Dissertations / Theses on the topic 'Embryo segmentation'

During vertebrate embryo development, the body axis is subdivided into repeated structures, called somites. Somites bud off from an un-segmented tissue called the pre-somitic mesoderm (PSM) in a sequential and periodic manner, tightly controlled by an in built molecular clock, called the "segmentation clock". According to current understanding, the clock is comprised of: (i) an autonomous cellular oscillator consisting of an intracellular negative feedback loop of Her genes within the PSM cells, (ii) Delta-ligand and Notch-receptor coupling that facilitates synchronization of oscillators among the PSM cells, (iii) Tissue level waves of gene expression that emerge in the posterior PSM and move coherently towards anterior, leading to global arrest of oscillations in the form of somites. However, the movement of cellular oscillators within the PSM before the formation of somitic furrows, a prominent feature of the tissue as observed through this work has not been experimentally considered as a constituent of the segmentation clock so far. Our work aims to incorporate movement of cellular oscillators in the framework of the segmentation clock. It is well known from theoretical studies that the characteristics of relative motion of oscillators affect their synchronization properties and the patterns of oscillations they form. Particularly, theoretical studies by Uriu et al., PNAS (2010) suggest that cell movements promotes synchronization of genetic oscillations. Here, we established experimental techniques and image analysis tools to attain quantitative insight on (i) diffusion co-efficient of cellular oscillators, (ii) dynamics of a population of oscillators, within the PSM aiming towards concomitant understanding of the relationship between movement and synchronization of cellular oscillators. In order to quantitatively relate cellular oscillators and their motion within the PSM, I established imaging techniques that enabled visualization of fluorescenctly labeled nuclei as readouts of cell positions in live embryo, and developed dedicated segmentation algorithm and implemented tracking protocol to obtain nuclei positions over time in 3D space. Furthermore, I provide benchmarking techniques in the form of artificial data that validate segmentation algorithm efficacy and, for the first time proposed the use of transgenic embryo chimeras to validate segmentation algorithm performance within the context of in vivo live imaging of embryonic tissues. Preliminary analysis of our data suggests that there is relatively high cell mixing in the posterior PSM, within the same spatial zone where synchronous oscillations emerge at maximum speed. Also, there are indications of gradient of cell mixing along the anterior-posterior axis of the embryo. By sampling single cell tracks with the help of nuclei markers, we have also been able to follow in vivo protein oscillations at single cell resolution that would allow quantitative characterization of coherence among a population of cellular oscillators over time. Our image analysis work flow allows testing of mutant embryos and perturbation of synchrony dynamics to understand the cause-effect relationship between movement and synchronization properties at cellular resolution. Essentially, through this work, we hope to bridge the time scales of events and cellular level dynamics that leads to highly coordinated tissue level patterns and thereby further our understanding of the segmentation clock mechanism.

