Hox genes encode a family of transcription factors that play highly conserved regulatory roles in specifying the properties of tissues in developing embryos. Very little is known about how HOX proteins control the cellular and developmental processes governing morphogenesis through regulation of down-stream target genes. The goal of this research was to investigate on a genome-wide basis, the rules and principles which underlie the binding of different HOX proteins to target sites and understand the basis for their distinct specificities. I utilized the programmed differentiation of mouse embryonic stem cells into a neural fate with retinoids and genomic technologies to systematically investigate binding properties of two HOX proteins, HOXA1 and HOXBI and their cofactors PBX and MEIS. I analyzed the induction properties of the cells and the transcriptional dynamics and epigenetic states in Hox clusters to explore the differentiation process. An extensive and dynamic pattern of transcriptional activity indicates that Hox clusters generate a large number of non-coding RNAs which may impact their activation and chromatin states. Global identification of HOXB 1, HOXA1, PBX and MEIS binding regions by chromatin immune precipitation and high throughout sequencing (ChIP-seq) has generated insight into many potential Hox target genes. HOXA1 binding peaks generally overlapped with those of PBX and MEIS, supporting their roles as HOX co-factors. The sites bound by HOXBl uncovered new classes of binding motifs. Regulatory assays demonstrated that many of these novel motifs functioned as neuronal enhancers. Many HOXB I binding peaks have closely associated REST motifs and bind the REST repressor complex, which is important in neuronal differentiation. The close association of REST and HOXB 1 binding sites provides a mechanism for coordinating cell differentiation programs in neurogenesis. This research has uncovered novel properties of HO X proteins and their co-factors that underlie their role as master regulators of patterning and morphogenesis

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No description available.

A method was developed for recording the development of moss protonema using time-lapse video microscopy. This has provided a detailed record of the time-course of development from spore germination to the production of gametophores. Detailed records of the growth of primary and secondary chloronema, the transition of primary chloronema to caulonema, and the development of side-branches were obtained. Filaments were found to undergo the transition to caulonema earlier than previously thought. The majority of caulonemas ide-branches were found to begin as chloronema and switch to caulonema after one or two cell cycles. The early cell divisions of bud formation were found to follow a distinct pattern, which was upset by high concentrations of cytokinin and lanthanum. The response of caulonema apical cells to polarotropic light was recorded and compared to the gravitropic response. The time-lapse studies provided the basis for the further development of the quantitative analysis of protonemal branching patterns to include second and third side-branches of a sub-apical cell, and transitional caulonema. Analysing side-branch patterns should allow the detection of developmental mechanisms underlying the determination of side-branch fate. The potential of this method for assessing the effect of hormone treatments and for analysing more precisely mutant phenotypes was explored. An analysis of bud spacing was carried out to determine if the formation of a bud on a filament was inhibitory to other buds forming on the same filament. It was found, to the contrary, that buds tended to form in clusters. The hypothesis that the primary mode of action of cytokinin is an enhanced influx of calcium ions into the cell was investigated. Classical electrophysiology was used in order to detect any change in membrane potential suggestive of ionic fluxes in response to cytokinin treatment. No definitive changes in membrane potential were detected in response to cytokinin. This appeared to rule out the involvement of voltage-regulated channels in cytokinin action. The effects of some inhibitors used in studies of calcium on the moss protonemal system were examined. It is suggested that the concentrations commonly used had toxic effects that were not specific to calcium channels. The ionophore A23187 was used to characterise the protonemal response to a sustained influx of calcium. Some mutant strains were found to have a differential response to the ionophore. This may mean that they have mutations affecting their calcium regulatory system. Two new techniques of imaging calcium were used in order to detect changes in intracellular calcium in response to cytokinin. A method was developed for loading the dual wavelength fluorescent dye Indo-1 into moss protonema using iontophoretic microinjection, and intracellular calcium was imaged using ratio-image technology. Wild-type moss and some mutant strains were also successfully transformed with the gene for apoaequorin, and calcium luminescence measured in response to cold-shock and plant hormones. Some different responsesto temperatures hock were apparent in one of the transformed mutants. Preliminary experiments did not reveal any aequor independent calcium luminescence in response to cytokinin.

