The focus of this study was to elucidate the molecular and cellular mechanisms whereby fibroblast growth factors (FGFs) mediate outgrowth of the limb. Specifically, we examined the epistatic relationship between FGF and Wnt/Planar cell polarity (PCP) signaling in establishing cell polarity as a mechanism for outgrowth. By implanting beads into embryonic limbs and lateral plate mesoderm, we established that FGF activates Wnt5a in a gradient fashion. Once it was established that Wnt5a was expressed at the right time and place to turn on PCP signaling, we investigated the ability of Wnt5a to influence cell migration and/or cell polarity. Our analysis revealed that there was no difference in cell migration when cells were exposed to an exogenous Wnt5a source. However, this did not rule out the possibility that cells were responding in a more mild fashion and polarizing toward a Wnt5a source. Live cell imaging was performed to observe the movement and morphology of limb mesenchyme cell cultures in the presence or absence of a Wnt5a expressing cell bolus. It appears as though the cells orient and move in a random fashion regardless of Wnt5a. However, this in vitro method may not truly recapitulate in vivo events. Future studies aim to develop better methods of observing cell polarization in vitro, including developing better methods to tract the movement of cells and observe “PCP” events. Due to the lack of information gathered from our in vitro studies, an in vivo study was conducted to test if FGF is necessary to polarize limb mesenchyme cells. If FGF is turning on Wnt5a and Wnt/PCP signaling is directing cell polarization, then FGF mutant clones will not migrate toward the AER. Therefore, it is expected that these mutant clones would be unable to undergo directed cell movement and/or cell divisions. Early clonal analysis indicates that a response to FGFs appears to be necessary to direct polarized outgrowth of limb mesenchyme.

Despite over 60 years of study, the molecular pathways and mechanisms governing limb outgrowth and patterning remain poorly understood. Fgfs expressed in the AER are known to be necessary and sufficient for proximodistal limb outgrowth and have been proposed to have a chemoattractive role. Wnt5a is a secreted factor which is expressed in a gradient in the distal limb with the highest concentration next to the AER. The presence of the AER is necessary to establish this gradient. Expression of Wnt5a in a concentration dependant manner can be induced in the limb through the implantation of a bead soaked in recombinant Fgf4 protein. This indicates that Fgfs from the AER may establish the gradient of Wnt5a in the limb mesenchyme. Wnt5a-/- mutants exhibit severe shortening of the face, limbs, and body axis, with limbs being progressively truncated proximally to distally. In normal limb proximodistal outgrowth, cells are seen to grow directionally toward the AER. Previous studies done in the Barrow lab, as well as those done by myself, have shown that if a portion of the AER is removed and the cells proximal to this area are labeled, those which are close enough to intact AER will redirect their growth toward this intact AER. When Wnt5a secreting cells are implanted in the limb mesenchyme of the chick this ectopic source of Wnt5a is sufficient to redirect the growth of the mesenchyme cells toward the Wnt5a source. This indicates that the AER may mediate directed growth of limb mesenchyme cells through the establishment of the Wnt5a gradient which provides positional information to the cells. This Wnt5a gradient results in the recruitment of the mesenchyme cells toward the AER. The Ror2 receptor has been found to be involved in several different pathways involving Wnt5a which are involved in changes in polarity and migration. This makes Ror2 a likely candidate for causing changes in cell polarity and migration during distal outgrowth in the limb. To test whether Ror2 is necessary for the polarizing response of limb mesenchyme cells to the Wnt5a gradient in vivo I co-transfected a dominant-negative Ror2 (Ror2ΔC) and a GFP expression vector in the embryonic chick limb using sonoporation. Limb mesenchyme cells transfected with dominant-negative Ror2 grew as radial clones in contrast to the directional outgrowth of the control limb mesenchyme cells along the proximodistal axis. This indicates that cells expressing the dominant-negative Ror2 could no longer respond to the Wnt5a gradient in the limb mesenchyme. This supports a role for Ror2 as a receptor or co-receptor for Wnt5a in mediating directional growth and movement during proximodistal outgrowth and patterning in the limb.

