Publications

2016

An integrated modelling framework from cells to organism based on a cohort of digital embryos

Scientific Reports 6 2016 37438

  • Paul Villoutreix
  • Julien Delile
  • Barbara Rizzi
  • Louise Duloquin
  • Thierry Savy
  • Paul Bourgine
  • René Doursat
  • Nadine Peyriéras

We conducted a quantitative comparison of developing sea urchin embryos based on the analysis of five digital specimens resulting from the automated 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 organization 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 characterizing 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 modeling and simulation, which allowed a systematic exploration and optimization 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.

An algorithmic workflow for the automated processing of 3D+time microscopy imaging of developing organisms and reconstruction of their cell lineage

Nature Communications 7 2016 8674

  • Emmanuel Faure
  • Thierry Savy
  • Barbara Rizzi
  • Camilo Melani
  • Olga Drblíková
  • Dimitri Fabrèges
  • Róbert Špir
  • Mark Hammons
  • Róbert Čunderlík
  • Gaëlle Recher
  • Benoît Lombardot
  • Louise Duloquin
  • Ingrid Colin
  • Jozef Kollár
  • Sophie Desnoulez
  • Pierre Affaticati
  • Benoît Maury
  • Adeline Boyreau
  • Jean-Yves Nief
  • Pascal Calvat
  • Philippe Vernier
  • Monique Frain
  • Georges Lutfalla
  • Yannick Kergosien
  • Pierre Suret
  • Mariana Remešíková
  • René Doursat
  • Alessandro Sarti
  • Karol Mikula
  • Nadine Peyriéras
  • Paul Bourgine

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.

2015

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

Journal of Visualized Experiments (JoVE) 95 2015 70-81

  • Estelle Hirsinger
  • João Emanuel Carvalho
  • Christine Chevalier
  • Georges Lutfalla
  • Jean-François Nicolas
  • Nadine Peyriéras
  • Michael Schubert

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Biocompatible photoresistant far-red emitting, fluorescent polymer probes, with near-infrared two-photon absorption, for living cell and zebrafish embryo imaging

Biomaterials 46 2015 70-81

  • Salim Adjili
  • Arnaud Favier
  • Guillaume Fargier
  • Audrey Thomas
  • Julien Massin
  • Karine Monier
  • Cyril Favard
  • Christophe Vanbelle
  • Sylvia Bruneau
  • Nadine Peyriéras
  • Chantal Andraud
  • Delphine Muriaux
  • Marie-Thérèse Charreyrea

Exogenous probes with far-red or near-infrared (NIR) two-photon absorption and fluorescence emission are highly desirable for deep tissue imaging while limiting autofluorescence. However, molecular probes exhibiting such properties are often hydrophobic. As an attractive alternative, we synthesized water-soluble polymer probes carrying multiple far-red fluorophores and demonstrated here their potential for live cell and zebrafish embryo imaging. First, at concentrations up to 10 μm, these polymer probes were not cytotoxic. They could efficiently label living HeLa cells, T lymphocytes and neurons at an optimal concentration of 0.5 μm. Moreover, they exhibited a high resistance to photobleaching in usual microscopy conditions. In addition, these polymer probes could be successfully used for in toto labeling and in vivo two-photon microscopy imaging of developing zebrafish embryos, with remarkable properties in terms of biocompatibility, internalization, diffusion, stability and wavelength emission range. The near-infrared two-photon absorption peak at 910 nm is particularly interesting since it does not excite the zebrafish endogenous fluorescence and is likely to enable long-term time-lapse imaging with limited photodamage.

2014

De nouveaux senseurs bioluminescents pour l'observation de l'activité cérébrale in toto chez la souris

New bioluminescent sensors for murine brain activity imaging in toto

Thesis in Neuroscience Cerveau, cognition et comportement (Paris 6) Supervised by Nadine Peyriéras 2014

  • Sandrine Picaud

English version : Monitoring calcium fluxes in the brain of freely moving animals requires the development ofnew probes. We use the bioluminescence of the protein aequorin observed in the jellyfishAequorea victoria. A fusion between aequorin and GFP fluorescent protein (GA) provides abioluminescent calcium sensor whose stability and spectral properties are adequate for manyapplications. The emission of bioluminescence is suitable to track in real-time the propagationof nervous impulses from cell to cell in neural networks.We first showed by transgenic murine lines construction that the chimeric protein GA can beexpressed in cell microdomains to analyze neuronal activity. Our results show for the firsttime it is possible to record in vivo and non-invasively in the moving animal, changes in theconcentration of mitochondrial calcium. We then performed a post synaptic targeting of thesensor by fusion with the protein PSD95. This permits to detect the entry of calcium next tothe NMDA receptor.Meanwhile, we tried to improve the properties of the GA sensor to detect calcium activity inthe animal in toto and mor specifically through the skull. We identified a new analogue ofcoelenterazine leading to a shift in the spectrum of aequorin bioluminescence to red. We thentested the activity of aequorin fused with different fluorescent proteins. This leads to newbioluminescent sensors for monitoring the brain activity in behavioral studies.

French version : Le suivi des flux calciques dans le cerveau de l’animal en mouvement nécessite de nouvellessondes. Nous utilisons la bioluminescence observée chez la méduse Aequorea victoria etimpliquant la protéine aequorine. Une fusion entre l’aequorine et la protéine fluorescente GFP(GA) permet d’obtenir un senseur calcique bioluminescent dont la stabilité et les propriétésspectrales sont adéquates pour de nombreuses applications. L’émission de bioluminescencepermet de suivre la propagation de l’information nerveuse de cellule en cellule, en temps réeldans des réseaux de neurones.Nous avons tout d’abord montré au moyen de construction de lignées transgéniques murinesque la protéine chimère GA peut être exprimée dans des microdomaines cellulaires pouranalyser l’activité neuronale. Nos résultats montrent pour la première fois qu’il est possibled’enregistrer in vivo et de manière non invasive dans l’animal en mouvement deschangements dans la concentration de calcium mitochondrial. Nous avons ensuite réalisé unciblage post synaptique du senseur par fusion à la protéine PSD95. Ce ciblage permet dedétecter l'entrée de calcium au niveau du récepteur NMDA.Parallèlement, nous avons cherché à améliorer les propriétés du senseur GA pour détécterl’activité calcique dans l’animal in toto. Nous identifié un nouvel analogue de lacoelenterazine conduisant à un décalage du spectre de bioluminescence de l’aequorine vers lerouge. Et par ailleurs, nous avons testé l’activité de l’aequorine en fusion avec diversesprotéines fluorescentes. Nous disposons avec ce travail de nouveaux senseurs bioluminescentspour le suivi de l’activité cérébrales dans des études comportementales.

Vers une modélisation multi-échelle de la variabilité biologique?

Toward a multi-scale modeling of the biological variability?