De nombreuses substances chimiques sont utilisées par l’industrie cosmétique pour entrer dans la composition de formules. En dehors de la nécessité d’évaluer leur efficacité, l’industrie cosmétique se doit surtout d’évaluer la sécurité de leurs substances pour l’humain. L'évaluation toxicologique des substances chimiques est réalisée dans le but de révéler un effet toxique potentiel de la substance testée. Parmi les effets potentiels que l’on souhaite détecter, la toxicité du développement (tératogénicité), c’est-à-dire la capacité d’une substance à provoquer l’apparition d’anomalies lors du développement embryonnaire, est fondamentale. En accord avec les législations internationales qui interdisent à l’industrie cosmétique d’avoir recours à des tests sur animaux de laboratoire pour l’évaluation de leurs substances, l’évaluation toxicologique de ces substances se base sur les résultats de tests in silico, in vitro et de tests faits sur des modèles alternatifs aux animaux de laboratoire. Pour le moment cependant, peu de méthodes alternatives existent et ont été validées pour la toxicologie du développement. Le développement de nouvelles méthodes alternatives est donc requis. D'autre part, en plus de l’évaluation de la sécurité des substances chez l’humain, l’évaluation de la toxicité pour l’environnement est nécessaire. L’usage de la plupart des produits cosmétiques et d’hygiène corporelle conduit, après lavage et rinçage, à un rejet à l’égout et donc dans les cours d’eau. Il en résulte que les environnements aquatiques (eaux de surface et milieux marins côtiers) sont parfois exposés aux substances chimiques incluses dans les formules cosmétiques. Ainsi, l’évaluation toxicologique environnementale des cosmétiques et de leurs ingrédients nécessite de connaître leur toxicité sur des organismes représentatifs de chaînes alimentaires aquatiques. Dans ce contexte, le modèle embryon de poisson présente un double avantage pour l’industrie cosmétique. Ce modèle, jugé par les législations internationales comme étant éthiquement acceptable pour les évaluations toxicologiques réalisées par l’industrie cosmétique, est représentatif des organismes aquatiques. Il est donc pertinent pour évaluer la toxicité environnementale des substances chimiques. D'autre part, ce modèle apparaît prometteur pour évaluer l’effet tératogène de substances chimiques chez l’humain. Pour ces raisons, un test d’analyse de la tératogénicité des substances chimiques est actuellement développé. Ce test se base sur l’analyse d’embryons de medaka (Oryzias Latipes) à 9 jours post fertilisation, après exposition des embryons par balnéation à des substances à concentrations déterminées. L’analyse de paramètres fonctionnels et morphologiques conduit au calcul d’un indice tératogène, qui permet de tirer une conclusion quant à l’effet tératogène de la substance testée. Cet indice est calculé à partir des mesures du taux de mortalité et du taux de malformations chez les embryons. L’objectif de ce projet est d’automatiser le test d’analyse de la tératogénicité, par classification automatique des embryons faite à partir d’image et de vidéo. La première méthode développée concerne la détection des battements cardiaques à partir de séquences vidéos, dans le but de calculer le taux de mortalité. Nous nous sommes ensuite concentrés sur deux types de malformations courantes qui sont les malformations axiales, et l'absence de vessie natatoire, en utilisant une méthode d'apprentissage automatique. Cette analyse doit être complétée par l'analyse d'autres malformations et conduire à un calcul du taux de malformations et de l’indice tératogène pour la substance testée
Numerous chemicals are used as ingredients by the cosmetics industry and are included in cosmetics formula. Aside from the assessment of their efficacy, the cosmetics industry especially needs to assess the safety of their chemicals for human. Toxicological screening of chemicals is performed with the aim of revealing the potential toxic effect of the tested chemical. Among the potential effects we want to detect, the developmental toxicity of the chemical (teratogenicity), meaning its capability of provoking abnormalities during the embryonic development, is crucial. With respect to the international regulations that forbid the use of animal testing for the safety assessment of cosmetics, the toxicological assessment of chemicals must base on an ensemble of in silico assays, in vitro assays and alternative models based assays. For now, a few alternative methods have been validated in the field of developmental toxicology. The development of new alternative methods is thus required. In addition to the safety assessment, the environmental toxicity assessment is also required. The use of most of cosmetics and personal care products leads to their rejection in waterways after washing and rince. This results in the exposition of some aquatic environments (surface waters and coastal marine environments) to chemicals included in cosmetics and personal care products. Thus, the environmental assessment of cosmetics and of their ingredients requires the knowledge of their toxicity on organisms that are representative of aquatic food chains. In this context, the fish embryo model, which is ethically acceptable according to international regulations, presents a dual advantage for the cosmetics industry. Firstly, as a model representative of aquatic organisms, it is accurate for the environmental assessment of chemicals. Secondly, this model is promising for the assessment of the teratogenic effect of chemicals on human. For this reason, a teratogenicity assessment test is developed. This test is based on the analysis of medaka fish embryos (Oryzias Latipes) at 9 days post fertilization, after balneation in a predetermined concentration of the chemical under study. The analysis of functional and morphological parameters allows to calculate a teratogenicity index, that depends on both rates of dead and malformed embryos. This index allows to to draw a conclusion concerning the teratogenic effect of the chemical.The objective of this project is to automate the teratogenicity test, by automated image and video classification. A first method is developed that aims to automatically detect embryo heart beats from acquired video sequences. This method will allow to calculate the proportion of dead embryos. We then focus on the detection of two common malformations: axial malformations and absence of a swim bladder, based on a machine learning classification. This analysis must be completed by the detection of other malformations so that we can measure the rate of malformed embryos and thus, calculate the teratogenicity index of the tested chemical

During amniote embryogenesis, the segmented pattern characteristic of the vertebral column appears early during development through the sequential formation of multipotent structures called somites. Somites differentiate subsequently into dermomyotome (giving rise later to skin and skeletal muscles) and sclerotome (giving rise to vertebral bone structures and cartilage). In addition, sclerotomes subdivide following their rostro-caudal intrasegmental boundary into an axon growth-permissive region (anterior half) and an axon growth-repulsive region (posterior half). This binary system instructs motor and sensory axon navigation, as well as neural crest cell migration, to ensure that the peripheral nervous system develops without obstruction by the future cartilage and bones of the vertebral column. Repellent cues are expressed in posterior half-sclerotomes in order to exclude navigating axons from “no-go” areas and restrict their growth to specific exit points of the future vertebral column. Interestingly, similar repellent cues (e.g. Eph/Ephrins) are expressed in the adult central nervous system (CNS) and have been shown to control connectivity and plasticity throughout life. Following brain or spinal cord injury, these repellent molecules are upregulated by reactive astrocytes accumulating at the lesion site, and may impede axon regeneration in this region. In this dissertation, I am presenting the results of a differential gene expression analysis of anterior and posterior half-sclerotomes, based on RNA-sequencing data and using the chick embryo as a model organism. This study led to the identification of molecules, previously uncharacterized in this system, that may play a role in adhesive and mechanical properties of somites and in axon guidance and fasciculation. I focused on the functional analysis of one molecule of the posterior half-sclerotome, the extracellular matrix protein Fibulin-2. To look at its role in the segmentation of spinal axons, I used ectopic misexpression in a subset of segments based on somite electroporation. The width of spinal nerve bundle growth was restricted by Fibulin-2 overexpression in posterior and anterior half-sclerotomes, suggesting a role in sharpening/controlling the path of spinal axon growth. In addition, I showed that this could occur via an interaction with the axon growth repellent Semaphorin 3A. Then I looked at the expression of Fibulin-2 in two models of CNS injury: mouse cerebral cortical stab injury and rat dorsal crush spinal cord injury. In both cases, I observed an increase in Fibulin-2 protein level compared to control. I also used primary cultures of rat cortical astrocytes to show that the expression of Fibulin-2 after inflammatory cytokine-induced activation is increased. Finally, I studied a candidate axon growth repellent previously identified in the laboratory. I explored the hypothesis that this repellent molecule is an O-glycosylated, spliced variant form of a known protein. To characterize this repellent molecule, I used RNA-sequencing data from chick embryonic somites and 2D gel electrophoresis of an astrocytic cell line protein extract. Together, these results suggested that the developing vertebral column and the adult CNS share molecular features to control axon growth and plasticity. This type of study could lead to the characterization of molecular systems that regulate axon growth, and to the identification of novel therapeutic targets in brain or spinal cord injury.

