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.

During neuronal development, the axonal and dendritic projections are polarized and oriented along specific body axis. To further explore the molecular basis of neuritogenesis in vivo, we used the nematode Caenorhabditis elegans as a developmental model and performed a forward genetic screen to identify genes that specify the polarity of neurite outgrowth. We examined the VC4 and VC5 neurons, members of the six VC motor neurons using the Pcat-1

Les éponges (Porifera) sont l'une des premières lignées d'animaux à avoir émergé. De ce fait, elles sont considérées comme des espèces clés pour retracer l’origine et l'évolution des gènes et des voies de signalisation qui ont sous-tendu l'apparition de la pluricellularité chez les métazoaires. Entre autres, les voies Wnt ont été décrites comme des cascades génétiques essentielles du control de nombreux mécanismes cellulaires (prolifération, communication, adhésion, motilité, etc.) au cours du développement précoce des bilatériens et des cnidaires. C’est pourquoi, l'étude de ces voies, chez les lignées d’émergences plus anciennes sont essentielles afin de comprendre l'origine des plans d’organisation des animaux.J’ai alors entrepris de nombreuses analyses bioinformatiques sur différentes bases de données d’éponges. Il apparait alors que l’ancêtre commun des éponges possédait déjà certainement tous les composants des voies Wnt. Néanmoins, à ce jour, puisque l’intégralité de ces composants n’a été identifiée que dans le genre Oscarella (lignée des Homoscleromorpha), différentes pertes secondaires sembleraient s’être produites chez les démosponges, les éponges calcaires et les hexactinellides. Afin de tester si ces gènes orthologues sont impliqués dans la mise en place du plan d’organisation des éponges, des études fonctionnelles ont été mises en œuvre. Ces approches fonctionnelles réalisées sur deux lignées d’éponges différentes tendent alors à confirmer la conservation des voies de signalisation Wnt dans les processus de mise en place des plans d’organisation des animaux, à la fois au cours de l'embryogenèse mais aussi lors du renouvellement cellulaire chez l'adulte. / Sponges (Porifera) are one of the earliest emerged animal lineages. They are thus considered as key species to retrace early evolution of genes and pathways underlying the emergence of multicellularity in metazoans. Among others, the Wnt pathways have been described as crucial modules controlling cell proliferation, cell communication, cell adhesion and cell motility during the early development of Bilaterians and Cnidarians. Therefore the study of these signaling pathways in more basally branching lineages is essential for unraveling the origin of animal body plans. I performed numerous bioinformatic analyses on different poriferan databases. One of my main results is that the last common ancestor of Porifera probably already possessed all the components of the Wnt pathways. Nevertheless, because, to date, all these components were only retrieved in the Oscarella genus (Homoscleromorpha lineage), several secondary gene losses would have occurred in other sponge lineages, namely Demospongia, Calcarea and Hexactinellida.In order to test whether or not these retrieved orthologous genes, are involved in patterning sponge body plan (as they do in Bilateria and Cnidaria), functional studies were implemented. These functional studies performed on two different lineages tend to confirm that Wnt signaling pathways were conserved from sponges to vertebrates to pattern animal body plan during both embryogenesis and cell renewal in adult.

The development of organs such as the lung and kidney occurs by branching morphogenesis. Changes in the cytoskeletal architecture, cell-cell adhesion and cell polarity are necessary for the formation of new branches. Interactions and reciprocal signalling between epithelial and mesenchymal cells mediate these organised cell movements that give rise to a complex system of tubes suitable for the transport of gas or fluids. Mutations that disrupt formation of either the correct number, or shape of epithelial branches, affect lung function. This, in turn, can lead to congenital abnormalities such as cystadenomatoid malformations, pulmonary hypertension or lung hypoplasia. Defects in lung architecture are also associated with adult lung disease, particularly in cases of idiopathic lung fibrosis. Identifying the signaling pathways that drive epithelial tube formation will likely shed light on both congenital and adult lung disease. This study shows that mutations in the planar cell polarity (PCP) genes: Celsr1; Vangl2 and Scribble, lead to disrupted lung development and defects in lung architecture. Examination of Vangl2 mutant kidneys reveals similar impairment of branching morphogenesis. Detailed histological and immunocytochemical analysis reveals that lungs from Celsr1Crsh/Crsh, Vangl2Lp/Lp and ScribbleCrc/Crc mice are small and misshapen with fewer branches, and by late gestation exhibit thickened interstitial mesenchyme and defective saccular formation. Moreover, epithelial integrity is disrupted, cytoskeletal remodeling perturbed and mutant endoderm does not branch normally in response to the chemoattractant FGF10. In ex-vivo culture, inhibition of Rho kinase, an important downstream effector of the PCP signaling pathway, can mimic the branching defects observed in these three mouse mutants. Furthermore, all three proteins are present in restricted spatial domains within lung epithelium. ScribbleCrc/Crc lungs, the most severely affected line, exhibit additional defects in components of the tight and adherens junctions; this in turn affects lumen diameter. These findings show that components of the PCP pathway: Celsr1; Vangl2 and Scribble are required for normal foetal lung development, thereby revealing a novel signalling pathway critical for this process. Examination of postnatal mice was not possible as homozygous mutations result in embryonic lethality. However, an assessment of Vangl2Lp/+ mice reveals that loss of a single copy of Vangl2 is enough to cause defects in embryonic lung development that persist into adult life, affecting lung function. Similarly, Vangl2Lp/+ mice show a small but significant reduction in kidney glomeruli.