Modéliser & Simuler. Epistémologies et pratiques de la modélisation et de la simulation. Tome 2 Chap 19 (p643-664) Editions matériologiques 2014

  • Paul Villoutreix

Modélisation multi-agent de l’embryogenèse animale

Multi-agent modeling of animal embryogenesis

Modéliser & Simuler. Epistémologies et pratiques de la modélisation et de la simulation. Tome 2 Chap 17 (p581-624) Editions matériologiques 2014

  • Julien Delile
  • René Doursat
  • Nadine Peyriéras

Phenotypic Variation and Resilience in Sea Urchin Morphogenesis

Sea Urchins: Habitat, Embryonic Development and Importance in the Environment Edgar Raymond Banks 2014

  • Dimitri Fabrèges

Sea urchins have long been used as model organisms for investigations in embryology. Thanks to their availability, accessibility and transparency, sea urchin eggs and embryos helped deciphering processes underlying fertilization, cell division and other generic morphogenetic events such as epithelium to mesenchyme transition or cell apical constriction in gastrulation. This is also in sea urchin models that the paradigm of gene regulatory network (GRN) architecture and dynamics underlying early morphogenesis was developed and the most systematically explored. In addition, sea urchin embryos display extensive regulative capacities as shown by their response to experimental manipulations. Hans Driesch showed that the separation of 2-cell stage blastomeres leads to perfect twins. Theodore Boveri observed the development of triploid embryos and Giovanni Giudice the reaggregation and further development of fully dissociated early embryos. These seminal studies opened the way to decipher processes underlying variation, robustness and resilience. Current approaches in developmental biology in the context of complex system science rely on the in vivo multiscale observation of biological processes to achieve their multilevel reconstruction. In this context, sea urchin model organisms will serve an emerging integrative biology and will help achieving the ultimate synthesis of genetics, embryology and evolution.

Computational Modeling and Simulation of Animal Early Embryogenesis with the MecaGen Platform

Computational Systems Biology Second Edition 548 pages Academic Press 2014

  • Julien Delile
  • René Doursat
  • Nadine Peyriéras

We propose a theoretical, yet realistic agent-based model and simulation platform of animal embryogenesis, called MecaGen,1 centered on the physico-chemical coupling of cell mechanics with gene expression and molecular signaling. This project aims to investigate the multiscale dynamics of the early stages of biological morphogenesis. Here, embryonic development is viewed as an emergent, self-organized phenomenon based on a myriad of cells and their genetically regulated, and regulating, biomechanical behavior. Cells’ mechani- cal properties (such as division rate, adhesion strength, or intrinsic motility) are closely correlated with their spatial location and temporal state of genetic and molecular dynamics (such as internal protein and external ligand concentrations) and affect each other concurrently. In a second part, we illustrate our model on artificial data (gene regulation motifs and cell sorting), then demonstrate a customization and application to a real bio- logical case study in the zebrafish early development. We use as an example the episode of intercalation pat- terns appearing during the first phase of epiboly and the movements of the deep cells between the yolk and the enveloping layer. A domain of the model’s multidimensional parameter space is explored systematically, while experimental data obtained from microscopy imaging of live embryos is used to measure the “fitness” of the virtual embryo and validate our hypotheses.

Numerical algorithm for tracking cell dynamics in 4D biomedical images

Discrete and Continuous Dynamical Systems Series S 2014 1-15

  • Karol Mikula
  • Nadine Peyriéras
  • Róbert Špir

The paper presents numerical algorithms leading to an automated cell tracking and reconstruction of the cell lineage tree during the first hours of animal embryogenesis. We present results obtained for large-scale 3D+time two-photon laser scanning microscopy images of early stages of zebrafish (Danio rerio) embryo development. Our approach consists of three basic steps – the image filtering, the cell centers detection and the cell trajectories extraction yielding the lineage tree reconstruction. In all three steps we use nonlinear partial differential equations. For the filtering the geodesic mean curvature flow in level set formulation is used, for the cell center detection the motion of level sets by a constant speed regularized by mean curvature flow is used and the solution of the eikonal equation is essential for the cell trajectories extraction. The core of our new tracking method is an original approach to cell trajectories extraction based on finding a continuous centered paths inside the spatio-temporal tree structures representing cell movement and divisions. Such paths are found by using a suitably designed distance function from cell centers detected in all time steps of the 3D+time image sequence and by a backtracking in the steepest descent direction of a potential field based on this distance function. We also present efficient and naturally parallelizable discretizations of the aforementioned nonlinear PDEs and discuss properties and results of our new tracking method on artificial and real 4D data.

Nonlinear PDE based numerical methods for cell tracking in zebrafish embryogenesis

Applied Numerical Mathematics 2014

  • Karol Mikula
  • Róbert Špir
  • Michal Smíšek
  • Emmanuel Faure
  • Nadine Peyriéras

The paper presents numerical algorithms leading to an automated cell tracking and reconstruction of the cell lineage tree during the first hours of animal embryogenesis. We present results obtained for large-scale 3D+time two-photon laser scanning microscopy images of early stages of zebrafish (Danio rerio) embryo development. Our approach consists of three basic steps – the image filtering, the cell centers detection and the cell trajectories extraction yielding the lineage tree reconstruction. In all three steps we use nonlinear partial differential equations. For the filtering the geodesic mean curvature flow in level set formulation is used, for the cell center detection the motion of level sets by a constant speed regularized by mean curvature flow is used and the solution of the eikonal equation is essential for the cell trajectories extraction. The core of our new tracking method is an original approach to cell trajectories extraction based on finding a continuous centered paths inside the spatio-temporal tree structures representing cell movement and divisions. Such paths are found by using a suitably designed distance function from cell centers detected in all time steps of the 3D+time image sequence and by a backtracking in the steepest descent direction of a potential field based on this distance function. We also present efficient and naturally parallelizable discretizations of the aforementioned nonlinear PDEs and discuss properties and results of our new tracking method on artificial and real 4D data.

A Digital Framework to Build, Visualize and Analyze a Gene Expression Atlas with Cellular Resolution in Zebrafish Early Embryogenesis

PLoS Computational Biology Vol. 10 No. 6 2014 e1003670

  • Carlos Castro-González
  • Miguel A. Luengo-Oroz
  • Louise Duloquin
  • Thierry Savy
  • Barbara Rizzi
  • Sophie Desnoulez
  • René Doursat
  • Yannick L. Kergosien
  • María J. Ledesma-Carbayo
  • Paul Bourgine
  • Nadine Peyriéras
  • Andrés Santos

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.

The Bcl-2 Homolog Nrz Inhibits Binding of IP3 to Its Receptor to Control Calcium Signaling During Zebrafish Epiboly

Science Signaling Vol. 7 No. 312 2014 ra14

  • Benjamin Bonneau
  • Adrien Nougarède
  • Julien Prudent
  • Nikolay Popgeorgiev
  • Nadine Peyriéras
  • Ruth Rimokh
  • Germain Gillet

Members of the Bcl-2 protein family regulate mitochondrial membrane permeability and also localize to the endoplasmic reticulum where they control Ca2+ homeostasis by interacting with inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs). In zebrafish, Bcl-2–like 10 (Nrz) is required for Ca2+ signaling during epiboly and gastrulation. We characterized the mechanism by which Nrz controls IP3-mediated Ca2+ release during this process. We showed that Nrz was phosphorylated during early epiboly, and that in embryos in which Nrz was knocked down, reconstitution with Nrz bearing mutations designed to prevent its phosphorylation disrupted cyclic Ca2+ transients and the assembly of the actin-myosin ring and led to epiboly arrest. In cultured cells, wild-type Nrz, but not Nrz with phosphomimetic mutations, interacted with the IP3 binding domain of IP3R1, inhibited binding of IP3 to IP3R1, and prevented histamine-induced increases in cytosolic Ca2+. Collectively, these data suggest that Nrz phosphorylation is necessary for the generation of IP3-mediated Ca2+ transients and the formation of circumferential actin-myosin cables required for epiboly. Thus, in addition to their role in apoptosis, by tightly regulating Ca2+ signaling, Bcl-2 family members participate in the cellular events associated with early vertebrate development, including cytoskeletal dynamics and cell movement.

