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.

Sonic hedgehog (Shh) is an important signalling protein expressed extensively in development, throughout tissues of the central nervous system, gut and the posterior of the limb bud. The complicated expression pattern of Shh is regulated by a series of long-range enhancers located flanking and dispersed throughout a 1 Mb genomic desert. Disruption of SHH as a result of mutations within the gene or its enhancers has been implicated in two developmental conditions. These are Holoproencephaly (HPE3) a common developmental defect of the forebrain and frequently the mid-face in humans, and preaxial polydactyly (PPD), a congenital limb abnormality encompassing a varied phenotype affecting the digits on the anterior side of the hands and feet that has been attributed to misexpression of Shh. In order to investigate the Shh regulatory landscape and survey regulatory activity, a transposon-based chromosomal engineering strategy known as the local hopping enhancer detection (LHED) system was employed. Using this method a targeting vector containing a LacZ reporter gene as well as LoxP sites was inserted within the Shh region. The ‘hopping’ nature of the transposable element was then exploited to scatter it throughout the region. Tetraploid complementation embryos derived entirely from ES cells were generated and examined in order to gain an insight into enhancer activity. The region was found to be in an open conformation over its length and is generally susceptible to all Shh enhancers. Genes within the regulatory domain, such as the widely expressed Rnf32 gene, were found to resist Shh enhancer activities by a process of regulatory evasion by the promoter, a mechanism that may be common in large regulatory domains. Finally, at the boundaries of the region Shh activity was found to be lost incrementally at a number of genomic positions. Mouse lines were also generated to look at both enhancer activity and loss of function effects and three deletions of increasing size were generated between Shh and the furthest enhancer, the Zrs. These in turn, delete firstly a gut and pharyngeal epithelial enhancer, secondly the gut, pharyngeal enhancers as well an oral epithelial enhancer and finally all three epithelial enhancers as well as three forebrain enhancers. Reporter gene expression was found to be lost incrementally from those enhancers deleted without disrupting the rest of the region. Previously unidentified notochord enhancer(s) were found to lie within the region 100-530 kb upstream of Shh. Examination of the resultant phenotypes showed that gut and craniofacial defects were found to occur as a result of the loss of enhancers which drive expression within these tissues. Variable phenotypes were found to occur potentially as a result of temporal changes to Shh expression or as a result of threshold levels of HH being required for normal development. Other enhancers within the Shh region and outwith the deletions were not found to be disrupted by these modifications suggesting the enhancers within the region act independently of each other. The largest deletion resulted in bringing the Zrs (which drives Shh limb expression) within 170 kb of the gene, however limb development; was not, found to be affected suggesting distance is not required for Zrs function. Overall, the LHED transposon system has been utilised in order to examine the Shh region in more detail, allowing mapping of enhancer function by reporter gene expression and examination of phenotypes generated by deletion of enhancers.

The Hedgehog signalling pathway is an essential developmental pathway present in all bilaterians that is involved in embryogenesis, body patterning and stem cell homeostasis. Dysregulation of the Hh pathway leads to various kinds of cancer, such as basal cell carcinoma and medulloblastoma. Smoothened (SMO), a Frizzled-type G-protein coupled receptor (GPCR), is the essential transmembrane signal transducer within the Hh pathway, conveying the signal from the upstream transmembrane protein, Patched1 (Ptc1), to the downstream intracellular proteins. The mechanisms by which SMO transmits the Hh signal from the extracellular environment, through the plasma membrane and to the intracellular proteins are not known. In this thesis, I present my work into the structural and functional characterisation of the extracellular and transmembrane domains (TMD) of human SMO in order to better understand the molecular mechanisms of its signal transduction. The extracellular region of SMO contains a highly conserved cysteine-rich domain (CRD) and a linker domain (LD). I present the first crystal structure of the CRD, LD and TMD of SMO, which is also the first crystal structure of a GPCR with a large functional extracellular domain. This structure revealed a domain architecture for SMO that enables regulation of its transmembrane domain by its extracellular domains. It also revealed a cholesterol molecule bound to the CRD, which we subsequently determined to be a new endogenous small-molecule agonist for SMO. I present five further structures of SMO bound to different small molecule agonists and antagonists. Together, these structures demonstrate that the position of the CRD relative to the TMD reflects the activation state of SMO. We also generated nanobodies against the extracellular region of SMO in order to stabilise its conformation. These studies not only improve our understanding of the workings of a key transmembrane protein within a fundamental signalling pathway but will also aid efforts to develop better therapeutics for an important cancer target.