Planar cell polarity (PCP) is a developmental signaling mechanism that establishes a polarity within the plane of an epithelium. PCP has been shown to play a role in guiding numerous neurodevelopmental processes such as convergent extension, neuron migration, and axon pathfinding. Certain commissural neurons in the dorsal spinal cord make a series of guidance decisions en route to the brain: first, a ventral projection along the D-V axis, followed by a midline crossing, and after exiting the floorplate, a dorso-anterior turn along the A-P axis. Here, we provide in vivo evidence that the axons of the Commissural Primary Ascending (CoPAs) neurons in zebrafish require the PCP genes fzd3a, vangl2, and scribble for rostral pathfinding both before and after crossing the midline. Dorsoventral guidance of CoPA axons is unaltered in fzd3a, vangl2, and scribble mutants, suggesting that the PCP signaling pathway only controls A-P guidance of CoPAs. Our results have provided evidence for two potential non- mutually exclusive models: (i) A-P axon guidance is achieved by cell-autonomous Wnt-Frizzled signaling or that (ii) A-P axon guidance is achieved by non-cell-autonomous PCP signaling in the neuroepithelial environment. The single-cell nature of the CoPA axon system allows for simple genetic manipulation and visualization, which will potentially elucidate the validity of either model. Scribble (Scrib), a member of the LAP family, plays a critical role in establishing and regulating cell polarization in epithelia and during cell migration. In zebrafish, Scrib mutants have defects in convergent extension (CE) cell movements and facial branchiomotor neuron (FBMN) migration. Despite our understanding of Scrib’s genetic role in neurodevelopment, little is known about the subcellular localization of endogenous Scrib in vivo during CE and FBMN migration. We have generated a monoclonal antibody against the C-terminus of zebrafish Scrib and have shown that this antibody is specific against endogenous Scrib in both western blot and immunocytochemical applications. Confocal microscopy of Scrib immunocytochemistry shows that at various developmental stages, Scrib distinctly localizes to basolateral membranes of non polarized epithelium, to the membrane in mesodermal cells undergoing CE, and to the membrane of migrating FBMNs. Furthermore, the distribution of Scrib puncta along membranes of FBMN- FBMN contact is significantly altered in the PCP mutant pk1b. Further application of our newly generated Scrib antibody will potentially lead to new insight on Scrib’s role in neurodevelopment.

In zebrafish, CoPA (Commissural Primary Ascending) is the first among ascending commissural axons to pathfind anteriorly and form the spinal commissure. One pathway that guides their anterior growth is the planar cell polarity (PCP) signaling pathway, but it is not fully known how PCP signaling regulates anterior guidance. We examined CoPA pathfinding in various PCP mutants to determine if anterior-posterior (A-P) guidance of CoPAs is dependent on PCP signaling. We found that certain PCP mutants exhibited anterior pathfinding defects, with approximately half of all affected CoPAs migrating incorrectly posteriorly. By using a translation-blocking DCC (Deleted in Colorectal Cancer) morpholino to prevent CoPA midline crossing, we discovered that CoPA axons in Fzd3a and Scribble mutants show severe defects in A-P guidance, which suggest that PCP influences A-P guidance of CoPAs prior to and after midline crossing.

