Cell cycle exit (“birth”) of retinal progenitor cells (RPCs) is considered a watershed that is preceded by changing levels of cell cycle regulators, and followed rapidly by induction of a post M-phase differentiation cascade. Yet the actual dynamics of these events are largely unclear, thus whether mitosis separates pre- and post- birth differentiation cascades is unproven. We characterized the regulation of many division and differentiation markers relative to each other and final mitosis. Unexpectedly, classic “cell cycle” markers were present well beyond exit (e.g. Ki67, Pcna), early embryonic RPCs expressed “differentiation” markers that later labeled post-mitotic neurons exclusively (e.g. Brn3b, Tubb3, Ptf1a), and factors detected just after cell birth in the embryo were induced well beyond M-phase post-natally (e.g. Nrl, Crx). Thus, the dynamics of birth-associated events shift dramatically during development, even to either side of mitosis. Instead of mitosis behaving as a cog that activates post-exit differentation events we suggest that a common trigger induces both the exit and differentiation programs in RPCs, precisely coordinating their startpoints, but that each subsequent cascade unfolds independently. This model explains the convergence of birth and differentiation but also their temporal maliability. This view fits with our observation that in the absence of the Rb tumor suppressor, differentiation still initiates even without cell cycle exit. Finally, neoplastic transformation in the mouse retina requires loss of Rb and its relative p107, and emerging tumor features suggest an amacrine cell-of-origin. We studied Rb/p107 null clones, and noted two striking features. First, despite initial expansion of aberrantly dividing differentiating cells, apoptosis pruned clones precisely to wild type sizes. “Cell competition” maintains tissue size by selecting fitter over weaker progenitors; our data provide a unique example of competition among differentiating cells. Second, despite normal numbers of amacrine cells per Rb/p107 null clone, more clones contained amacrine cells and fewer had bipolar cells. Both this effect and ectopic division were E2f1-dependent. Thus, the oncogenic initiation event in mouse retinoblastoma triggers a very early fate switch, even before neoplastic transformation, broadening the possibilities for the cell-of-origin of retinoblastoma, and arguing that even very early stage tumors cannot be used to define cancer origin.

Cell cycle exit (“birth”) of retinal progenitor cells (RPCs) is considered a watershed that is preceded by changing levels of cell cycle regulators, and followed rapidly by induction of a post M-phase differentiation cascade. Yet the actual dynamics of these events are largely unclear, thus whether mitosis separates pre- and post- birth differentiation cascades is unproven. We characterized the regulation of many division and differentiation markers relative to each other and final mitosis. Unexpectedly, classic “cell cycle” markers were present well beyond exit (e.g. Ki67, Pcna), early embryonic RPCs expressed “differentiation” markers that later labeled post-mitotic neurons exclusively (e.g. Brn3b, Tubb3, Ptf1a), and factors detected just after cell birth in the embryo were induced well beyond M-phase post-natally (e.g. Nrl, Crx). Thus, the dynamics of birth-associated events shift dramatically during development, even to either side of mitosis. Instead of mitosis behaving as a cog that activates post-exit differentation events we suggest that a common trigger induces both the exit and differentiation programs in RPCs, precisely coordinating their startpoints, but that each subsequent cascade unfolds independently. This model explains the convergence of birth and differentiation but also their temporal maliability. This view fits with our observation that in the absence of the Rb tumor suppressor, differentiation still initiates even without cell cycle exit. Finally, neoplastic transformation in the mouse retina requires loss of Rb and its relative p107, and emerging tumor features suggest an amacrine cell-of-origin. We studied Rb/p107 null clones, and noted two striking features. First, despite initial expansion of aberrantly dividing differentiating cells, apoptosis pruned clones precisely to wild type sizes. “Cell competition” maintains tissue size by selecting fitter over weaker progenitors; our data provide a unique example of competition among differentiating cells. Second, despite normal numbers of amacrine cells per Rb/p107 null clone, more clones contained amacrine cells and fewer had bipolar cells. Both this effect and ectopic division were E2f1-dependent. Thus, the oncogenic initiation event in mouse retinoblastoma triggers a very early fate switch, even before neoplastic transformation, broadening the possibilities for the cell-of-origin of retinoblastoma, and arguing that even very early stage tumors cannot be used to define cancer origin.

