La dégénérescence discale lombaire (DDL) est caractérisée par un vieillissement prématuré du disque intervertébral (DIV) et une déshydratation progressive du nucleus pulposus (NP) entrainant in fine des lombalgies. L'objectif général de ce travail est d'établir des données précliniques afin de régénérer le DIV en cas de DDL modérée. Dans un premier chapitre, nous avons déterminé une association de facteur de croissance et un mode de culture visant à obtenir une prédifférentiation nucléopulpogénique de cellules stromales mésenchymateuses (CSMs) humaines issues de la moelle osseuse. Nos résultats montrent que la culture tridimensionnelle des CSMs en billes d'alginate en présence de TGF-β3, GDF-5 et BMP-7 les oriente vers un phénotype cartilagineux. Dans un deuxième chapitre, nous avons élaboré un modèle porcin de DDL induite par cryolésion et nous l'avons comparé aux techniques de référence. L'évaluation de l'importance de la DDL a été effectuée par scanner, IRM et histologiquement. Un score histologique de DDL porcine a été décrit et validé. La cryolésion a permis d'obtenir une DDL plus importante que les autres techniques. Dans un troisième chapitre, nous avons injecté les CSMs préorientées dans les DIV lésés. L'analyse IRM a montré une amélioration de l'intensité du signal et de la surface du NP après injection des cellules. L'analyse immunohistologique a montré une survie des CSMs dans les DIV porcins à 2 mois. Dans un quatrième chapitre, nous avons comparé les taux de fusion et de complication pour la RhBMP-2 et la greffe spongieuse autologue dans les arthrodèses lombaires par voie antérieure dans une même cohorte de patients. La RhBMP-2 était associée à un taux de fusion inférieur et un taux de complications radiologiques supérieur à l'autogreffe spongieuse. / Degenerative disc disease (DDD) is characterized by premature aging of the intervertebral disc (IVD) and gradual dehydration of the nucleus pulposus (NP), ultimately causing back pain. The general objective of this work is to establish preclinical data to regenerate the IVD in moderate DDD. In the first chapter, we have identified a growth factor association and a culture method to achieve nucleopulpogenic prédifférentiation of mesenchymal stromal cells (MSCs) derived from human bone marrow. Our results show that the three-dimensional culture of MSCs in alginate beads in the presence of TGF-β3, GDF-5 and BMP-7 directs them to a cartilaginous phenotype. In the second chapter, we developed a porcine model of DDD, induced by cryoinjury, and compared it to reference techniques. Assessing the importance of DDD was performed by CT, MRI and histologically. A histological score of porcine DDD has been described and validated. Cryoinjury yielded a higher DDD that other techniques. In a third chapter, we injected preoriented MSCs in cryo-injured IVDs. MRI analysis showed an improvement in the signal intensity and the surface of the NP after the injection. The immunohistological analysis showed a survival of the MSCs in the porcine IVD 2 months after injection. In a fourth chapter, we compared the rate of fusion and complication for rhBMP-2 and autologous cancellous graft in the anterior lumbar interbody fusion, in the same cohort of patients. RhBMP-2 was associated with a lower fusion rate and a higher rate of radiological complications than the cancellous autograft.

