Thesis (Ph. D.)--University of Hawaii at Manoa, 2003. / Includes bibliographical references (leaves 210-214).

Thesis (Ph. D.)--University of Missouri-Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 276-280). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 276-280). Also available on the Internet.

Our laboratory has identified two secreted proteins, Wnt7a and Sonic hedgehog (Shh), that regulate satellite cell (SC) fate, during muscle differentiation. While Wnt7a stimulates symmetric SC division through the planar cell polarity (PCP) pathway, Shh activates Myf5 expression in the committed SC following asymmetric division through cilia-mediated Hedgehog (Hh) signaling. Crosstalk between these pathways has been well characterized during development, and is likely to be conserved in muscle regeneration. Indeed, accumulating evidence suggests the PCP pathway influences primary cilia formation, an organelle required for proper Hh signal transduction. Here we show that Wnt7a treatment in primary myoblasts increases the presence of primary cilia. Additionally, using myofiber culture, we demonstrate that Wnt7a increases myogenin (MyoG) expression. Removal of primary cilia through a small interfering RNA (siRNA) targeted towards IFT88 impedes Wnt7a mediated MyoG expression, suggesting crosstalk between the PCP and Hh pathways facilitates muscle differentiation. Furthermore, through siRNA knockdown we have identified the downstream PCP effectors, Inturned and Fuzzy as the main candidates responsible for this crosstalk. Knockdown of either Inturned or Fuzzy impedes Wnt7a-mediated MyoG expression. Taken together our data demonstrates crosstalk between the PCP pathway and Hh signaling regulates the differentiation of SCs.

Elucidating the molecular events that integrate the patterning, morphogenesis, and differentiation of epithelial progenitor cells into complex tissues is a primary focus of epithelial developmental biology research. Expansion and maintenance of epithelial progenitor populations is crucial for developmental events, but growth must be tightly coupled to consequent cellular differentiation and specialization. The Hippo pathway has surfaced as an important regulator of epithelial progenitor identity: nuclear activity of the Hippo effector Yap maintains epithelial progenitor status while Hippo-mediated nuclear exclusion of Yap by the Lats1/2 kinases induces differentiation. Extending this general theme into an additional organ system, the submandibular gland (SMG), as well as identifying upstream regulators of Yap and Lats1/2 in the developing lung was the goal of this work. Here, we describe important roles for Yap in the morphogenesis and patterning of lung and SMG epithelium, both of which are composed of highly organized branched structures. Epithelial-specific genetic ablation of Yap as well as its upstream negative regulators Lats1/2 was used to interrogate loss- and gain-of-function phenotypes, whereby Lats1/2 ablation is known to result in unrestricted nuclear Yap activity. Loss of Yap in the SMG resulted in a striking deficiency of Krt5/Krt14-positive epithelial progenitor populations accompanied by impaired branching morphogenesis. Deletion of Lats1/2 in the SMG resulted in a massive expansion of Krt5/Krt14-positive epithelial progenitor populations that failed to terminally differentiate. As epithelial progenitors in the lung and SMG begin to differentiate, they also acquire distinct morphologies. In both the lung and the SMG, Krt5-positive basal cells lie beneath a layer of Krt8/Krt19-positive luminal cells. We observed that luminal cells exhibited a columnar morphology while basal cells retained a cuboidal morphology, and that this difference correlated with the expression of the polarity protein Crb3. After ablating Crb3 in the developing lung epithelium, luminal cells were unable to polarize, exhibited aberrant nuclear Yap activity, and remained in a progenitor state. Crb3 functions to initiate Lats1/2 activity, promoting Yap phosphorylation and its consequent nuclear exclusion, which drives differentiation. Taken together, this work identifies essential roles for polarity/Hippo pathway-mediated control of Yap activity in epithelial progenitor expansion and differentiation. / 2018-07-09T00:00:00Z

