The exact mechanisms that regulate limb outgrowth the mouse embryo are unknown. Although there are several models, we favor a hypothesis where cells become polarized by signals secreted from the AER which orient their cell migration and/or divisions causing limb outgrowth. Clonal analysis has provided a mechanism to study cell behavior. We have generated a targeting construct containing the Fgf8 inhibitor, Sprouty2, in order to generate mutant clones for behavioral analyses in the limb. In order to more effectively study clonal behavior we report the modification of a novel clonal analysis approach, exo-utero surgery. We have modified, enhanced and proven that this technique can be used successfully in mouse embryos in which we directly apply 4-OHtamoxifen to the limb to induce YFP or β-gal reporter genes in limb mesenchyme. Using this method, we can closely control the timing and location of the induced clones and observe cell behavior during embryonic limb development. Phenotypes of Wnt5a-/- and Ror2-/- exhibit shortened limbs suggesting they function in a similar pathway. Wnt5a and Ror have been found to "colocalize" in the growing limb bud and have also been shown to bind in vitro. Here we show preliminary results about Wnt5a and Ror2 in vivo association by immunoprecipitation of limb bud extracts.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24506号 / 医博第4948号 / 新制||医||1064(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 万代 昌紀, 教授 松田 秀一, 教授 中本 裕士 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

In this thesis we combine quantitative measurements of mouse limb morphogenesis and computer modelling to test a well established theory about the cellular mechanisms promoting limb elongation. A distally directed gradient of cellular proliferation was believed to be the driving mechanism for limb outgrowth. We find that the empirically measured spatial proliferation pattern fails to promote normal development - a reverse engineering algorithm was applied and revealed a proliferation pattern that could indeed carry out normal development. The differences between those patterns is dramatic and suggests that isotopic cellular proliferation alone has very little impact on limb morphogenesis and other – non isotropic - mechanisms need to be involved. / En esta tesis tratamos de testar una bien establecida teor´ıa sobre los mecanismos celulares que promueven de la elongaci´on de las extremidades. Para eso combinamos mediciones cuantitativas del proceso morfogen ´etico de la extremidad del rat´on con modelos computacionales. Se cre´ıa que la fuerza conductora del crecimiento de las extremidades era un gradiente en sentido distal del incremento de la proliferaci´on celular. Descubrimos que el patr´on de proliferaci´on celular basado en medidas emp´ıricas no consegu´ıa promover un desarrollo normal, mientras que un algoritmo de ingenier´ıa inversa aplicado al proceso revel´o un patr´on que si podr´ıa. La diferencia entre estos dos patrones es inmensa y sugiere que la proliferaci´on celular isotropica por si sola tiene muy poco impacto sobre la morfog´enesis de las extremidades, indicando as´ı la necesidad de que otros procesos no isotr´opicos se hallen involucrados.

This thesis covers broad topics concerning limb growth and development and their effects on locomotion in the brown kiwi (Apteryx mantelli). I begin by describing the morphological features of a collection of unknown-age wild kiwi embryos from early development to point of hatch. Using these features, I assign developmental stages to each embryo and compare the progress of development to the same-staged ostrich and chicken embryos. Measurements of the hindlimb, bill and crown-rump length are used to develop an aging scheme based on comparisons with the ostrich and the chicken. The ostrich model and chicken model create age predictions for the unknown aged kiwi embryos. One kiwi embryo was of known age and both models gave identical predictions for this marker embryo, but gave differing predictions for all other kiwi embryos. Using captive-reared kiwi chicks, I characterise hindlimb, bill and bodyweight growth from the time of hatch to 3 months of age. Growth patterns are very linear within this time period for all measurements but bodyweight. Female kiwi hatch with longer bills than males, but the growth of both sexes converges by the end of the 3-month period. Growth of bodyweight in the males slows earlier than in females. Bodyweight and bill length were then compared to a wild population of kiwi. Captive-reared chicks were found to hatch with shorter bills than the wild birds and to increase in bodyweight at a faster rate than wild birds. Rapid weight gain has been implicated in developmental limb deformities in other precocial and long-legged birds and has the potential to produce similar results in captive kiwi. I further studied the movement of the hindlimb during locomotion in two adults and one juvenile kiwi by filming them while they were walking on a treadmill. Kinematic parameters were measured from the video recordings and compared to overground parameters from another study. Similarity between the treadmill and overground locomotor parameters validates the use of a treadmill in studying kiwi locomotion. None of the birds achieved the theoretical transition from a walk to a run at a duty factor of 0.5. After normalising for size, the juvenile showed a longer stride length and lower stride frequency with increasing speed than the adults. Lateral head oscillations were observed during the stride cycle, which I propose having a sensory function as well as a biomechanical one.