Les p21-activated kinases (PAK) du sous-groupe I sont impliquées dans de nombreux processus cellulaires tels la prolifération, les mouvements cellulaires, l’adhérence et l’apoptose. Ces kinases sont des effecteurs des Rho-GTPases Rac1 et Cdc42 et participent à la régulation du cytosquelette d’actine. Les deux kinases neuronales PAK1 et PAK3, qui présentent de fortes identités de séquence, régulent le cytosquelette d’actine, contrôlant ainsi la dynamique des épines dendritiques, et la plasticité synaptique.Les mutations du gène pak3, localisé sur le chromosome X, sont responsables de déficience intellectuelle chez l’homme, et les mécanismes moléculaires et cellulaires associés aux défauts cognitifs sont mal connus. Il a été montré que PAK3 participe à la voie proneurale au cours de l’embryogénèse précoce du xénope en favorisant la sortie du cycle cellulaire et la différenciation neuronale. Cependant, le rôle de PAK3 dans la neurogenèse adulte n’a pas été étudié. Or depuis maintenant une quinzaine d’années, il est admis que la neurogenèse perdure à l’âge adulte et participe aux processus de mémorisation et d’apprentissage. Nous nous sommes donc intéressés à l’implication de PAK3 dans la régulation de la neurogenèse adulte, posant l’hypothèse qu’un défaut de neurogenèse serait responsable, au moins en partie, des défauts cognitifs chez les patients. Nous avons montré que PAK3 n’est pas exprimée dans les cellules souches neurales/progéniteurs prolifératifs mais son expression augmente fortement dès le retrait des facteurs de croissance, ex vivo, suggérant un rôle dans la neurogenèse adulte. Nous avons montré que l’invalidation de pak3 provoque une augmentation de la fréquence de neurosphères primaires formées ainsi qu’un accroissement de leur taille, ceci sans affecter la taille du réservoir de cellules souches ni les propriétés cardinales de celles-ci (multipotence, auto-renouvellement et prolifération). Toutefois, les cellules progénitrices pak3- poursuivent leur prolifération dans des conditions de culture induisant normalement la différenciation, suggérant un défaut de sortie du cycle cellulaire.Nous nous sommes ensuite demandé si les mutations de déficience intellectuelle du gène pak3 altèrent la neurogenèse adulte. Nous avons créé pour cela un modèle murin portant la mutation R67C, responsable chez l’homme de la forme la plus sévère de déficience intellectuelle associée aux mutations de ce gène. Nous mettons en évidence, dans cette souris knock-in, une forte diminution du nombre de cellules nouveau-nées dans les deux zones neurogéniques du cerveau (la zone sous-ventriculaire et le gyrus denté de l’hippocampe) et une augmentation de la proportion de neurones nouveau-nés immatures. Ces données suggèrent que la mutation R67C n’induit pas une perte de fonction de la kinase mais un changement de fonction dépendante d’une activation préférentielle par la GTPase Rac1.En conclusion, ce travail de thèse montre que PAK3 participe à la régulation de la neurogenèse adulte chez les mammifères, contrôle la sortie du cycle cellulaire des progéniteurs neuraux et que la mutation R67C impacte la maturation des neurones nouveau-nés. L’ensemble de ces données suggère que les défauts de neurogenèse adulte dus aux mutations de déficience intellectuelle du gène pak3 sont à l’origine de certains dysfonctionnements cognitifs. / The group I p21-activated kinases (PAK) are involved in many cellular processes such as proliferation, cell movement, adhesion and apoptosis. These kinases are effectors of Rho GTPases Rac1 and Cdc42, and participate in the regulation of the actin cytoskeleton. Both neuronal kinase PAK1 and PAK3, which exhibit high sequence identities, regulate the actin cytoskeleton, thereby controlling the dynamics of dendritic spines and synaptic plasticity. Mutations of the X-linked pak3 are responsible for intellectual disability (ID) in humans, and the molecular and cellular mechanisms associated with cognitive defects are poorly described. It was shown that PAK3 participates in the proneural pathway during early Xenopus embryogenic development, by promoting cell cycle exit and neuronal differentiation of neural precursors. However, the role of PAK3 in the adult neurogenesis has not been studied in mammals. It is now generally accepted that neurogenesis persists during human adulthood and is involved in learning and memory. We are therefore interested in the involvement of PAK3 in the regulation of adult neurogenesis, on the assumption that defects in neurogenesis may be responsible, at least in part, for cognitive defects in ID patients.We showed that PAK3 is not expressed in proliferative neural stem/progenitor cells but its expression increased significantly upon growth factor removal, suggesting a role in adult neurogenesis. We showed that the invalidation of pak3 gene causes an increase in the frequency and in size of primary neurospheres. However Pak3 invalidation does not affect the size of the stem cell reservoir nor the NCS cardinal properties (pluripotency, self-renewal and proliferation). However, the pak3- progenitor cells continue their proliferation in culture conditions normally inducing differentiation, suggesting a defect in cell cycle exit. We then asked whether pak3 ID mutations affect adult neurogenesis. We created a knock-in model expressing the pak3-R67C mutation responsible in humans for a severe form of intellectual impairment. We observed in the knock-in mice, a significant decrease in the number of newborn cells in both neurogenic areas of the brain (the subventricular zone inforebrain, and the dentate gyrus of the hippocampus) and an increase in the proportion of immature newborn neurons. These data suggest that the R67C mutation does not induce a loss of function of the kinase but a change of a function dependent on preferential activation by the Rac1 GTPase.In conclusion, we show that PAK3 play an important role in the regulation of adult neurogenesis in mammals by controlling the cell cycle exit of neural progenitors. The R67C ID mutation impacts both newborn cell proliferation and their maturation. Taken together, these data suggest that defects in adult neurogenesis caused by ID mutations in the pak3 gene may be involved in some cognitive dysfunctions.

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