This diploma thesis deals with a segmentation of soft tissues in facial part of mouse embryos in Matlab. Segmentation of soft tissues of mouse embryos was not fully automated and every case needs a specific solution. Solving parts of this issues can provide valuable data for evolutionary biologists. Issues about staining and segmentation techniques are described. On the basis of accessible literature otsu thresholding, region growing, k-means clustering and segmentation with atlas were tested. In the end of this paper are those methods tested and evaluated on 3D microtomography data.

The focus of this thesis is the smoothing of manually segmented 3D models of mouse embryo craniofacial cartilege. During the process of manual segmentation, artefacts and other imperfections appear in the final models and need to be repaired. Firstly, manual segmentation is corrected using gradients and thresholding. Subsequent smoothing methods are constructed based on theoretical research. Algorithmizing is executed in the MATLAB environment. All the designed algorithms are then tested on selected models. Statistical evaluation is determined using the Srensen–Dice coefficient, where manually smoothened models cleared of all artefacts are used as the gold standard.

Les cellules sont les éléments constitutifs de base de tout organisme vivant. Tous les organismes vivants partagent des processus vitaux tels que croissance, développement, mouvement, nutrition, excrétion, reproduction, respiration et réaction à l’environnement. En biologie cellulaire, comprendre la structure et fonction des cellules est essentielle pour développer et tester de nouveaux médicaments. Par ailleurs, cela aide aussi à l’étude du développement embryonnaire. Enfin, cela permet aux chercheurs de mieux comprendre les effets des mutations et de diverses maladies. La vidéo-microscopie (Time Lapse Fluorescence Microscopie) est l’une des techniques d’imagerie les plus utilisées afin de quantifier diverses caractéristiques des processus cellulaires, à savoir la survie, la prolifération, la migration ou la différenciation cellulaire. En vidéo-microscopie, non seulement les informations spatiales sont disponibles, mais aussi les informations temporelles en réitérant l’acquisition de l’échantillon, et enfin les informations spectrales, ce qui génère des données dites « cinq dimensions » (X, Y, Z + temps + canal). En règle générale, les jeux de données générés consistent en plusieurs (centaines ou milliers) d’images, chacune contenant des centaines ou milliers d’objets à analyser. Pour effectuer une quantification précise et à haut débit des processus cellulaires, les étapes de segmentation et de suivi des noyaux cellulaires doivent être effectuées de manière automatisée. Cependant, la segmentation et le suivi des noyaux sont des tâches difficiles dû notamment au bruit intrinsèque dans les images, à l’inhomogénéité de l’intensité, au changement de forme des noyaux ainsi qu’à un faible contraste des noyaux. Bien que plusieurs approches de segmentation des noyaux aient été rapportées dans la littérature, le fait de traiter le bruit intrinsèque reste la partie la plus difficile de tout algorithme de segmentation. Nous proposons un nouvel algorithme de débruitage 3D, basé sur l’apprentissage d’un dictionnaire non supervisé et une représentation parcimonieuse, qui à la fois améliore la visualisation des noyaux très peu contrastés et bruités, mais aussi détecte simultanément la position de ces noyaux avec précision. De plus, notre méthode possède un nombre limité de paramètres, un seul étant critique, à savoir la taille approximative des objets à traiter. Le cadre de la méthode proposée comprend le débruitage d’images, la détection des noyaux et leur segmentation. Dans l’étape de débruitage, un dictionnaire initial est construit en sélectionnant des régions (patches) aléatoires dans l’image originale, puis une technique itérative est implémentée pour mettre à jour ce dictionnaire afin d’obtenir un dictionnaire dont les éléments mis à jour présentent un meilleur contraste. Ensuite, une carte de détection, basée sur le calcul des coefficients du dictionnaire utilisés pour débruiter l’image, est utilisée pour détecter le centre approximatif des noyaux qui serviront de marqueurs pour la segmentation. Ensuite, une approche basée sur le seuillage est proposée pour obtenir le masque de segmentation des noyaux. Finalement, une approche de segmentation par partage des eaux contrôlée par les marqueurs est utilisée pour obtenir le résultat final de segmentation des noyaux. Nous avons créé des images synthétiques 3D afin d’étudier l’effet des paramètres de notre méthode sur la détection et la segmentation des noyaux, et pour comprendre le mécanisme global de sélection et de réglage de ces paramètres significatifs sur différents jeux de données
Cells are the basic building blocks of all living organisms. All living organisms share life processes such as growth and development, movement, nutrition, excretion, reproduction, respiration and response to the environment. In cell biology research, understanding cells structure and function is essential for developing and testing new drugs. In addition, cell biology research provides a powerful tool to study embryo development. Furthermore, it helps the scientific research community to understand the effects of mutations and various diseases. Time-Lapse Fluorescence Microscopy (TLFM) is one of the most appreciated imaging techniques which can be used in live-cell imaging experiments to quantify various characteristics of cellular processes, i.e., cell survival, proliferation, migration, and differentiation. In TLFM imaging, not only spatial information is acquired, but also temporal information obtained by repeating imaging of a labeled sample at specific time points, as well as spectral information, that produces up to five-dimensional (X, Y, Z + Time + Channel) images. Typically, the generated datasets consist of several (hundreds or thousands) images, each containing hundreds to thousands of objects to be analyzed. To perform high-throughput quantification of cellular processes, nuclei segmentation and tracking should be performed in an automated manner. Nevertheless, nuclei segmentation and tracking are challenging tasks due to embedded noise, intensity inhomogeneity, shape variation as well as a weak boundary of nuclei. Although several nuclei segmentation approaches have been reported in the literature, dealing with embedded noise remains the most challenging part of any segmentation algorithm. We propose a novel 3D denoising algorithm, based on unsupervised dictionary learning and sparse representation, that can both enhance very faint and noisy nuclei, in addition, it simultaneously detects nuclei position accurately. Furthermore, our method is based on a limited number of parameters, with only one being critical, which is the approximate size of the objects of interest. The framework of the proposed method comprises image denoising, nuclei detection, and segmentation. In the denoising step, an initial dictionary is constructed by selecting random patches from the raw image then an iterative technique is implemented to update the dictionary and obtain the final one which is less noisy. Next, a detection map, based on the dictionary coefficients used to denoise the image, is used to detect marker points. Afterward, a thresholding-based approach is proposed to get the segmentation mask. Finally, a marker-controlled watershed approach is used to get the final nuclei segmentation result. We generate 3D synthetic images to study the effect of the few parameters of our method on cell nuclei detection and segmentation, and to understand the overall mechanism for selecting and tuning the significant parameters of the several datasets. These synthetic images have low contrast and low signal to noise ratio. Furthermore, they include touching spheres where these conditions simulate the same characteristics exist in the real datasets. The proposed framework shows that integrating our denoising method along with classical segmentation method works properly in the context of the most challenging cases. To evaluate the performance of the proposed method, two datasets from the cell tracking challenge are extensively tested. Across all datasets, the proposed method achieved very promising results with 96.96% recall for the C.elegans dataset. Besides, in the Drosophila dataset, our method achieved very high recall (99.3%)