From cell behavior to tissue deformation: Mechanogenetic modeling and computational simulation of animal early embryogenesis

Computational Systems Biology Second Edition 548 pages Academic Press 2013

  • Julien Delile
  • Nadine Peyriéras
  • René Doursat

Systems Biology is concerned with the quantitative study of complex biosystems at the molecular, cellular, tissue, and systems scales. Its focus is on the function of the system as a whole, rather than on individual parts.

This exciting new arena applies mathematical modeling and engineering methods to the study of biological systems. This book is the first of its kind to focus on the newly emerging field of systems biology with an emphasis on computational approaches. The work covers new concepts, methods for information storage, mining and knowledge extraction, reverse engineering of gene and metabolic networks, as well as modelling and simulation of multi-cellular systems. Central themes include strategies for predicting biological properties and methods for elucidating structure-function relationships.

2013

Des comportements cellulaires à la morphogenèse embryonnaire : modélisation mécano-génétique et simulations computationnelles du développement animal précoce

From cell behavior to embryonic morphogenesis : mechanogenetic modeling and computational simulation of early animal development

Frontière du vivant (Paris 7) Supervised by Nadine Peyriéras & René Doursat 2013

  • Julien Delile

English version : We present a theoretical model of animal morphogenesis construed as a self-organized phenomenon emerging from a complex system made of a myriad of individual cell behaviors. It is implemented in an agent-based simulation centered on the mechanic-chemical coupling between cellular and genetic dynamics. The goal is to integrate the collective motion of cells and the dynamics of their gene expression underlying the patterning of morphogenetic fields. We also investigate the causal bottom-up link from local cell behavior to global tissue deformation. Each cell's mechanical behavior is mapped from its molecular and genetic identity. Among these behaviors, we focus particularly on cell intercalation as an active process driving tissue deformation. We operate this model to explore the different morphogenetic episode occuring through the first 10 hours of the zebrafish development : cell segmentation, enveloping layer formation, epiboly, internalization and convergence-extension. For each specific episode, a case study is realized to decipher the respective role of the different tissue involved. Quantitative measures reconstructed from both the simulated and the experimental data are compared to automatically explore the multi-dimensional parameter spaces of our hypotheses and their interpretation. Various state of the art computational reconstruction will be presented, including global 4D (3D + time) displacement fields from in toto data of the developing zebrafish embryos. A waddingtonian interactive timeline tool to specifiy intra and inter tissue induction and mechanical behaviors is also proposed.

French version : Ce travail présente un modèle théorique de morphogenèse animale, sous la forme d'un système complexe émergeant de nombreux comportements cellulaires. Son implémentation repose sur un système multi-agents fondé sur le couplage mécano-génétique entre les dynamiques de la mécanique cellulaire et de la régulation génétique et moléculaire. Notre objectif est l'intégration des mouvements collectifs de myriades de cellules avec les dynamiques d'expression génétique sous-jacentes aux motifs des champs morphogénétiques. Nous examinons aussi les relations de causalité ascendante reliant les comportements cellulaires locaux aux déformations tissulaires globales. Le comportement mécanique de chaque cellule est associé à leur constitution moléculaire et génétique. Nous nous intéressons particulièrement au phénomène d'intercalation cellulaire induit par des comportements de protrusion active. Différents épisodes morphogénétiques se déroulant au cours des 10 premières heures de développement du poisson zébré sont explorés : la période de clivage, la formation de la couche épithéliale externe l'épibolie, l'internalisation du mésendoderme et la convergence-extension de l'axe antéro-postérieur. Pour chacun de ces phénomènes, une étude de cas examine le rôle respectif des différents tissus impliqués. Les hypothèses que nous proposons sont discutées au moyen de comparaisons automatisées entre les mesures reconstruites issues des données d'imagerie microscopique et des simulations computationnelles. Les reconstructions présentées incluent les champs de déplacements cellulaires in toto en 4 dimensions (3D + temps) de l'embryon de poisson zébré. Le modèle est paramétré à travers une interface inspirée du concept de paysage épigénétique de Waddington, permettant de spécifier les phénomènes d'induction et d'interaction mécanique à l'échelle des champs morphogénétiques.

Inhibitory signalling to the Arp2/3 complex steers cell migration

Nature Vol. 14 No. 503 2013 281-4

  • Irene Dang
  • Roman Gorelik
  • Carla Sousa-Blin
  • Emmanuel Derivery
  • Christophe Guérin
  • Joern Linkner
  • Maria Nemethova
  • Julien G. Dumortier
  • Florence A. Giger
  • Tamara A. Chipysheva
  • Valeria D. Ermilova
  • Sophie Vacher
  • Valérie Campanacci
  • Isaline Herrada
  • Anne-Gaelle Planson
  • Susan Fetics
  • Véronique Henriot
  • Violaine David
  • Ksenia Oguievetskaia
  • Goran Lakisic
  • Fabienne Pierre
  • Anika Steffen
  • Adeline Boyreau
  • Nadine Peyriéras
  • Klemens Rottner
  • Sophie Zinn-Justin
  • Jacqueline Cherfils
  • Ivan Bièche
  • Antonina Y. Alexandrova
  • Nicolas B. David
  • J. Victor Small
  • Jan Faix
  • Laurent Blanchoin
  • Alexis Gautreau

Cell migration requires the generation of branched actin networks that power the protrusion of the plasma membrane in lamellipodia. The actin-related proteins 2 and 3 (Arp2/3) complex is the molecular machine that nucleates these branched actin networks. This machine is activated at the leading edge of migrating cells by Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein (WAVE, also known as SCAR). The WAVE complex is itself directly activated by the small GTPase Rac, which induces lamellipodia. However, how cells regulate the directionality of migration is poorly understood. Here we identify a new protein, Arpin, that inhibits the Arp2/3 complex in vitro, and show that Rac signalling recruits and activates Arpin at the lamellipodial tip, like WAVE. Consistently, after depletion of the inhibitory Arpin, lamellipodia protrude faster and cells migrate faster. A major role of this inhibitory circuit, however, is to control directional persistence of migration. Indeed, Arpin depletion in both mammalian cells and Dictyostelium discoideum amoeba resulted in straighter trajectories, whereas Arpin microinjection in fish keratocytes, one of the most persistent systems of cell migration, induced these cells to turn. The coexistence of the Rac-Arpin-Arp2/3 inhibitory circuit with the Rac-WAVE-Arp2/3 activatory circuit can account for this conserved role of Arpin in steering cell migration.

