The term "pattern formation" refers to the process by which order unfolds in development. The present thesis deals with a particular aspect of molecular pattern formation during vertebrate embryogenesis. The model system in the focus of this study is the zebrafish, Danio rerio. In the early developmental phases of the zebrafish, Fibroblast growth factors (Fgfs) are involved in the molecular patterning of various tissues, including two regions of the brain, the forebrain and the midbrain-hindbrain region, affecting cellular processes as diverse as cell proliferation, differentiation, and axonal targeting. The goal of this study was to better understand the mechanisms by which Fgf signaling regulates pattern formation and embryogenesis. I addressed this question on several levels, investigating the extent of intracellular signaling (MAPK activation) relative to sources of Fgf expression, and the transcriptional responses of cells to Fgf signaling during embryogenesis. By a macroarray analysis, I identified putative transcriptional targets of Fgf signaling in late gastrulation, providing a set of molecules that are likely to act as functional players in relaying the patterning information encoded by Fgf signals. Among those are the secreted signaling molecules Chordin and Wnt8, as well as Isthmin, a novel secreted molecule that I found capable to interfere with anterior embryonic patterning. In addition, I identified two ETS domain transcription factors, Erm and Pea3, which constitute bona fide integrators of FgfR signaling. By gain- and loss-of-function studies, I demonstrate that transcript levels of erm and pea3 are tightly regulated by Fgf signaling. Detailed analysis of the expression patterns of erm and pea3 along with other Fgf target genes also provides evidence for a differential read-out of Fgf concentration in the embryo, consistent with a role of Fgf as a vertebrate morphogen. The discovery of novel molecular components downstream of Fgf receptor activity paves a way to characterize previously unknown or underestimated developmental roles of Fgfs in the molecular patterning of the forebrain, the eye and parts of the neural crest.

Les hydrocarbures aromatiques polycycliques (HAP) sont des polluants organiques persistants qui sont présents en mélange complexe dans l’environnement et notamment dans les milieux aquatiques. Ils s’adsorbent sur les particules en suspension, s’accumulent dans les sédiments et dans les premiers maillons de la chaîne trophique. L’objectif de cette étude était de mesurer les effets à long terme d’une contamination par des HAP chez un modèle de vertébrés : le poisson zèbre. Dans un premier temps, des embryons ont été exposés pendant les 4 premiers jours de leur développement sur du sédiment naturel enrobé de 3 HAP individuel (phénanthrène, pyrène et benzo[a]pyrène) puis transférés et élevés en eau propre. Dans un deuxième temps, des poissons ont été exposés par voie alimentaire tout au long du cycle de vie et dès le premier repas à 3 fractions de HAP à doses environnementales enrobées sur les aliments : une fraction pyrolytique (PY) extraite d’un sédiment échantillonné dans la Seine et deux fractions pétrogéniques (un fuel lourd (HO) provenant de l’Erika et un pétrole brut type Arabian light (LO)). Les effets à long terme sur la croissance, la survie, la reproduction et le comportement ont été évalués à différents âges. Ces effets ont pu être observés à plusieurs échelles, i.e. de la molécule, aux tissus et organes puis à l’échelle de l’individu. Globalement, chez les poissons contaminés, la croissance est ralentie, la survie larvaire diminuée chez les HO, la fonction reproduction est altérée fortement à la fois au niveau hormonal, tissulaire et phénotypique ainsi que les réponses comportementales léthargiques ou hyper-réactives selon les cas. L’exposition par voie sédimentaire a révélé des effets visibles lorsque les individus atteignaient 6 mois soulignant l’existence d’effets tardifs, notamment sur les réponses comportementales, d’une exposition très précoce y compris chez la descendance. Quant à l’exposition par voie alimentaire, elle a montré une gradation des effets. Toutes fonctions confondues, les résultats obtenus permettent d'établir une hiérarchie dans la toxicité des fractions étudiées : PY < LO ≈ HO. L'approche multi-échelle utilisée dans ce travail a permis, en partant d'informations acquises au niveau moléculaire, tissulaire et individuel, d'aboutir à une compréhension intégrée des conséquences qu'une exposition à des HAP peut avoir sur l'aptitude à la survie des poissons dans leur milieu naturel ainsi qu'à leur capacité à participer au renouvellement des générations futures. / Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants (POPs) which are found in complex mixtures in the environment including aquatic ecosystems. They adsorb on particles, accumulate in sediment and in the first link of the food web. The aim of this study was to measure long term effects of PAHs contamination on a vertebrate model : the zebrafish. In a first study, embryos were exposed on natural sediment spiked with 3 individual PAH (phenanthrene, pyrene and benzo[a]pyrene) during the first four days of their development. In a second study, to evaluate the consequences of long-term chronic exposure to PAHs, zebrafish were exposed, from their first meal (5 days post fertilisation) until they became