Au cours de ma thèse, mes projets de recherche ont visé à mieux comprendre les mécanismes moléculaires contrôlant la prolifération et la spécification des cellules progénitrices dans la rétine du xénope à travers trois projets principaux. Le réseau de régulation qui contrôle la spécification des cellules progénitrices vers les sous-types neuronaux est à ce jour très peu connu. C’est dans ce contexte que j’ai étudié le rôle du facteur de transcription à domaine bHLH, Ascl1, dans la détermination des sous-types rétiniens au cours du développement. Par des approches in vivo de gain et perte de fonction d’Ascl1, des expériences d’épistasie et la recherche de ses cibles transcriptionnelles, j’ai pu mettre en évidence qu’Ascl1 (i) est impliqué dans la genèse des neurones GABAergiques rétiniens, (ii) qu’il est épistatique sur des facteurs glutamatergiques tels que Neurog2, NeuroD1 ou Atoh7, (iii) que son activité GABAergique est conférée par son domaine basique de liaison à l’ADN et (iv) que cette activité implique la régulation directe du facteur de transcription Ptf1a. Ces données ajoutent donc une nouvelle pièce au réseau transcriptionnel gouvernant la spécification des sous-types GABAergiques au cours du développement de la rétine. La mise en place correcte des types et sous-types cellulaires de la rétine nécessite une coordination avec le moment de sortie du cycle cellulaire des progéniteurs rétiniens. Dans ce contexte, j’ai contribué à l’avancée d’un projet visant à étudier le réseau de signalisation contrôlant la prolifération des précurseurs de la rétine. Par des approches in vivo, génétiques et pharmacologiques, cette étude a montré que les voies Wnt et Hedgehog s’antagonisent pour réguler l’activité proliférative des cellules souches et progénitrices rétiniennes. Nos données préliminaires suggèrent que ces voies agissent de façon opposée à la fois sur la sortie et sur la cinétique du cycle cellulaire. Ce travail nous a conduit à proposer un modèle selon lequel ces voies Wnt et Hedgehog réguleraient la balance entre prolifération et différenciation dans la rétine post-embryonnaire. Enfin, dans le but d’élargir nos connaissances sur les réseaux de signalisation et les réseaux transcriptionnels impliqués dans le contrôle de la prolifération et de la détermination cellulaire dans la rétine, j’ai également contribué à la recherche de nouveaux marqueurs spécifiques des différentes populations cellulaires rétiniennes au travers d’un crible à grande échelle par hybridation in situ. De nombreux gènes spécifiquement exprimés dans les cellules souches ou les cellules progénitrices constituent des gènes candidats pour de futures approches fonctionnelles. / My thesis research work aimed to better understand the molecular mechanisms underlying proliferation and specification of retinal progenitors in Xenopus through three main projects. As the mechanisms governing specification of retinal progenitors towards the different neuronal subtypes are still poorly understood, I focused my work on the role of Ascl1, a bHLH transcription factor, in cell-subtype determination during retinogenesis. Using in vivo gain- and loss-of-function experiments, I have investigated Ascl1’s epistatic relationships with other bHLH factors and identified its transcriptional targets. My results indicate that Ascl1 (i) is implicated in the genesis of retinal GABAergic neurons (ii) is epistatic to glutamatergic factors such as Neurog2, NeuroD1 and Atoh7 (iii) that its basic DNA-biding domain is sufficient for its GABAergic-inducing activity (iv) and that this activity involves a direct regulation of the Ptf1a transcription factor. The correct order of neural cell types and subtypes formation is tightly coordinated with the timing of cell-cycle exit of retinal progenitors. Ongoing work in the laboratory, to which I have contributed, was therefore investigating the role of signaling pathways controlling retinal precursor proliferation in this process. Using in vivo genetic and pharmacological tools, we have shown that an antagonistic cross-regulation between Wnt and Hedgehog signaling governs stem cell and progenitor proliferation in post-embryonic retina. Preliminary data shows that Wnt and Hedgehog have opposite effects on both cell cycle exit and kinetics and may therefore regulate the proliferation/differentiation balance in the post-embryonic retina. Lastly, in order to broaden our knowledge on the transcriptional and signaling networks which govern proliferation and cell fate determination in the retina, I have participated in a large scale screen by in situ hybridization aiming to identify new molecular markers of different retinal cell population. Many genes that are exclusively expressed in retinal stem cells or progenitors are promising candidates for future functional studies.