Orientadores: Carmen Verissima Ferreira Halder, Silvia Mika Shishido / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T22:59:08Z (GMT). No. of bitstreams: 1 Machado_Daisy_D.pdf: 7339714 bytes, checksum: b126414072e5dc8abcca091c7af0fdbf (MD5) Previous issue date: 2012 / Resumo: Melanoma é o tipo mais agressivo de tumor de pele e uma das principais causas de morte por tumor de pele, devido a sua alta capacidade metastática. Em termos de estratégias terapêuticas de combate ao melanoma tem-se dado ênfase no controle da resistência e da metástase. Nosso grupo de pesquisa observou que a riboflavina irradiada (RFi) induz apoptose de células de câncer de próstata, renal e leucemia mielóide. Portanto, o objetivo geral deste trabalho foi utilizar a RFi para modular química e geneticamente as vias de transdução de sinal associadas com a sobrevivência, resistência e agressividade do melanoma. Assim, neste trabalho estão apresentados os dados sobre a influência da RFi em diferentes aspectos metabólicos das células de melanoma murino (B16F10), tais como: citotoxicidade, adesão, invasão, migração, capacidade de formação de colônia e em mediadores de transdução de sinal: Src quinase, mTOR e componentes da via sonic hedgehog. Em todos os experimentos a riboflavina (RF) foi previamente irradiada com UVA (dose de 9 J/cm²). Foi observado inibição da proliferação celular com valor de IC50 de 50 ?M. De forma interessante, RFi na faixa de concentração de nanomolar foi eficiente na inibição da formação de colônias. Além disso, causou a redução da adesão das células B16F10, quando utilizada na concentração de 1?M. A capacidade de migração e invasão das células de melanoma foi reduzida na presença da RFi, nas concentrações de 1 e 30 ?M respectivamente, porém a resposta foi independente da dose. A atividade e expressão das metaloproteinases foram diminuídas na presença de RFi, indicando inibição na capacidade de invasão. Sob o contexto de sinalização a RFi modulou negativamente a via sonic hedgehog, PI3K/mTOR e aumentou a expressão da p53 e PTEN. O conjunto de resultados obtidos nesse trabalho mostra que flavinas são candidatas promissoras para a intervenção farmacológica do melanoma / Abstract: Melanoma is the most aggressive type of skin disorder and a major cause of death by skin's disease due to its highly metastatic ability. In terms of melanoma therapeutic strategies has given emphasis on control of resistance and metastasis. Our group observed that irradiated riboflavin (RF) induces apoptosis of prostate cancer cells, kidney cancer cells and myeloid leukemia. Therefore, the goal of this study was to employ irradiated RF for modulating chemical and genetically signal pathways associated with melanoma survival, resistance and aggressiveness. Thus, in this manuscript data about the influence of RF in different cellular metabolic aspects of murine melanoma (B16F10) such as cytotoxicity, adhesion, invasion, migration, colony formation and signal transduction mediators Src kinase, mTOR and sonic hedgehog components, will be presented. In all experiments the RF was previously irradiated with UVA (dose of 9 J/cm²). Inhibition of cell proliferation was observed with IC50 value of 50 ?M. Interestingly, RF in a nanomolar concentration inhibited the formation of colonies. In addition, 1 ?M irradiated RF caused a reduction of B16F10 cells adhesion. The ability of migration and invasion of melanoma cells was reduced in the presence of RF, however, those cells response was dose-independent. The activity and expression of metalloproteinases were diminished indicating reduction of cellular invasiveness capacity. Sonic hedgehog and PI3K/mTOR pathways were negatively modulated and the expression of p53 and PTEN were increased in melanoma cells treated with irradiated RF. The findings showed in this study brought out flavins as promising candidates for pharmacological intervention of melanoma / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Correct number and pattern of digits is determined in a time and concentration-dependent manner by a gradient of Sonic Hedgehog (SHH) across the anterior-posterior axis of the embryonic