reproducing adults, to diets spiked with three PAHs fractions at environmentally relevant concentrations (0.3X, 1X and 3X; with the 1X concentration being in the range of 4.6-6.7 μg.g-1 for total quantified PAHs). The fractions used were representative of PAHs of pyrolytic (PY) origin or of two different oils of differing compositions (a heavy fuel (HO) and a light crude oil (LO)). Long term effects on growth, survival, reproduction and behavior were evaluated at different ages. Effects were observed at molecule, tissue, organ and individual scales. In general, for contaminated fish, growth was reduced, larval survival decreased in HO, and reproduction was strongly impaired at hormonal, tissue and phenotypic levels, and behavioral responses were lethargic or hyperactive depending on fractions and stages. Sediment exposure still revealed visible effects when fish were 6 month old. These results hereby showed significant late effects, especially in behavioral responses after an early exposure, including in larvae issued from contaminated fish. In spiked diets contaminated fish, we observed a gradation of effects. Combining all functions, we established of hierarchy in toxicity in the studied fractions: PY < LO ≈ HO. Finally, our results gathered at a multiscale from molecule, to tissue and individuals, indicate that PAH mixtures of different compositions, representative of situations encountered in the wild, can promote lethal and sublethal effects which are likely to be detrimental for fish survival and recruitment into future generations.

Nous proposons dans cette thèse de caractériser quantitativement la variabilité à différentes échelles au cours de l'embryogenèse. Pour ce faire, nous utilisons une combinaison de modèles mathématiques et de résultats expérimentaux. Dans la première partie, nous utilisons une petite cohorte d'oursins digitaux pour construire une représentation prototypique du lignage cellulaire, reliant les caractéristiques des cellules individuelles avec les dynamiques à l'échelle de l'embryon tout entier. Ce modèle probabiliste multi-niveau et empirique repose sur les symétries des embryons et sur les identités cellulaires; cela permet d'identifier un niveau de granularité générique pour observer les distributions de caractéristiques cellulaires individuelles. Le prototype est défini comme le barycentre de la cohorte dans la variété statistique correspondante. Parmi plusieurs résultats, nous montrons que la variabilité intra-individuelle est impliquée dans la reproductibilité du développement embryonnaire. Dans la seconde partie, nous considérons les mécanismes sources de variabilité au cours du développement et leurs relations à l'évolution. En nous appuyant sur des résultats expérimentaux montrant une pénétrance incomplète et une expressivité variable de phénotype dans une lignée mutante du poisson zèbre, nous proposons une clarification des différents niveaux de variabilité biologique reposant sur une analogie formelle avec le cadre mathématique de la mécanique quantique. Nous trouvons notamment une analogie formelle entre l'intrication quantique et le schéma Mendélien de transmission héréditaire. Dans la troisième partie, nous étudions l'organisation biologique et ses relations aux trajectoires développementales. En adaptant les outils de la topologie algébrique, nous caractérisons des invariants du réseaux de contacts cellulaires extrait d'images de microscopie confocale d'épithéliums de différentes espèces et de différents fonds génétiques. En particulier, nous montrons l'influence des histoires individuelles sur la distribution spatiales des cellules dans un tissu épithélial. / We propose in this thesis to characterize variability quantitatively at various scales during embryogenesis. We use a combination of mathematical models and experimental results. In the first part, we use a small cohort of digital sea urchin embryos to construct a prototypical representation of the cell lineage, which relates individual cell features with embryo-level dynamics. This multi-level data-driven probabilistic model relies on symmetries of the embryo and known cell types, which provide a generic coarse-grained level of observation for distributions of individual cell features. The prototype is defined as the centroid of the cohort in the corresponding statistical manifold. Among several results, we show that intra-individual variability is involved in the reproducibility of the developmental process. In the second part, we consider the mechanisms sources of variability during development and their relations to evolution. Building on experimental results showing variable phenotypic expression and incomplete penetrance in a zebrafish mutant line, we propose a clarification of the various levels of biological variability using a formal analogy with quantum mechanics mathematical framework. Surprisingly, we find a formal analogy between quantum entanglement and Mendel’s idealized scheme of inheritance. In the third part, we study biological organization and its relations to developmental paths. By adapting the tools of algebraic topology, we compute invariants of the network of cellular contacts extracted from confocal microscopy images of epithelia from different species and genetic backgrounds. In particular, we show the influence of individual histories on the spatial distribution of cells in epithelial tissues.