The vertebrate body is segmented along the anteroposterior axis into repetitive structures, the vertebrae, which derive from embryonic precursors called somites. During development, periodic somite formation is driven by a molecular oscillator, the segmentation clock. Segmentation and elongation of the body axis depend on a population of progenitor cells located at the tail end of the embryo that contributes to axial tissues, including somitic tissue, until the entire embryonic body and the correct number of somites is produced. Although much is known about somite production, it is not known how segmentation and axial elongation come to an end. In this thesis, I show that termination of chick axial elongation is associated with decline of signals required for maintenance of progenitor cells, implying that downregulation of these signals triggers depletion of the progenitors. I also show that somite formation decreases as axial elongation comes to an end, suggesting that slow down of the segmentation clock causes somite formation to cease. I have also explored whether the dose of specific genes is limiting in determining the final somite number in mouse, and I have found that heterozygous mutations of selected genes of the Wnt signalling pathway form fewer somites, indicating that Wnt gene activity might be limiting in controlling the definitive somite number. I have also investigated the role of Greb1, a gene that our laboratory identified as being selectively expressed in the tail region where progenitors reside. I provide evidence that Greb1 controls axial morphogenesis of the zebrafish embryo by regulating movements required for normal convergence and extension of the embryonic axis during gastrulation. My results possibly provide a link between progenitor contribution to axial elongation and cell movements in the tail.

Une demande croissante d'outils pour construire et décrypter des réseaux génétiques contrôlant des processus cellulaires est ressentie en biologie. Nous soutenons que l'utilisation de l'approche déclarative est pertinente et applicable pour répondre aux questions des biologistes sur ces réseaux, en général partiellement connus. L'idée principale est de modéliser des connaissances portant à la fois sur la structure et la dynamique d'un réseau par un ensemble de contraintes représentant l'ensemble des solutions, de vérifier sa cohérence, de réparer une incohérence éventuelle par un relâchement automatique, et d'inférer des propriétés sur la structure et la dynamique du réseau. Pour montrer la faisabilité de l'approche, nous formalisons les réseaux discrets de R. Thomas et les propriétés biologiques pertinentes, proposons un outil reposant sur la programmation logique par contraintes en coopération avec un solveur SAT, et la validons sur des applications biologiques significatives
A growing demand for tools to build and decrypt genetic networks that control cellular processes is felt in biology. We argue that the use of the declarative approach is relevant and applicable to answer questions from biologists about these networks, which are in general partially known. The main idea is to model knowledge about both the structure and the dynamic of a network by a set of constraints representing all the solutions, to check its consistency, to repair a possible inconsistency by an automatic constraint removal, and to infer properties on the structure and dynamic of the network. To demonstrate the feasibility of the approach, we formalize the discrete networks of R. Thomas and relevant biological properties, offer a tool based on constraint logic programming in cooperation with a SAT solver, and validate it on significant biological applications