limb bud. Owing to the potent morphogenic/mitogenic capabilities of SHH, transcription of the SHH gene in the limb is tightly regulated by feedback loops with other signalling pathways and by the Zone of Polarising Activity regulatory sequence (ZRS). The ZRS is a long-range, cis-regulatory limb-specific enhancer of SHH, and is essential for correct limb SHH expression. The Silkie, a polydactylous breed of chicken, possesses a C > A mutation in the ZRS, resulting in ectopic SHH expression in the anterior limb and hindlimb-specific polydactyly. We employ the Silkie mutant to investigate how SHH is regulated by the ZRS, and how Hedgehog signalling can modulate SHH expression in an autoregulatory manner. We further characterise the effects that the Silkie mutation has on subsequent limb development; investigating the dependence of increased posterior SHH, increased Hedgehog-dependent growth and necessary genotype in both the posterior and anterior limb bud. Several fundamental questions regarding SHH during limb development have yet to be fully addressed: how much SHH protein is present, and does it form a gradient as hypothesised by Wolpert’s Morphogen Gradient Model? By developing a standard curve-based method to assess absolute quantities of processed SHH protein, N-SHH, we find that the quantity of N-SHH protein increases through limb development, and does indeed form a quantifiable gradient across the posterior limb. By comparing quantity of N-SHH protein in equivalently staged mouse, rat, emu and chicken limbs, we find that there is no significant link between N-SHH protein quantity and digit number between mammalian and avian species, and investigate how digit number is modulated in the late limb. A number of species exhibit reduced numbers of digits, including the wings of the emu, cassowary and kiwi. Unlike in mammalian examples of digit loss (i.e. cow, pig) the emu wing has delayed and significantly reduced SHH expression. Through sequencing and functional in vivo testing of ZRS sequences of ratite bird species, we investigate whether the ZRS has a role in evolutionary digit loss. We also demonstrate the aspects of digit loss and Hh signalling are shared with examples of mammalian digit loss. This thesis presents novel research into multiple aspects of genetic regulation, limb development, and evolutionary developmental biology; elucidating both long held dogmas and upcoming areas of limb development.

Nematic liquid crystals are mesogenic materials that are popular working materials for optical displays. There has been an increased interest in bistable liquid crystal devices which support two optically distinct stable equilibria. These devices typically exploit a complex geometry or anchoring conditions, which often induces defects in the equilibria. There remains a great deal to be understood about the structure of the defects and how they stabilize multiple equilibria in modern devices. This thesis focuses on four problems: the first three explore the effect of confinement and defects on nematic equilibria in simple geometries, with the aim of exploring multistability in these geometries; the fourth problem concerns the fine structure of point defects, essential for future modelling of nematic equilibria in more complex geometries. Firstly, we study nematic liquid crystals confined to two-dimensional rectangular wells using the Oseen-Frank theory. Secondly, we study equilibria within a semi-infinite rectangular domain with weak tangential anchoring on the surfaces. Thirdly, we study nematic equilibria within two-dimensional annuli. We derive explicit expressions for the director fields and free energies of equilibria within these geometries and discuss the stability of the predicted states. These three problems are motivated by the experimental work on colloidal nematic liquid crystals, which we interpret in the context of our results. Finally, we study the fine structure and stability of the radial hedgehog defect in the Landau-de Gennes theory with a sixth order bulk potential, relevant to the observability of global biaxial phases in a model with higher order potential terms.

