Prenatal alcohol exposure is known to have many profound detrimental effects on human fetal development (fetal alcohol spectrum disorders), which may manifest into lifelong disabilities. Although hearing deficiency is a recognized effect, how alcohol affects the auditory/vestibular systems has not been well studied. This is the first study that used the zebrafish, Danio rerio, as a model organism to investigate morphological effects of alcohol on the developing octavolateral system (auditory, vestibular and lateral line). Zebrafish embryos of two hours post fertilization (hpf) were treated in 2% alcohol for 48 hours and screened at 72 hpf for morphological defects of the inner ear and lateral line neuromasts. Inner ear size and otoliths of zebrafish from both alcohol-treated and control groups were examined using light microscopy. Zebrafish were stained with fluorescent vital dyes to visualize lateral line hair cells using confocal microscopy. The size of neuromasts and length of kinocilia were measured using scanning electron microscopy. Results reveal that alcohol treatment during the early development impairs the formation of otoliths, neuromasts and their hair cells, as well as ear size and kinocilium length. We observed several otolith phenotypes including zero, one, two abnormal, two normal and multiple otoliths for alcohol-treated zebrafish. Fetal alcohol exposure appears to severely impact the size of both anterior and posterior lateral line neuromasts. Our results indicate that early fetal alcohol exposure most likely results in functional defects of the octavolateral system due to inner ear and lateral line dysmorphology.

Cette thèse est basée sur deux projets principaux: (1) l'étude de la réponse innée antivirale du poisson zèbre, en particulier des voies de signalisation des interférons de type I et (2) l'étude de leucocytes particuliers localisés au voisinage des neuromastes, structures permettant au poisson de percevoir le flux d'eau qu'il traverse et constituant potentiellement des brèches dans la peau de l'animal. La voie des IFN de type I est le principal composant de l'immunité antivirale innée. Dans cette thèse, deux types de protéines de poisson-zèbre capables d'augmenter l'induction des IFN de type I ont été étudiés. Nous avons montré que les deux orthologues chez le poisson zèbre du facteur de transcription à domaine BTB/POZ nommé PLZF (Promyelocytic leukemia zinc finger) augmentent l'induction de l'Ifn par différents stimuli. Ce travail montre que l'implication de PLZF dans la régulation de la voie IFN est ancienne et peut intervenir à différents niveaux de la voie Ifn. Le second modèle étudié est le gène Ftr83 (finTRIM83), qui appartient à un groupe de TRIM très diversifié et spécifique des poissons. L'expression de cette protéine TRIM induit une très forte induction des Ifn de type I et une protection contre différents virus, via la surexpression de différents ISGs. Ftr83 est exprimé dans la peau et dans les branchies, régions très exposées aux pathogènes, et son niveau d'expression est fortement corrélé au niveau d'expression de l'Ifn. Dans cette thèse, une lignée transgénique où les cellules spécifiquement fluorescentes évoquent des leucocytes localisés à proximité des neuromastes a été étudiée. Ces cellules ont été observées, leurs mouvements suivis et leur transcriptome analysé par séquençage profond après tri au FACS. Cette analyse a identifié des marqueurs typiques de cellules myéloides (macrophages, dendritiques); ces observations sont cohérentes avec l'idée de cellules sentinelles autour des neuromastes. / This thesis is based on the studies of two aspects of innate immunity in zebrafish: 1) proteins involved in the regulation of type I interferon (Ifn) and 2) specialized myeloid cells that patrol neuromasts – mechano-sensory organs embed in the skin that could be pathogen entry sites. In this thesis two different proteins are described for the capability to enhance Ifn production. In one part, two zebrafish orthologues of mammalian transcription factor PLZF (Promyelocytic leukemia zinc finger) are shown to augment type I Ifn and ISG in response to double-stranded RNA viruses. PLZF is a BTB/POZ transcription factor that was recently shown to induce a subset of ISG, in human and mouse. Thus, zebrafish Plzf proteins can operate at multiple steps in the Ifn system. Furthermore, their activity was not dependent on the presence of BTB-domain implying that the underlying mechanism is different from the usual mode of action of BTB/POZ transcription factors. In the second part, fish-specific TRIM ubiquitin ligase - Ftr83 (Fish novel tripartite motif protein 83), mounted a strong anti-viral protection through the upregulation of Ifn. Interestingly a strong correlation between the expression of Ftr83 and Ifn was seen in the gills suggesting that Ftr83 might maintain a low basal level of Ifn signalling in organs constantly exposed to pathogens. In the second part, a GFP reporter transgenic line called medaktin:EGFP has been characterized. It marks leukocytes in the skin surrounding neuromasts. Deep sequencing revealed that these cells express several macrophage and dendritic cell markers, including genes involved in autophagy, microbicidial functions and antigen presentation, thus highlighting them as possible sentinel cells.