Les "Bone Morphogenetic Proteins" (BMPs) jouent un rôle clef dans la régulation de cellules précurseurs du muscle prénatal et de cellules souches musculaires adultes dénommées "cellules satellites". Les objectifs principaux de ma thèse étaient d'une part de déterminer si la signalisation BMP joue un rôle pendant la phase de croissance du muscle postnatale/juvénile dépendante des cellules satellites, et d'autre part d'investiguer si cette voie est impliquée dans la maintenance de la masse musculaire squelettique adulte. J'ai trouvé que les composants de cette voie de signalisation sont exprimés dans les cellules satellites de souris néonatales, juvéniles et adultes. Par ailleurs, j'ai utilisé des lignées de souris transgéniques pour surexprimer, de manière conditionnelle, l'inhibiteur Smad6 de la cascade de signalisation BMP dans les cellules satellites ou dans le muscle squelettique. J'ai pu ainsi démontrer que cette signalisation est requise pour une prolifération correcte des cellules satellites et pour leur différentiation en myonuclei, assurant que les fibres musculaires en croissance atteignent une taille finale normale. Par ailleurs, mes travaux révèlent que le nombre final de cellules satellites est établis pendant la phase de croissance postnatale/juvénile et que celle-ci dépend de la cascade de signalisation BMP. Enfin, je fournis des preuves montrant que la signalisation BMP est un puissant signal hypertrophique dans le muscle squelettique adulte et que sa présence est indispensable pour le maintien du tissu musculaire. En résumé, mes résultats de recherche démontrent que les BMPs sont des facteurs de croissance essentiels pour le muscle squelettique postnatal. / Bone Morphogenetic Proteins (BMPs), a subfamily of TGF-β growth factors, have been shown to be key signals that regulate embryonic and fetal muscle precursors during prenatal myogenesis, as well as the stem cells of adult muscle, termed ‘satellite cells’, when activated during muscle regeneration. The main aims of my thesis were to elucidate whether BMP signaling plays a role during postnatal/juvenile satellite cell-dependent muscle growth as well as for maintenance of adult muscle mass. I found that components of BMP signaling pathway are expressed in muscle satellite cells of neonatal, juvenile and adult mice. I used transgenic mouse lines to conditionally overexpress the BMP signaling cascade inhibitor Smad6 in muscle satellite cells and in differentiated skeletal muscle. I show that BMP signaling is required for correct proliferation of muscle satellite cells and their differentiation into myonuclei, thereby ensuring that the growing muscle fibers reach the correct final size. Moreover, I demonstrated that the final number of muscle stem cells is established during the postnatal/juvenile growth phase and this also depends on the BMP signaling cascade. Finally, I provide evidence that BMP signaling is a strong hypertrophic signal for the adult skeletal muscle and its presence is indispensable for muscle tissue maintenance. In summary, my findings demonstrate that BMPs are essential growth factors for postnatal skeletal muscle.

O câncer de pâncreas é considerado um dos adenocarcinomas mais agressivos, ocupando o quarto lugar de mortes devidas a câncer, isto é dado principalmente a seu desenvolvimento silencioso e a sua complexidade genética, tornando-o de difícil detecção. Consequentemente, o diagnóstico desta doença ocorre apenas em fase tardia, quando o tratamento é apenas para fins paliativos. As anomalias genéticas mais frequentes no câncer de pâncreas invasivo estão relacionadas à ativação por mutações do gene KRAS e à inativação dos genes supressores de tumor CDKN2A, TP53, SMAD4 e BRCA2. Além destas conhecidas vias de sinalização, fatores de crescimento como o TGF-? e EGF também apresentam papel fundamental na progressão e metástase do câncer de pâncreas. Interessantemente, TGF-? e EGF são também indutores do processo denominado transição epitelial-mesenquimal (EMT), onde células epiteliais normais, durante a embriogênese, ou células cancerosas durante a progressão tumoral e metástase, perdem seus contatos intracelulares e adquirem caráter migratório. Desta forma a EMT, induzida por altos níveis de TGF-? e/ou EGF, é considerada como um dos mecanismos de progressão tumoral em adenocarcinomas. No presente estudo, foram estudados os proteomas e o fosfoproteoma da EMT do PanCa. Assim, células de câncer de pâncreas PANC- 1 foram induzidas à EMT em cultura com os fatores de crescimento TGF-?1 ou TGF-?2 e EGF, e após a indução, marcadores moleculares e propriedades funcionais de migração e invasão foram confirmados. Duas condições