<p>Induction of vulval fates in the C. elegans hermaphrodite is mediated by a conserved RTK/Ras/MAP kinase signalling pathway, in which the core components can be placed into a linear genetic and biochemical pathway. However, the events that occur downstream of this pathway are not yet well understood. This thesis describes studies on three genes, lin-1, lin-25 and sur-2 that function genetically downstream of the RTK/Ras/MAP kinase pathway in vulva induction. lin-1 encodes an ETS protein that appears to be a direct target of the RTK/Ras/MAP kinase pathway during the induction of vulval fates. To understand more in detail how Ras signalling in C. elegans affects cell fate specification we have analysed the effects of lin-1 mutations on various Ras-mediated cell fate specification events. Our results show that lin-1, besides its function in vulval induction, functions in most other Ras-mediated cell fate specification events in C. elegans, and that lin-1 appears to have a negative function in a majority of these events. Two other genes, lin-25 and sur-2, also function genetically downstream of the RTK/Ras/MAP kinase pathway during induction of vulval fates. Previously, two different models have been proposed for the function of these genes (I) that they function together with a gene in the homeotic cluster to specify the identity of the vulval precursor cells or (II) that they constitute components of the RTK/Ras/MAP kinase signalling pathway. To help clarify the role of lin-25 and sur-2, we have caried out studies of the effects of lin-25 and sur-2 mutations on other cells in the worm in which the RTK/Ras/MAP kinase pathway functions. The results exclude the possibility that lin-25 and sur-2 solely function in vulva induction and suggest that the two genes are intimately involved in Ras-mediated signalling. In addition we show that the major focus for lin-25 during vulval induction is in the vulva precursor cells themselves. Furthermore, results presented here suggest that LIN-25 and SUR-2 function together in the same process in the cell. We show here by both genetic and immunological experiments that LIN-25 is associated with Mediator in C. elegans, a multiprotein complex required for transcriptional regulation. Taken together, these results suggest that lin-25 and sur-2 function in regulating transcription of genes in response to Ras signalling.</p>

Induction of vulval fates in the C. elegans hermaphrodite is mediated by a conserved RTK/Ras/MAP kinase signalling pathway, in which the core components can be placed into a linear genetic and biochemical pathway. However, the events that occur downstream of this pathway are not yet well understood. This thesis describes studies on three genes, lin-1, lin-25 and sur-2 that function genetically downstream of the RTK/Ras/MAP kinase pathway in vulva induction. lin-1 encodes an ETS protein that appears to be a direct target of the RTK/Ras/MAP kinase pathway during the induction of vulval fates. To understand more in detail how Ras signalling in C. elegans affects cell fate specification we have analysed the effects of lin-1 mutations on various Ras-mediated cell fate specification events. Our results show that lin-1, besides its function in vulval induction, functions in most other Ras-mediated cell fate specification events in C. elegans, and that lin-1 appears to have a negative function in a majority of these events. Two other genes, lin-25 and sur-2, also function genetically downstream of the RTK/Ras/MAP kinase pathway during induction of vulval fates. Previously, two different models have been proposed for the function of these genes (I) that they function together with a gene in the homeotic cluster to specify the identity of the vulval precursor cells or (II) that they constitute components of the RTK/Ras/MAP kinase signalling pathway. To help clarify the role of lin-25 and sur-2, we have caried out studies of the effects of lin-25 and sur-2 mutations on other cells in the worm in which the RTK/Ras/MAP kinase pathway functions. The results exclude the possibility that lin-25 and sur-2 solely function in vulva induction and suggest that the two genes are intimately involved in Ras-mediated signalling. In addition we show that the major focus for lin-25 during vulval induction is in the vulva precursor cells themselves. Furthermore, results presented here suggest that LIN-25 and SUR-2 function together in the same process in the cell. We show here by both genetic and immunological experiments that LIN-25 is associated with Mediator in C. elegans, a multiprotein complex required for transcriptional regulation. Taken together, these results suggest that lin-25 and sur-2 function in regulating transcription of genes in response to Ras signalling.