Contour extraction of Drosophila embryos is an important step to build a computational system for pattern matching of embryonic images which aids in the discovery of genes. Automatic contour extraction of embryos is challenging due to several image variations such as size, shape, orientation and neigh- boring embryos such as touching and non-touching embryos. In this thesis, we introduce a framework for contour extraction based on the connected components in the gaussian scale space of an embryonic image. The active contour model is applied on the images to refine embryo contours. Data cleaning methods are applied to smooth the jaggy contours caused by blurred embryo boundaries. The scale space theory is applied to improve the performance of the result. The active contour adjusts better to the object for finer scales. The proposed framework contains three components. In the first component, we find the connected components of the image. The second component is to find the largest component of the image. Finally, we analyze the largest component across scales by selecting the optimal scale corresponding to the largest component having largest area. The optimal scale at which maximum area is attained is assumed to give information about the feature being extracted. We tested the proposed framework on BDGP images, and the results achieved promising accuracy in extracting the targeting embryo.

Manual segmentation of cartilage tissue in micro CT images of mouse embryos is a very time consuming process and significantly increases the time required for the research of mammal facial structure development. This problem might be solved by using a fully-automatic segmentation algorithm. In this diploma thesis a fully-automatic segmentation method is proposed using a convolutional neural network trained on manually segmented data. The architecture of the proposed convolutional network is based on the U-Net architecture with it's encoding part substituted for the encoding part of the VGG16 classification convolutional neural network pretrained on the ImageNet database of labeled images. The proposed network achieves Dice coefficient 0.8731 ± 0.0326 in comparison to manually segmented images.

Automatická segmentace biologických struktur v mikro-CT datech je stále výzvou, protože často objekt zájmu (v našem případě obličejová chrupavka) není charakterizovaný unikátním jasem či ostrými hranicemi. V posledních letech se konvoluční neuronové sítě (CNNs) staly mimořádně populárními v mnoha oblastech počítačového vidění. Konkrétně pro segmentaci biomedicínských obrazů je široce používaná architektura U-Net. Nicméně v případě mikro-CT dat vyvstává otázka, zda by nebylo výhodnější použít 3D CNN. Diplomová práce navrhla CNN architekturu založenou na síti V-Net včetně metodologie pro předzpracování a postprocessing dat. Základní architektura byla dále optimalizována pomocí pokročilých architektonických modifikací jako jsou pyramidální modul dilatovaných konvolucí (ASPP modul), škálovatelná exponenciálně-lineární jednotka (SELU aktivační funkce), víceúrovňová kontrola učení (multi-output supervision) či bloky s hustými propojeními (Dense blocks). Pro učení sítě byly použity moderní přístupy jako zahřívání kroku učení (learning rate warmup) či AdamW optimalizátor. I přes to, že 3D CNN v úloze segmentace obličejové chrupavky nepřekonala U-Net, optimalizace zvýšila medián Dice koeficientu z 69,74 % na 80,01 %. Používání těchto pokročilých architektonických modifikací v dalším výzkumu je proto vřele doporučováno, jelikož můžou být přidány do libovolné architektury typu U-Net a zároveň výrazně zlepšit výsledky.

Heparan sulfate Proteoglycans (HSPG) are ubiquitous molecules with indispensable functions in various biological processes. Glypicans are a family of HSPG’s, characterized by a Gpi-anchor which directs them to the cell surface and/or extracellular matrix where they regulate growth factor signaling during development and disease. We report the identification and expression pattern of glypican genes from zebrafish. The zebrafish genome contains 10 glypican homologs, as opposed to six in mammals, which are highly conserved and are phylogenetically related to the mammalian genes. Some of the fish glypicans like Gpc1a, Gpc3, Gpc4, Gpc6a and Gpc6b show conserved synteny with their mammalian cognate genes. Many glypicans are expressed during the gastrulation stage, but their expression becomes more tissue specific and defined during somitogenesis stages, particularly in the developing central nervous system. Existence of multiple glypican orthologs in fish with diverse expression pattern suggests highly specialized and/or redundant function of these genes during embryonic development.

Heparan sulfate Proteoglycans (HSPG) are ubiquitous molecules with indispensable functions in various biological processes. Glypicans are a family of HSPG’s, characterized by a Gpi-anchor which directs them to the cell surface and/or extracellular matrix where they regulate growth factor signaling during development and disease. We report the identification and expression pattern of glypican genes from zebrafish. The zebrafish genome contains 10 glypican homologs, as opposed to six in mammals, which are highly conserved and are phylogenetically related to the mammalian genes. Some of the fish glypicans like Gpc1a, Gpc3, Gpc4, Gpc6a and Gpc6b show conserved synteny with their mammalian cognate genes. Many glypicans are expressed during the gastrulation stage, but their expression becomes more tissue specific and defined during somitogenesis stages, particularly in the developing central nervous system. Existence of multiple glypican orthologs in fish with diverse expression pattern suggests highly specialized and/or redundant function of these genes during embryonic development.