In the vertebrate retina, bipolar interneurons consist of at least 13 distinct subtypes, which are classified based on their morphology, behavior and gene expression. The mechanisms underlying the formation of these subtypes is poorly understood. Our previous unpublished work has implicated Sonic Hedgehog (Shh) in the formation of cone and rod bipolar cell subtypes. In this thesis, I characterized the relationship between Hh signaling and bipolar subtype cell development in greater detail. Using an in vivo plasmid-based reporter approach, I show that Hh signaling is active in both retinal progenitor cells (RPCs) and bipolar cells of the postnatal retina. Next, to address function, I used a conditional gene targeting approach to show that activation of Smoothened (Smo), a downstream Hh signaling component, is both necessary and sufficient in postnatal RPCs to promote the formation of cone but not rod bipolar cells. In contrast, activation of Smo in postmitotic bipolar cells that are greater than 24 hours old from cell birth, does not affect bipolar subtype formation. Together, these results suggest that Hh signaling functions in postnatal RPCs (and potentially in early bipolar cell precursors) to promote cone bipolar cell formation. / Graduate / 2018-06-12

Sonic Hedgehog (SHH) is a secreted morphogen that is an essential regulator of patterning and growth. The SHH protein requires cleavage of its full-length precursor (SHHFL) for secretion of biologically active SHH (SHHNp). Mutations in SHH that affect SHH processing are associated with human disease, which highlights the importance of processing for patterning in vivo. We identified Sortilin (SORT1), a member of the VPS10P receptor family, as a novel SHH interacting protein. SORT1 preferentially associates with SHHFL and SORT1 levels correlate inversely with cleavage of SHHFL. Consistent with an antagonistic relationship between SORT1 and SHH processing, loss of SORT1 results in an increase in SHH levels in axons and a partial rescue of Hedgehog-associated patterning defects in a mouse model of deficient SHH processing. Finally, we demonstrate a functional requirement for SORT1-mediated trafficking on SHH-dependent signaling from axons in the developing visual system in vivo. Our findings identify a novel role for SORT1 in the regulation of SHH processing and trafficking.

Medulloblastoma (MB), a cancer of the cerebellum, is the most common solid tumor affecting children. In the cerebellum, Sonic Hedgehog (Shh) drives the proliferative expansion of granule neuron progenitors (GNP). These cells are located in the external granule layer (EGL) and are the cells of origin of Shh-MB. We recently identified Norrie Disease Pseudoglioma (Ndp) as a novel downstream target of Hh signaling in the developing retina. Ndp encodes an X-linked cysteine-rich secreted protein called Norrin, which is best known for its role in angiogenesis and blood brain barrier (BBB) maintenance in the developing retina and cerebellum, respectively. Norrin mediates this effect by binding to its receptor Frizzled4 (Fzd4) and co-receptors LRP5/6 and Tpsan12 to activate the canonical, β-catenin-dependent Wnt signaling pathway in endothelial cells (ECs). We detected the expression of Ndp and all required receptors in mouse GNPs and MB samples. To investigate a potential role for Ndp in Hh-driven MB, we genetically and pharmacologically inactivated Ndp/Fzd4 signaling in Ptch+/- mice (a mouse model for human Gorlin syndrome), which dramatically increased the incidence and reduced the latency of MB. This accelerated rate of tumorigenesis was caused by an increase in the number of preneoplastic lesions (PNLs), the precursor lesions to MB, and a faster conversion of these lesions to MB. We showed that Ndp mediates this increase in tumorigenesis by signaling through endothelial cell receptor Fzd4 to alter the GNP stroma, which is characterised by 5 major alterations: 1) activated angiogenic program, 2) open BBB, 3) aberrant deposition of extracellular matrix, 4) aberrant lymphocyte recruitment and 5) reduction in meningeal lymphatic vasculature. We propose that these stromal alterations are associated with a pro-tumor microenvironment that promotes DNA damage in GNPs and leads to enhanced lesion formation and progression towards MB. This research highlights 1) an unanticipated role for Ndp/Fzd4 signaling in Shh-MB initiation and progression, 2) a role for stromal signaling in the regulation of MB development and 3) a previously undescribed role for Ndp signaling in maintaining meningeal cerebellum lymphatic vessels.