The vertebrate embryonic limb is a key model in elucidating the genetic basis underlying the three dimensional morphogenesis of structures. Despite the wealth of insights that have been generated from this model, many long-standing questions remain. For example, it has been known for over 70 years that the apical ectodermal ridge (AER) of the embryonic limb is essential for distal outgrowth and patterning of the adjacent limb mesenchyme. The mechanisms whereby the AER does accomplish outgrowth and patterning are still poorly understood. We propose that secreted FGFs from the AER activate Wnt5a expression in gradient fashion, which in turn provides an instructional cue to direct outgrowth in the direction of increasing Wnt5a expression (i.e. toward the distal tip of the limb). In vivo and in vitro models were used to test this hypothesis. We placed Wnt5a expressing L-cell implants into stage 23 chick limb buds and demonstrate that labeled mesenchyme cells grow toward the source of Wnt5a. Purified Wnt5a soaked heparin bead implants have only a marginal effect on directed growth of the adjacent mesenchyme, whereas a greater effect was seen with beads soaked in Wnt5a conditioned media. Using an in vitro model where cultured limb mesenchyme cells were subjected to a gradient of conditioned Wnt5a media or purified Wnt5a, we show no specific migratory direction. However, clusters of cells tended to move toward the source of Wnt5a indicating that it might be necessary for the cells to be in complete contact to respond to the Wnt5a signal. Taken together, our results suggest that Wnt5a is sufficient to direct limb mesenchyme. This finding has given support to a new model of limb development proposed by our lab and referred to as the Mesenchyme Recruitment Model.

No description available.

Bien que partageant une homologie structurelle évidente, les membres antérieurs (MA) sont toujours différents des membres postérieurs (MP). Ceci suggère l’existence d’un programme générique de formation d’un membre, un bauplan, qui doit être modulé de façon spécifique pour engendrer cette différence antéro-postérieure de l’identité. Nous avons donc voulu identifier les mécanismes déployés durant l’évolution pour permettre la mise en place de l’identité des membres. Le laboratoire avait précédemment caractérisé, chez les souris où le gène Pitx1 est inactivé, une transformation partielle des MP en MA couplée à une perte de croissance. Nous avons donc cherché à comprendre les mécanismes en aval de Pitx1 dans la détermination de l’identité postérieure. Notre démarche nous a permis d’identifier les gènes affectés par la perte de Pitx1 dans les MP, où nous avons confirmé une dérégulation de l’expression de Tbx4. Tbx4 et Tbx5 sont des candidats évidents pour déterminer l’identité, leur expression étant restreinte aux MP et MA, respectivement, mais leur implication dans ce processus était sujette à controverse. Nous avons donc évalué l’apport de Tbx4 en aval de Pitx1 dans les processus d’identité en restaurant son expression dans les MP des souris Pitx1-/-. Ce faisant, nous avons pu montrer que Tbx4 est capable de pallier la perte de Pitx1 dans le MP, en rétablissant à la fois les caractères d’identité postérieure et la croissance. En parallèle, nous avons montré que Tbx5 était capable de rétablir la croissance mais non l’identité des MP Pitx1-/-, démontrant ainsi de façon définitive une propriété propre à Tbx4 dans la détermination de l’identité des membres postérieure. La caractérisation de l’activité transcriptionnelle de Tbx4 et Tbx5 nous a permis de mettre en évidence un domaine activateur conservé mais aussi un domaine spécifique à Tbx4, répresseur de la transcription. Par ailleurs, une mutation faux-sens de TBX4 dans les patients atteints du syndrome