Trois études ont été réalisées sur l’adaptation d'une souche aviaire H9N2 de faible pouvoir pathogène sur trois modèles différents : embryons de poulet, poulet de chair puis hamster. La première visait à évaluer l'influence du passage embryonnaire du virus H9N2 sur la stabilité de la séquence d'acides aminés de l'Hémagglutinine 1 (HA1) et sa relation avec la pathogénicité. Elle a montré une augmentation significative de la pathogénicité du virus suite au passage embryonnaire, malgré la présence du même motif au site de clivage. La deuxième partie du travail a consisté à évaluer l'effet de trois passages consécutifs du virus H9N2 chez les poulets de chair sur sa pathogénicité et les variations moléculaires qui en sont responsables. Nous avons montré qu'un passage d'H9N2 faiblement pathogène dans les poulets de chair provoque une augmentation de la pathogénicité, associée à une stabilité de la séquence d'acides aminés de l'Hémagglutinine au site de clivage, et une variabilité dans la séquence de la tige de la neuraminidase. Le risque d'évolution d'une souche peu pathogène vers une souche hautement pathogène a été démontré. La troisième étude, menée sur des hamsters, a mis en évidence l'adaptation de la pathogénicité lors du franchissement de la barrière inter-espèces et a signalé le risque potentiel de maladies zoonotiques dans certains élevages
Three studies were carried out for studying the adaptation of avian H9N2 virus after embryonic, broilers or hamster passaging. The first study have shown that the pathogenicity increased significantly upon passaging in chicken embryos in spite of the presence of the same motif at the HA1 cleavage site. The second study assessed the impact of H9N2 viral passaging in broilers on amino acid sequences of the hemagglutin cleavage site and neuraminidase stalk, and their relatedness to pathogenicity. In that case, we observed that passaging leads to a trand of increase in pathogenic adaptability, associated with a conserved a. A. Sequence of the hemagglutinin cleavage site, and a variability in the sequence of the neuraminidase stalk. The third study demonstrates the impact of avian-H9N2 viral passaging in hamsters on its cross species-pathogenic adaptability, and variability of amino acid sequences of the hemagglutinin and neuraminidase stalk of the original and the differently passaged H9N2 viruses. The adaptation of avian virus to mammalian cells raise a concern of a possible public health threat since the mixing of avian with mammalian animal species is a common practise on farms and petshops of most developing countries