Embryonic stem cells (ESCs) are characterized by their unlimited capacity for self-renewal and the ability to contribute to every lineage of the developing embryo. The promoters of developmentally regulated loci within these cells are marked by coincident epigenetic modifications of gene activation and repression, termed bivalent domains. Trithorax group (TrxG) and Polycomb Group (PcG) proteins respectively place these epigenetic marks on chromatin and extensively colocalize with Oct4 in ESCs. Although it appears that these cells are poised and ready for differentiation, the switch that permits this transition is critically held in check. The derepression of bivalent domains upon knockdown of Oct4 or PcG underscores their respective roles in maintaining the pluripotent state through epigenetic regulation of chromatin structure. The mechanisms that facilitate the recruitment and retention of Oct4, TrxG, and PcG proteins at developmentally regulated loci to maintain the pluripotent state, however, remain unknown. Oct4 may function as either a transcriptional activator or repressor. Prevailing thought holds that both of these activities are required to maintain the pluripotent state through activation of genes implicated in pluripotency and cell-cycle control with concomitant repression of genes required for differentiation and lineage-specific differentiation. More recent evidence however, suggests that the activator function of Oct4 may play a more critical role in maintaining the pluripotent state (Hammachi et al., 2012). The purpose of the studies described in this dissertation was to clarify the underlying mechanisms by which Oct4 functions in transcriptional activation and repression. By so doing, we wished to contextualize its role in pluripotent cells, and to provide insight into how changes in Oct4 function might account for its ability to facilitate cell fate transitions. As a result of our studies we find that Oct4 function is dependent upon post-translational modifications (PTMs). We find through a combination of experimental approaches, including genome-wide microarray analysis, bioinformatics, chromatin immunoprecipitation, functional molecular, and biochemical analyses, that in the pluripotent state Oct4, Akt, and Hmgb2 participate in a regulatory feedback loop. Akt-mediated phosphorylation of Oct4 facilitates interaction with PcG recruiter Hmgb2. Consequently, Hmgb2 functions as a context dependent modulator of Akt and Oct4 function, promoting transcriptional poise at Oct4 bound loci. Sumoylation of Oct4 is then required to maintain Hmgb2 enrichment at repressed loci and to transmit the H3K27me3 mark in daughter progeny. The expression of Oct4 phosphorylation mutants however, leads to Akt inactivation and initiates the DNA Damage Checkpoint response. Our results suggest that this may subsequently facilitate chromatin reorganization and cell fate transitions. In summary, our results suggest that controlled modulation of Oct4, Akt, and Hmgb2 function is required to maintain pluripotency and for the faithful induction of transcriptional programs required for lineage specific differentiation.