coxo-podo-patellaire, TBX4Q531R, inactive le domaine répresseur, empêchant la compensation de l’identité mais non de la croissance des MP dépourvus de Pitx1, démontrant l’importance de cette fonction dans l’identité postérieure. La caractérisation de l’activité répressive de Tbx4, qui se manifeste seulement dans les membres postérieurs démontre l’importance de cette fonction dans l’identité postérieure. Nous avons aussi été en mesure d’identifier un corépresseur qui est suffisant pour supporter cette activité de Tbx4. Enfin, nous avons pu aussi démontrer l’activité transcriptionnelle d’un représentant du gène ancestral, présent chez Amphioxus, qui se comporte strictement comme un activateur et semble dépourvu du domaine répresseur. En somme, nous avons précisé le rôle de Tbx4 et Tbx5, ainsi que leur mécanisme, dans la détermination de l’identité des membres. Globalement, nos travaux permettent d’élaborer une théorie où une divergence d’activité transcriptionnelle de Tbx4 et Tbx5 est responsable de l’identité des membres et même entrevoir que cette divergence d’activité soit à la base de son apparition durant l’évolution. / Forelimbs and hindlimbs are a classical example of serial homology, suggesting they share an evolutionary common generic program that directs their formation. Identity is presumably derived from specific modulations of that program in different limb type. Transcription factors are prime candidates to link these structural differences to specific modulations and three factors with limb-specific expression have been identified. Pitx1 and Tbx4 expression is restricted to the hindlimbs while Tbx5 is restricted to the forelimbs and they have all been ascribed functions in both growth and identity from knockout and overexpression studies. Recent studies have produce evidence that Tbx4 and Tbx5 are interchangeable, sharing identical properties to support growth but not identity of the limbs, the latter being a direct consequence of Pitx1 expression. Indeed, Pitx1 deficient mice have been previously described as undergoing a hindlimb-to-forelimb transformation in addition of growth defects. To better assess the shared and specific properties of Tbx4 and Tbx5, we assessed their capacities to rescue identity and growth defects by expression studies in Pitx1-/- hindlimbs. Specifically, previous studies had shown that Pitx1 deficiency causes the loss of hindlimb features, the transformation of hindlimb features toward forelimb-like morphology, the gain of a forelimb feature and the asymmetric loss of growth at the level of the femur. Targeted expression in the limbs of both Tbx4 and Tbx5 rescued the growth defects similarly. Interestingly, only Tbx4 was able to restore identity features affected in absence of Pitx1. To further assess these shared and specific properties, we conducted transcriptional assays that revealed the presence of a shared and conserved transactivating domain in the C-terminal moiety of these proteins. Moreover, we could identify a repressor domain specific to Tbx4. Human small patella syndrome maps to TBX4 and a coding mutation, TBX4Q531R, that specifically inactivates the repressive properties of Tbx4 prevents it from rescuing identity to the Pitx1-/- hindlimbs but not from rescuing growth. We also conducted a yeast two-hybrid assay that allowed the identification of putative co-factors of Tbx4, of which one seems to act as a co-repressor. Together, our results support the presence of a Tbx4/Tbx5 conserved activating domain required for limb outgrowth that is an integral part of the limb bauplan. Importantly, we identified a molecular basis for the determination of limb identity through the Tbx4-specific repressor domain and reveal a novel path through which limb identity may have emerged during evolution.