Οι πρωτεογλυκάνες, μόρια της εξωκυττάριας ουσίας, αλληλεπιδρούν μεταξύ τους και με άλλα μορφορυθμιστικά μόρια (γλυκοπρωτεΐνες, ιντεγκρίνες) καθώς επίσης και με αυξητικούς παράγοντες (FGF, TGF-β, Wnt, hedgehog). Οι πρωτεογλυκάνες ενεργοποιούν ποικίλα μονοπάτια μεταγωγής σήματος και επάγουν πλειοτροπικές αποκρίσεις από τα κύτταρα καθώς συμμετέχουν στον έλεγχο της κυτταρικής προσκόλλησης, μετανάστευσης και πολλαπλασιασμού, διαφοροποίησης και επιβίωσης κυττάρων και της μορφογένεσης ιστών και οργάνων. Η agrin, πρωτεογλυκάνη θειικής ηπαράνης, έχει ανιχνευτεί στις βασικές μεμβράνες των εγκεφάλου, νευρομυϊκής σύναψης, πνεύμονα και νεφρού. Έκφραση της agrin δεν έχει μελετηθεί στο πρώιμο έμβρυο. Στην παρούσα εργασία μας, με τη μέθοδο της RT-PCR και του ανοσοφθορισμού, μελετήσαμε πότε αρχίζει να εκφράζεται και πού εκφράζεται η agrin κατά τη διάρκεια της ανάπτυξης του πρώιμου εμβρύου όρνιθας από το στάδιο Χ (μορίδιο) μέχρι το ΗΗ17 (29 σωμίτες/οργανογένεση). Το mRNA της agrin πρωτοανιχνεύτηκε, σε χαμηλά επίπεδα, στο στάδιο ΗΗ1-ΗΗ2 (προχωρημένο βλαστίδιο) και η έκφρασή του ρυθμίζεται αναπτυξιακά. Η έκφραση του mRNA της agrin ήταν υψηλή στο στάδιο ΗΗ3 (μέσα σταδίου γαστριδίου), χαμηλότερη στο στάδιο ΗΗ4-5 (πρώιμο νευρίδιο), υψηλή στο στάδιο ΗΗ8-9 (5-6 σωμίτες) και χαμηλότερη στο στάδιο ΗΗ10 (10 σωμίτες). Ένταση φθορισμού της πρωτεΐνης agrin ανιχνεύτηκε και στις τρεις βλαστικές στιβάδες στο στάδιο ΗΗ4 (γαστρίδιο), καθώς και στο νευροεπιθήλιο στο στάδιο ΗΗ8 (4 σωμίτες). Στο στάδιο ΗΗ11 (13 σωμίτες) ο φθορισμός της agrin ήταν ισχυρός στο νευρικό σωλήνα και στους σωμίτες, ενώ ανιχνεύτηκε αξιοσημείωτη ένταση στο άνω τοίχωμα του εντέρου και στα προκαρδιακά κύτταρα. Στο στάδιο ΗΗ17, ο φθορισμός της agrin ήταν έντονος στο νευροεπιθήλιο του νευρικού σωλήνα, ενώ ανιχνεύτηκε και στις βασικές μεμβράνες του διεγκεφάλου και μυελεγκεφάλου. Ένταση έκφρασης της agrin εντοπίστηκε στο φακό και στον αμφιβληστροειδή του οφθαλμού και στο μυοτόμο, αλλά όχι στο δερμοτόμο και σκληροτόμο των σωμιτών. Έντονη ήταν η έκφραση της agrin στο ενδοκάρδιο και στο μυοκάρδιο στην καρδία και στα τοιχώματα του εντέρου. Επίσης, μελετήσαμε το ρόλο της agrin, μέσω αναστολής της λειτουργίας της, με τη χρήση μονοκλωνικών αντισωμάτων έναντι αυτής. Τα αποτελέσματά μας έδειξαν ότι η απουσία της πρωτεΐνης agrin προκαλεί ανωμαλίες στην επιθηλιοποίηση κυρίως των σωμιτών, του καρδιακού σωλήνα και του εντέρου. Επίσης, τα αποτελέσματά μας έδειξαν ότι η agrin φαίνεται να συμμετέχει στα σηματοδοτικά μονοπάτια που καθοδηγούν τα κύτταρα των νευρικών κρηπίδων κατά τη μετανάστευσή τους. Η agrin αρχίζει να εκφράζεται με την έναρξη των μορφογενετικών κινήσεων της γαστριδίωσης, ρυθμίζεται αναπτυξιακά και είναι σημαντικός ο ρόλος της στη διαφοροποίηση κυττάρων και στη μορφογένεση ιστών και οργάνων στο πρώιμο έμβρυο. / Agrin is a heparan sulfate proteoglycan and has been studied extensively in the late embryonic and postnatal nervous system and muscle. Agrin seems to play a critical role in the mediation of axonal growth and path finding. We studied the expression of agrin by RT-PCR and immunofluorescence in the chick embryo from stages X (morula) to HH17 (29 somites). Agrin mRNA was first detectable at low levels at stage HH1-HH2 (late blastula) and its expression was developmentally regulated. Expression of agrin mRNA was high at stage HH3 (intermediate streak), lower at stage HH4-5 (head process), high at stage HH8-9 (5-6 somites) and lower at stage HH10 (10 somites). Agrin protein fluorescence was strong in the three germ layers at stage HH4 (definitive streak) and was strong in the neuroepithelium at stage HH8 (4 somites). At stage HH11 (13 somites), agrin fluorescence was strong in the neural tube and somites and was intense in the gut roof plate and in the heart primordia. By stage HH17, agrin fluorescence was strong in the neuroepithelium of neural tube and was detected in the laminal surface of the diencephalon and myelencephalon. Agrin expression was strong in the lens and retina in the eye, was strong in the myotome but was not expressed in the dermotome and sclerotome in the somites, was strong in myocardium and endocardium in the heart and in the gut walls. Immunodetection of agrin was intense in neuroepithelium and mesenchymal tissues as they epithelialize. Inhibition of function of agrin by blocking antibodies showed that agrin is crucial for maintaining basement membrane integrity which mediates the epithelialization especially in somites, heart tube and gut. It is also possible that agrin participates in the signaling pathways that guide neural crest cell migration.

