In this work, we identified and characterized developmentally regulated aspects to cell division in the Xenopus laevis. We found that cells in the early neural plate divide in an oriented manner. This orientation is established by Cdc42 controlled maintenance of stable interactions between the spindle and the cell cortex. This role of Cdc42 is developmentally regulated and cells dividing later in a related tissue, the tail epidermis, are not under this control. Moreover, we find that the cell divisions in the early neural plate are further specialized in their mechanisms of cell division. Cells in the early neural plate exhibit exaggerated anaphase-B movements, a delayed onset of cytokinesis, low density of midzone microtubules and a rapid cytokinetic furrow ingression as compared to the late tail epidermis, another ectodermally derived tissue. These modifications to the mechanism of cell division appear to be because of a reduced level of PRC1, a microtubule bundling protein, and thus modifications to the central spindle structure. Finally, we find that cytokinetic mechanisms may be functionally related to the process of ciliogenesis. We find proteins known to localize to the central spindle localized to the rootlet of the basal body of cilia in multiciliated cells of the mucociliary epidermis. This localization may be related to vesicle transport during both these processes. This work reveals unexpected plasticity to fundamental mechanisms of cell division. / text

Neurulation is critical for the proper development of the central nervous system during embryogenesis. This process requires coordinated morphogenetic movements driven by localized cell movements. The key morphogenetic process responsible for lengthening the neural plate is convergent extension. During convergent extension medially oriented cell polarity, protrusive activity, and motility are thought to generate forces through cell intercalation resulting in stiffer elongating tissues. My research determines that forces that help shape the neural plate arise from morphogenetic movements in the neural tissue and determines PCP signaling regulates tissue stiffness in the neural ectoderm. We have established an experimental system sensitive enough to evaluate the stiffness of Xenopus neural tissue. Stiffness is measured by gluing two fine wires onto neural explants from an early gastrula stage Xenopus laevis embryo. The wires stretch the tissue at a constant strain rate using a real-time image-based feedback system and stiffness is determined by measuring the deflection of one wire. Measurements obtained from control embryos prior to neurulation estimate tissue stiffness at approximately 12.7 ± 0.53 mN/m in both mediolateral and anteroposterior directions. Stiffness measurements double in early neurula embryos (P < 0.05). Mediolateral stiffness, 24.9 ±6.2 mN/m, is significantly greater than anteroposterior stiffness, 21.4 ±5.3 mN/m (P < 0.05). These trends are strengthened in normalized data to reduce clutch-to-clutch variation. Expressions of dominant-negative Wnt11, Fz7, and Dsh constructs successfully disrupt neurulation by interfering with the PCP pathway. Changes in stiffness of the neural plate were measured and show reduced stiffness at early neurula stage in both mediolateral and anteroposterior directions suggesting mechanical forces are generated within the neural plate.

Neurulation is critical for the proper development of the central nervous system during embryogenesis. This process requires coordinated morphogenetic movements driven by localized cell movements. The key morphogenetic process responsible for lengthening the neural plate is convergent extension. During convergent extension medially oriented cell polarity, protrusive activity, and motility are thought to generate forces through cell intercalation resulting in stiffer elongating tissues. My research determines that forces that help shape the neural plate arise from morphogenetic movements in the neural tissue and determines PCP signaling regulates tissue stiffness in the neural ectoderm. We have established an experimental system sensitive enough to evaluate the stiffness of Xenopus neural tissue. Stiffness is measured by gluing two fine wires onto neural explants from an early gastrula stage Xenopus laevis embryo. The wires stretch the tissue at a constant strain rate using a real-time image-based feedback system and stiffness is determined by measuring the deflection of one wire. Measurements obtained from control embryos prior to neurulation estimate tissue stiffness at approximately 12.7 ± 0.53 mN/m in both mediolateral and anteroposterior directions. Stiffness measurements double in early neurula embryos (P < 0.05). Mediolateral stiffness, 24.9 ±6.2 mN/m, is significantly greater than anteroposterior stiffness, 21.4 ±5.3 mN/m (P < 0.05). These trends are strengthened in normalized data to reduce clutch-to-clutch variation. Expressions of dominant-negative Wnt11, Fz7, and Dsh constructs successfully disrupt neurulation by interfering with the PCP pathway. Changes in stiffness of the neural plate were measured and show reduced stiffness at early neurula stage in both mediolateral and anteroposterior directions suggesting mechanical forces are generated within the neural plate.