de indução da EMT foram estabelecidas, para as quais foram desenvolvidas as análises proteômicas quantitativas. A abordagem foi baseada em marcação isotópica de células em cultura (SILAC), em conjunto com fracionamento celular e de proteínas intactas, e cromatografia líquida acoplada à espectrometria de massas para identificação de proteínas em larga escala. No total, aproximadamente 5.000 proteínas foram identificadas, e a maioria delas quantificadas com precisão nas duplicatas experimentais. Foram selecionadas 37 proteínas com expressão diferencial estatisticamente significativa nos experimentos proteômicos, as quais participam principalmente em processos de biogênese, adesão e apoptóticos. A análise de redes de interação revelou que as proteínas alteradas estavam principalmente localizadas em vias de sinalização que controlam processos de organização da matriz extracelular, splicing alternativo e regulação da apoptose. A análise do fosfoproteoma foi feita usando TGF-?1 como agente indutor da EMT nas células PANC-1, usando a estratégia ERLIC para o enriquecimento de fosfopeptídeos. No total, foram identificados 5.965 fosfopeptídeos não redundantes, correspondendo a um total de 2.250 fosfoproteínas analisadas, sendo quantificadas 2.053 ao menos em duas replicatas, e destas foram identificados 61 fosfopeptídeos regulados pertencentes a 55 fosfoproteínas, relacionados com processos de regulação do mRNA e vias de sinalização ligadas à adesão celular. Em conclusão, nosso estudo elucida potenciais novos alvos para inibição da EMT, controle da metástase, ou para auxiliar no diagnóstico da doença, quando devidamente validada. / Pancreatic cancer kills more than 200 thousand people worldwide every year. Also, pancreatic cancer is considered one of the most aggressive adenocarcinomas and difficult to diagnose since it develops silently and presents a high genetic complexity. Consequently, the diagnostic is often late, when the pancreatic cancer has already metastasized and the treatment has only palliative purposes. The most frequent genetic alterations observed in pancreatic cancer are related to mutations in KRAS oncogene and CDKN2A, TP53, SMAD4 and BRCA2 tumor suppressor genes. In addition to these known frequent mutations, growth factors such as TGB- ? and EGF play important roles in pancreatic cancer progression and metastasis. Interestingly, TGB- ? and EGF are also inducers of the Epithelial to Mesenchymal Transition (EMT), in which epithelial cells lose their intracellular contacts and acquire migratory capacities. Therefore, EMT is considered one of the mechanisms responsible for tumor progression and metastasis in adenocarcinomas in addition of being correlated to the process of generating cancer stem cells. In our present study, pancreatic cancer cell line PANC-1 was induced to EMT by using growth factors TGF-?1 or TGF-?2 and EGF. Both molecular and functional properties, such as invasion and migration, were evaluated in PANC-1 cells undergoing EMT and confirm the induction. Two conditions of EMT induction were properly established and in-depth quantitative proteomic analysis based on stable isotope labeling in cell culture (SILAC) followed by cellular and protein fractionation were assessed. In total, 5.000 proteins were identified and most of them were accurately quantified in duplicate experiments. Thirty-seven proteins were selected as differentially expressed with statistical significance, and were related mainly with biogenesis, adhesion and apoptosis processes. Interaction network analysis showed that regulated proteins were predominantly participating in signaling pathways linked to extracellular matrix organization, alternative splicing and apoptosis regulation. Phosphoproteome analysis was done using TGF-?1 as EMT-inductor agent on PANC-1 cells and ERLIC strategy for phosphopeptides enrichment. In total, were identified 5.965 non-redundant phosphopeptides corresponding to approximately 2.250 analyzed phosphoproteins, thereof 2.053 were quantified in at least two replicates. At comparison, were identified 61 regulated phosphopeptides belonging to 55 phosphoproteins, which were related with mRNA regulation processes and signialing pathways linked to cellular adhesion. In conclusion, our study highlighted potential new targets for EMT inhibition, metastasis control or to help in pancreatic cancer diagnosis, when careful validated.