A necklace algorithm to determine the growth function of trinucleotide circular codes

Journal of Applied Mathematics & Bioinformatics Vol. 3 No. 3 2013 1-40

  • Matthieu Herrmann
  • Christian J. Michel
  • Benoît Zugmeyer

Circular codes are mathematical objects studied in combinatorics - theoretical computer science, and theoretical biology. So far, there is no close formulas allowing to determine the growth function (number and list) of circular codes. This combinatorial problem can only be solved by an algorithmic approach. We propose a new algorithm based on a necklace proposition to determine the growth function of trinucleotide circular codes, a trinucleotide being a word of 3 letters on a 4-letter alphabet. This necklace algorithm, unique in its class, can be extended in future to the analysis of codes, e.g. circular codes, containing words greater than 3 letters and also over larger alphabets.

Zebrafish midbrain slow-amplifying progenitors exhibit high levels of transcripts for nucleotide and ribosome biogenesis

Development Vol. 140 No. 24 2013 4860-9

  • Gaëlle Recher
  • Julia Jouralet
  • Alessandro Brombin
  • Aurélie Heuzé
  • Emilie Mugniery
  • Jean-Michel Hermel
  • Sophie Desnoulez
  • Thierry Savy
  • Philippe Herbomel
  • Franck Bourrat
  • Nadine Peyriéras
  • Françoise Jamen
  • Jean-Stéphane Joly

Investigating neural stem cell (NSC) behaviour in vivo, which is a major area of research, requires NSC models to be developed. We carried out a multilevel characterisation of the zebrafish embryo peripheral midbrain layer (PML) and identified a unique vertebrate progenitor population. Located dorsally in the transparent embryo midbrain, these large slow-amplifying progenitors (SAPs) are accessible for long-term in vivo imaging. They form a neuroepithelial layer adjacent to the optic tectum, which has transitory fast-amplifying progenitors (FAPs) at its margin. The presence of these SAPs and FAPs in separate domains provided the opportunity to data mine the ZFIN expression pattern database for SAP markers, which are co-expressed in the retina. Most of them are involved in nucleotide synthesis, or encode nucleolar and ribosomal proteins. A mutant for the cad gene, which is strongly expressed in the PML, reveals severe midbrain defects with massive apoptosis and sustained proliferation. We discuss how fish midbrain and retina progenitors might derive from ancient sister cell types and have specific features that are not shared with other SAPs.

Towards 3D in silico modeling of the sea urchin embryonic development

Journal of Chemical Biology Vol. 7 No. 1 2013 17-28

  • Barbara Rizzi
  • Nadine Peyriéras

Embryogenesis is a dynamic process with an intrinsic variability whose understanding requires the integration of molecular, genetic, and cellular dynamics. Biological circuits function over time at the level of single cells and require a precise analysis of the topology, temporality, and probability of events. Integrative developmental biology is currently looking for the appropriate strategies to capture the intrinsic properties of biological systems. The “–omic” approaches require disruption of the function of the biological circuit; they provide static information, with low temporal resolution and usually with population averaging that masks fast or variable features at the cellular scale and in a single individual. This data should be correlated with cell behavior as cells are the integrators of biological activity. Cellular dynamics are captured by the in vivo microscopy observation of live organisms. This can be used to reconstruct the 3D + time cell lineage tree to serve as the basis for modeling the organism's multiscale dynamics. We discuss here the progress that has been made in this direction, starting with the reconstruction over time of three-dimensional digital embryos from in toto time-lapse imaging. Digital specimens provide the means for a quantitative description of the development of model organisms that can be stored, shared, and compared. They open the way to in silico experimentation and to a more theoretical approach to biological processes. We show, with some unpublished results, how the proposed methodology can be applied to sea urchin species that have been model organisms in the field of classical embryology and modern developmental biology for over a century.

Automated Processing of Zebrafish Imaging Data: A Survey

Zebrafish Vol. 10 No. 3 2013 401-421

  • Ralf Mikut
  • Thomas Dickmeis
  • Wolfgang Driever
  • Pierre Geurts
  • Fred A. Hamprecht
  • Bernhard X. Kausler
  • María J. Ledesma-Carbayo
  • Raphaël Marée
  • Karol Mikula
  • Periklis Pantazis
  • Olaf Ronneberger
  • Andrés Santos
  • Rainer Stotzka
  • Uwe Strähle
  • Nadine Peyriéras

Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines.

Recording and statistical analysis of early zebrafish developmental patterns using in vivo multiphoton microscopy

The FASEB Journal (Joint Annual Meeting of the ASPET/BPS at Experimental Biology) Vol. 27 No. 3 2013 312.2

  • Nadine Peyriéras

We take the cell lineage tree of living organisms as the basis for the reconstruction of their spatio-temporal multiscale dynamics. Indeed, embryonic development leading from the egg cell to the whole organism can be fully described by the spatio temporal deployment of the cell lineage tree annotated with quantitative parameters for cell membrane and nucleus shape. This data can be automatically extracted from in toto 3D+time imaging of the developing organism and allows answering most of the questions of classical embryology such as cell fate, presumptive organs clonal history, cell proliferation rates, contribution of cell division and its characteristic features to shaping tissues and organs. These questions can be answered by using the digital specimen corresponding to the validated phenomenological reconstruction of 3D+time image data sets. Exploring cohorts of individuals with different genetic and environmental conditions allows to integrate the cellular and molecular levels of organisation and to assess the basis for the robustness and variability of embryonic development. The phenomenological reconstruction of multimodal and multiscale 3D+time data is a first step toward the theoretical modelling and numerical simulation of morphogenetic processes. Virtual specimens derived from simulations are compared with digital ones reconstructed from the microscopy data to explore parameter spaces. This paradigm is used for investigating the early embryonic development of the teleostean Danio rerio.

Dissection of a Krox20 positive feedback loop driving cell fate choices in hindbrain patterning

Molecular Systems Biology Vol. 9 No. 1 2013 690

  • Yassine X. Bouchoucha
  • Jürgen Reingruber
  • Charlotte Labalette
  • Michel A. Wassef
  • Elodie Thierion
  • Carole Desmarquet-Trin Dinh
  • David Holcman
  • Pascale Gilardi-Hebenstreit
  • Patrick Charnay

Although feedback loops are essential in development, their molecular implementation and precise functions remain elusive. Using enhancer knockout in mice, we demonstrate that a direct, positive autoregulatory loop amplifies and maintains the expression of Krox20, a transcription factor governing vertebrate hindbrain segmentation. By combining quantitative data collected in the zebrafish with biophysical modelling that accounts for the intrinsic stochastic molecular dynamics, we dissect the loop at the molecular level. We find that it underpins a bistable switch that turns a transient input signal into cell fate commitment, as we observe in single cell analyses. The stochasticity of the activation process leads to a graded input–output response until saturation is reached. Consequently, the duration and strength of the input signal controls the size of the hindbrain segments by modulating the distribution between the two cell fates. Moreover, segment formation is buffered from severe variations in input level. Finally, the progressive extinction of Krox20 expression involves a destabilization of the loop by repressor molecules. These mechanisms are of general significance for cell type specification and tissue patterning.

