The avian embryo is a popular animal model because it is widely available (Antin et al., 2004), it is easily manipulated, and it can provide important insights into normal and abnormal embryo development (Kulesa, 2004). While in vivo and in vitro cultures of chick embryos are common, in ovo cultures are rarer, and none have been designed where the egg did not have to be resealed afterwards. The present study aimed to develop a set-up in which the egg would be windowed without resealing the egg so that the embryo would remain accessible for experimental manipulation. As well, this study aimed to track cell movement during neurulation by microinjecting points of dye along the embryo. Two prototypes were developed based on the concept that temperature and moisture controlled air passing over the windowed egg would serve as a blanket. When these prototypes were unable to keep the embryo alive, a protocol developed by Kulesa and Fraser (2004) was adapted for the study. This protocol involved the construction of a Teflon window which was placed in the windowed egg and sealed with beeswax. Initial microinjection tests with Fast Green FCF showed that the dye dissipated quickly after injection, most likely because of the hydrophilicity of the dye. Therefore, a list of non-fluorescent, hydrophobic dyes were chosen and tested for suitability to cell tracking. Time restrictions prevented the actual cell tracking experiments from taking place, but it was found that Oil Red O fulfilled the criteria. As Oil Red O is usually used to identify lipids in static experiments, it remains to be seen whether it would function as a vital dye. Future experiments include expanding the set-up for use with a confocal microscope for a 4-D rendering of cell movement, and taking advantage of the symmetrical nature of neurulation in the chick embryo to examine perturbations to the normal progress of development, via drugs such as valproic acid.

The avian embryo is a popular animal model because it is widely available (Antin et al., 2004), it is easily manipulated, and it can provide important insights into normal and abnormal embryo development (Kulesa, 2004). While in vivo and in vitro cultures of chick embryos are common, in ovo cultures are rarer, and none have been designed where the egg did not have to be resealed afterwards. The present study aimed to develop a set-up in which the egg would be windowed without resealing the egg so that the embryo would remain accessible for experimental manipulation. As well, this study aimed to track cell movement during neurulation by microinjecting points of dye along the embryo. Two prototypes were developed based on the concept that temperature and moisture controlled air passing over the windowed egg would serve as a blanket. When these prototypes were unable to keep the embryo alive, a protocol developed by Kulesa and Fraser (2004) was adapted for the study. This protocol involved the construction of a Teflon window which was placed in the windowed egg and sealed with beeswax. Initial microinjection tests with Fast Green FCF showed that the dye dissipated quickly after injection, most likely because of the hydrophilicity of the dye. Therefore, a list of non-fluorescent, hydrophobic dyes were chosen and tested for suitability to cell tracking. Time restrictions prevented the actual cell tracking experiments from taking place, but it was found that Oil Red O fulfilled the criteria. As Oil Red O is usually used to identify lipids in static experiments, it remains to be seen whether it would function as a vital dye. Future experiments include expanding the set-up for use with a confocal microscope for a 4-D rendering of cell movement, and taking advantage of the symmetrical nature of neurulation in the chick embryo to examine perturbations to the normal progress of development, via drugs such as valproic acid.

How is it that some cells become neurons? And how is it that neurons become organized in the spinal cord and brain to allow us to walk and talk, to see, recall events in our lives, feel pain, keep our balance, and think? The cells that are specified to form the brain and spinal cord are originally located on the outside surface of the embryo. They loop inward to form the neural tube in a process called neurulation. Structures that are nearby send signals to the posterior neural tube to form and pattern the spinal cord so that the dorsal side receives sensory input and the ventral side sends motor signals from neurons to muscles. In the brain, stem cells near the center of the neural tube migrate out to form a mantel zone, and a set of dividing cells from the mantle zone migrate further to produce a second set of neurons at the outer surface of the brain. These neurons will form the cerebral cortex, which contains six discrete layers. Each layer has different connections and different functions. WIREs Dev Biol 2017, 6:e215. doi: 10.1002/wdev.215 For further resources related to this article, please visit the WIREs website.

Les transitions de cadhérines sont souvent impliquées dans des phénomènes de ségrégation cellulaire mettant en jeu un phénomène de Transition Epithélium-Mésenchyme (TEM). Cependant, lors de la formation du système nerveux central, la transition E-/N-cadhérine n'entraîne pas de TEM et, contrairement au modèle en vigueur, nos résultats montrent que celle-ci n'est absolument pas un prérequis nécessaire aux mouvements morphogénétiques de la neurulation. Le point important lors de la formation du système nerveux central semble surtout être le contrôle de la cinétique de cette transition E-/N-Cadhérine. Le système nerveux central d'oiseau se forme au cours du développement selon des modes bien distincts : dans la région antérieure de l'embryon, la neurulation primaire ; dans la région postérieure, la neurulation dite secondaire conduit à un tube nerveux généré par accrétion cellulaire dont la lumière centrale est créée par cavitation. Dans la région thoracique, le tube neural se forme selon un mode totalement original ayant certaines caractéristiques des deux modes classiques, c'est la neurulation intermédiaire. Les précurseurs neuraux du tube neural intermédiaire et secondaire effectuent une TEM puis migrent postérieurement de manière coordonnée et dirigée grâce au dépôt polarisé de fibronectine induit par la protéine de la polarité planaire, Prickle, puis se ré-épithélialisent. Les Cellules de la Crête Neurale (CCN) constituent un tissu à part du tube neural. Nous montrons que ces cellules se distinguent du reste du neuroépithélium par un répertoire d'expression de cadhérines spécifiques