The ophthalmic trigeminal placode (opV) exclusively gives rise to sensory neurons. A number of signaling pathways including Wnt, PDGF, FGF, and Notch are all involved in the progression of an undifferentiated cell in the opV placode to a proneural cell in the condensing opV ganglion. However, the regulatory relationships between these signal transduction pathways are still unknown. To determine if FGF activation acts to modulate Notch signaling in the sensory neurogenesis pathway, a novel multifactorial approach was employed: FGF signaling was inhibited in individual cells and globally with simultaneous inactivation of Notch signaling in chick embryos to investigate if FGF activation downregulates Notch thereby driving neurogenesis. These experiments resulted in few differentiating opV cells in the mesenchymal region of future ganglion formation suggesting an alternate regulatory relationship between FGF and Notch where either reduced Notch activity allows for FGFR4 expression (leading to FGF signaling and neurogenesis), or a parallel relationship where FGF and Notch act independently of one another to induce neurogenesis. To distinguish between these two possibilities Notch signaling was inhibited with DAPT, a gamma-secretase inhibitor, and assayed for FGFR4 mRNA expression. These results indicated FGFR4 is not upregulated by reduced Notch activity, suggesting that FGF and Notch act in parallel to promote neurogenesis. During these experiments it was observed that Notch inhibition resulted in an undefined ectoderm in the opV placode region. To investigate this, FGF and Notch were inhibited by SU5402, an FGF antagonist, and DAPT, and later sectioned and stained for Laminin. In DAPT treated embryos the basement membrane became highly fragmented, a remarkable observation not yet reported. From these data a proposed mechanism was established where activation of FGF with parallel downregulation of Notch leads to disruption of extracellular matrix proteins in the basement membrane resulting in fragmentation and subsequent delamination of differentiating opV placode cells.

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Ce travail a été mené afin d’étudier l’effet du FGF 2 et du TGF-β1 sur les étapes précoces de la régénération dentinaire en utilisant la micro-encapsulation de ces facteurs dans une matrice pour les protéger et contrôler leur libération et ensuite l’application des microparticules obtenues en coiffage pulpaire direct dans un modèle de culture de dents entières. Ce travail consiste d’abord à l’optimisation des moyens techniques mis en oeuvre pour réaliser l'encapsulation du TGFβ1, FGF-2 à l'aide de l'acide poly (lactique-glycolique) PLGA. Les études de la caractérisation colloïdal et physico chimique des microparticules montre que les microparticules gardent leurs caractéristiques physicochimiques après séchage et resuspension dans l’eau. La procèdes optimisé a été ensuite utilisé pour encapsuler les facteurs de croissance. L’encapsulation de FGF-2 et TGF-β1 a été obtenue avec une taille, une efficacité d’encapsulation et une profile de libération adaptés au type d’application choisi. Les études biologiques ne montrent aucun effet toxique des particules sur les fibroblastes pulpaires. Les facteurs de croissance ont gardé leur activité biologique spécifique. Un modèle de culture de dent entier humain a été utilisé pour réaliser l’application de nos microparticules comme un matériau de coiffage dentaire pour confirmer leurs activités biologiques ex-vivo. Ces microparticules peuvent être utiles dans les études des étapes précoces de la régénération dentinaire, l'activation et la migration des cellules progénitrices de la pulpe dentaire / The purpose of this work was to investigate the effect of FGF 2 and TGF-β1 on the early steps of dentin regeneration using microencapsulation