The term &amp;quot;pattern formation&amp;quot; refers to the process by which order unfolds in development. The present thesis deals with a particular aspect of molecular pattern formation during vertebrate embryogenesis. The model system in the focus of this study is the zebrafish, Danio rerio. In the early developmental phases of the zebrafish, Fibroblast growth factors (Fgfs) are involved in the molecular patterning of various tissues, including two regions of the brain, the forebrain and the midbrain-hindbrain region, affecting cellular processes as diverse as cell proliferation, differentiation, and axonal targeting. The goal of this study was to better understand the mechanisms by which Fgf signaling regulates pattern formation and embryogenesis. I addressed this question on several levels, investigating the extent of intracellular signaling (MAPK activation) relative to sources of Fgf expression, and the transcriptional responses of cells to Fgf signaling during embryogenesis. By a macroarray analysis, I identified putative transcriptional targets of Fgf signaling in late gastrulation, providing a set of molecules that are likely to act as functional players in relaying the patterning information encoded by Fgf signals. Among those are the secreted signaling molecules Chordin and Wnt8, as well as Isthmin, a novel secreted molecule that I found capable to interfere with anterior embryonic patterning. In addition, I identified two ETS domain transcription factors, Erm and Pea3, which constitute bona fide integrators of FgfR signaling. By gain- and loss-of-function studies, I demonstrate that transcript levels of erm and pea3 are tightly regulated by Fgf signaling. Detailed analysis of the expression patterns of erm and pea3 along with other Fgf target genes also provides evidence for a differential read-out of Fgf concentration in the embryo, consistent with a role of Fgf as a vertebrate morphogen. The discovery of novel molecular components downstream of Fgf receptor activity paves a way to characterize previously unknown or underestimated developmental roles of Fgfs in the molecular patterning of the forebrain, the eye and parts of the neural crest.

Die salzhaltigen Abwässer des Kalibergbaues führen in natürlichen Süßwassersystemen zu Ionenimbalanzen und einer sekundären Versalzung der Werra in Deutschland. Die Auswirkungen dieser Ionenungleichgewichte auf die Reproduktion von Süßwasserfischen wurden unter Verwendung der Modellfischart Danio rerio untersucht. Angepasst an die aktuellen Grenzwerte für die Einleitung der Abwässer aus dem Kalibergbau wurden fünf verschiedene Kombinationen erhöhter Ionenkonzentrationen getestet. Während eines partiellen Lebenszyklustests wurden adulte Fische 35 Tage lang in den Salzkombinationen exponiert. Anschließend wurden die Nachkommen dieser Fische bis zum 8. Tag nach der Befruchtung den gleichen Salzkonzentrationen ausgesetzt. Zusätzlich wurden Early-Life-Stage-Tests (ELST) mit den Nachkommen von nicht exponierten Eltern durchgeführt. Im Vergleich zu natürlich vorkommenden Ionenkonzentrationen und -verhältnissen in Süßwassersystemen war die Befruchtungsrate der Eier für alle Ionenkombinationen signifikant niedriger, die Koagulations- und Deformationsrate jedoch signifikant höher. Die ELST ergaben bei den Embryonen und Larven u.a. vorzeitige und verlängerte Schlupfzeiten, verringerte Überlebensraten, erhöhte Deformationsraten und Herzschlagfrequenzen sowie Unregelmäßigkeiten des Ganzkörpergehalts von K, Mg, Na und Ca und des Ganzkörper-Ionenverhältnisses von Ca:Mg bei erhöhten Ionenkonzentrationen und Ionenimbalanzen. Im Vergleich zu den Effekten auf die Fortpflanzung und Entwicklung der Nachkommen waren die Effekte auf die adulten Tiere moderat. Die Ergebnisse dieser Dissertation zeigen, dass Teillebenszyklus-Tests besser als Fischeitests oder ELST geeignet sind, die Effekte von durch Abwasser verursachten Ionenimbalanzen, auf die Fortpflanzung und frühe Entwicklung von Süßwasserfischen, zu untersuchen. Weder die momentan gültigen noch die zukünftig herabgesetzten Grenzwerte bis 2027 sind danach als unschädlich, für die Reproduktion von Süßwasserfischen, zu betrachten. / The potash mining industry discharges saline effluents which generate ion imbalances in natural freshwater systems and cause severe secondary salinization in the river Werra in Germany. The effects of these ion imbalances on reproduction of freshwater fish were investigated using the fish model species Danio rerio. Five different combinations of elevated ion concentrations adjusted to the current threshold values for the discharge of potash mining effluents in Germany were tested. During a partial life cycle test, adult