rôles des voies de signalisation Wnt et VEGF au cours de l'embryogenèse de l'oursin

Au cours de ma thèse, nous nous sommes penchés sur deux évènements particulièrement important du développement de l embryon d oursin : la formation du tube digestif et la morphogenèse du squelette larvaire. Nous avons pu mettre en évidence le rôle crucial de deux voies de signalisation dans ces processus : celui de la voie Frizzled/wnt dans l invagination du tube digestif, et celui de la voie VEGF dans la migration et la différenciation des cellules du mésenchyme primaire. La fonction maternelle de la voie Wnt/beta-caténine dans la mise en place de l axe antéropostérieur était bien connue et nous avons pu décrire une fonction zygotique de la voie Wnt, fondamentale pour la formation du tube digestif. Nous avons montré que l action de Wnt dans ce processus passait par l activation, via un récepteur frizzled, de la voie PCP (planar cell polarity) (Croce et al., 2006a). Vascular Endothelial Growth Factor (VEGF) est le facteur clef de la formation des vaisseaux sanguins chez les vertébrés. Il est conservé chez l oursin et exprimé de façon localisée dans deux régions de l ectoderme. Les transcrits codant pour son récepteur à activité tyrosine kinase sont quant à eux restreints à une sous population de cellules mésodermiques, les cellules du mésenchyme primaire (PMCs). Ces PMCs sont les cellules qui forment le squelette de la larve d oursin. L influence de l ectoderme sur ces cellules est connue pour être nécessaire à la correcte morphogenèse du squelette mais les bases moléculaires de l interaction entre ces deux tissus étaient inconnues. Nous avons démontré par l étude fonctionnelle du ligand ectodermique VEGF et de son récepteur mésodermique VEGFR, que la source localisée de VEGF consiste en un repaire spatial nécessaire aux PMCS pour leur correcte migration à l intérieur du blastocoele et leur organisation spatiale. L activation de cette voie VEGF/VEGFR est également un facteur clef de la différenciation des PMCs (Duloquin et al., en préparation). Le laboratoire a également participé à l annotation du génome de l oursin Strongylocentrotus purpuratus, et je suis plus particulièrement attachée à caractériser les gènes de la voie Wnt (Croce et al., 2006b) et les récepteurs à activité tyrosine kinase (Lapraz et al., 2006).NICE-BU Sciences (060882101) / SudocSudocFranceF

Frizzled5/8 is required in secondary mesenchyme cells to initiate archenteron invagination during sea urchin development

International audienceWnt 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

The Drosophila RNA-binding protein ELAV is required for commissural axon midline crossing via control of commissureless mRNA expression in neurons.

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

Combined third-harmonic generation and four-wave mixing microscopy of tissues and embryos.

International audienceNonlinear 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

Combining sea urchin embryo cell lineages by error-tolerant graph matching.

International audienceObtaining the complete cell lineage tree of an embryo's development is a very appealing and ambitious goal, but fortunately recent developments both in optical imaging and digital image processing are bringing it closer. However, when imaging the embryos (sea urchin embryos for this work) with high enough spatial resolution and short enough time-step to make cell segmentation and tracking possible, it is currently not possible to image the specimen throughout its all embryogenesis. For this reason it is interesting to explore how cell lineage trees extracted from two different embryos of the same species and imaged for overlapping periods of time can be concatenated, resulting in a single lineage tree covering both embryos' development time frames. To achieve this we used an error-tolerant graph matching strategy by selecting a time point at which both lineage trees overlap, and representing the information about each embryo at that time point as a graph in which nodes stand for cells and edges for neighborhood relationships among cells. The expected output of the graph matching algorithm is the minimal-cost correspondence between cells of both specimens, allowing us to perform the lineage combination

Can Voronoi diagram model cell geometries in early sea-urchin embryogenesis ?

5th IEEE International Symposium on Biomedical Imaging, Paris, FRANCE, MAY 14-17, 2008International audienceWe test the hypothesis that cell membranes in early sea- urchin embryos can be modeled as a Voronoi diagram from nuclei centers. In order to obtain a model of the cell geometry against which to test our Voronoi model hypothesis, we developed a viscous watershed framework that allows segmenting 3D images of living sea-urchin embryos obtained by biphoton laser scanning microscopy. Measurements of the differences between segmented cells and the Voronoi model, show an interesting high correlation that can serve for developing more accurate methods of segmentation and modelling cell geometries

Cell tracking in fluorescence images of embryogenesis processes with morphological reconstruction by 4D-tubular structuring elements.

International audienceWe present a simple and parameter-free nuclei tracking method for reconstructing cell dynamics in fluorescence 3D+t images of embryogenesis. The strategy is based on the use of the mathematical morphology operators directly in the 4D image. The morphological reconstruction of a marker -manually or automatically selected- in an initial spatio-temporal position generates a connected path over the time representing the cell migration. Thus, the processing provides a coherent spatiotemporal estimation of cell movement. The algorithm has been validated on in vivo images of early zebrafish and sea urchin embryogenesis acquired with two-photon laser scanning microscopy providing mean tracking rates above 98% per time step