2012

Wavelet-based image fusion in multi-view three-dimensional microscopy

Bioinformatics Vol. 28 No. 2 2012 238-245

  • Jose Luis Rubio-Guivernau
  • Vasily Gurchenkov
  • Miguel A. Luengo-Oroz
  • Louise Duloquin
  • Paul Bourgine
  • Andrés Santos
  • Nadine Peyriéras
  • María J. Ledesma-Carbayo

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.

Assembling models of embryo development: Image analysis and the construction of digital atlases.

Revised on September 2012, Birth Defects Res, 96(3):271

Birth Defects Research Part C: Embryo Today: Reviews Vol. 96 No. 2 2012 109-120

  • Carlos Castro-González
  • María J. Ledesma-Carbayo
  • Nadine Peyriéras
  • Andrés Santos

Digital atlases of animal development provide a quantitative description of morphogenesis, opening the path toward processes modeling. Prototypic atlases offer a data integration framework where to gather information from cohorts of individuals with phenotypic variability. Relevant information for further theoretical reconstruction includes measurements in time and space for cell behaviors and gene expression. The latter as well as data integration in a prototypic model, rely on image processing strategies. Developing the tools to integrate and analyze biological multidimensional data are highly relevant for assessing chemical toxicity or performing drugs preclinical testing. This article surveys some of the most prominent efforts to assemble these prototypes, categorizes them according to salient criteria and discusses the key questions in the field and the future challenges toward the reconstruction of multiscale dynamics in model organisms.

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

IEEE Transactions on Image Processing Vol. 21 No. 8 2012 3518-3530

  • Miguel A. Luengo-Oroz
  • David Pastor-Escuredo
  • Carlos Castro-González
  • Emmanuel Faure
  • Thierry Savy
  • Benoît Lombardot
  • Jose Luis Rubio-Guivernau
  • Louise Duloquin
  • María J. Ledesma-Carbayo
  • Paul Bourgine
  • Nadine Peyriéras
  • Andrés Santos

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.

Methodology for Reconstructing Early Zebrafish Development from In Vivo Multiphoton Microscopy

IEEE Transactions on Image Processing Vol. 21 No. 4 2012 2335-2340

  • Miguel A. Luengo-Oroz
  • Jose Luis Rubio-Guivernau
  • Emmanuel Faure
  • Thierry Savy
  • Louise Duloquin
  • Nicolas Olivier
  • David Pastor
  • María J. Ledesma-Carbayo
  • Delphine Débarre
  • Paul Bourgine
  • Emmanuel Beaurepaire
  • Nadine Peyriéras
  • Andrés Santos

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.

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

9th IEEE International Symposium on Biomedical Imaging 2012 1312-1315

  • David Pastor-Escuredo
  • Miguel A. Luengo-Oroz
  • Louise Duloquin
  • Benoît Lombardot
  • María J. Ledesma-Carbayo
  • Paul Bourgine
  • Nadine Peyriéras
  • Andrés Santos

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.

2011

Automated inference of 4-D cell polarization fields in an agent-based model of early vertebrate embryogenesis

The FASEB Journal Vol. 25 No. 9.4 2011

  • Julien Delile
  • Taras Kowaliw
  • Nadine Peyrieras
  • René Doursat

We present a theoretical model of animal morphogenesis construed as a self-organized phenomenon emerging from a complex system made of a myriad of individual cell behaviors. It is implemented in an agent-based simulation centered on the mechanic-chemical coupling between cellular and genetic dynamics. The goal is to integrate the collective motion of cells and the dynamics of their gene expression underlying the patterning of morphogenetic fields. Within this larger framework, we examine here cell intercalation, a generic process underlying tissue elongation in vertebrate embryogenesis, in particular the convergence and extension movements shaping the embryonic axes during gastrulation. Cell intercalation is thought to arise from polarized cell movements. In our model, we include monopolar and bipolar protrusive activity of polarized cells to investigate the causal bottom-up link from local cell behavior to global tissue deformation. Conversely, this model also allows reverse inference, e.g., by evolutionary optimization, of the local quantitative features of cell adhesion and polarization from a global 4 D (3 D + time) computational reconstruction of the cell lineage tree, itself based on in toto imaging data of developing zebrafish embryos.

Comparison of distribution and activity of nanoparticles with short interfering DNA (Dbait) in various living systems

Cancer Gene Therapy Vol. 18 No. 11 2011 695–706

  • Nathalie Berthault
  • Benoît Maury
  • Céline Agrario
  • Aurélie Herbette
  • Jian-Sheng Sun
  • Nadine Peyrieras
  • Marie Dutreix

Introducing small DNA molecules (Dbait) impairs the repair of damaged chromosomes and provides a new method for enhancing the efficiency of radiotherapy in radio-resistant tumors. The radiosensitizing activity is dependent upon the efficient delivery of Dbait molecules into the tumor cells. Different strategies have been compared, to improve this key step. We developed a pipeline of assays to select the most efficient nanoparticles and administration protocols before preclinical assays: (i) molecular analyses of complexes formed with Dbait molecules, (ii) cellular tests for Dbait uptake and activity, (iii) live zebrafish embryo confocal microscopy monitoring for in vivo distribution and biological activity of the nanoparticles and (iv) tumor growth and survival measurement on mice with xenografted tumors. Two classes of nanoparticles were compared, polycationic polymers with linear or branched polyethylenimine (PEI) and covalently attached cholesterol (coDbait). The most efficient Dbait transfection was observed with linear PEI complexes, in vitro and in vivo. Doses of coDbait ten-fold higher than PEI/Dbait nanoparticles, and pretreatment with chloroquine, were required to obtain the same antitumoral effect on xenografted melanoma. However, with a 22-fold lower ‘efficacy dose/toxicity dose’ ratio as compared with Dbait/PEI, coDbait was selected for clinical trials.

Image analysis for understanding embryo development: a bridge from microscopy to biological insights

Current Opinion in Genetics & Development Vol. 5 2011 630-637

  • Miguel Luengo-Oroz
  • María J. Ledesma-Carbayo
  • Nadine Peyriéras
  • Andrés Santos

The digital reconstruction of the embryogenesis of model organisms from 3D+time data is revolutionizing practices in quantitative and integrative Developmental Biology. A manual and fully supervised image analysis of the massive complex data acquired with new microscopy technologies is no longer an option and automated image processing methods are required to fully exploit the potential of imaging data for biological insights. Current developments and challenges in biological image processing include algorithms for microscopy multiview fusion, cell nucleus tracking for quasi-perfect lineage reconstruction, segmentation, and validation methodologies for cell membrane shape identification, single cell gene expression quantification from in situ hybridization data, and multidimensional image registration algorithms for the construction of prototypic models. These tools will be essential to ultimately produce the multilevel in toto reconstruction that combines the cell lineage tree, cells, and tissues structural information and quantitative gene expression data in its spatio-temporal context throughout development.