fish were exposed to the salt combinations for 35 days. Subsequently, the offspring were exposed to the same concentrations until hatch, and the larvae were further reared at the exposure concentrations from hatch until the 8th day post fertilization. Additionally, a standard early life stage test with offspring from unexposed parents was performed. Compared to naturally occurring ion concentrations and ratios in freshwater systems, the fertilization rate of the eggs was significantly lower for all ion combinations, while coagulation and deformation rates were significantly higher. Early life stage tests on embryos and larvae revealed premature and prolonged hatching times, reduced survival rates, increased deformation and heart rates and irregularities in whole body content of K, Mg, Na and Ca and whole body Ca:Mg ratios at elevated ion concentrations and imbalances of ion ratios. Compared to effects on reproduction and development of the offspring, effects on the parental generation were moderate. The results of this dissertation indicate that partial life cycle tests instead of fish egg tests or ELST are needed to examine most sensitively the effects of ion imbalances caused by potash mining effluents on reproduction and early development of freshwater fish. Neither the recent German threshold values nor the future reduced values until 2027 are safe for the reproduction of freshwater fish.

Submitted by Hellen Luz (hellencrisluz@gmail.com) on 2017-09-18T19:58:53Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_ParametrosEscototaxiaModelo.pdf: 1500226 bytes, checksum: 275abf0aa72799c079b3c856bd268f80 (MD5) / Rejected by Irvana Coutinho (irvana@ufpa.br), reason: Aguardar as orientações on 2017-10-10T16:49:03Z (GMT) / Submitted by Hellen Luz (hellencrisluz@gmail.com) on 2017-10-17T17:42:47Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_ParametrosEscototaxiaModelo.pdf: 1500226 bytes, checksum: 275abf0aa72799c079b3c856bd268f80 (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-11-22T18:35:53Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_ParametrosEscototaxiaModelo.pdf: 1500226 bytes, checksum: 275abf0aa72799c079b3c856bd268f80 (MD5) / Made available in DSpace on 2017-11-22T18:35:53Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_ParametrosEscototaxiaModelo.pdf: 1500226 bytes, checksum: 275abf0aa72799c079b3c856bd268f80 (MD5) Previous issue date: 2011-02-24 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Determinados peixes teleósteos apresentam um caractere comportamental de escototaxia, a preferência por ambientes escuros a ambientes claros. O presente trabalho buscou avaliar alguns parâmetros do comportamento exploratório do paulistinha (Danio rerio Hamilton 1822) na caixa claro-escuro. A evitação do compartimento branco apresenta um padrão bifásico, com um aumento na evitação precedendo uma diminuição (Experimento 1). Essa mesma evi- tação não habitua frente à exposição repetida, independentemente do intervalo inter-sessões, ao contrário da locomoção total (Experimentos 2 e 3); a exposição forçada ao compartimento branco não altera a exploração subseqüente nem a locomoção total (Experimento 4). Esses resultados sugerem que a novidade do aparato não é a dimensão controladora da escototaxia; além disso, esses resultados também sugerem que a preferência pelo compartimento preto não é causada simplesmente pela esquiva ao compartimento branco, ainda que essa certamente tenha um papel importante. O papel da aversividade do compartimento branco foi investigado na segunda série de experimentos. A iluminação sobre o compartimento branco é um fator ansiogênico, já que aumentá-la diminui o tempo que os animais passam sobre este sem afetar a locomoção total (Experimento 5). Esse fenômeno parece se dever a uma diminuição na ca- pacidade de camuflagem com o substrato (cripse), já que alterar a cor do compartimento bran- co para cinza aumenta o tempo passado neste, enquanto alterar a cor do compartimento preto para cinza aumenta o tempo passado no compartimento branco, ambas as alterações não afe- tando a locomoção total (Experimento 6). Além disso, o aumento da proporção ocupada pelo compartimento branco no aparato (de 50% para 75%) diminui o tempo passado neste sem afetar a locomoção total (Experimento 7). Esses resultados sugerem que o compartimento branco é aversivo, e portanto a preferência pelo compartimento preto não é causada simples- mente por propriedades reforçadoras positivas desse ambiente. Tomados em conjunto, os re- sultados das duas séries de experimentos sugerem que a escototaxia resulta do conflito apro- ximação-evitação. O Experimento 8 representa uma manipulação ambiental comum que altera a ansiedade