Objetivo: Avaliar por meio de análises radiográfica e histológica os efeitos da associação do laser de diodo e do fator de crescimento de fibroblastos no tratamento de dentes de ratos reimplantados tardiamente. Método: Cinquenta ratos (Rattus norvegicus albinus, Wistar) tiveram seus incisivos superiores direitos extraídos, a papila dental removida, e por via retrógrada, o preparo e preenchimento dos canais radiculares com pasta de hidróxido de cálcio. Em seguida, os espécimes foram aleatoriamente divididos em cinco grupos: C+ (controle positivo)- reimplante imediato; C- (controle negativo)- reimplante tardio, mantidos em meio ambiente seco por sessenta minutos, sem tratamento adicional; LA (laser de alta potência)-reimplante tardio associado ao tratamento prévio da superfície radicular com irradiação por varredura com laser diodo de alta potência (810nm, modo contínuo, fibra óptica de 600&#956;m, 1,5W input, 30s); FGF (Fator de crescimento de fibroblastos)- reimplante tardio associado à aplicação do fator de crescimento de fibroblastos 50 &#956;g na concentração de 0,2% FGF-2 em gel de hidroxipropilmetilcelulose (HPMC) a 3% sobre a superfície radicular lingual e no interior do alvéolo dental de cada espécime; LA+FGF (Laser de alta potência e FGF)- reimplante tardio, com tratamento prévio da superfície radicular com laser diodo nos mesmos parâmetros do grupo LA, associado à aplicação do FGF-2 da mesma forma que no grupo FGF. A eutanásia dos animais ocorreu após sessenta dias. Os espécimes foram radiografados digitalmente e processados para análise descritiva dos eventos histológicos e análise da porcentagem de fibras colágenas do tipo I por meio do método Picrosirius Red. Resultados: Na análise radiográfica, o grupo LA+FGF apresentou o menor número e porcentagem de áreas reabsorvidas (p<0,05). Com a análise dos eventos histológicos por meio de escores, o grupo LA apresentou as menores médias de reabsorção substitutiva e inflamatória e anquilose, seguido pelo grupo LA+FGF e não diferindo estatisticamente do grupo controle positivo. O grupo LA+FGF apresentou o melhor reparo periodontal entre os grupos experimentais, com a maior quantidade de fibras colágenas espessas, (p<0,05). Conclusões: A irradiação da superfície radicular com laser de diodo de alta potência associada ou não ao FGF-2 reduziu a incidência de reabsorções radiculares externas e anquilose. A aplicação do FGF-2 favoreceu o reparo do ligamento periodontal, embora a reinserção das fibras não tenha ocorrido em todos os espécimes. / Aim: The aim of this study was to evaluate, by radiographic and histological analyses, the effects of high-power diode laser irradiation and fibroblast growth factor (FGF) in the treatment of delayed replanted rat teeth. Methods: Fifty rats ratos (Rattus norvegicus albinus, Wistar) had their maxillary right incisors extracted, the dental papilla removed, and by a retrograde way, the root canals were prepared and filled with calcium hydroxide paste. Subsequently, the specimens were randomly assigned to five groups: C+ (positive control)- immediate replantation; C- (negative control)- delayed replantation, left on the bench for sixty minutes, without additional treatment; LA (Diode laser high potency)- delayed replantation and root surface treatment scanning irradiation with a high-power diode laser (810nm, continuous mode, optic fiber 600&#956;m, 1.5W input, 30s); FGF (Fibroblast Growth Factor) - delayed replantation and topical application of 50&#956;g 0,2% FGF-2 gel in 3% hydroxylpropyl methylcellulose matrix (HPMC) over the lingual root surface and into dental alveolus of each specimen; LA+FGF (Diode laser high potency + Fibroblast Growth Factor)- delayed replantation and root surface treatment irradiation with a diode laser using the same parameters as those used for LA group, associated to topical application of FGF-2 in the same way as FGF group. The rats were euthanized after sixty days of replantation. The specimens were digitally radiographed and processed for descriptive histological events analysis and percentage analysis of type I collagen fibers by the Picrosírius red method. Results: By radiographic analysis, the minnor number and percentage of resorpted areas were seen in LA+FGF group (p<0.05). In the histological analysis through scores, the LA group showed lower means of replacement and inflammatory resorption and ankylosis and followed by LA+FGF group and they were not statistically different of positive control group. The LA+FGF group showed the best periodontal healing of all experimental groups, with the greater number of thick collagen fibers stained (p<0,05). Conclusions: The irradiation of root surface treatment with high-power diode laser associated or not to FGF-2 reduced the occurrence of external root resorption and ankylosis. The FGF-2 application favored the periodontal ligament healing, but the fibers reinsertion not occurred in all the specimens.