La formation du tube neural et l'induction de la crête neurale sont des processus importants qui se passent au cours de la neurulation. Le système nerveux central se forme à partir du tube neural, alors que le système nerveux périphérique, le squelette cranio-facial et les mélanocytes se forment à partir de la crête neurale. Cependant, plusieurs maladies génétiques liées au développement anormal du tube neural et de la crête neurale existent chez l'homme. Donc, notre équipe s'est intéressée à faire une recherche au niveau moléculaire dans ce domaine. Le développement du tube neural et des cellules de la crête neurale est influencé par la présence de plusieurs voies de signalisation y compris la voie Wnt. Nous avons focalisé notre étude sur la transcription du gène Pax3 qui code pour un facteur de transcription qui s'exprime dans le tube neural et les cellules pré-migratoires de la crête neurale. L'induction de Pax3, des cellules de la crête neurale ainsi que l'expression des gènes Cdx (Cdx1, Cdx2 et Cdx4) se font par la voie de signalisation Wnt. Le chevauchement de l'expression des gènes Pax3 et Cdx se passe lors de l'induction de la crête neurale à la plaque neurale postérieure aussi bien que pendant la formation du tube neural et des cellules de la crête neurale. Les résultats préliminaires par ChIP ont montré la présence physique de Flag-Cdx1 et Flag-EnRCdx1 sur le promoteur proximal de Pax3. Notre étude par RT-PCR sur la régulation de l'expression de Pax3 par les facteurs de transcription Cdx a démontré la régulation endogène de Pax3 par ces protéines dans les cellules Neuro2a. L'essai Luciférase montre l'induction de l'expression de Pax3 par la surexpression des protéines Cdx et la diminution de son expression par la surexpression du dominant négatif EnRCdx1 dans les cellules Neuro2a. Après avoir identifié les sites potentiels de liaison pour les protéines Cdx sur le promoteur proximal de Pax3, nous avons procédé à une délétion des différentes régions de ce promoteur. On a remarqué la forte induction du promoteur au niveau de NCE (Neural Crest Enhancer). Pourtant, la mutation en combinaison de ces sites n'a pas montré la diminution de l'activité de ce promoteur. Étant donné la présence d'un site potentiel de liaison pour Brn1 sur le promoteur proximal de Pax3, notre étude par l'essai Luciférase a mis en évidence une synergie entre les co-facteurs Cdx2 et Brn1 dans les cellules Neuro2a. Le même phénomène a été observé dans les cellules P19. Pourtant, la mutation du site de Brn1 n'a pas diminué l'activité du promoteur proximal de Pax3. En conclusion, nos résultats mettent en évidence la non fonctionnalité des sites de liaison pour les facteurs de transcription Cdx sur le promoteur proximal de Pax3 ainsi que la synergie entre la protéine Cdx2 et son co-facteur Brn1 dans les cellules Neuro2a ce qui suggère que la régulation directe de Pax3 par les protéines Cdx n'implique pas de liaison des Cdx à l'ADN. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Neurulation, tube neural, crête neurale, neuroectoderme, système nerveux, voie de signalisation Wnt, transcription, facteur de transcription

Debate continues on the merits of strictly limiting alcohol consumption during all of pregnancy, and whether “safe” consumption levels and/or times exist. Only a relatively few experimental studies have been conducted that limit the timing of exposure to specific events during development and the exposure level to one that might model sporadic, incidental drinking during pregnancy. In the present study, the effects of two acute gavage exposures to low and moderate levels of ethanol (peak blood ethanol concentrations (BEC) of 104 and 177 mg/dl, respectively) either during gastrulation on gestational day (GD) 7 (at GD7:0 h and GD7:4 h) or during neurulation on GD8 (at GD8:6 h and GD8:10 h) on the spatial learning and memory abilities of adult mice in the radial arm maze (RAM) were examined. Mice were selected from a prenatal ethanol exposure (PAE) cohort that had been tested as neonates for their sensorimotor development (Schambra et al., 2015) and as juveniles and young adults for open field activity levels and emotionality (Schambra et al., 2016). Mice exposed on either of the two gestational days to acute, low or moderate levels of ethanol were deficient in overall performance in the RAM in adulthood. Importantly, mice in ethanol exposed groups took longer to reach criterion in the RAM, and many mice in these groups failed to do so after 48 trials when testing was terminated. Exposure to a low level of ethanol on either GD7 or GD8, or a moderate level on GD7, resulted in significant impairment in spatial reference (long-term) memory, while only mice exposed on GD7 to the low level of ethanol were significantly impaired in spatial working (short-term) memory. Mice exposed to the low ethanol level on either day had significantly shorter response latencies, which may reflect impairment of processes related to response inhibition or executive attention in these mice. For all measures, distributions of individual scores revealed a relatively small subset of mice in each PAE group who scored well outside the range of the control group, which skewed the population distributions to varying degrees in the direction of worse performance for the PAE groups. Overall the data suggest that after acute, low level ethanol exposure early in gestation, the likelihood that an individual mouse embryo experienced measureable ill-effects due to the exposure was rather low, but in a few of the embryos, damage occurred that resulted in significant deficits in later performance. The overall characteristics of our cohort of PAE mice, including delayed sensorimotor development, mild hypoactivity and increased emotionality, as shown in previous studies, together with deficits in spatial learning and memory as shown here, resemble those in a subset of human Fetal Alcohol Spectrum Disorder (FASD) diagnoses, specifically ADHD-Inattentive type (ADHD-I) and/or Sluggish Cognitive Tempo (SCT). Although possible correspondences between mechanisms underlying PAE-induced deficits in mice and those operating in humans remain undefined, further study with this mouse PAE model may ultimately help advance understanding of the causes of these conditions in affected children. This study highlights the possibility of risk associated with low to moderate sporadic alcohol consumption during the first month of human pregnancy.