em roedores, o enriquecimento; aqui, animais criados em ambiente enriquecido por duas semanas apresentam menor evitação do compartimento branco. Conclui-se que a escototaxia apresenta bom valor preditivo. / Certain teleost fishes present a behavioral trait of scototaxis, the preference for dark environ- ments and not bright ones. The present work tried to evaluate some parameters of the explora- tory behavior of the zebrafish (Danio rerio Hamilton 1822) in the black/white tank, aiming to establish the reliability of measures in different contexts. White compartment avoidance presents a biphasic pattern, with an increase in avoidance preceding a decrease (Experiment 1). This same avoidance does not habituate to repeated exposure, independently of the inter- session interval, on the contrary of total locomotion (Experiments 2 and 3); forced exposure to the white compartment does not alter the subsequent exploratory behavior (Experiment 4). These results suggest that novelty is not the controlling dimension of scototaxis; besides, these results also suggest that the preference for the Black compartment is not caused simply by avoidance of the white compartment, although this certainly has an important role. The role of the aversiveness of the white compartment was assessed in a second series of experi- ments. The illumination above the white compartment is an anxiogenic factor, since increas- ing it decreases the time spent in the white compartment without affecting total locomotion (Experiment 5). This phenomenon seems to be due to a decrease in the capacity to camouf- lage with the substrate (crypsis), since altering the color of the white compartment to grey increases its exploration, while altering the color of the black compartment to grey increases the time spent in the white compartment (Experiment 6). Besides, the increase in the propor- tion of the apparatus occupied by the white compartment (from 50% to 75%) decreases the time spent in it (Experiment 7). These results suggest that the white compartment is aversive, and therefore the preference for darkness is not caused simply by positive reinforcing proper- ties of the black compartment. Taken together, the results of both series of experiments sug- gest that scototaxis results from an approach-avoidance conflict. Experiment 8 represents a common environmental manipulation which alters anxiety in rodents, environmental enrich- ment; here, animals raised in an enriched aquarium for two weeks present less white avoid- ance. It is concluded that scototaxis has good construct validity.

Tesis por compendio de publicaciones / La luz y los patrones de alimentación influyeron sobre el comportamiento, fisiología y reloj molecular de dos peces teleósteos. Se clonaron dos criptocromos de lubina (cry1 y cry2), expresándose en todos los tejidos estudiados y mostrando ritmos diarios de expresión en cerebro, hígado y corazón (excepto cry2). La fase (día/noche) de alimentación a demanda en lubina afecta a los patrones diarios de actividad amilasa intestinal y expresión de period1 en tejidos periféricos (i.e. hígado), pero no en cerebro. Durante las inversiones estacionales de alimentación de la lubina, mostraron mayores valores de glucosa sanguínea durante la fase de alimentación, revelando también los mayores valores medios en lubinas nocturnas invierno. El pez cebra utilizó un nuevo comedero a demanda, mostrando independencia de fase entre ritmos circadianos de alimentación (nocturno) y locomoción (diurno), con diferentes periodicidades. Además la actividad y posición del pez cebra resultó espectro-dependiente, provocando mayores efectos las longitudes de onda cortas. / Thesis summary in English Light and feeding patterns influence on the behaviour, physiology and molecular clock of two teleost fish. Two cryptochromes (cry1 and cry2) were cloned, being expressed in all studied tissues and showing daily rhythm of expression in brain, liver and heart (except cry2). The self-seabass feeding phase (day/night) affected on daily patterns of intestinal amylase activity and on the period1 expression in peripheral tissues (i.e. liver), but not in brain. During the seasonal feeding inversions in seabass, the highest blood glucose levels were shown during their feeding phase, revealing also the highest values in nocturnal seabass in winter. Zebrafish used a new self-feeding system, displaying phase-independence between feeding (nocturnal) and locomotor (diurnal) circadian rhythms, with different periodicities. Moreover, the zebrafish activity and vertical position were spectrum-dependent, being the main effects provoked by short-wavelengths.

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