Chez les vertébrés, la vitesse de la conduction nerveuse dépend du processus de myélinisation. Dans le système nerveux périphérique, ce sont les cellules de Schwann (CS) qui en s’enroulant autour de l’axone, constituent les gaines de myéline, séparés par des nœuds de Ranvier. La succession de ces gaines augmente la vitesse de conduction nerveuse car les potentiels d’action sont forcés de « sauter » d’un nœud de Ranvier à un autre, ce qui accélère leur vitesse de propagation. La géométrie (l’épaisseur et la longueur) de la gaine de myéline est donc un paramètre essentiel de la conduction de l’influx. Une publication à laquelle j’ai participé, a mis en évidence la polarisation cellulaire de la cellule de Schwann myélinisante. Notre hypothèse est que ce processus est capital pour la formation d’une gaine de myéline fonctionnelle. Comme trois complexes protéiques, conservés au cours de l’évolution, établissent et maintiennent la polarisation cellulaire (ces complexes sont: aPKC/Par3/Par6, Pals1/Patj/Crb3 et Dlg1/Lgl/Scrib chez les mammifères), mon travail consiste à étudier le rôle fonctionnel des protéines de la polarité Dlg1 et Crb3 lors de la myélinisation. Comme l’altération de la géométrie de la myéline est la cause d’un grand nombre de pathologies du système nerveux périphérique mais aussi central. Mon travail sur la mise en lumière des mécanismes qui préside à ce phénomène permet d’envisager de nouvelles voies thérapeutiques. / In the mammalian nervous system, the nerve conduction velocity depends on the myelin sheath. Myelin is produced by Schwann cells in the peripheral nervous system. The myelin sheath, together with the highly specialized nodes of Ranvier that are regulary arrayed along the myelinated fibers, is responsible for efficient and rapid propagation of action potentials along the nerve. Optimal conduction is obtained by adjusting the geometry (length and thickness) of the myelin sheath When I arrived in the laboratory, the team just showed the polarization of the myelinating Schwann cell ( mSC). We hypothesized then that cell polarity proteins are key players for the formation of the myelin sheath. Three complexes, well conserved among species, organize polarized cellular processes. In mammals, these complexes are aPKC/Par3/Par6, Pals1/Patj/Crb3 et Dlg1/Lgl/Scrib. Using an approch allowing the in vivo transduction of mSC, I investigate the relevance of Dlg1 and Crb3 in myelin formation. Changes in the myelin geometry is linked to several human neuropathies in the central and peripheral nervous system. This work highlights mechanisms which control correct myelin formation and allow designing strategies for their treatment.

Abstract Bone resorbing osteoclasts require tight attachment of their plasma membrane to the bone surface in order to retain the specific microenvironment and thus to be able to dissolve the bone matrix underneath. Cadherins are transmembrane glycoproteins usually mediating homophilic calcium-dependent cell-cell adhesion. In the present work we have studied the effects of the cadherin CAR sequence HAV-containing hexapeptide AHAVSE on osteoclasts. The primary attachment of osteoclasts to bone surface is not affected by the peptide, suggesting that it is not mediated by cadherins. Treatment of osteoclast cultures with AHAVSE decreased the number of resorption pits and the total resorbed area. Furthermore, we show rapid inactivation of osteoclasts with AHAVSE, which is seen as a decrease in the percentage of osteoclasts with actin rings. Pan-cadherin antibodies localized cadherin-like molecule in the sealing zone area of osteoclasts. These results suggest that cadherin-like molecules may mediate the tight attachment of osteoclasts in the sealing zone area and that the decrease of resorption in AHAVSE-treated osteoclast cultures is due to prevention of sealing zone formation. We studied the polarity of mesenchymal osteoblasts using osteosarcoma cell line UMR-108 and endosteal osteoblasts in situ in bone tissue cultures. Immunofluorescence confocal microscopy revealed that the vesicular stomatitis virus glycoprotein (VSV G) was targeted to the culture medium-facing surface. In endosteal osteoblasts, VSV G protein was found in the surface facing the bone marrow and circulation. On the contrary, Influenza virus hemagglutinin (HA) was localized to the bone substrate-facing surface of the UMR-108 cells. Electron microscopy showed that VSV particles were budding from the culture medium-facing surface, whereas Influenza viruses budded from the bone substrate-facing plasma membrane. These findings suggest the bone attaching plasma membrane of osteoblasts is apical, and the circulation or bone marrow facing plasma membrane is basolateral in nature. Gap junctions often mediate communication between different cells and cell types. In the present work, we demonstrate that rat osteoclasts show connexin-43 staining localizing in the plasma membrane of the cells in cell-cell contacts and over the basolateral membrane of osteoclasts. The effects of heptanol and Gap 27, known gap- junctional inhibitors, were studied using the well-characterized pit formation assay. The inhibitors decreased the number and activity of osteoclasts, suggesting a defect in the fusion of mononuclear osteoclast precursors to multinucleated mature osteoclasts. Furthermore, the total resorbed area and the number of resorption pits also decreased in the cultures. These results suggest that gap-junctional connexin-43 plays a functional role in osteoclasts, and that the blocking of gap junctions decreases both the number and the activity of osteoclasts.