Ce travail présente un modèle théorique de morphogenèse animale, sous la forme d’un système complexe émergeant de nombreux comportements, processus internes, expressions et interactions cellulaires. Son implémentation repose sur un automate cellulaire orienté système multi-agents avec un couplage énergico-génétique entre les dynamiques cellulaires et les ressources.Notre objectif est de proposer des outils permettant l’étude numérique du développement de tissus cellulaires à travers une approche hybride (discrète/continue et qualitative/quantitative) pour modéliser les aspects génétiques, énergétiques et comportementaux des cellules. La modélisation de ces aspects s’inspire des principes de la théorie de la viabilité et des données expérimentales sur les premiers stades de division de l’embryon du poisson-zèbre.La théorie de la viabilité appliquée à la morphogenèse pose cependant de nouveaux défis en informatique pour pouvoir implémenter des algorithmes dédiés aux dynamiques morphologiques. Le choix de données biologiques pertinentes à considérer dans le modèle à proposer, la conception d’un modèle basé sur une théorie nouvelle, l’implémentation d’algorithmes adaptés reposant sur des processeurs puissants et le choix d’expérimentations pour éprouver nos propositions sont les enjeux fondamentaux de ces travaux. Les hypothèses que nous proposons sont discutées au moyen d’expérimentations in silico qui ont porté principalement sur l’atteignabilité et la capturabilité de formes de tissus ; sur la viabilité de l’évolution d’un tissu pour un horizon de temps ; sur la mise en évidence de nouvelles propriétés de tissus et la simulation de mécanismes tissulaires essentiels pour leur contrôlabilité face à des perturbations ; sur de nouvelles méthodes de caractérisation de tissus pathologiques, etc. De telles propositions doivent venir en appoint aux expérimentations in vitro et in vivo et permettre à terme de mieux comprendre les mécanismes régissant le développement de tissus. Plus particulièrement, nous avons mis en évidence lors du calcul de noyaux de viabilité les relations de causalité ascendante reliant la maintenance des cellules en fonction des ressources énergétiques disponibles et la viabilité du tissu en croissance. La dynamique de chaque cellule est associée à sa constitution énergétique et génétique. Le modèle est paramétré à travers une interface permettant de prendre en compte le nombre de coeurs à solliciter pour la simulation afin d’exploiter la puissance de calcul offerte par les matériels multi-coeurs. / This work presents a theoretical model of animal morphogenesis, as a complex system from which emerge cellular behaviors, internal processes, interactions and expressions. Its implementation is based on a cellular automaton oriented multi-agent system with an energico-genetic coupling between the cellular dynamics and resources. Our main purpose is to provide tools for the numerical study of tissue development through a hybrid approach (discrete/continuous and qualitative/quantitative) that models genetic, behavioral and energetic aspects of cells. The modeling of these aspects is based on the principles of viability theory and on experimental data on the early stages of the zebrafish embryo division. The viability theory applied to the morphogenesis, however, raises new challenges in computer science to implement algorithms dedicated to morphological dynamics. The choice of relevant biological data to be considered in the model to propose, the design of a model based on a new theory, the implementation of suitable algorithms based on powerful processors and the choice of experiments to test our proposals are fundamental issues of this work. The assumptions we offer are discussed using in silico experiments that focused on the reachability and catchability of tissue forms ; on the viability of the evolution of a tissue for a time horizon ; on the discovery of new tissue properties and simulation of tissue mechanisms that are fondamental for their controllability face to disruptions ; on new pathological tissue characterization methods, etc. Such proposals must come extra to support experiments in vitro and in vivo and eventually allow a better understanding of the mechanisms governing the development of tissues.In particular, we have highlighted through the computing of viability kernels the bottom causal relationship between the maintenance of cells according to available energy resources and the viability of the tissue in growth. The model is set through an interface that takes into account the number of cores to solicit for simulation in order to exploit the computing power offered by multicore hardware.