A manutenção do estado progenitor durante o cultivo de células mesenquimais progenitoras derivadas do tecido adiposo (MSCs-TA), caracterizado pelo potencial de diferenciação e da capacidade de autorrenovação, é atualmente um dos maiores desafios da terapia celular. Sabendo da influência da angiogênese no desenvolvimento de tecidos de origem mesenquimal, avaliamos se um ambiente pro-angiogênico mimetizado em cultura forneceria condições para manutenção de um estado progenitor durante o processo de expansão celular. Utilizando como modelo de um ambiente pró-angiogênico o cultivo no meio EGM-2, o qual é suplementado pelos fatores de crescimento EGF, FGF-2, IGF e VEGF, nós demonstramos que a presença de tais fatores pró-angiogênicos é fundamental para a manutenção do estado progenitor de MSCs-TA em cultura. Verificamos que a presença de tais fatores de crescimento possibilitaram às MSCs-TA apresentarem um alto potencial de diferenciação adipogênico e osteogênico em comparação ao meio convencional DMEM/F12 e ao meio EBM, ausente de fatores. Além disso, o cultivo na presença de fatores pró-angiogênicos aumentou o potencial clonogênico das MSCs-TA, ao mesmo tempo em que aumentou a capacidade proliferativa destas células. Dentre os fatores de crescimento, EGF e FGF-2 foram responsáveis pelos efeitos mais robustos. Ao mesmo tempo, células cultivadas nas presença destas citocinas foram capazes de manter a morfologia fibroblastóide e apresentaram alta expressão do fator de pluripotência Klf-4. Em concordância com estes achados, o transplante subcutâneo de MSCs-TA cultivadas nestas condições mostrou que aquelas mantidas em EGM-2 geram um tecido semelhante ao tecido formado pela fração estromal vascular não cultivada. Estes resultados reforçam o papel do ambiente pró-angiogênico na manutenção do estado progenitor de MSCs-TA, e que tal estado foi proporcionado pela ação dos fatores de crescimento pró-angiogênicos EGF, FGF-2, IGF e VEGF nas células em cultivo, com destaque para as citocinas EGF e FGF-2. Em conclusão, o uso do ambiente pró-angiogênico no cultivo de MSCs-TA mostrou-se como uma abordagem promissora para a manutenção do estado progenitor destas células in vitro. / The maintenance of the progenitor state in the culture of adipose tissue derived- mesenchymal progenitor cell (TA-MSCs), characterized by the differentiation potential and self-renewal capability, is currently one of the major challenges of cell therapy. The information that the angiogenesis influences the development of mesenchymal tissues, has led us to evaluate how a pro-angiogenic environment mimicked in culture would provide conditions for maintaining a progenitor state during the cell expansion process. We designe a model for a pro-angiogenic environment in which cells grown in EGM-2 supplemented with the following growth factors: EGF, FGF-2, IGF and VEGF, and demonstrated that the presence of such pro-angiogenic growth factors was crucial for maintenance of the progenitor of AT-MSCs in culture. We observed that the presence of such growth factors allowed to AT-MSCs a high potential of adipogenic and osteogenic differentiation compared to conventional DMEM/F12 medium and the EBM medium, in the absence of the factors. Furthermore, the culture in presence of pro-angiogenic growth factors increased the clonogenic potential of AT-MSCs and increased the proliferative capability of these cells. Among the growth factors, EGF and FGF-2 were responsible for most robust effects. At the same time, cells cultured in the presence of these cytokines were able to maintaining the fibroblastoid morphology and presented high expression levels of Klf-4 pluripotency factor. In agreement with these observations, the subcutaneous transplantation of AT-MSCs cultured under these conditions showed that those cells kept in EGM-2 generated a tissue-like to tissue formed by the stromal vascular fraction uncultivated. These results reinforce the role of the pro-angiogenic environment in the maintenance of the progenitor state of AT-MSCs, and that such a state was provided by the action of the pro-angiogenic growth factors EGF, FGF-2, IGF and VEGF in cultured cells, highlighting EGF and FGF-2 cytokines. In conclusion, we showed that the use of a pro-angiogenic environment in AT-MSCs culture is a promising approach to the maintain the progenitor state of these cells in vitro.