Abstract Changes of cellular polarity in osteoclasts during resorption cycle was studied. Targeting of vesicular stomatitis virus (VSV) G-protein has earlier been studied in epithelial cells and in neurons, in which it serves as a basolateral or dendritic marker protein, respectively. In osteoclasts it occupied only the peripheral parts of the circulation facing basal membrane, but not ruffled border membrane or sealing zone area. Apical or axonal markers including Influenza A haemagglutinin were neither targeted to ruffled border area. Instead, they were transported to a limited area in the middle of the osteoclast basal surface. This membrane domain also showed staining for organic bone matrix components. Further works were done to find out the route of degraded bone matrix components to this membrane domain. It is shown in the confocal laser scanning and transmission electron microscopic level that osteoclasts take both organic and inorganic bone matrix dissolution products into intracellular vesicles which then are transcytosed to basal surface and finally exocytosed. One biological function to the new membrane domain seems to be to serve as an endpoint for intracellular handling of degraded bone matrix components, and as a final secretion point to release these components to circulation. This specialized membrane area is named as functional secretory domain (FSD) in this study. Membrane associated fine structures on the FSD area showed some novel membrane associsted structures. Their appearance and amount correlated to the resorption activity of osteoclasts suggesting that these new structures, termed clastosomes and debris, could be directly involved in the handling of bone degradation products during resorption. It is also shown that the bone matrix itself has effect on the resorption activity of cultured osteoclasts.

La formation des neurites constitue une étape cruciale dans le processus de différenciation neuronale. Cependant, les mécanismes qui permettent de déterminer comment et à quelle position les neurites émergent sont toujours largement méconnus. Nous avons postulé qu'une marque moléculaire pouvait préfigurer la différenciation morphologique. Au cours de ma thèse, j'ai cherché à identifier de nouvelles molécules capables de s'accumuler aux sites d'initiation des neurites et d'en contrôler la protrusion. De manière intéressante, chez la levure, la marque moléculaire qui contrôle le site de protrusion du bourgeon a été caractérisée. Parmi les centaines de protéines contrôlant le site d'initiation chez la levure, les Septines constituent une famille de protéines bien conservée chez les vertébrés. Ces GTPases forment des filaments qui agissent comme barrière de diffusion ou « échafaudage » moléculaire. Au cours de ma thèse, je me suis donc intéressée au rôle des Septines lors de l'initiation axonale dans le modèle des neurones sensoriels de DRG chez l'embryon de poulet. Nous avons pu démontrer qu'aux stades précoces de leur développement, ces neurones formaient deux axones, un au pôle ventral et l'autre au pôle dorsal, indiquant que le nombre et la position des sites d'initiation des axones sont bien contrôlés dans ces neurones. Nous avons, ensuite, démontré que les Septines étaient bien exprimées dans les DRG aux stades précoces du développement. Mes analyses en vidéo-microscopie de la localisation de la septine 7 au cours de la différentiation des neurones de DRG montrent que les Septines s'accumulent au site d'émergence de l'axone, juste avant ou lors de sa formation. L'inhibition des Septines induite par une construction dominant-négative (DN) ou par ajout d'un inhibiteur pharmacologique bloque la formation des axones. De plus, cette inhibition entraine une modification précoce de la morphologie, qui se traduit par l'apparition de cellules multipolaires complexes et de cellules rondes sans prolongement suggérant que, conformément à notre hypothèse, les Septines sont impliquées dans l'initiation des neurites. L'ensemble de ces résultats montre que les Septines régulent la différenciation morphologique précoce des neurones sensoriels / Neurite formation is a crucial step of neuronal differentiation. However, the mechanisms that determine how and at which position neurites emerge in the soma are still poorly understood. We postulated that a molecular polarity could prefigure the morphological differentiation, with some molecules that could accumulate at the future site of axon initiation. Interestingly, such molecular polarity has been evidenced in the contest of yeast budding, with bud forming at specific position relatively to the previous bud site. Genome-wide screen identified hundreds of proteins that control bud site location. Among the vertebrate molecules homologous to those involved in budding site selection, we selected the Septins as promising candidates. These GTP-ases form filaments that act as diffusion barriers and molecular scaffolds. We investigated the contribution of Septins to axon initiation using the chick dorsal root ganglion (DRG) neurons as a model. Monitoring of cell morphology in nascent ganglia indicates that DRG neurons form a single axon at the ventral pole and a second one at the dorsal pole and that these axons seem to emerge directly after their last division. This suggests that two initiation sites are selected at opposite pole of the soma.We found that Septins homologous with those controlling budding are expressed in the early DRG developmental stages. My analyses by time-lapse video-microscopy showed that Septin7 accumulate at the site of axon emergence, just before or during its formation.We observed that a pharmacological inhibitor and a dominant-negative construct block axon formation both in vitro and in vivo respectively. Furthermore, blocking Septin function leads to the appearance of uncommon round or sea urchin-like neurons. Thus, Septins appear to regulate early step of morphological differentiation of DRG neurons, possibly by controlling axon initiation site selection