No description available.

The vertebrate nervous system is extremely complex and contains a wide diversity of cell types. The formation of a functional nervous system requires the differential specification of progenitor cells at the right time and place. The generation of many different types of neurons along the rostro-caudal axis of the CNS begins with the initial specification of a few progenitor domains. This initial coarse pattern is refined by so-called secondary organizers arising at boundaries between these domains. The Isthmic Organizer (IsO) is a secondary organizer located at the boundary between the midbrain and the hindbrain. Although the function and maintenance of the IsO are well understood, the processes underlying its initial specification have remained elusive. In the present work we provide evidence that convergent Wnt and FGF signals initiate the specification of the IsO during late gastrulation as part of the neural caudalization process. The initial step in the generation of the nervous system is the division of the embryonic ectoderm into three cell populations: neural cells giving rise to the CNS, neural plate border cells giving rise to the peripheral nervous system, and epidermal cells giving rise to the outer layer of the skin. While the choice between neural and epidermal fate has been well studied, the mechanism by which neural plate border cells are generated is less well understood. At rostral levels of the neuraxis, the neural plate border gives rise to the olfactory and lens placodes, thickenings of the surface ectoderm from which sensory organs are derived. More caudally, the neural plate border generates neural crest cells, a transient population that migrates extensively and contributes to neurons and glia of the peripheral nervous system. How the early patterning of the central and peripheral nervous systems are coordinated has remained poorly understood. Here we show that the generation of neural plate border cells is initiated at the late blastula stage and involves two phases. During the first phase, neural plate border cells are exposed to Wnt signals in the absence of BMP signals. Simultaneous exposure to Wnt and BMP signals at this early stage leads to epidermal induction. Wnt signals induce expression of Bmp4, thereby regulating the sequential exposure of cells to Wnt and BMP signals. During the second phase, at the late gastrula stage, BMP signals play an instructive role to specify neural plate border cells of either placodal or neural crest character depending on the status of Wnt signaling. At this stage, Wnt signals promote caudal character simultaneously in the neural plate border and in the neural ectoderm. Thus, the choice between epidermal and neural plate border specification is mediated by an interplay of Wnt and BMP signals that represents a novel mechanism involving temporal control of BMP activity by Wnt signals. Moreover, the early development of the central and peripheral nervous systems are coordinated by simultaneous caudalization by Wnt signals.

Thesis (Ph.D. in Neuroscience) -- University of Colorado Denver, 2008. / Includes bibliographical references (leaves 112-120). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第22283号 / 理博第4597号 / 新制||理||1659(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)准教授 佐藤 ゆたか, 教授 高橋 淑子, 准教授 秋山 秋梅 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Morphogen gradients impart positional information to cells in a homogenous tissue field. Fgf8a, a highly conserved growth factor, has been proposed to act as a morphogen during zebrafish gastrulation. However, technical limitations have so far prevented direct visualization of the endogenous Fgf8a gradient and confirmation of its morphogenic activity. Here, we monitor Fgf8a propagation in the developing neural plate using a CRISPR/Cas9-mediated EGFP knock-in at the endogenous fgf8a locus. By combining sensitive imaging with single-molecule fluorescence correlation spectroscopy, we demonstrate that Fgf8a, which is produced at the embryonic margin, propagates by diffusion through the extracellular space and forms a graded distribution towards the animal pole. Overlaying the Fgf8a gradient curve with expression profiles of its downstream targets determines the precise input-output relationship of Fgf8a-mediated patterning. Manipulation of the extracellular Fgf8a levels alters the signaling outcome, thus establishing Fgf8a as a bona fide morphogen during zebrafish gastrulation. Furthermore, by hindering Fgf8a diffusion, we demonstrate that extracellular diffusion of the protein from the source is crucial for it to achieve its morphogenic potential.