Embryonic stem cells (ESCs) are characterized by their unlimited capacity for self-renewal and the ability to contribute to every lineage of the developing embryo. The promoters of developmentally regulated loci within these cells are marked by coincident epigenetic modifications of gene activation and repression, termed bivalent domains. Trithorax group (TrxG) and Polycomb Group (PcG) proteins respectively place these epigenetic marks on chromatin and extensively colocalize with Oct4 in ESCs. Although it appears that these cells are poised and ready for differentiation, the switch that permits this transition is critically held in check. The derepression of bivalent domains upon knockdown of Oct4 or PcG underscores their respective roles in maintaining the pluripotent state through epigenetic regulation of chromatin structure. The mechanisms that facilitate the recruitment and retention of Oct4, TrxG, and PcG proteins at developmentally regulated loci to maintain the pluripotent state, however, remain unknown. Oct4 may function as either a transcriptional activator or repressor. Prevailing thought holds that both of these activities are required to maintain the pluripotent state through activation of genes implicated in pluripotency and cell-cycle control with concomitant repression of genes required for differentiation and lineage-specific differentiation. More recent evidence however, suggests that the activator function of Oct4 may play a more critical role in maintaining the pluripotent state (Hammachi et al., 2012). The purpose of the studies described in this dissertation was to clarify the underlying mechanisms by which Oct4 functions in transcriptional activation and repression. By so doing, we wished to contextualize its role in pluripotent cells, and to provide insight into how changes in Oct4 function might account for its ability to facilitate cell fate transitions. As a result of our studies we find that Oct4 function is dependent upon post-translational modifications (PTMs). We find through a combination of experimental approaches, including genome-wide microarray analysis, bioinformatics, chromatin immunoprecipitation, functional molecular, and biochemical analyses, that in the pluripotent state Oct4, Akt, and Hmgb2 participate in a regulatory feedback loop. Akt-mediated phosphorylation of Oct4 facilitates interaction with PcG recruiter Hmgb2. Consequently, Hmgb2 functions as a context dependent modulator of Akt and Oct4 function, promoting transcriptional poise at Oct4 bound loci. Sumoylation of Oct4 is then required to maintain Hmgb2 enrichment at repressed loci and to transmit the H3K27me3 mark in daughter progeny. The expression of Oct4 phosphorylation mutants however, leads to Akt inactivation and initiates the DNA Damage Checkpoint response. Our results suggest that this may subsequently facilitate chromatin reorganization and cell fate transitions. In summary, our results suggest that controlled modulation of Oct4, Akt, and Hmgb2 function is required to maintain pluripotency and for the faithful induction of transcriptional programs required for lineage specific differentiation.

Receptores purinérgicos e canais de cálcio voltagem-dependentes estão envolvidos em diversos processos biológicos como na gastrulação, durante o desenvolvimento embrionário, e na diferenciação neural. Quando ativados, canais de cálcio voltagem-dependentes e receptores purinérgicos do tipo P2, ativados por nucleotídeos, desencadeiam transientes de cálcio intracelulares controlando diversos processos biológicos. Neste trabalho, nós estudamos a participação de canais de cálcio voltagem-dependentes e receptores do tipo P2 na geração de transientes de cálcio espontâneos e sua regulação na expressão de fatores de transcrição relacionados com a neurogênese utilizando como modelo células tronco (CTE) induzidas à diferenciação em células tronco neurais (NSC) com ácido retinóico. Descrevemos que CTE indiferenciadas podem ter a proliferação acelerada pela ativação de receptores P2X7, enquanto que a expressão e a atividade desse receptor precisam ser inibidas para o progresso da diferenciação em neuroblasto. Além disso, ao longo da diferenciação neural, por análise em tempo real dos níveis de cálcio intracelular livre identificamos 3 padrões de oscilações espontâneas de cálcio (onda, pico e unique), e mostramos que ondas e picos tiveram a frequência e amplitude aumentadas conforme o andamento da diferenciação. Células tratadas com o inibidor do receptor de inositol 1,4,5-trifosfato (IP3R), Xestospongin