No description available.

O sistema látero-sensorial dos peixes é responsável, dentre outras funções, pela percepção de vibrações e de movimentos na água. A unidade funcional desse sistema é denominada neuromasto, o qual pode estar presente na superfície ou em ranhuras da pele, ou também em canais que percorrem ossos dérmicos, alcançando a superfície externa da pele por intermédio de poros. O padrão de ossificação dos canais sensoriais cefálicos é conservado em diversas linhagens de peixes de tal forma que esse complexo morfológico representa uma rica fonte de informações filogenéticas, sendo amplamente utilizado em análises cladísticas de vários grupos de Ostariophysi, como, por exemplo, em Siluriformes. No entanto, o sistema láterosensorial cefálico de Characiformes, uma das mais diversificadas ordens de teleósteos e também um membro de Ostariophysi, nunca havia sido descrito em detalhes, sendo suas implicações filogenéticas pouco investigadas. O presente trabalho teve como objetivo realizar uma análise exploratória da homologia dos diversos elementos que compõem o sistema látero-sensorial em Characiformes, especificamente de: 1) levantar informações sobre os padrões de ramificação e de ossificação dos canais sensoriais cefálicos de todas as suas principais linhagens; 2) mapear as terminações destes ramos, os poros sensoriais, que se abrem na superfície da pele; e 3) identificar as principais implicações filogenéticas relacionadas a este sistema dentre os Characiformes e demais Ostariophysi. Para tal, foram analisados um total de trinta e seis táxons da ordem Characiformes, englobando representantes de todas as suas famílias e principais subfamílias. Além destes, outros treze componentes da ordem Ostariophysi, bem como sete demais Teleostei foram incluidos na análise como representantes do grupo-externo. Um dos principais resultados do presente estudo foi a constatação de que a disposição dos canais do sistema látero-sensorial cefálico, e o número de poros associados a cada um destes canais pouco variou dentre as diferentes famílias de Characiformes, mantendo-se também relativamente contantes nos demais Ostariophysi, e apresentando-se altamente informativas em um contexto filogenético. A complexidade morfológica desse sistema deixa implícito que o mesmo pode representar uma grande fonte de informações anatômicas combinando-os com demais caracteres morfológicos. Os resultados apresentados no presente trabalho indicam que os canais e poros da linha látero-sensorial cefálica constituem mais uma evidência de que novas fontes de caracteres morfológicos, apesar de não tradicionais, têm muito a contribuir com o entendimento das relações de parentesco entre peixes. / The laterosensory system of fishes is responsible, among other functions, for the perception of water movement and vibration. The functional unit of this system is the neuromast, which can be present on the skin surface or in grooves on it, or also in canals that are distributed along dermic bones, reaching the skin surface by pores. The ossification pattern related to the laterosensory canals is conserved among distinct fish lineages so that this morphological complex may represent a rich source of phylogenetic information. This system has been extensively used in cladistics analysis on several Ostariophysans, such as in Siluriformes and Cypriniformes. However, the laterosensory system of Characiformes, one of the most diverse order of teleost and also a member of the superorder Ostariophysi, has not been described in details yet, and its phylogenetic implications are poorly investigated. The present study aimed on performing an investigation on the laterosensory system components of Characiformes, having as its main goals: 1) a description of the cephalic laterosensory canals and branches for all main lineages of the order; 2) a characterization the canals ending points, i.e. the pores that open on the skin surface; and 3) a interpretation of the main philogenetical implications related to the laterosensory system among Characiformes and Ostariophysi. In order to accomplish these goals, a total of thirty-six Characiformes taxa were analysed, encompassing all families and main subfamilies of this order. Added to that, another thirteen representatives of other Ostariophysans, as well as seven taxa of other Teleostei were included to the comparative analysis as out-group material. As one of the main results of this survey is the acknowledgement that the distribution of the canals os the laterosensory system, as well as the number of branches and pores in association to this complex show little variation between the different lineages of Characiformes, keeping relatively stable among other Ostariophysi. This is interpreted as evidence that morphological traits in relation to this system may be very informative on a phylogenetical context when combined to other sources of characters. Therefore, these results indicate that features of the canals and pores of the laterosensory system may be one more source of anatomical characters, that even not traditionally used, are potentially informative on what regards the the relationship of fishes.