Bien que partageant une homologie structurelle évidente, les membres antérieurs (MA) sont toujours différents des membres postérieurs (MP). Ceci suggère l’existence d’un programme générique de formation d’un membre, un bauplan, qui doit être modulé de façon spécifique pour engendrer cette différence antéro-postérieure de l’identité. Nous avons donc voulu identifier les mécanismes déployés durant l’évolution pour permettre la mise en place de l’identité des membres. Le laboratoire avait précédemment caractérisé, chez les souris où le gène Pitx1 est inactivé, une transformation partielle des MP en MA couplée à une perte de croissance. Nous avons donc cherché à comprendre les mécanismes en aval de Pitx1 dans la détermination de l’identité postérieure. Notre démarche nous a permis d’identifier les gènes affectés par la perte de Pitx1 dans les MP, où nous avons confirmé une dérégulation de l’expression de Tbx4. Tbx4 et Tbx5 sont des candidats évidents pour déterminer l’identité, leur expression étant restreinte aux MP et MA, respectivement, mais leur implication dans ce processus était sujette à controverse. Nous avons donc évalué l’apport de Tbx4 en aval de Pitx1 dans les processus d’identité en restaurant son expression dans les MP des souris Pitx1-/-. Ce faisant, nous avons pu montrer que Tbx4 est capable de pallier la perte de Pitx1 dans le MP, en rétablissant à la fois les caractères d’identité postérieure et la croissance. En parallèle, nous avons montré que Tbx5 était capable de rétablir la croissance mais non l’identité des MP Pitx1-/-, démontrant ainsi de façon définitive une propriété propre à Tbx4 dans la détermination de l’identité des membres postérieure. La caractérisation de l’activité transcriptionnelle de Tbx4 et Tbx5 nous a permis de mettre en évidence un domaine activateur conservé mais aussi un domaine spécifique à Tbx4, répresseur de la transcription. Par ailleurs, une mutation faux-sens de TBX4 dans les patients atteints du syndrome coxo-podo-patellaire, TBX4Q531R, inactive le domaine répresseur, empêchant la compensation de l’identité mais non de la croissance des MP dépourvus de Pitx1, démontrant l’importance de cette fonction dans l’identité postérieure. La caractérisation de l’activité répressive de Tbx4, qui se manifeste seulement dans les membres postérieurs démontre l’importance de cette fonction dans l’identité postérieure. Nous avons aussi été en mesure d’identifier un corépresseur qui est suffisant pour supporter cette activité de Tbx4. Enfin, nous avons pu aussi démontrer l’activité transcriptionnelle d’un représentant du gène ancestral, présent chez Amphioxus, qui se comporte strictement comme un activateur et semble dépourvu du domaine répresseur. En somme, nous avons précisé le rôle de Tbx4 et Tbx5, ainsi que leur mécanisme, dans la détermination de l’identité des membres. Globalement, nos travaux permettent d’élaborer une théorie où une divergence d’activité transcriptionnelle de Tbx4 et Tbx5 est responsable de l’identité des membres et même entrevoir que cette divergence d’activité soit à la base de son apparition durant l’évolution. / Forelimbs and hindlimbs are a classical example of serial homology, suggesting they share an evolutionary common generic program that directs their formation. Identity is presumably derived from specific modulations of that program in different limb type. Transcription factors are prime candidates to link these structural differences to specific modulations and three factors with limb-specific expression have been identified. Pitx1 and Tbx4 expression is restricted to the hindlimbs while Tbx5 is restricted to the forelimbs and they have all been ascribed functions in both growth and identity from knockout and overexpression studies. Recent studies have produce evidence that Tbx4 and Tbx5 are interchangeable, sharing identical properties to support growth but not identity of the limbs, the latter being a direct consequence of Pitx1 expression. Indeed, Pitx1 deficient mice have been previously described as undergoing a hindlimb-to-forelimb transformation in addition of growth defects. To better assess the shared and specific properties of Tbx4 and Tbx5, we assessed their capacities to rescue identity and growth defects by expression studies in Pitx1-/- hindlimbs. Specifically, previous studies had shown that Pitx1 deficiency causes the loss of hindlimb features, the transformation of hindlimb features toward forelimb-like morphology, the gain of a forelimb feature and the asymmetric loss of growth at the level of the femur. Targeted expression in the limbs of both Tbx4 and Tbx5 rescued the growth defects similarly. Interestingly, only Tbx4 was able to restore identity features affected in absence of Pitx1. To further assess these shared and specific properties, we conducted transcriptional assays that revealed the presence of a shared and conserved transactivating domain in the C-terminal moiety of these proteins. Moreover, we could identify a repressor domain specific to Tbx4. Human small patella syndrome maps to TBX4 and a coding mutation, TBX4Q531R, that specifically inactivates the repressive properties of Tbx4 prevents it from rescuing identity to the Pitx1-/- hindlimbs but not from rescuing growth. We also conducted a yeast two-hybrid assay that allowed the identification of putative co-factors of Tbx4, of which one seems to act as a co-repressor. Together, our results support the presence of a Tbx4/Tbx5 conserved activating domain required for limb outgrowth that is an integral part of the limb bauplan. Importantly, we identified a molecular basis for the determination of limb identity through the Tbx4-specific repressor domain and reveal a novel path through which limb identity may have emerged during evolution.