C, apresentaram picos mas não ondas, indicando que ondas dependem exclusivamente de cálcio oriundo do retículo endoplasmático pela ativação de IP3R. NSC de telencéfalo de embrião de camundongos transgênicos ou pré-diferenciadas de CTE tratadas com Bz-ATP, o agonista do receptor P2X7, e com 2SUTP, agonista de P2Y2 e P2Y4, aumentaram a frequência e a amplitude das oscilações espontâneas de cálcio do tipo pico. Dados, obtidos por microscopia de luminescência, da expressão em tempo real de gene repórter luciferase fusionado à Mash1 e Ngn2 revelou que a ativação dos receptores P2Y2/P2Y4 aumentou a expressão estável de Mash1 enquanto que ativação do receptor P2X7 levou ao aumento de Ngn2. Além disso, células na presença do quelante de cálcio extracelular (EGTA) ou do depletor dos estoques intracelulares de cálcio do retículo endoplasmático (thapsigargin) apresentaram redução na expressão de Mash1 e Ngn2, indicando que ambos são regulados pela sinalização de cálcio. A investigação dos canais de cálcio voltagem-dependentes demonstrou que o influxo de cálcio gerado por despolarização da membrana de NSC diferenciadas de CTE é decorrente da ativação de canais de cálcio voltagem-dependentes do tipo L. Além disso, esse influxo pode controlar o destino celular por estabilizar expressão de Mash1 e induzir a diferenciação neuronal por fosforilação e translocação do fator de transcrição CREB. Esses dados sugerem que os receptores P2X7, P2Y2, P2Y4 e canais de cálcio voltagem-dependentes do tipo L podem modular as oscilações espontâneas de cálcio durante a diferenciação neural e consequentemente alteram o padrão de expressão de Mash1 e Ngn2 favorecendo a decisão do destino celular neuronal. / Purinergic receptors and voltage gated Ca2+ channels have been attributed with developmental functions including gastrulation and neural differentiation. Upon activation, nucleotide-activated P2 purinergic receptor and voltage-gated Ca2+ channel subtypes trigger intracellular calcium transients controlling cellular processes. Here, we studied the participation of voltage-gated calcium channels and P2 receptor activity in spontaneous calcium transients and consequent regulation expression of transcription factors related to retinoic acid-induced neurogenesis of mouse neural stem and embryonic stem cells (ESC). In embryonic pluripotent stem cells, proliferation is accelerated by P2X7 receptor activation, while receptor expression / activity needs to be down-regulated for the progress of neuroblast differentiation. Moreover, along neural differentiation time lapse imaging with means of a cytosolic calcium-sensitive fluorescent probe provided different patterns of spontaneous calcium transients (waves and spikes) showing that both, frequency and amplitude increased along differentiation. Cells treated with the inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor Xestospongin C showed spikes but not waves, indicating that waves exclusively depended on calcium release from endoplasmic reticulum by IP3R activation. Cells treated with the P2X7 receptor subtype agonist Bz-ATP and the P2Y2 and P2Y4 receptor 2-S-UTP increased frequency and amplitudes of calcium transients, mainly spikes, in embryonic telencephalon neural stem cells (NSC) and NSC pre-differentiated from ESC. Data obtained by luminescence time lapse imaging of stable transfected cells with Mash1 or Ngn2 promoter-protein fusion to luciferase reporter construct revealed increased Mash1 expression due to activation of P2Y2/P2Y4 receptor subtypes, while increased expression of Ngn2 was observed following P2X7 receptor activation. In addition, cells imaged in presence of the extracellular calcium chelator EGTA or following endoplasmic reticulum calcium store depletion by thapsigargin showed a decrease in Mash1 and Ngn2 expression, indicating that both are regulated by calcium signaling. Investigation of the roles of voltage gated Ca2+ channels in neural differentiation showed that Ca2+ influx in NSC pre-differentiated from ESC is due to membrane depolarization and L-type voltage gated Ca2+ channel activation, thereby controlling cell fate decision, by stabilizing the expression of MASH1 and inducing differentiation, by phosphorylation of the transcription factor CREB. Altogether these data suggest that P2X7, P2Y2, P2Y4 receptors and L-type voltage gated Ca2+ channels can modulate spontaneous calcium oscillations during neural differentiation and consequently change the Mash1 and Ngn2 expression patterns, thus favoring the cell fate decision to the neuronal phenotype.