Le laboratoire d’Embryologie Moléculaire étudie les mécanismes moléculaires contrôlant le développement embryonnaire et utilise comme système expérimental l’embryon de xénope. En collaboration le laboratoire du Dr Daniel Christophe, nous avons abordé l’étude du gène XHRT1 chez le xénope. Ce gène est l’orthologue du gène HRT-1/Hey1/Hesr-1/HERP2/CHF2 de souris. Celui-ci, avec deux autres protéines apparentées (HRT2 et HRT3) forme une sous-famille de facteurs de transcription de type bHLH-O qui se différencient des autres facteurs bHLH-O par l’absence d’un motif carboxy-terminal de séquence « WRPW » et par la présence d’un nouveau motif carboxy-terminal conservé de séquence « TEI/VGAF ». Le rôle de ces facteurs HRT dans le développement est encore actuellement mal connu. Dans un premier temps, nous avons déterminé le profil d’expression de XHRT1 au cours de l’embryogenèse. Nous avons observé que ce gène est fortement exprimé au stade neurula dans le plancher du tube neural, et que plus tardivement celui-ci est exprimé dans différentes régions du système nerveux, dans les somites et le dans le pronéphros. Comme attendu pour un membre de la famille des facteurs bHLH-O, nous avons également observé que l’expression précoce de HRT1 au niveau du plancher du tube neural est bien régulée par la voie de signalisation Notch. Dans un deuxième temps, nous nous sommes intéressés au rôle et au mode d’action du facteur XHRT1 dans le développement du plancher du tube neural. Nous avons pu montrer que XHRT1 agit comme répresseur transcriptionnel et que cette répression nécessite la présence du domaine bHLH et de séquences en aval de celui-ci. Nous avons montré en embryon que la surexpression précoce de XHRT1 induit un blocage de l’expression des marqueurs du mésoderme et une augmentation de marqueur du plancher du tube neural, ce qui est en accord avec le modèle selon lequel la voie de signalisation Notch interviendrait dans le choix de la destinée des cellules de la région médiane en inhibant la différenciation des cellules en notocorde et en favorisant leur différenciation en cellules du plancher du tube neural. XHRT1 n’étant cependant activé qu’à partir du stade neurula, nous avons conclu que les effets observés n’étaient probablement pas dus à XHRT1 mais à un autre facteur bHLH-O apparenté exprimé plus précocement dans les cellules de la ligne mediane de l’embryon. Afin d’éviter ces effets non spécifiques précoces, nous avons utilisé un vecteur d’expression de XHRT1 permettant un contrôle temporel de l’activité de la protéine. Nous avons ainsi montré que l’activation de XHRT1 au stade neurula dans l’ectoderme inhibe la différenciation des cellules précurseurs neurales en neurones et qu’il pourrait ainsi jouer un rôle important dans le développement du plancher du tube neural. Nos résultats ont montré également que XHRT1 est capable d’homo- et hétérodimériser in vivo avec les facteurs Xhairy1 et Xhairy2b coexprimés avec XHRT1 dans le plancher du tube neural. Enfin, nous avons montré que les propriétés de dimérisation de XHRT1 sont dépendantes non seulement du domaine bHLH, mais aussi du domaine Orange et des séquences situées en aval, séquences jouant un rôle important dans le choix du partenaire. Des travaux récents ayant montré que la voie de signalisation Notch joue un rôle important dans le développement du rein, nous avons voulu déterminer l’importance de XHRT1 dans le développement du pronéphros. Nos résultats ont montré que XHRT1 ainsi que d’autres facteurs bHLH-O sont exprimés de manière dynamique, d’abord dans le glomus puis dans la partie dorso-antérieure de l’ébauche du pronéphros à l’origine des tubules proximaux, et que leur expression est régulée positivement par Notch. La surexpression de XHRT1 à la fin de la neurulation inhibe la formation du canal et du tubule distal, tandis que l’inhibition de la traduction de la protéine entraîne une réduction de l’expression de marqueurs spécifiques des tubules proximaux et du glomus. Ces résultats démontrent que XHRT1 joue un rôle important comme médiateur de la voie de signalisation Notch dans le pronéphros.

No description available.