The correct polarity of a neuron underlies its ability to integrate precise circuitries in the nervous system. The goal of my thesis was to investigate the pathways that establish and maintain neuron polarity/orientation in vivo. To accomplish this, I used bipolar VC4/5 motor neurons, which innervate the C. elegans egg-laying musculature, as a model system. Vulval proximal VC4/5 neurons extend axons in the left-right (LR) orientation, around the vulva; whereas vulval distal VC1-3,6 neurons extend axons along the anterior-posterior (AP) axis. A previous study showed that vang-1, a core planar cell polarity (PCP) gene, suppresses AP axon growth in VC4/5 neurons. In order to identify new components of this pathway we performed genetic screens for mutants with abnormal VC4/5 polarity/morphology. We isolated and mapped alleles of farnesyl transferase b (fntb-1) and of core PCP genes, prickle- 1 (prkl-1) and dishevelled-1 (dsh-1); all of which display tripolar VC4/5 neurons, similar to vang-1 lof. In prkl-1 and dsh-1 mutants, primary LR and ectopic AP VC4/5 axons are born simultaneously, suggesting an early role in establishing polarity. In addition, prkl-1 and dsh-1 act persistently to maintain neuron morphology/orientation. Genetic analysis of double mutants suggests that prkl-1 interacts with vang-1 in a common PCP pathway to prevent AP axon growth, while dsh-1 also acts in a parallel pathway. Furthermore, prkl-1 functions cell autonomously in neurons, whereas dsh-1 acts both cell autonomously and cell nonautonomously in epithelial cells. Notably, prkl-1 overexpression results in unipolar VC4/5 neurons, in a dose-dependent manner. In contrast, dsh-1 overexpression in VC4/5 neurons results in a lof phenotype, similar to vang-1 lof and overexpression phenotype. Remarkably, prkl-1 overexpression restores normal VC4/5 polarity in dsh-1 and vang-1 mutants, which is suggestive of a downstream role for prkl-1. Both PRKL-1 and DSH-1 are expressed in iii uniformly distributed puncta at the plasma membrane of VC4/5, similar to VANG-1; suggesting that their asymmetric distribution is not critical for neuron polarity. Furthermore, we found that the vulva epithelium induces prkl-1 expression in VC4/5; indicating a functional relationship between the egg-laying organ and neuron morphology. Moreover, a structure-function analysis of PRKL-1 revealed that the conserved PET domain and the Cterminal region are crucial to prevent AP axon growth, whereas the three LIM domains are dispensable for this role. In addition, we showed that dsh-1 also regulates the morphology of AP-oriented PDE neurons. dsh-1 promotes the formation of PDE posterior axons, contrary to its function in VC5 neurons; which indicates a context-dependent role for dsh-1 in neuronal polarity. Altogether, this thesis implicates the PCP signalling pathway in a previously unknown role, in establishing and maintaining neuronal polarity, by controlling AP axon growth in response to organ-derived polarizing cues.