Tese de doutoramento em Ciências da Saúde
All vertebrate species present a segmented articulated body, which is easily observed at the vertebral column level. This segmented nature can be detected quite early during embryonic development with the periodic formation of repeated segments, the somites, along the anteriorposterior embryo body axis. These are formed as blocks of cells that bud off from the rostral tip of the mesenchymal presomitic mesoderm (PSM), which flanks the embryo midline structures, notochord and neural tube. Somites will later originate all segmented structures of the adult body such as vertebrae, intervertebral disks, ribs, the dermis of the back and all skeletal muscles, except those of the head. Somitogenesis occurs in a highly controlled and coordinated fashion and both the number of somites and the periodicity with which they are formed are constant and species specific. In the trunk region of the chick embryo a new pair of somites is formed every 90min. Underlying somite segmentation periodicity is an intrinsic molecular oscillator designated segmentation molecular clock. It was first described in the chick embryo with the demonstration of hairy1 cyclic expression in the PSM with a periodicity of 90min, which corresponds to the time required to form a pair of somites in the chick. It is now known that several genes belonging to distinct signaling pathways such as Notch, Wnt and Fgf present a similar oscillatory behavior. Periodic gene transcription has been described to occur in other vertebrates, other tissues and also in cultured cells. This suggests that the molecular events underlying somitogenesis are highly conserved and that gene oscillations may be a widespread mechanism experienced by many cells and tissue types. A second molecular regulation has been described to account for period somite formation, the wavefront of differentiation. PSM maturation is defined by two opposing gradients with crossregulatory activities: high Fgf/Wnt levels maintain the posterior PSM in an undetermined state and are counteracted by an anterior gradient of Retinoic Acid (RA). The confrontation between these opposing gradients and the molecular clock oscillations regulates somite formation in the anterior PSM. Thus, although the molecular clock operates along the entire PSM, only its anterior third is determined to form somites. Within this PSM region, only the medial-most PSM (M-PSM) possess intrinsic information for both somite formation and molecular clock gene expression, suggesting that M-PSM and lateral PSM (L-PSM) cells are differently committed to segment. Both somite formation and somitogenesis molecular clock are thought to operate independently of the embryo midline structures, notochord and neural tube, and the signaling molecules produced therein, namely Sonic hedgehog (Shh). Shh is the most studied member of the Hedgehog family, which has been implicated in several mechanisms during embryo development but has never been associated with somitogenesis regulation. However, quail/chick grafting experiments have suggested that the midline structures regulate symmetrical bilateral somite formation. Moreover, Shh knock-out mice lack the entire vertebrate column except for five to six ribs. In the present study, we have investigated the role of midline derived Shh in somitogenesis regulation. We show that chick PSM cells express both Shh receptors smoothened and patched, enabling them to respond to notochord-derived Shh. Upon notochord ablation, we observe a delay in somite formation accompanied by an increased period of the molecular clock oscillations. These alterations are recapitulated by Shh chemical inhibitors and rescued by an exogenous Shh source, indicating that Shh is the notochord-derived signal responsible for those perturbations. Segmentation rate recovers over time, accompanied by raldh2 overexpression. Accordingly, external RA supply rescues somite formation. Shh absence leads to an upregulation in the PSM of its downstream effectors, the Glis, in a repressor form and RA is thought to counteract their activity. We have also addressed the role of Shh in the differential specification of M- and LPSM. We show that a diffusible signal travels along the M-L anterior PSM axis and that Shh pathway is responsible for the recruitment of lateral cells by medial ones for timely somite formation. Quail/chick grafts experiments indicate that prospective L-PSM can be re-specified into a medial fate when placed into a PM-PSM position and we suggest that this is also mediated by Shh. A model for Shh activity during PSM specification and somitogenesis, as well as interactions with the diverse pathways operating in the PSM is proposed. Altogether, the results presented here provide concluding evidence that Shh signaling is a component of the intricate molecular machinery responsible for temporal control of somite formation, implicating this molecule in the somitogenesis machinery for the first time.
Os vertebrados são animais segmentados, o que é evidenciado cedo no desenvolvimento embrionário com o aparecimento de estruturas metaméricas, os sómitos, ao longo do eixo anteriorposterior do embrião. Estes formam-se periodicamente como blocos de células a partir da parte rostral da mesoderme pré-somítica (MPS), que surge como duas bandas de tecido mesenquimatoso a ladear as estruturas axiais do embrião, a notocorda e o tubo neural. Os sómitos originam todas as estruturas segmentadas presentes no animal adulto: vértebras, discos intervertebrais, costelas, a derme das costas e todos os músculos esqueléticos do tronco e membros. A somitogénese é um processo coordenado e tanto o número total de sómitos como o tempo necessário para a formação de cada par é constante e específico de cada espécie. Na região do tronco da galinha, um novo par de sómitos é formado a cada 90min. A regular a surpreendente periodicidade da formação de sómitos está o relógio molecular da segmentação, que foi primeiramente descrito em galinha aquando da observação de que o gene hairy1 apresenta um padrão de expressão cíclico. Este tem uma periodicidade de 90min, o que corresponde ao tempo necessário para se formar um novo par de sómitos na galinha. Actualmente sabe-se que diversos genes pertencentes a vias de sinalização como Notch, Wnt e Fgf apresentam também comportamento oscilatório. Esta transcrição periódica foi igualmente descrita noutros vertebrados, noutros tecidos e em linhas celulares, sugerindo que os mecanismos moleculares subjacentes à somitogénese são conservados e que este comportamento oscilatório pode ser um evento generalizado, ocorrendo em diferentes células e tecidos. A formação periódica de sómitos é também regulada por uma frente de maturação observada na MPS e definida por dois gradientes opostos: a MPS posterior é mantida num estado indiferenciado por elevados níveis de Fgf/Wnt, que são contrapostos por um gradiente anterior de ácido retinóico (AR). O confronto entre este gradiente de maturação e as oscilações do relógio regulam a formação de sómitos na MPS anterior. Deste modo, apesar de o relógio da somitogénese estar activo em toda a MPS, apenas a sua porção anterior está determinada para segmentar. Nesta região, verifou-se também que apenas a porção mais mediana da MPS (M-MPS) contém informação intrínseca para a formação de sómitos e a expressão de genes do relógio, o que sugere que a MPS-M e a MPS lateral (MPS-L) são diferentes no que diz respeito à sua capacidade de segmentação. Considera-se que tanto a formação de sómitos como o relógio molecular são processos independentes das estruturas axiais do embrião, a notocorda e o tubo neural, e das moléculas sinalizadoras aí produzidas, nomeadamente Sonic hedgehog (Shh). Esta faz parte da família de proteínas Hedgehog envolvida na regulação de diversos processos embrionários, mas que nunca foi implicada na somitogénese. Contudo, experiências usando quimeras codorniz/galinha indicam que as estruturas axiais regulam a formação bilateral e simétrica dos sómitos. Na verdade, nos ratinhos mutantes para Shh a coluna vertebral está ausente, apresentando apenas cinco a seis costelas. Com este trabalho pretendeu-se estudar o papel de Shh proveniente das estruturas axiais na regulação da somitogénese. A análise da expressão dos receptores de Shh smoothened e patched indica que a MPS está apta para responder à sinalização de Shh vinda da notocorda. Após remoção da notocorda, verificou-se um atraso na formação periódica de sómitos, que foi acompanhado por um aumento na periodicidade das oscilações moleculares. Foi possível recapitular estas alterações usando inibidores de Shh e restaurá-las com a adição de Shh, sugerindo que esta molécula é responsável pelas perturbações observadas após ablação da notocorda. Verifica-se que a periodicidade de formação de sómitos é recuperada ao longo do tempo, ao mesmo tempo que se observa uma sobre-expressão de raldh2. De facto, a adição de AR exógeno permite a recuperação da formação de sómitos. Na ausência de Shh, há um aumento da expressão na PSM dos seus efectores moleculares, os Glis, na sua forma repressora e pensa-se que o AR inibe a sua actividade. O papel de Shh na especificação da MPS M e L foi também avaliado. Verificámos a existência de um sinal difusível que percorre a MPS anterior ao longo do seu eixo M-L e mostramos que Shh é responsável pelo recrutamento de células laterais da MPS para integrar o sómito em formação. Para além disso, o uso de quimeras codorniz/galinha permitiu verificar que o território L da MPS prospectiva adquire um destino mediano quando posicionado na região M prospectiva, o que provavelmente se deve também a Shh. Apresentamos um modelo explicativo da actividade de Shh durante a especificação da MPS e na somitogénese e também da sua interacção com outras moléculas sinalizadoras que actuam na MPS. Os resultados aqui apresentados levam-nos a concluir que Shh é um componente adicional da complexa rede molecular subjacente ao controlo temporal da formação de sómitos, implicando esta via de sinalização na somitogénese pela primeira vez.
Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/27796/2006, PTDC/SAUOBD/099758/2008, PTDC/SAU-OBD/105111/2008)