Pas de résumé / Production of new neurons in the brain decreases dramatically with age due to progressive physiological depletion of stem and progenitor cell populations (NSCs). Recent studies indicate that circulating factors constitute a systemic aging milieu regulating the birth of new cells. Interestingly, some long-lived mouse strains such as Ames dwarf mutants, with low circulating levels of GH and IGF-1, show increased neurogenesis and preserved hematopoietic stem cell pool. Thus, the possibility that genes regulating lifespan and aging also quantitatively modulate stem cells in mammals is more and more explored. IGF-1 plays a pivotal role in aging in different species, and I am asking whether some of the well-known longevity effects resulting from down-regulation of this signaling pathway could be explained by local regulation of stem and progenitor cell compartments. To validate this hypothesis, I pursued a dual approach based on biological experiments and mathematical modeling. Using a novel triple transgenic mouse model, I inactivated IGF-1 signaling specifically in adult NSCs, and traced knockout cell lineages with a fluorescent reporter transgene. By analyzing the phenotype at different time points after KO induction, I could distinguish between short and long-term effects of IGF signaling on cellular regeneration and identify cumulative physiological consequences of down-regulation of this pathway using behavioral tests. In my mathematical models, the dynamics of regenerative cell populations were described by a set of differential equations depending on circulating “growth-factor like molecules” (GFs). My results suggest that in aging tissues, the optimal distribution of GFs is a function that decreases with time. In the olfactory system, I showed that inactivation of IGF signaling in adult NSCs enhanced long-term maintenance of neuroblasts and increased the overall production of neurons. Mutants started with the same number of adult-born neurons as controls one month after KO induction at 4 months of age, but ended up having significantly more differentiated cells integrating the olfactory bulb at long-term, i.e. at 16 months of age. This highly increased neurogenic activity occurred without depletion of neural stem/progenitor cell compartments. In contrast, IGF-1R deletion in adult hippocampal stem cells did not change neurogenesis dynamics, pointing out a niche-dependent effect of IGFs. The important cellular changes in the olfactory bulb led to improved olfactory memory and odor discrimination in aged mutants. Strikingly, mutants also displayed altered energy homeostasis and increased sensitivity to metabolic hormones, namely leptin and insulin. This metabolic shift could be linked to enhanced olfactory function, and to changes in hypothalamic neurogenesis. Indeed, we observed that IGF-1R deletion in hypothalamic stem cells (HySC) protected α-tanycyte pool from age-related decline and increased the number of newborn neurons in the hypothalamus. Taken together, my results validate the hypothesis that life-long inhibition of IGF signaling in adult NSCs delays age-related decline of neurogenesis, in a niche-dependent manner. These data also show that local modulation of neural cell replacement has important physiological effects at the level of the whole organism, pointing out a novel pathophysiological role for adult neurogenesis.

Fibroblast growth factor (Fgf) and Retinoic acid (RA) are known to be involved in patterning the posterior embryo. Work has shown that Fgf can convert anterior tissue into posterior fates and that embryos deficient in Fgf signaling lack posterior trunk and tail structures. Likewise, studies performed on RA have shown that overexpression of RA posteriorizes anterior tissue, while disrupting RA signaling yields a loss of posterior fates. While it appears these signals are necessary for posterior development, the role Fgf and RA play in development of the hindbrain is still enigmatic. A detailed study of the requirements for Fgf and RA in the early vertebrate hindbrain are lacking, namely due to a deficiency in gene markers for the presumptive hindbrain at early developmental stages. In this study, we make use of recently isolated genes, which are expressed in the presumptive hindbrain region at early developmental stages, to explore Fgf and RA regulation of the early vertebrate hindprain. We employed both overexpression and loss of function approaches to explore the role of Fgf in early vertebrate development with an emphasis on the presumptive hindbrain region in zebrafish embryos. By loss of function analysis, we show that Fgf regulates genes expressed exclusively in the hindbrain region (meis3 and hoxbla) as well as genes whose expression domains encompass both the hindbrain and more caudal regions (nlz and hoxb1b), thus demonstrating a requirement for Fgf signaling throughout the anteroposterior axis of the hindbrain (rostral to caudal hindbrain) by mid-gastrula stages. To further characterize early gene regulation by Fgf, we utilized an in vitro system and found that Fgf is sufficient to induce nlz directly and hoxb1b indirectly, while it does not induce meis3 or hoxb1a. Furthermore, in vivo work demonstrates that Fgf soaked beads can induce nlz and hoxb1b adjacent to the bead and meis3at a distance. Given the regulation of these genes in vitro and in vivo by Fgf and their position along the rostrocaudal axis of the embryo, our results suggest an early acting Fgf resides in the caudal end of the embryo and signals at a distance to the hindbrain. We detect a similar regulation of hindbrain genes by RA at gastrula stages as well, suggesting that both factors are essential for early hindbrain development. Interestingly however, we find that the relationship between Fgf and RA is dynamic throughout development. Both signals are required at gastrula stages as disruption of either pathway alone disrupts hindbrain gene expression, but a simultaneous disruption of both pathways at later stages is required to disrupt the hindbrain. We suggest that Fgf and RA are present in limiting concentrations at gastrula stages, such that both factors are required for gene expression or that one factor is necessary for activation of the other. Our results also reveal a changing and dynamic relationship between Fgf and RA in the regulation of the zebrafish hindbrain, suggesting that at segmentation stages, Fgf and RA may no longer be limiting or that they are no longer interdependent. As we have demonstrated that an early Fgf signal is required for gastrula stage hindbrain development, we next questioned which Fgf performed this function. We have demonstrated that the early Fgf signal required for hindbrain development is not Fgf3 or Fgf8, two Fgfs known to be involved in signaling centers at the mid-hindbrain boundary (MHB) and rhombomere (r) 4. We further show that two recently identified Fgfs, Fgf4 and Fgf24 are also insufficient alone or in combination with other known Fgfs to regulate hindbrain gene expression. However, as Fgfs may act combinatorially, we do not rule out the possibility of their involvement in early hindbrain gene regulation. However, as time passes and additional Fgfs are isolated and cloned, the elusive Fgf signal required for early hindbrain development will likely be identified. Taken together, we propose that an early acting Fgf residing in the caudal end of the embryo regulates hindbrain genes together with RA at gastrula stages. We suggest that both Fgf and RA are required for gene expression at gastrula stages, but this requirements changes over time as Fgf and RA become redundant. We also demonstrate that the Fgf required for gastrula stage hindbrain development has yet to be identified.