O sistema látero-sensorial dos peixes é responsável, dentre outras funções, pela percepção de vibrações e de movimentos na água. A unidade funcional desse sistema é denominada neuromasto, o qual pode estar presente na superfície ou em ranhuras da pele, ou também em canais que percorrem ossos dérmicos, alcançando a superfície externa da pele por intermédio de poros. O padrão de ossificação dos canais sensoriais cefálicos é conservado em diversas linhagens de peixes de tal forma que esse complexo morfológico representa uma rica fonte de informações filogenéticas, sendo amplamente utilizado em análises cladísticas de vários grupos de Ostariophysi, como, por exemplo, em Siluriformes. No entanto, o sistema láterosensorial cefálico de Characiformes, uma das mais diversificadas ordens de teleósteos e também um membro de Ostariophysi, nunca havia sido descrito em detalhes, sendo suas implicações filogenéticas pouco investigadas. O presente trabalho teve como objetivo realizar uma análise exploratória da homologia dos diversos elementos que compõem o sistema látero-sensorial em Characiformes, especificamente de: 1) levantar informações sobre os padrões de ramificação e de ossificação dos canais sensoriais cefálicos de todas as suas principais linhagens; 2) mapear as terminações destes ramos, os poros sensoriais, que se abrem na superfície da pele; e 3) identificar as principais implicações filogenéticas relacionadas a este sistema dentre os Characiformes e demais Ostariophysi. Para tal, foram analisados um total de trinta e seis táxons da ordem Characiformes, englobando representantes de todas as suas famílias e principais subfamílias. Além destes, outros treze componentes da ordem Ostariophysi, bem como sete demais Teleostei foram incluidos na análise como representantes do grupo-externo. Um dos principais resultados do presente estudo foi a constatação de que a disposição dos canais do sistema látero-sensorial cefálico, e o número de poros associados a cada um destes canais pouco variou dentre as diferentes famílias de Characiformes, mantendo-se também relativamente contantes nos demais Ostariophysi, e apresentando-se altamente informativas em um contexto filogenético. A complexidade morfológica desse sistema deixa implícito que o mesmo pode representar uma grande fonte de informações anatômicas combinando-os com demais caracteres morfológicos. Os resultados apresentados no presente trabalho indicam que os canais e poros da linha látero-sensorial cefálica constituem mais uma evidência de que novas fontes de caracteres morfológicos, apesar de não tradicionais, têm muito a contribuir com o entendimento das relações de parentesco entre peixes. / The laterosensory system of fishes is responsible, among other functions, for the perception of water movement and vibration. The functional unit of this system is the neuromast, which can be present on the skin surface or in grooves on it, or also in canals that are distributed along dermic bones, reaching the skin surface by pores. The ossification pattern related to the laterosensory canals is conserved among distinct fish lineages so that this morphological complex may represent a rich source of phylogenetic information. This system has been extensively used in cladistics analysis on several Ostariophysans, such as in Siluriformes and Cypriniformes. However, the laterosensory system of Characiformes, one of the most diverse order of teleost and also a member of the superorder Ostariophysi, has not been described in details yet, and its phylogenetic implications are poorly investigated. The present study aimed on performing an investigation on the laterosensory system components of Characiformes, having as its main goals: 1) a description of the cephalic laterosensory canals and branches for all main lineages of the order; 2) a characterization the canals ending points, i.e. the pores that open on the skin surface; and 3) a interpretation of the main philogenetical implications related to the laterosensory system among Characiformes and Ostariophysi. In order to accomplish these goals, a total of thirty-six Characiformes taxa were analysed, encompassing all families and main subfamilies of this order. Added to that, another thirteen representatives of other Ostariophysans, as well as seven taxa of other Teleostei were included to the comparative analysis as out-group material. As one of the main results of this survey is the acknowledgement that the distribution of the canals os the laterosensory system, as well as the number of branches and pores in association to this complex show little variation between the different lineages of Characiformes, keeping relatively stable among other Ostariophysi. This is interpreted as evidence that morphological traits in relation to this system may be very informative on a phylogenetical context when combined to other sources of characters. Therefore, these results indicate that features of the canals and pores of the laterosensory system may be one more source of anatomical characters, that even not traditionally used, are potentially informative on what regards the the relationship of fishes.