Embryonic stem cells (ESCs) are characterized by their unlimited capacity for self-renewal and the ability to contribute to every lineage of the developing embryo. The promoters of developmentally regulated loci within these cells are marked by coincident epigenetic modifications of gene activation and repression, termed bivalent domains. Trithorax group (TrxG) and Polycomb Group (PcG) proteins respectively place these epigenetic marks on chromatin and extensively colocalize with Oct4 in ESCs. Although it appears that these cells are poised and ready for differentiation, the switch that permits this transition is critically held in check. The derepression of bivalent domains upon knockdown of Oct4 or PcG underscores their respective roles in maintaining the pluripotent state through epigenetic regulation of chromatin structure. The mechanisms that facilitate the recruitment and retention of Oct4, TrxG, and PcG proteins at developmentally regulated loci to maintain the pluripotent state, however, remain unknown. Oct4 may function as either a transcriptional activator or repressor. Prevailing thought holds that both of these activities are required to maintain the pluripotent state through activation of genes implicated in pluripotency and cell-cycle control with concomitant repression of genes required for differentiation and lineage-specific differentiation. More recent evidence however, suggests that the activator function of Oct4 may play a more critical role in maintaining the pluripotent state (Hammachi et al., 2012). The purpose of the studies described in this dissertation was to clarify the underlying mechanisms by which Oct4 functions in transcriptional activation and repression. By so doing, we wished to contextualize its role in pluripotent cells, and to provide insight into how changes in Oct4 function might account for its ability to facilitate cell fate transitions. As a result of our studies we find that Oct4 function is dependent upon post-translational modifications (PTMs). We find through a combination of experimental approaches, including genome-wide microarray analysis, bioinformatics, chromatin immunoprecipitation, functional molecular, and biochemical analyses, that in the pluripotent state Oct4, Akt, and Hmgb2 participate in a regulatory feedback loop. Akt-mediated phosphorylation of Oct4 facilitates interaction with PcG recruiter Hmgb2. Consequently, Hmgb2 functions as a context dependent modulator of Akt and Oct4 function, promoting transcriptional poise at Oct4 bound loci. Sumoylation of Oct4 is then required to maintain Hmgb2 enrichment at repressed loci and to transmit the H3K27me3 mark in daughter progeny. The expression of Oct4 phosphorylation mutants however, leads to Akt inactivation and initiates the DNA Damage Checkpoint response. Our results suggest that this may subsequently facilitate chromatin reorganization and cell fate transitions. In summary, our results suggest that controlled modulation of Oct4, Akt, and Hmgb2 function is required to maintain pluripotency and for the faithful induction of transcriptional programs required for lineage specific differentiation.

On admet communément que les tumeurs proviennent de cellules échappant aux contrôles homéostatiques qui sous-tendent les structures histologiques saines et que le phénotype d’une cellule n’est pas le résultat de processus génétiques et biochimiques déterministes mais la conséquence stochastique de réseaux de régulation intra- et intercellulaires. Ce doctorat vise à étudier quantitativement l’homéostasie phénotypique de populations cellulaires et à présenter une approche à la question fondamentale, mais jusqu’alors jamais étudiée, concernant l’autonomie versus le contrôle collectif du devenir des cellules. Nous avons étudié sur le long terme, par cytométrie de flux et dans des conditions 2D puis 3D, le niveau d’expression de CD24 et CD44 de deux lignées cellulaires de cancer du sein (SUM149-PT et SUM159-PT). Trois phénotypes ont été isolés (CD24-/CD44+, CD24+/CD44+, CD24-/CD44-), ce dernier n’avait pour le moment pas été documenté dans la littérature. Le comportement phénotypique des sous-populations CD44-low et CD44-high a été caractérisé en évaluant leur proportion et en analysant leur spectre de fluorescence. Ainsi nous avons observé des comportements périodiques d’apparition et de disparition de pool de cellules caractéristiques