of theses factors into a microparticles matrix to ensure growth factors protection and to provide bioactive sustained release in contact with dental pulp cells and then the application of the obtained microparticles in direct pulp capping using a culture model of entire tooth. This work involves the optimization of technical means used to achieve encapsulation of TGFβ1, FGF-2 using the poly (lactic-glycolic acid) PLGA. Physicochemical and colloidal characterization of microspheres shows that the microparticles retain their physicochemical characteristics after drying and re-suspended in water. The double emulsion method was used to separately encapsulate (FGF-2) and (TGFβ1). Microparticles morphology, loading, shelf life, potential toxicity and release kinetics were studied. Then the proliferation of dental pulp cells was examined in contact with microparticles. Biological studies show no toxic effect of particles on pulp fibroblasts. Growth factors have kept their specific biological activity. A culture model of human entire tooth was used to achieve the application of microparticles as a dental direct capping material to confirm their biological activities ex vivo. These microparticles can be useful in studies of early steps of dentin regeneration, activation and migration of progenitor cells in dental pulp

Descrevemos, recentemente, que FGF2 parácrino dispara senescência nas linhagens celulares murinas Y1 e 3T3-B61, transformadas malignamente por Ras, mas sem ativação das vias apoptóticas (Costa et al., 2008). Nesta tese, estudamos os mecanismos celulares e moleculares desta resposta de estresse irreversível, disparada por FGF2. Focalizamos, principalmente, a linhagem Y1, que carrega uma amplificação do oncogene Kras, mas apresenta um controle parcial da transição G0/G1 → S do ciclo celular. Por estas características fenotípicas, as células Y1 foram utilizadas no estudo dos mecanismos das ações antagônicas de FGF2, isto é, a atividade mitogênica clássica e a nova ação citotóxica que causa senescência. Análises de citometria de fluxo e marcação com BrdU mostraram que FGF2 promove a transição G0/G1 → S (atividade mitogênica), mas bloqueia a progressão através de S e G2/M (atividade antimitogênica). Ensaios de viabilidade celular (MTS e Cyto-Tox) demonstraram que, durante o bloqueio do ciclo celular por FGF2, as células permanecem íntegras e metabolicamente ativas, embora exibam alterações morfológicas, que sugerem estresse celular. Além disso, experimentos de tomada de 3H-timidina em DNA evidenciaram que, já nas primeiras horas de G1, FGF2 dispara um processo antimitogênico que só tardiamente vai se manifestar na fase S, bloqueando a síntese de DNA. Verificamos ainda, que o inibidor específico da Tyr-quinase dos receptores de FGF, PD173074, abole completamente, tanto os efeitos mitogênicos como os antimitogênicos de FGF2 nas células Y1, demonstrando que ambos os processos iniciam-se com a ativação da Tyr-quinase dos FGFRs. Por outro lado, inibidores específicos das vias de sinalização de MEK/ERK, PI3K/AKT e PKC (todas mitogênicas e à jusante dos FGFRs) bloqueiam a progressão no ciclo celular, sem proteger as células Y1 de FGF2, evidenciando que estas vias mitogênicas não participam dos mecanismos moleculares citotóxicos disparados por FGF2. Entretanto, inibidores das Tyr-quinases Src protegem parcialmente as células Y1 de FGF2, implicando a família Src na transformação maligna destas células. Para buscar novos genes pertinentes à ação citotóxica de FGF2 analisamos expressão gênica por RT-qPCR, dando continuidade a estudos anteriores desenvolvidos no laboratório, através de microarranjos de cDNA (Asprino & Armelin, 2006). Desta busca, resultaram genes codificantes de proteínas envolvidas no controle de ciclo celular, adesão e citoesqueleto, com destaque para proteínas