La détection des stimuli sensoriels est assurée par des cellules réceptrices spécialisées souvent grâce à des protrusions membranaires apicales telles que les microvillosités. La forme finale des extensions apicales microvilliées conditionne de nombreuses propriétés de la transduction sensorielle mais leur formation reste méconnue. Quels sont les facteurs moléculaires responsables de l’initiation et de l’élongation des filaments d’actine chez les cellules sensorielles microvilliées (CSMs) ? Peut-on décrire des éléments clés de la morphogenèse en commun ? Quel est le rôle structurel des microvillosités dans la fonction sensorielle ?J’ai étudié deux types sensoriels microvilliés : les neurones contactant le liquide cérébrospinal (NcLCS) et les cellules sensorielles des neuromastes (CSn). Mon projet visait à investiguer les mécanismes moléculaires sous-jacents à la morphogenèse des CSMs par l’étude des NcLCS. J’ai décrit les étapes critiques menant à la formation de l’extension apicale des NcLCS auxquelles j’ai pu associer des candidats potentiels grâce l’analyse transcriptomique des NcLCS. J’ai démontré le rôle critique de l’interaction entre Espin et Myo3b dans l’élongation de l’extension apicale des NcLCS et j’ai établi un lien direct entre structure et fonction en montrant que le raccourcissement de l’extension apicale aboutissait à la réduction de la réponse sensorielle.Mon travail a permis d’apporter des éléments de réponse quant à la formation de l’organe sensoriel des NcLCS. L’analyse transcriptomique des CSn a par ailleurs révélé des facteurs de morphogenèse communs avec les NcLCS, suggérant que toutes les CSMs partagent des propriétés de différenciation conservées. / Sensory systems use specialized receptor cells, many of which detect sensory cues through specialized apical membrane protrusions, such as microvilli. The final shape of the microvilliated apical extension requires specific molecular machinery and determines many of the properties of sensory transduction. The establishment of this structure remains however elusive. What molecular factors orchestrate the initiation and elongation of actin filaments in microvilliated sensory cells (MSCs)? Can we find key elements of morphogenesis common to MSCs? What is the precise role of microvilli structure in sensory function? I investigated two sensory cell types harboring microvilli: spinal cerebrospinal fluid-contacting neurons (CSF-cNs) and neuromast hair cells (nHCs). The primary goal was to unravel the molecular mechanisms underlying morphogenesis of MSCs by focusing on CSF-cNs. I was able to describe critical steps leading to the development of CSF-cN apical extension. My participation to the transcriptome analysis of CSF-cNs revealed candidate molecular factors associated with each of these steps. I demonstrated the importance of the interaction between Espin and Myo3b to ensure the proper lengthening of CSF-cN apical extension. In this system, I established a direct link between morphology and function by showing that shorter apical extensions lead to reduced sensory response. Altogether, my work shed light on the formation of CSF-cN sensory organelle and its functional role. In parallel, the establishment of the nHC transcriptome dataset revealed similar morphogenetic factors with CSF-cNs, supporting the idea that all MSCs share conserved features for their differentiation.