During vertebrate embryo development, the body axis is subdivided into repeated structures, called somites. Somites bud off from an un-segmented tissue called the pre-somitic mesoderm (PSM) in a sequential and periodic manner, tightly controlled by an in built molecular clock, called the "segmentation clock". According to current understanding, the clock is comprised of: (i) an autonomous cellular oscillator consisting of an intracellular negative feedback loop of Her genes within the PSM cells, (ii) Delta-ligand and Notch-receptor coupling that facilitates synchronization of oscillators among the PSM cells, (iii) Tissue level waves of gene expression that emerge in the posterior PSM and move coherently towards anterior, leading to global arrest of oscillations in the form of somites. However, the movement of cellular oscillators within the PSM before the formation of somitic furrows, a prominent feature of the tissue as observed through this work has not been experimentally considered as a constituent of the segmentation clock so far. Our work aims to incorporate movement of cellular oscillators in the framework of the segmentation clock. It is well known from theoretical studies that the characteristics of relative motion of oscillators affect their synchronization properties and the patterns of oscillations they form. Particularly, theoretical studies by Uriu et al., PNAS (2010) suggest that cell movements promotes synchronization of genetic oscillations. Here, we established experimental techniques and image analysis tools to attain quantitative insight on (i) diffusion co-efficient of cellular oscillators, (ii) dynamics of a population of oscillators, within the PSM aiming towards concomitant understanding of the relationship between movement and synchronization of cellular oscillators. In order to quantitatively relate cellular oscillators and their motion within the PSM, I established imaging techniques that enabled visualization of fluorescenctly labeled nuclei as readouts of cell positions in live embryo, and developed dedicated segmentation algorithm and implemented tracking protocol to obtain nuclei positions over time in 3D space. Furthermore, I provide benchmarking techniques in the form of artificial data that validate segmentation algorithm efficacy and, for the first time proposed the use of transgenic embryo chimeras to validate segmentation algorithm performance within the context of in vivo live imaging of embryonic tissues. Preliminary analysis of our data suggests that there is relatively high cell mixing in the posterior PSM, within the same spatial zone where synchronous oscillations emerge at maximum speed. Also, there are indications of gradient of cell mixing along the anterior-posterior axis of the embryo. By sampling single cell tracks with the help of nuclei markers, we have also been able to follow in vivo protein oscillations at single cell resolution that would allow quantitative characterization of coherence among a population of cellular oscillators over time. Our image analysis work flow allows testing of mutant embryos and perturbation of synchrony dynamics to understand the cause-effect relationship between movement and synchronization properties at cellular resolution. Essentially, through this work, we hope to bridge the time scales of events and cellular level dynamics that leads to highly coordinated tissue level patterns and thereby further our understanding of the segmentation clock mechanism.

Dissertation presented as the partial requirement for obtaining a Master's degree in Data Science and Advanced Analytics, specialization in Data Science
Nos últimos anos, a aprendizagem profunda tem se tornado cada vez mais bem-sucedida quando aplicada para lidar com diferentes questões em diversos campos. Na análise de bioimagem, tem sido usada para extrair informações significativas de imagens microscópicas, onde aplicamos aprendizagem profunda a dados de microscopia de light-sheet para compreender o desenvolvimento inicial do sistema nervoso. Atualmente, sabe-se que o cérebro é responsável pela maioria de nossas ações voluntárias e involuntárias e que regula os processos fisiológicos em todo o corpo. No entanto, as barreiras técnicas deixaram muitas questões em aberto em relação ao desenvolvimento e função dos circuitos neuronais. Imagiologia provou ser uma técnica poderosa para responder a essas perguntas, embora as dificuldades em segmentar e rastrear neurônios individuais tenham retardado o progresso. Danionella translucida foi recentemente introduzida como um poderoso organismo modelo para estudos neurocientíficos devido a ter o menor cérebro de vertebrado conhecido e não desenvolver um crânio completo na idade adulta, tornando-a facilmente acessível para estudos de imagem. No entanto, o surgimento da atividade neural e subsequente montagem de circuitos neurais no desenvolvimento inicial do embrião não foi ainda caracterizado. Esta dissertação pretende fornecer uma descrição inicial de todo o processo de resolução celular, utilizando técnicas avançadas de microscopia e um método de inteligência artificial para segmentar e analisar os dados. Usamos microscopia de fluorescência de light-sheet para obter imagens do início e da coordenação da atividade neuronal da medula espinhal da Danionella translucida com alta resolução temporal e por longos períodos de tempo. Além disso, analisamos os dados com um algoritmo baseado em aprendizagem profunda para detetar, segmentar e rastrear no espaço e no tempo o sinal de cada neurônio. Focamos nossa análise nos picos de intensidade do sinal, ou seja, no momento em que os neurónios estavam a disparar, e encontramos mais atividade na região inferior do embrião, sugerindo uma correspondência com a extensão da cauda. Este trabalho demonstra que a combinação de métodos utilizados foi capaz de gerar imagens e analisar os dados com sucesso. Abre as possibilidades para um estudo mais aprofundado da rede neuronal da Danionella translucida, e para estudar sinais de imagens aglomeradas com resolução de célula única que, de outra forma, seriam muito complexas para serem analisadas.