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

Biomedical Optics Express Vol. 2 No. 10 2011 2837-2849

  • Pierre Mahou
  • Nicolas Olivier
  • Guillaume Labroille
  • Louise Duloquin
  • Jean-Marc Sintes
  • Nadine Peyriéras
  • Renaud Legouis
  • Delphine Debarre
  • Emmanuel Beaurepaire

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 chi((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 |Deltachi((3))(-3omega;omega,omega,omega)| with remarkable sensitivity while FWM directly reflects the distribution of chi((3))(-2omega(1) + omega(2);omega(1), -omega(2), omega(1)). We show that FWM images provide chi((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.

Segmentation of 3D cell membrane images by PDE methods and its applications

Comput Biol Med Vol. 41 No. 6 2011 326-339

  • By alphabetic order:
  • Karol Mikula
  • Nadine Peyriéras
  • Mariana Remesikova
  • O. Stasova

We present a set of techniques that enable us to segment objects from 3D cell membrane images. Particularly, we propose methods for detection of approximate cell nuclei centers, extraction of the inner cell boundaries, the surface of the organism and the intercellular borders--the so called intercellular skeleton. All methods are based on numerical solution of partial differential equations. The center detection problem is represented by a level set equation for advective motion in normal direction with curvature term. In case of the inner cell boundaries and the global surface, we use the generalized subjective surface model. The intercellular borders are segmented by the advective level set equation where the velocity field is given by the gradient of the signed distance function to the segmented inner cell boundaries. The distance function is computed by solving the time relaxed eikonal equation. We describe the mathematical models, explain their numerical approximation and finally we present various possible practical applications on the images of zebrafish embryogenesis--computation of important quantitative characteristics, evaluation of the cell shape, detection of cell divisions and others.

Image processing challenges in the creation of spatiotemporal gene expression atlases of developing embryos

Engineering in Medicine and Biology Society - Annual International Conference of the IEEE 2011 6841-6844

  • Carlos Castro-González
  • Miguel A. Luengo-Oroz
  • Louise Duloquin
  • Thierry Savy
  • Camillo Melani
  • Sophie Desnoulez
  • Paul Bourgine
  • Nadine Peyriéras
  • María J. Ledesma-Carbayo
  • Andrés Santos

To properly understand and model animal embryogenesis it is crucial to obtain detailed measurements, both in time and space, about their gene expression domains and cell dynamics. Such challenge has been confronted in recent years by a surge of atlases which integrate a statistically relevant number of different individuals to get robust, complete information about their spatiotemporal locations of gene patterns. This paper will discuss the fundamental image analysis strategies required to build such models and the most common problems found along the way. We also discuss the main challenges and future goals in the field.

Amphioxus spawning behavior in an artificial seawater facility

Journal of Experimental Zoology Part B: Molecular and Developmental Evolution Vol. 316B No. 4 2011 263-275

  • Maria Theodosiou
  • Audrey Colin
  • Jasmin Schulz
  • Vincent Laudet
  • Nadine Peyriéras
  • Jean-François Nicolas
  • Michael Schubert
  • Estelle Hirsinger

Owing to its phylogenetic position at the base of the chordates, the cephalochordate amphioxus is an emerging model system carrying immense significance for understanding the evolution of vertebrate development. One important shortcoming of amphioxus as a model organism has been the unavailability of animal husbandry protocols to maintain amphioxus adults away from the field. Here, we present the first report of successful maintenance and spawning of Branchiostoma lanceolatum adults in a facility run on artificial seawater. B. lanceolatum has been chosen for this study because it is the only amphioxus species that can be induced to spawn. We provide a step-by-step guide for the assembly of such a facility and discuss the day-to-day operations required for successful animal husbandry of B. lanceolatum adults. This work also includes a detailed description of the B. lanceolatum spawning behavior in captivity. Our analysis shows that the induced spawning efficiency is not sex biased, but increases as the natural spawning season progresses. We find that a minor fraction of the animals undergo phases of spontaneous spawning in the tanks and that this behavior is not affected by the treatment used to induce spawning. Moreover, the induced spawning efficiency is not discernibly correlated with spontaneous spawning in the facility. Last, we describe a protocol for long-term cryopreservation of B. lanceolatum sperm. Taken together, this work represents an important step toward further establishing amphioxus as a laboratory animal making it more amenable to experimental research, and hence assists the coming of age of this emerging model.

2010

Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy

Science Vol. 329 No. 5994 2010 967-971

  • Nicolas Olivier
  • Miguel A. Luengo-Oroz
  • Louise Duloquin
  • Emmanuel Faure
  • Thierry Savy
  • Israël Veilleux
  • Xavier Solinas
  • Delphine Débarre
  • Paul Bourgine
  • Andrés Santos
  • Nadine Peyriéras
  • Emmanuel Beaurepaire

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.

4D Embryogenesis image analysis using PDE methods of image processing

Kybernetika Vol. 46 No. 2 2010 226-259

  • By alphabetic order:
  • Paul Bourgine
  • Róbert Čunderlík
  • Olga Drblíková-Stašová
  • Karol Mikula
  • Mariana Remešíková
  • Nadine Peyriéras
  • Barbara Rizzi
  • Alessandro Sarti

In this paper, we introduce a set of methods for processing and analyzing long time series of 3D images representing embryo evolution. The images are obtained by in vivo scanning using a confocal microscope where one of the channels represents the cell nuclei and the other one the cell membranes. Our image processing chain consists of three steps: image filtering, object counting (center detection) and segmentation. The corresponding methods are based on numerical solution of nonlinear PDEs, namely the geodesic mean curvature flow model, flux-based level set center detection and generalized subjective surface equation. All three models have a similar character and therefore can be solved using a common approach. We explain in details our semi-implicit time discretization and finite volume space discretization. This part is concluded by a short description of parallelization of the algorithms. In the part devoted to experiments, we provide the experimental order of convergence of the numerical scheme, the validation of the methods and numerous experiments with the data representing an early developmental stage of a zebrafish embryo.

Methods toward in vivo measurement of zebrafish epithelial and deep cell proliferation

Computer Methods and Programs in Biomedicine Vol. 98 No. 2 2010 103-117

  • Matteo Campana
  • Benoit Maury
  • Marie Dutreix
  • Nadine Peyriéras
  • Alessandro Sarti

We present a strategy for automatic classification and density estimation of epithelial enveloping layer (EVL) and deep layer (DEL) cells, throughout zebrafish early embryonic stages. Automatic cells classification provides the bases to measure the variability of relevant parameters, such as cells density, in different classes of cells and is finalized to the estimation of effectiveness and selectivity of anticancer drug in vivo. We aim at approaching these measurements through epithelial/deep cells classification, epithelial area and thickness measurement, and density estimation from scattered points. Our procedure is based on Minimal Surfaces, Otsu clustering, Delaunay Triangulation, and Within-R cloud of points density estimation approaches. In this paper, we investigated whether the distance between nuclei and epithelial surface is sufficient to discriminate epithelial cells from deep cells. Comparisons of different density estimators, experimental results, and extensively accuracy measurements are included.

An automatic quantification and registration strategy to create a gene expression atlas of zebrafish embryogenesis

Conference Proceedings of the IEEE Engineering in Medicine and Biology Society Vol. 2009 2010 1469-1472

  • Carlos Castro-González
  • Miguel A. Luengo-Oroz
  • Sophie Desnoulez
  • Louise Duloquin
  • L. Fernandez-de-Manuel
  • S. Montagna
  • María J. Ledesma-Carbayo
  • Paul Bourgine
  • Nadine Peyriéras
  • Andrés Santos

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.