No description available.

Serotonin ist ein Neurotransmitter im zentralen Nervensystem (ZNS), die eine Vielzahl von Funktionen in der menschlichen Physiologie hat. Serotonergen Neuronen in der Raphe-Kerne des Gehirns Unser Ziel war die Leitung der Differenzierung von embryonalen Stammzellen (ES-Zellen) eingeengt und pluripotenten Stammzellen (iPS) Zellen in eine angereicherte Population von Serotonin-produzierenden Zellen, neuartige Gene, die wesentlich für die Entwicklung zu identifizieren und die Funktion des serotonergen Systems. Zu diesem Zweck haben wir differenzierten ES-Zellen in Serotonin-produzierenden Neuronen. Verwendung von RNA zu verschiedenen Zeitpunkten im Verlauf der ES-Zelldifferenzierung wir Gene spezifisch in serotonergen Linie von Affymetrix Genarray angereichert identifiziert isoliert. Um Kandidatengene bewerten wir neu programmiert Maus und Ratte embryonale Fibroblasten zu iPS-Zellen und anschließend differenziert sie serotonergen Neuronen. Wir haben uns für Cacna2d1, für eine alpha2 / delta-Untereinheit von spannungsabhängigen Calciumkanäle als prominentesten Kandidaten unter diesen Genen kodiert. Zur Analyse der Rolle des Proteins Cacna2d1 wir verwendet Cacna2d1 Knockout-Mäusen und Morpholino-Knockdown im Zebrafisch. Wir versäumt, direkte Beteiligung der Cacna2d1 mit serotonergen Systems sehen. Allerdings Immunfärbung für Cacna2d1 in Zebrafisch zeigte zeitabhängige Muster während der frühen Entwicklung. Cacna2d1 Expression wurde in seitlichen Mittellinie Stamm gesehen; vermutlich in Neuromasten Zellen. Übereinstimmend mit ihrer Charakterisierung als Neuromasten werden diese Cacna2d1-positiven Zellen in Richtung der Schwanz der Migration. Darüber hinaus zeigte Zebrafisch gestörten Migrationsverhalten der Neuromasten nach Morpholino-Knockdown von Cacna2d1. So ist diese Studie stellte klar, dass Cacna2d1 ist für Zebrafisch Seitenlinie Entwicklung aber keinen Einfluss auf die Einrichtung des serotonergen Systems. / Serotonin is a neurotransmitter in the central nervous system (CNS), which has a wide range of functions in human physiology. Serotonergic neurons are concentrated in the raphe nuclei of the brain We aimed at directing the differentiation of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells into an enriched population of serotonin producing cells to identify novel genes that are essential for the development and function of serotonergic system. To this purpose we differentiated ES cells into serotonin producing neurons. Using RNA isolated at different time points during the course of ES cell differentiation we identified genes specifically enriched in the serotonergic lineage by Affymetrix gene array. To evaluate candidate genes we reprogrammed mouse and rat embryonic fibroblast to iPS cells and subsequently differentiated them to serotonergic neurons. We selected Cacna2d1, coding for an alpha2/delta subunit of voltage dependent calcium channels as a most prominent candidate among these genes. To analyse the role of the Cacna2d1 protein we used Cacna2d1 knockout mice and morpholino-knockdown in zebrafish. We failed to see direct involvement of Cacna2d1 with serotonergic system. However immunostaining for Cacna2d1 in zebrafish revealed time-dependent pattern during early development. Cacna2d1 expression was seen in lateral midline trunk; presumably in neuromast cells. Concordantly with their characterization as neuromasts, these Cacna2d1-positive cells are migrating towards the tail. Moreover, zebrafish showed disturbed migration behaviours of neuromasts after morpholino-knockdown of Cacna2d1. Thus, this study clarified that Cacna2d1 is essential for zebrafish lateral line development but does not affect the establishment of the serotonergic system.