reguladoras de RhoGTPases e os receptores de FGF. Procuramos também examinar especificidades entre os FGFRs, quanto ao disparo das ações antagônicas de FGF2. As células Y1 expressam FGFR1IIIc, FGFR2IIIc e FGFR5 enquanto as 3T3-B61 expressam FGFR1IIIc e FGFR5. A redução da expressão de FGFR2 por RNAi, em células Y1, não impediu a ação citotóxica de FGF2, mas a redução de FGFR1 protegeu as células da ação morfológico-estressante de FGF2. Como FGFR5 não possui domínio de Tyr-quinase, concluímos que o FGFR1 é o receptor mais relevante para o efeito citotóxico de FGF2, em ambas as células. Em conclusão, FGF2 ativa a Tyr-quinase dos FGFRs, disparando mecanismos moleculares antagônicos, paralelos e independentes, onde o efeito final é o bloqueio do ciclo celular nas fases S e G2/M e, consequentemente, senescência celular. / We have recently described that paracrine FGF2 triggers senescence in Ras-driven murine cell lines Y1 and 3T3-B61, without activation of apoptotic pathways (Costa et al., 2008). On this thesis, we studied the molecular and cellular mechanisms of this irreversible stress response triggered by FGF2. We have mainly focused on the Y1 cell line, which carries Kras oncogene amplification, but presents a certain control of the G0/G1 → S cell cycle transition. Because of these phenotypic features, the Y1 cells were utilized to study the mechanisms of FGF2 antagonic actions, i. e., the classical mitogenic activity and the new cytotoxic action, which causes senescence. Flow cytometer analysis and BrdU labeling have shown that FGF2 promotes the G0/G1 → S transition (mitogenic activity), but arrests the progression through S and G2/M (antimitogenic activity). Viability assays (MTS and Cyto-Tox) have shown that, during the cell cycle arrest by FGF2, cells remain intact and metabolically active, although exhibiting morphologic alterations, which suggested cellular stress. Moreover, 3H-thymidine uptake into DNA has evidenced that, within the first hours of G1, FGF2 triggers an antimitogenic process that only lately will manifest in S phase, blocking DNA synthesis. We have further verified that the specific Tyr-kinase inhibitor, PD173074, completely abolishes both FGF2 mitogenic and antimitogenic effects, showing that both processes start at FGFRs Tyr-kinase. On the other hand, specific inhibitors of MEK/ERK, PI3K/AKT and PKC signaling pathways (all mitogenic and downstream of FGFRs) block the cell cycle progression, but do not protect cells from FGF2, showing that these pathways do not participate of the cytotoxic molecular mechanisms triggered by FGF2. However, Src Tyr-kinases inhibitors partially protect Y1 cells from FGF2, implicating the Src family on malignant transformation of these cells. To search new genes pertinent to the cytotoxic action of FGF2, we analyzed gene expression by RT-qPCR, continuing previous studies developed in our laboratory through cDNA microarrays (Asprino & Armelin, 2006). This search revealed protein-coding genes involved on cell cycle control, cellular adhesion and cytoskeleton, in which we highlighted regulatory proteins of RhoGTPases and FGF receptors. We also examined specificities among FGFRs in relation to FGF2 antagonic actions. Y1 cells express FGFR1IIIc, FGFR2IIIc and FGFR5 whereas 3T3-B61 cells express FGFR1IIIc and FGFR5. In Y1 cells, the knockdown of FGFR2 expression by RNAi do not stop FGF2 cytotoxic actions, but knockdown of FGFR1 expression protects cells from the FGF2 morphologic-stressing action. Since FGFR5 lacks Tyr-kinase domain, we concluded that FGFR1 is the most relevant receptor for FGF2 cytotoxic effect in both cells. In conclusion, FGF2 activates FGFRs Tyr-kinase, triggering parallel and independent antagonic molecular mechanisms, in which the final effect is the S and G2/M cell cycle arrest and, consequently, cellular senescence.