Towards a digital model of zebrafish embryogenesis. Integration of cell tracking and gene expression quantification

Conference Proceedings of the IEEE Engineering in Medicine and Biology Society Vol. 2010 2010 5520-5523

  • Carlos Castro-Gonzalez
  • Miguel A. Luengo-Oroz
  • Louise Duloquin
  • Thierry Savy
  • Camillo Melani
  • Sophie Desnoulez
  • María J. Ledesma-Carbayo
  • Paul Bourgine
  • Nadine Peyriéras
  • Andrés Santos

We elaborate on a general framework composed of a set of computational tools to accurately quantificate cellular position and gene expression levels throughout early zebrafish embryogenesis captured over a time-lapse series of in vivo 3D images. Our modeling strategy involves nuclei detection, cell geometries extraction, automatic gene levels quantification and cell tracking to reconstruct cell trajectories and lineage tree which describe the animal development. Each cell in the embryo is then precisely described at each given time t by a vector composed of the cell 3D spatial coordinates (x; y; z) along with its gene expression level g. This comprehensive description of the embryo development is used to assess the general connection between genetic expression and cell movement. We also investigate genetic expression propagation between a cell and its progeny in the lineage tree. More to the point, this paper focuses on the evolution of the expression pattern of transcriptional factor goosecoid (gsc) through the gastrulation process between 6 and 9 hours post fertilization (hpf).

3D early embryogenesis image filtering by nonlinear partial differential equations

Medical Image Analysis Vol. 14 No. 4 2010 510-526

  • By alphabetic order:
  • Zuzana Krivá
  • Karol Mikula
  • Nadine Peyriéras
  • Barbara Rizzi
  • Alessandro Sarti
  • Olga Stašová

We present nonlinear diffusion equations, numerical schemes to solve them and their application for filtering 3D images obtained from laser scanning microscopy (LSM) of living zebrafish embryos, with a goal to identify the optimal filtering method and its parameters. In the large scale applications dealing with analysis of 3D+time embryogenesis images, an important objective is a correct detection of the number and position of cell nuclei yielding the spatio-temporal cell lineage tree of embryogenesis. The filtering is the first and necessary step of the image analysis chain and must lead to correct results, removing the noise, sharpening the nuclei edges and correcting the acquisition errors related to spuriously connected subregions. In this paper we study such properties for the regularized Perona-Malik model and for the generalized mean curvature flow equations in the level-set formulation. A comparison with other nonlinear diffusion filters, like tensor anisotropic diffusion and Beltrami flow, is also included. All numerical schemes are based on the same discretization principles, i.e. finite volume method in space and semi-implicit scheme in time, for solving nonlinear partial differential equations. These numerical schemes are unconditionally stable, fast and naturally parallelizable. The filtering results are evaluated and compared first using the Mean Hausdorff distance between a gold standard and different isosurfaces of original and filtered data. Then, the number of isosurface connected components in a region of interest (ROI) detected in original and after the filtering is compared with the corresponding correct number of nuclei in the gold standard. Such analysis proves the robustness and reliability of the edge preserving nonlinear diffusion filtering for this type of data and lead to finding the optimal filtering parameters for the studied models and numerical schemes. Further comparisons consist in ability of splitting the very close objects which are artificially connected due to acquisition error intrinsically linked to physics of LSM. In all studied aspects it turned out that the nonlinear diffusion filter which is called geodesic mean curvature flow (GMCF) has the best performance.

Cells segmentation from 3-D confocal images of early zebrafish embryogenesis

IEEE Trans Image Process Vol. 19 No. 3 2010 770-781

  • Cecilia Zanella
  • Matteo Campana
  • Barbara Rizzi
  • Camillo Melani
  • Gonzalo Sanguinetti
  • Paul Bourgine
  • Karol Mikula
  • Nadine Peyriéras
  • Alessandro Sarti

We designed a strategy for extracting the shapes of cell membranes and nuclei from time lapse confocal images taken throughout early zebrafish embryogenesis using a partial-differential-equation-based segmentation. This segmentation step is a prerequisite for an accurate quantitative analysis of cell morphodynamics during embryogenesis and it is the basis for an integrated understanding of biological processes. The segmentation of embryonic cells requires live zebrafish embryos fluorescently labeled to highlight sub-cellular structures and designing specific algorithms by adapting classical methods to image features. Our strategy includes the following steps: the signal-to-noise ratio is first improved by an edge-preserving filtering, then the cell shape is reconstructed applying a fully automated algorithm based on a generalized version of the Subjective Surfaces technique. Finally we present a procedure for the algorithm validation either from the accuracy and the robustness perspective.

2009

Extraction of the Intercellular Skeleton from 2D Images of Embryogenesis Using Eikonal Equation and Advective Subjective Surface Method

Scale Space and Variational Methods in Computer Vision - Second International Conference, SSVM 2009, Voss, Norway, June 1-5, 2009. Proceedings 38-49 Springer Berlin Heidelberg Vol. 5567 2009

  • Paul Bourgine
  • Peter Frolkovič
  • Karol Mikula
  • Nadine Peyriéras
  • Mariana Remešíková

We suggest an efficient method for automatic detection of the intercellular skeleton in microscope images of early embryogenesis. The method is based on the solution of two advective PDEs. First, we solve numerically the time relaxed eikonal equation in order to obtain the signed distance function to a given set – a set of points representing cell centers or a set of closed curves representing segmented inner borders of cells. The second step is a segmentation process driven by the advective version of subjective surface equation where the velocity field is given by the gradient of the computed distance function. The first equation is discretized by Rouy-Tourin scheme and we suggest a fixing strategy that significantly improves the speed of the computation. The second equation is solved using a classical upwind strategy. We present several test examples and we show a practical application - the intercellular skeleton extracted from a 2D image of a zebrafish embryo.

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

Engineering in Medicine and Biology Society - Annual International Conference of the IEEE 2009 5918-5921

  • Jose Luis Rubio-Guivernau
  • Miguel A. Luengo-Oroz
  • Louise Duloquin
  • Thierry Savy
  • Nadine Peyriéras
  • Paul Bourgine
  • Andrés Santos

Obtaining 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.

Region-Based PDEs for Cells Counting and Segmentation in 3D+Time Images of Vertebrate Early Embryogenesis

International Journal of Biomedical Imaging Vol. 2009 2009 9 pages

  • By alphabetic order:
  • Barbara Rizzi
  • Alessandro Sarti

This paper is devoted to the segmentation of cell nuclei from time lapse confocal microscopy images, taken throughout early Zebrafish embryogenesis. The segmentation allows to identify and quantify the number of cells in the animal model. This kind of information is relevant to estimate important biological parameters such as the cell proliferation rate in time and space. Our approach is based on the active contour model without edges. We compare two different formulations of the model equation and evaluate their performances in segmenting nuclei of different shapes and sizes. Qualitative and quantitative comparisons are performed on both synthetic and real data, by means of suitable gold standard. The best approach is then applied on a number of time lapses for the segmentation and counting of cells during the development of a zebrafish embryo between the sphere and the shield stage.