Neuromesodermal progenitors (NMPs) are bipotent progenitors, located at the caudal end of the embryo and are essential for axis formation. These stem cell-like progenitors possess the ability to self-renew and differentiate to both mesodermal and neural lineages, such as skeletal muscle and spinal cord derivatives. These progenitors arise at E8.5 and are localised in the caudal lateral epiblast (CLE), a posterior region of the embryo near the primitive streak. Later in development, they reside in the tail bud until cessation of axial elongation at E13.5. Throughout these stages NMPs are characteristically marked by co-expression of T(Bra) (Brachyury) and Sox2. This characteristic is also present in in vitro NMPs, which can be derived from Epiblast Stem Cells (EpiSCs) through treatment with Wnt/β-catenin signalling agonists and Fgf2, which simulates their in vivo environment. Protein and mRNA profiling of NMPs and mutant phenotypes in vivo supports the hypothesis that a non-canonical Wnt pathway, the Wnt/Planar Cell Polarity pathway (PCP) could be involved in NMP fate decision and/or maintenance. This thesis focuses on understanding more about the role of PCP by aiming to identify the spatio-temporal profile of Wnt/PCP pathway components in NMP regions during axial elongation, as well as determining its role in NMP behaviour through manipulation of this pathway via in vivo and in vitro assays Employing in situ hybridisation and immunohistochemistry techniques, key Wnt/PCP components, including Pk1, Vangl2 and Ptk7, were confirmed to be present in in vivo and in vitro NMPs, thus, providing strong evidence that Wnt/PCP may be involved regulating NMP behaviour. Disruption of Wnt/PCP signalling through overexpression of Wnt/PCP components was tested in refined in vivo and in vitro assays. Overexpression of Vangl2 and Ptk7, but not Pk1 in NMPs regions in vivo resulted in loss of contribution to neural lineages, as well as lower contribution to NMP regions themselves. Similarly, Wnt/PCP components were disrupted in vitro through generation of dox-inducible overexpression cells lines for Wnt/PCP components. These lines were used to generate NMPs from an optimised novel alternative source Epiblast-Like Cells (EpiLCs), however no clear affect to lineage was observed. Overall this work has successfully advanced our knowledge of Wnt/PCP mediated control of NMP differentiation and maintenance, and provided a finer grained description of the relationships between them.

Little is known about the molecular components and interactions of the planar cell polarity pathway that regulate neuronal polarity. This study uses a prkl-1 induced backwards locomotion defect as an array to perform a prkl-1 suppressor screen in C. elegans looking for new components of the planar cell polarity pathway involved in the neuronal polarization of VC4 and VC5. The screen discovered twelve new alleles of vang-1, one new allele of fntb-1 and five new mutations in unknown polarity genes. fntb-1 encodes for the worm ortholog of Farnesyltransferase β-subunit and is important for neuronal polarization. Acting cell and non-cell autonomously, fntb-1 regulates the function and localization of prkl-1 through the recognition of a CAAX motif. Therefore, fntb-1 modifies prkl-1 to regulate the neuronal polarity of VC4 and VC5.

Cilia are small microtubule based protrusions found on most cells of the vertebrate body. In humans, defects in the structure or function of cilia results in a large class of developmental and homeostatic diseases known collectively as the ciliopathies. Ciliogenesis is accomplished by the concerted action of a number of molecular pathways including the intraflagellar transport (IFT) system. IFT is a group of ~20 highly conserved proteins that assemble into large macromolecular complexes known as trains. These trains act to carry cargo bi-directionally between the cell body and ciliary tip, via interaction with the microtubule motors kinesin and dynein. IFT train dynamics are required for both cilia structure and function, however the controls on these dynamics are still incompletely understood. Here, I present the first platform for study of IFT dynamics within vertebrate multiciliated cells, an understudied population with critical functions in development and homeostasis. Using this platform, I demonstrate that the planar cell polarity effector protein Fuz is required for IFT dynamics via its control of the cytoplasmic localization of a subset of IFT proteins. Subsequently, I find that a Fuz binding partner, the putative small GTPase Rsg1, is also required for IFT protein localization and dynamics. Additionally, I describe a role for Rsg1 in basal body docking, one of the earliest events of ciliogenesis. Finally, I show that the poorly studied protein TTC29 is required for a specific subset of IFT dynamic behaviors. These data reveal novel regulatory motifs for ciliogenesis and demonstrate, specifically, the complexities of IFT regulation in the cytoplasm and within the cilium itself. Finally, they suggest that multiciliated cells provide a tractable platform for generating robust datasets for the investigation ciliary dynamics. Such studies are critical for informing our understanding of the molecular etiology of human ciliopathic diseases. / text

Little is known about the molecular components and interactions of the planar cell polarity pathway that regulate neuronal polarity. This study uses a prkl-1 induced backwards locomotion defect as an array to perform a prkl-1 suppressor screen in C. elegans looking for new components of the planar cell polarity pathway involved in the neuronal polarization of VC4 and VC5. The screen discovered twelve new alleles of vang-1, one new allele of fntb-1 and five new mutations in unknown polarity genes. fntb-1 encodes for the worm ortholog of Farnesyltransferase β-subunit and is important for neuronal polarization. Acting cell and non-cell autonomously, fntb-1 regulates the function and localization of prkl-1 through the recognition of a CAAX motif. Therefore, fntb-1 modifies prkl-1 to regulate the neuronal polarity of VC4 and VC5.