The caudal fin of zebrafish (danio rerio) is often used to study regeneration thanks to its extraordinary regenerative ability, easy access, and relative simplicity in structure. Branching morphogenesis is observed in many organs, including lungs and salivary glands in mammals, as well as the fin rays in zebrafish and is thought to follow unifying principles. An important developmental gene, sonic hedgehog a (shha), has been shown in other studies to play an essential role in the branch formation. Previous studies in our lab have shown that the transient depletion of the shha-expressing cells following laser ablation of the shha-expressing cells in the regenerating caudal fin results in a delay of fin rays branch formation. In order to study the long-term effect of ablating the shha-expressing cells, I generated a new zebrafish transgenic line (Tg)(2.4shha:CFP-NTR-ABC) to perform a conditional cell ablation using the Metronidazole/Nitroreductase (Mtz/NTR) system. Preliminary data suggest that cell ablation using the Mtz/NTR system is successful in the Tg(2.4shha:CFP-NTR-ABC) embryos. In addition, short-term ablation of the shha-expressing cells through Mtz/NTR system delays branch formation during caudal fin regeneration of the Tg(2.4shha:CFP-NTR-ABC) adult fish. Further work will involve the analysis of the effects of the long-term ablation of the shha-expressing cells and the involvement of other signaling pathways in the ray branching formation during zebrafish caudal fin regeneration. This study can provide insights into understanding of the molecular mechanisms underlying branching morphogenesis in various organs. During the course of the above project, I have observed an organ-wide response to local injury in the zebrafish caudal fin. In this study, I have shown, for the first time, an immediate organ-wide response to partial fin amputation characterized by the damage of blood vessels, nerve fibers and the activation of inflammatory response in the non-amputated tissues. I established that the adult zebrafish caudal fin serves as an excellent model for the study of the organ-wide response to local injury, and such study may provide new insights into the field of regenerative medicine in which stimulating regeneration locally may trigger responses in unintended locations.   Résumé La nageoire caudale du poisson zèbre (danio rerio) est souvent utilisée pour étudier les mécanismes de régénération à cause de son extraordinaire capacité de régénération, son accès facile, et sa relative simplicité structurale. La morphogenèse de branches est observée dans plusieurs organes incluant les poumons et les glandes salivaires chez les mammifères ainsi que les rayons des nageoires du poisson zèbre et est supposée suivre des principes communs. Un important gène de développement, sonic hedgehog a (shha), joue un rôle essentiel dans la formation des branches. Des études précédentes effectuées dans notre laboratoire ont montré que l’absence transitoire des cellules exprimant shha dans des expériences d’ablation au rayon laser induit un délai de la formation des branches dans les rayons au cours de la régénération de la nageoire caudale. Afin d’étudier les effets de l’ablation à long terme des cellules exprimant shha, j’ai fait un nouvelle lignée transgénique de poisson zèbre Tg(2.4shha:CFP-NTR-ABC) pour effectuer une ablation cellulaire conditionnelle à l’aide du système Métronidazole / Nitroréductase (Mtz/NTR). Mes données préliminaires suggèrent que l’ablation cellulaire à l’aide du système Mtz/NTR fonctionne sur les embryons Tg(2.4shha:CFP-NTR-ABC). De plus, l’ablation à court terme des cellules exprimant shha à l’aide du système Mtz/NTR induit un délai de la formation des branches au cours de la régénération des rayons la nageoire caudale des poissons adultes Tg(2.4shha:CFP-NTR-ABC). Des études supplémentaires incluront l’analyse des effets de l’ablation à long terme des cellules exprimant shha et le rôle d’autres cascades de signalisation dans la formation des branches des rayons au cours de la régénération de la nageoire caudale du poisson zèbre. Cette étude pourrait fournir des informations concernant la compréhension des mécanismes moléculaires sous-jacents à la formation de branches dans des organes variés. Au cours de l’étude décrite ci-dessus, j’ai fait l’observation d’une réponse globale de toute la nageoire caudale à une blessure locale. Dans cette étude, j’ai montré pour la première fois, une réponse immédiate et globale après amputation partielle de la nageoire. Cette réponse est caractérisée par des lésions des vaisseaux sanguins, des fibres nerveuses et par l’activation d’une réponse inflammatoire dans les tissus non-amputés. J’ai établi que la nageoire caudale du poisson zèbre adulte est un excellent modèle pour l’étude de la réponse globale d’un organe à une lésion locale. Une telle étude pourrait fournir de nouvelles informations pertinentes à la médecine régénérative qui, en visant à stimuler la régénération de façon locale, peut entraîner des réponses dans des domaines non voulus.