des lignées et une re-diversification des phénotypes pour chacune des sous-population. Seule la population issue de CD24-/CD44- re-diversifiée présente le même équilibre que la population initiale non triée. En 3D, le processus de re-diversification a été observé dans les tumorsphères issues de CD24-/CD44+ et CD24+/CD44+. Les cellules CD24-/CD44- n’ont pas ce potentiel mais survivent néanmoins à l’anoïkis. Ces comportements laissent penser qu’il existe une coordination intercellulaire régulant l’équilibre des proportions phénotypiques. Pour découvrir les règles sociales régissant l’organisation spatiale inter-phénotypique, nous avons mis en place un rapporteur des variations du niveau d’expression endogène des marqueurs d’intérêt et élaboré un modèle théorique d’interactions cellulaires. Ce travail a conforté notre hypothèse selon laquelle il s’établit des règles sociales inter-cellulaires déterminant l’expression phénotypique à l’échelle uni- et pluricellulaire. / It is commonly accepted that tumors arise from cells that escape the homeostatic controls which underlie the healthy histological structure and that cell phenotype is not the result of deterministic biochemical and genetic processes, but rather the stochastic and dynamic outcome of multiple intra- and intercellular regulation networks. This PhD aims to quantitatively study the phenotypic homeostasis of the cell populations and to present an approach to the fundamental question, never heretofore studied, regarding the autonomy versus collective control of cell fate. We studied in the long run, using flow cytometry and in 2D and 3D conditions, the level of expression of CD24 and CD44 of two breast cancer cell lines (SUM149-PT and SUM159-PT). Three phenotypes were isolated (CD24-/CD44+, CD24+/CD44+, CD24-/CD44-), the latter had not previously been documented in the literature. The phenotypic behavior of CD44-low and CD44-high subpopulations has been characterized by assessing their proportion and analyzing the fluorescence map. Thereby, we observed both a periodic behavior of appearance and disappearance of pool of cells characteristics of each cell lines and a phenotypic re-diversification for each subpopulation. Only the resulting population derived from CD24-/CD44- provided the same balance as the original unsorted population. 3D re-diversification process was observed in tumorspheres from CD24-/CD44+ and CD24+/CD44+. The cells CD24-/CD44did not have that potential but nonetheless outlived anoikis. These behaviors suggest that there is an inter-cell coordination regulating the balance of phenotypic proportions. To discover the social rules regulating inter-phenotypic spatial organization, we have set up a reporter of the endogenous variations of CD24 and CD44 and developed a theoretical model of cell interactions. This work has confirmed our hypothesis that inter-cellular social rules are determining the phenotypic expression at both the uni- and multicellular scales.

Der Gen-Knockdown mittels RNAi hat sich als essentiell erwiesen, um Inhibitoren der induzierten Transdifferenzierung in C. elegans zu identifizieren (Tursun et al., 2011). Bakterienstämme, die dsRNA exprimieren, das die Expression spezifischer Gene mindert, können dem Wurm direkt zugefüttert werden, um einen genomweiten RNAi-screen der insgesamt 20.000 Gene in C. elegans durchzuführen. Allerdings werden die meisten biologischen Prozese durch mehr als ein Gen reguliert, was den Bedarf nach einer Methode generiert, die es erlaubt, zwei oder mehr Gene gleichzeitig herunter zu regulieren, um die Steuerung biologischer Prozesse studieren zu können. Die derzeitig vorhandenen Methoden liefern entweder nicht reproduzierbare Ergebnisse oder sind nicht skalierbar. Wir nutzen baktierelle Konjugation, die es durch ein konjugatives Plasmid ermöglicht Bakterienzellen zu generieren, die zwei verschiedene RNAi-Plasmide enthalten. Das Ziel war es, modifizierte RNAi-Donor-Plasmide mittels bakterieller Konjugation an eine Vielzahl anderer Bakterienzellen zu übertragen, die bereits ein anderes RNAi-Plasmid enthalten und dies dann im Hochdurchsatzverfahren durchführen zu können. Um Enhancer induzierter Expression von unc-25::gfp in der Keimbahn, ermöglicht durch den Knockdown des Histonchaperons LIN-53 (RbAp46/48 in Menschen), zu finden, wurden RNAi-Klone generiert, die gleichzeitig lin-53 als auch eines von insgesamt 800 