<p>Early Xenopus laevis development involves highly complex morphogenic movements. Two key movements are gastrulation, which establishes germ layer spatial arrangement, and neurulation, which results in the folding and closure of the neural tube. Multiple signaling pathways are involved in regulating cell adhesion, migration, shape and polarity during these processes to ensure normal development. Two of the most characterized pathways are the canonical and non-canonical Wnt pathways. However, the roles of all the individual molecules involved are not fully understood. In this thesis I provide initial characterization of the POZ-ZF transcription factor xZnf131. Znf131 is a transcriptional activator and its binding partner Kaiso negatively regulates this function. Since Znf131 and Kaiso display antagonistic roles and Kaiso mediates Wnt signaling and morphogenesis during Xenopus gastrulation and neurulation I hypothesize that xZnf131 is also required to regulate morphogenesis during these key developmental events.</p> <p>Like other POZ-ZF proteins, xZnf131 contains an amino-terminal POZ domain and a carboxy-terminal ZF domain comprised of five zinc fingers. xZnf131 is continuously expressed through early Xenopus development but was spatially localized to the dorsal and anterior structures of the embryo, notably the neural plate. Morpholino oligonucleotide (MO) knockdown of xZnf131 resulted in severe defects in notochord and neural plate formation, with abnormal cell morphology, typical of non-canonical Wnt misregulation. Interestingly, xZnf131 overexpression produced phenotypes very similar to xZnf131 knockdown suggesting that xZnf131 protein levels need to be tightly maintained to regulate the correct/normal morphogenic movements during Xenopus gastrulation and neurulation.</p> <p>Our findings indicate that xZnf131 plays a role in the morphogenic movements during Xenopus gastrulation and neurulation. Our data provides a useful foundation for future experiments to elucidate the biological mechanism of xZnf131 action during these key developmental processes.</p> / Master of Science (MSc)

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京都大学 / 0048 / 新制・論文博士 / 博士(理学) / 乙第13353号 / 論理博第1571号 / 新制||理||1664(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 高橋 淑子, 教授 沼田 英治, 教授 曽田 貞滋 / 学位規則第4条第2項該当 / Doctor of Science / Kyoto University / DFAM

Human and animal studies show significant delays in neurobehavioral development in offspring after prolonged prenatal exposure to moderate and high ethanol doses resulting in high blood alcohol concentration (BECs). However, none have investigated the effects of lower ethanol doses given acutely during specific developmental time periods. Here, we sought to create a mouse model for modest and circumscribed human drinking during the 3rd and 4th weeks of pregnancy.We acutely treated mice during embryo gastrulation on gestational day (GD) 7 or neurulation on GD8 with a low or moderate ethanol dose given via gavage that resulted in BECs of 107 and 177. mg/dl, respectively. We assessed neonatal physical development (pinnae unfolding, and eye opening); weight gain from postnatal day (PD) 3-65; and neurobehavioral maturation (pivoting, walking, cliff aversion, surface righting, vertical screen grasp, and rope balance) from PD3 to 17. We used a multiple linear regression model to determine the effects of dose, sex, day of treatment and birth in animals dosed during gastrulation or neurulation, relative to their vehicle controls.We found that ethanol exposure during both time points (GD7 and GD8) resulted in some delays of physical development and significant sensorimotor delays of pivoting, walking, and thick rope balance, as well as additional significant delays in cliff aversion and surface righting after GD8 treatment. We also found that treatment with the low ethanol dose more frequently affected neurobehavioral development of the surviving pups than treatment with the moderate ethanol dose, possibly due to a loss of severely affected offspring. Finally, mice born prematurely were delayed in their physical and sensorimotor development.Importantly, we showed that brief exposure to low dose ethanol, if administered during vulnerable periods of neuroanatomical development, results in significant neurobehavioral delays in neonatal mice. We thus expand concerns about alcohol consumption during the 3rd and 4th weeks of human pregnancy to include occasional light to moderate drinking.