Os Fatores de Crescimento de Fibroblastos (\"Fibroblast Growth Factors\"; FGFs) participam de fenômenos biológicos de grande importância, tais como migração, divisão e diferenciação celulares. O presente trabalho teve como objetivo central a busca de compostos biologicamente ativos através de um desenho racional de peptídeos derivados do FGF-1 e do seu receptor (FGFR-1 ). A partir da análise dos dados disponíveis na literatura, aliada a técnicas de modelagem molecular, foram desenhados, sintetizados e testados dois grupos de peptídeos. O primeiro conjunto (R1 - R3) é constituído por peptídeos lineares derivados do FGFR-1. Os ensaios de atividade mitogênica dos FGFs 1 e 2 em presença dos peptídeos mostram que R1 e R2 foram capazes de inibir a ação mitogênica do FGF-1. Este efeito é seletivo, já que a atividade do FGF-2 não é afetada. A atividade inibitória é dose-dependente para ambos os peptídeos. Os resultados mostram ainda que o efeito é sequência-dependente, já que o peptídeo R3, correspondente à porção e-terminal de R2, é inativo. Por outro lado, o segmento N-terminal de R2 (representado por R1) é suficiente para desencadear o mesmo nível de inibição apresentado pelo peptídeo R2 inteiro. Os peptídeos sintéticos semi-cíclicos F1 - F3, correspondentes a um importante sítio de ligação no FGF-1, foram avaliados quanto à sua capacidade de estimular a síntese de DNA em fibroblastos em cultura. Os dados obtidos mostram que, na faixa de concentração testada (0,1 a 200 µM), o peptídeo F1 é inativo. O peptídeo F2 apresentou atividade mitogênica (ED50 = 60 -70 µM), estimulando a incorporação de timidina tritiada em até 66 % do valor máximo induzido por 10% de soro fetal bovino. Na mesma faixa de concentração, o peptídeo F3 apresentou atividade em níveis inferiores (ED50 > 100 µM) aos apresentados pelo peptídeo F2. Estes resultados indicam que os peptídeos F2 e F3 poderiam mimetizar a superfície correspondente a um sítio de ligação do FGF-1 ao receptor. Além disso, o fato de F2 ser mais ativo que F3 indica que, além dos resíduos hidrofóbicos Y e L (presentes em ambos), o resíduo R presente em F2 exerce um importante papel para a atividade mitogênica do peptídeo. Como já proposto por nós em trabalhos anteriores, os dados apresentados indicam que é possível obter compostos com atividade mitogênica através do desenho racional de estruturas peptídicas derivadas dos FGFs. A análise do conjunto de peptídeos estudados até o momento revela a existência de características químicas comuns a todos aqueles que se mostraram mitogênicos, ou seja, a presença de um núcleo hidrofóbico flanqueado por resíduos polares carregados. / The Fibroblast Growth Factors (FGFs) are involved in very important biological processes like cell migration, division and differentiation. The aim of this work was the search of biologically active compounds through a rational design of peptides derived from FGF-1 and its receptor (FGFR-1). On the basis on several data available in the literature and with the aid of molecular modeling techniques, we designed, synthesized and tested two sets of peptides. The first group (R1-R3) is composed by linear peptides derived from FGFR-1. The mitogenic activity assays of FGF-1 and FGF-2 in the presence of these peptides reveal that R1 and R2 were able to inhibit the mitogenic response elicited by FGF-1. This effect is dose-dependent and selective, since the FGF-2 activity was not affected. Also, the inhibitory activity is sequence-dependent since peptide R3, corresponding to the e-terminal stretch of R2, was inactive. On the other hand, the N-terminal segment of peptide R2, represented by R1, is sufficient to elicit about the same response observed for the longer peptide R2. The semi-cyclic synthetic peptides F1 - F3, corresponding to an important FGF-1 binding site, were tested for their ability to stimulate DNA synthesis on fibroblast cultures. The results show that F1 is inactive in the range tested (0.1 to 200 µM). Peptide F2 was able to elicit a mitogenic activity (ED50 = 60 - 70 µM), stimulating the incorporation of [methyl-3H] thymidine to a level corresponding to 66 % of the maximum response induced by 10 % fetal calf serum. In the same range, peptide F3 was less active (ED50 > 100 µM). These results suggest that peptides F2 and F3 could mimic a surface corresponding to a receptor binding site of FGF-1. Also, the better performance of F2 could be explained by the presence of the residue R (besides Y and L) that could be important to elicit a mitogenic response. These results, together with those presented in former papers, indicate that it is possible to obtain compounds with mitogenic activity through the rational design of peptides derived from the FGFs. The analysis of the assembly of peptides studied allow us to define a chemical pattern shared by all the mitogenic compounds obtained until now, namely the presence of a hydrophobic core flanked by polar charged residues.