Fibroblast growth factor (Fgf) signaling plays multiple inductive roles during development of vertebrates (Itoh 2007). Some Fgfs, such as Fgf8, are locally secreted and signal over a long range to provide positional information in the target tissue (Scholpp and Brand 2004). Fgf ligands signal in a receptor-dependent manner via tyrosine kinase receptors, four of which have been so far identified. Fgf8 signaling was shown to depend both on receptor activation as well as endocytosis. The specificity of Fgf ligands and receptors as well as the function of receptors in the control of the Fgf signaling range have been, however, largely unclear. In this study, we show that the putative Fgf8 receptor Fgfr1 is duplicated in zebrafish and that it acts redundantly in the formation of the posterior mesoderm. Also, in overexpression studies we confirm the notion that receptor endocytosis influences Fgf8 signaling range. Through TILLING mutant recovery and morpholino knockdown studies we also show that Fgfr2 is required for growth and skeletal development in zebrafish, whereas Fgfr4 is required for pectoral fin specification and growth.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Pancreatic ductal adenocarcinoma (PDAC), which comprises 85% of pancreatic cancers, is the 4th leading cause of cancer death in the United States with a 5-year survival rate of 8%. While human PDACs (hPDACs) are hypovascular, they also overexpress a number of angiogenic growth factors and receptors. Additionally, the use of anti-angiogenic agents in murine models of PDAC leads to reduced tumor volume, tumor spread, and microvessel density (MVD), and improved survival. Nonetheless, clinical trials using anti-angiogenic therapy have been overwhelmingly unsuccessful in hPDAC. On the other hand, pancreatic neuroendocrine tumors (PNETs) account for only 2% of pancreatic tumors, yet they are very vascular and classically angiogenic, respond to anti-angiogenic therapy, and confer a better prognosis than PDAC even in the metastatic setting. In an effort to compare and contrast the angiogenic transcriptomes of these two tumor types, we analyzed RNA-Sequencing (RNA-Seq) data from The Cancer Genome Atlas (TCGA) and found that a pro-angiogenic gene signature is present in 35% of PDACs and that it is mostly distinct from the angiogenic signature present in PNETs. The pro-angiogenic PDAC subgroup also exhibits a transcriptome that reflects active TGF-β signaling, less frequent SMAD4 inactivation than PDACs without the signature, and up-regulation of several pro-inflammatory genes, including members of JAK signaling pathways. Consequently, targeting the TGF-β receptor type-1 kinase with SB505124 and JAK1/2 with ruxolitinib blocks proliferative crosstalk between human pancreatic cancer cells (PCCs) and human endothelial cells (ECs). Additionally, treatment of the KRC (oncogenic Kras, homozygous deletion of Rb1) and KPC (oncogenic Kras, mutated Trp53) genetically engineered PDAC mouse models with ruxolitinib suppresses murine PDAC (mPDAC) progression only in the KRC model, which shows superior enrichment and differential expression of the human pro-angiogenic gene signature as compared to KPC tumors. These findings suggest that targeting both TGF-β and JAK signaling in the 35% of PDAC patients whose cancers exhibit an pro-angiogenic gene signature should be explored in a clinical trial.