2008

3D embryogenesis image segmentation by generalized subjective surfaces method using finite volume technique

Finite Volumes for Complex Applications Vol. V 2008 585-592

  • By alphabetic order:
  • Karol Mikula
  • Nadine Peyriéras
  • Mariana Remešíková
  • Alessandro Sarti

In this paper, an efficient finite volume method for image segmentation is introduced. The method is based on surface evolution governed by a nonlinear PDE, the generalized subjective surfaces equation. Our numerical method is based on semi-implicit time discretization and finite volume space approximation. We show examples of image segmentation - particularly, we deal with images of early embryogenesis of zebrafish obtained by a confocal microscope. We mention how the segmentation can be useful for analysis of the embryo images and reconstruction of the embryo evolution.

A Subjective Surfaces based Segmentation for the Reconstruction of Biological Cell Shape

VISAPP 2008: Proceedings of the Third International Conference on Computer Vision Theory and Applications 2008 555-558

  • Matteo Campana
  • Cecilia Zanella
  • Barbara Rizzi
  • Paul Bourgine
  • Nadine Peyriéras
  • Alessandro Sarti

Confocal laser scanning microscopy provides nondestructive in vivo imaging to capture specific structures that have been fluorescently labeled, such as cell nuclei and membranes, throughout early Zebrafish embryogenesis. With this strategy we aim at reconstruct in time and space the biological structures of the embryo during the organogenesis. In this paper we propose a method to extract bounding surfaces at the cellular-organization level from microscopy images. The shape reconstruction of membranes and nuclei is obtained first with an automatic identification of the cell center and then a subjective surfaces based segmentation is used to extract the bounding surfaces.

A Framework for 4-D Biomedical Image Processing, Visualization and Analysis

GRAPP 2008: Proceedings of the Third International Conference on Computer Graphics Theory and Applications 2008 403-408

  • Matteo Campana
  • Barbara Rizzi
  • Camilo Melani
  • Paul Bourgine
  • Nadine Peyriéras
  • Alessandro Sarti

Today, studies on biological systems are often realized acquiring, processing and analyzing 3D-time lapse images. Different structures of the organism can be simultaneously observed by acquiring multi-channel image datasets. In this paper we present a software framework that aims at providing support for managing these kinds of multidimensional images, designing and validating new image processing algorithms, and analyzing processed images through different visualization techniques. We present a real scenario where the framework has been used for the detection and segmentation of biological cell membranes and nuclei imaged from live zebrafish embryos.

2007

Morphogenèse animale

Translated as : Morphogenesis: Origins of Patterns and Shapes (Springer Complexity - 2010)

Morphogenèse : L'origine des formes 360 pages Belin Echelles 2007

  • Nadine Peyriéras

English version : What are the relations between the shape of a system of cities and that of fish school? Which events should happen in a cell in order that it participates to one of the finger of our hands? How to interpret the shape of a sand dune? This collective book written for the non-specialist addresses these questions and more generally, the fundamental issue of the emergence of forms and patterns in physical and living systems. It is a single book gathering the different aspects of morphogenesis and approaches developed in different disciplines on shape and pattern formation. Relying on the seminal works of D’Arcy Thompson, Alan Turing and René Thom, it confronts major examples like plant growth and shape, intra-cellular organization, evolution of living forms or motifs generated by crystals. A book essential to understand universal principles at work in the shapes and patterns surrounding us but also to avoid spurious analogies. How to interpret the shape of a sand dune? This collective book written for the non-specialist addresses these questions and more generally, the fundamental issue of the emergence of forms and patterns in physical and living systems.

French version : Quels sont les liens entre la forme d'un système de villes et celle d'un banc de poissons ? Quels événements doivent se produire dans une cellule pour qu'elle participe à la formation d'un des doigts de la main ? Comment interpréter la forme des dunes barchanes ?

Accessible pour l'essentiel au non-spécialiste, ce livre inédit pose la question fondamentale de l'émergence des formes dans les systèmes physiques et dans le vivant. S'appuyant sur les travaux fondateurs de d'Arcy Thompson, Alan Turing et René Thom, il met en regard des exemples emblématiques comme la croissance du tournesol, l'auto-organisation intracellulaire, l'évolution des formes vivantes ou les surprenants motifs créés par les cristaux liquides. Un ouvrage indispensable pour enfin comprendre les principes universels à l'origine des formes qui nous entourent et nous émerveillent, mais aussi pour éviter le cas échéant les analogies trompeuses.

3-D Zebrafish Embryo Image Filtering by Nonlinear Partial Differential Equations

Engineering in Medicine and Biology Society (EMBS 2007, 29th Annual International Conference of the IEEE) 2007 6251-6254

  • Barbara Rizzi
  • Matteo Campana
  • Cecilia Zanella
  • Camilo Melani
  • Robert Cunderlik
  • Zuzana Krivá
  • Paul Bourgine
  • Karol Mikula
  • Nadine Peyriéras
  • Alessandro Sarti

We discuss application of nonlinear PDE based methods to filtering of 3-D confocal images of embryogenesis. We focus on the mean curvature driven and the regularized Perona-Malik equations, where standard as well as newly suggested edge detectors are used. After presenting the related mathematical models, the practical results are given and discussed by visual inspection and quantitatively using the mean Hausdorff distance.

Cells tracking in a live zebrafish embryo

Engineering in Medicine and Biology Society (EMBS 2007, 29th Annual International Conference of the IEEE) 2007 1631-1634

  • Camilo Melani
  • Matteo Campana
  • Benoit Lombardot
  • Barbara Rizzi
  • Federico Veronesi
  • Cecilia Zanella
  • Paul Bourgine
  • Karol Mikula
  • Nadine Peyriéras
  • Alessandro Sarti

We designed a set of procedures for achieving the tracking of cell nuclei and the identification of cell divisions in live zebrafish embryos using 3D+time images acquired by confocal laser scanning microscopy (CLSM). Our strategy includes image signal enhancement with feature preserving denoising algorithm, automated identification of the nuclei position, extraction of the optical flow from 3D images sequences and tracking of nuclei.

Segmentation of Cells from 3-D Confocal Images of Live Zebrafish Embryo

Engineering in Medicine and Biology Society (EMBS 2007, 29th Annual International Conference of the IEEE) 2007 6027-6030

  • Cecilia Zanella
  • Barbara Rizzi
  • Matteo Campana
  • Paul Bourgine
  • Karol Mikula
  • Nadine Peyriéras
  • Alessandro Sarti

In this paper, we use partial-differential-equation-based segmentation to accurately extract the shapes of membranes and nuclei from time lapse confocal microscopy images, taken throughout early zebrafish embryogenesis. This strategy is a prerequisite for an accurate quantitative analysis of cell shape and morphodynamics during organogenesis and is the basis for an integrated understanding of biological processes. This data will also serve for the measurement of the variability between individuals in a population. The segmentation of cellular structures is achieved by first using an edge-preserving image filtering method for noise reduction and then applying an algorithm for cell shape reconstruction based on the subjective surfaces technique.