verschiedenen Chromatin-bezogenen Gene herunter regulieren. Dabei identifizierten wir RBBP-5, Mitglied des Set1/ MLL-Methyltransferase-Komplexes, als neuen Barrierefaktor der induzierten Transdifferenzierung. RBBP-5 agiert dabei mutmaßlich parallel zu LIN-53. Doppelte RNAi, ermöglicht durch bakterielle Konjugation, erlaubt den simultanen Knockdown zweier oder mehr Gene, um genetische Interaktionen studieren zu können und erweitert damit die Einsatzmöglichkeiten von RNAi-Screens, um untereinander verbundene biologische Prozesse zu studieren. / The knock down of genes by RNAi has been fundamental to identify inhibitors of induced cell transdifferentiation in C. elegans (Tursun et al., 2011). Bacteria strains expressing dsRNA that target specific genes can be fed to the worm allowing straightforward whole-genome RNAi screens of the 20,000 genes in theC. elegans genome. However, many biological processes are regulated by more than one gene raising the need for simultaneous knock down of two or more genes to more fully interrogate the regulation of complex biological processes. Two approaches are currently available for double RNAi knockdown, − two bacteria strains expressing specific dsRNA can be mixed and grown together and fed simultaneously, which gives highly variable results. Alternatively, a new bacterial clone can be generated carrying a plasmid on which two RNAi targets of interest are 'stitched' together, which is not scalable. To address this challenge, we have developed a protocol using bacterial conjugation mediated by the 'Fertility Factor' (F) Episome in order to combine two different RNAi plasmids in a single bacterium. The objective was to be able to transfer a single RNAi plasmid to a large number of bacterial cells carrying different RNAi clones in one step in a high-throughput manner for large scale 'double' or even 'triple' RNAi screens. To find enhancers of induced unc-25::gfp expression in the germ line enabled by the depletion of histone chaperone LIN-53 (RbAp46/48 in humans), double RNAi clones targeting lin-53 and a total of 800 chromatin-related genes were generated and screened. We identified the Set1/MLL methyltransferase complex member RBBP-5 as a novel reprogramming barrier that putatively acts in a parallel pathway to LIN-53. Double RNAi by conjugation permits to reliably knock down two genes simultaneously in order to study genetic interactions at a genome-wide level, thus further increasing the versatility of RNAi screens to investigate interconnected biological processes.

Intestinal cylindrical growth peaks in mice a few weeks after birth, simultaneously with crypt fission activity. It nearly stops after weaning and cannot be reactivated later. Transgenic mice expressing Cd97/Adgre5 in the intestinal epithelium develop a mega-intestine with normal microscopic morphology in adult mice. Here, we demonstrate premature intestinal differentiation in Cd97/Adgre5 transgenic mice at both the cellular and molecular levels until postnatal day 14. Subsequently, the growth of the intestinal epithelium becomes activated and its maturation suppressed. These changes are paralleled by postnatal regulation of growth factors and by an increased expression of secretory cell markers, suggesting growth activation of non-epithelial tissue layers as the origin of enforced tissue growth. To understand postnatal intestinal growth mechanistically, we study epithelial fate decisions during this period with the use of a 3D individual cell-based computer model. In the model, the expansion of the intestinal stem cell (SC) population, a prerequisite for crypt fission, is largely independent of the tissue growth rate and is therefore not spontaneously adaptive. Accordingly, the model suggests that, besides the growth activation of non-epithelial tissue layers, the formation of a mega-intestine requires a released growth control in the epithelium, enabling accelerated SC expansion. The similar intestinal morphology in Cd97/Adgre5 transgenic and wild type mice indicates a synchronization of tissue growth and SC expansion, likely by a crypt density-controlled contact inhibition of growth of intestinal SC proliferation. The formation of a mega-intestine with normal microscopic morphology turns out to originate in changes of autonomous and conditional specification of the intestinal cell fate induced by the activation of Cd97/Adgre5.