Limb outgrowth is controlled by a specialized group of cells called the apical ectodermal ridge (AER), a thickening of the limb epithelium, at its distal tip. This specialized thickening of ectodermal cells is responsible for maintaining the underlying mesenchymal cells in an undifferentiated and proliferative state, and its structure is preserved through a fine-tuned balance between proliferation and apoptosis. This equilibrium is genetically controlled but little is known about the molecules involved in this process. Several authors have been shown that both fibroblast growth factor (FGF) and Erk pathway activation are crucial for AER function. Recently, FLRT3, a transmembrane protein able to interact with FGF receptors, has been implicated in the triggering of ERK activity by FGFs. In this thesis, we show that flrt3 expression is restricted to the AER, co-localizing its expression with fgf8 and pERK activity. Loss-of-function studies demonstrate that silencing of flrt3 affects the integrity of the AER and, subsequently, its proper function during limb bud outgrowth. Our data also indicate that flrt3 expression is not regulated by FGF activity in the AER, whereas ectopic WNT3A is able to induce flrt3 expression. Overall, our findings confirm flrt3 as a key player during chicken limb development, being necessary but not sufficient for proper AER formation and maintenance under the control of BMP and WNT signalling. During limb bud development, AER structure is maintained through a fine-tuned balance between proliferation and programmed cell death and this equilibrium is genetically controlled, although little is known about the molecules involved in that process. In this thesis we present evidences involving oct4, required to establish and maintain the pluripotent cell population necessary for embryogenesis in mouse and human, in the control of the proliferative balance within the AER cells. Overexpression of otc4 in the limb ectoderm disrupts the ratio apoptosis/proliferation and, moreover, oct4 expression is under the control of wnt-canonical pathway. We also describe a special localization and behaviour of proliferating cells in the AER in response to oct4 activity. We, therefore, describe a role for oct4 as a factor able to maintain a niche of cells that is responsible for the renewal of the AER. / El crecimiento del esbozo de la extremidad está controlado por un grupo especializado de células denominado Cresta Ectodérmica Apical (CEA), un engrosamiento del epitelio del miembro en su borde más distal. Este engrosamiento es responsable del mantenimiento de las células del mesodermo distal en un estado indiferenciado y proliferativo. Diferentes estudios muestran que la actividad de los factores de crecimiento fibroblástico (FCF) y de la vía Erk son cruciales para la correcta funcionalidad de la CEA. Recientemente se ha implicado a FLRT3, una proteína transmembranal capaz de interaccionar con los receptores de los FCF, en la activación de la vía Erk por los mismos. En esta tesis describimos cómo la expresión de flrt3 se restringe a la CEA, colocalizándose su expresión con fgf8 y la actividad de la vía Erk. Los experimentos de pérdida de función demuestran que la inhibición de flrt3 afecta la integridad de la CEA y, consecuentemente, a su función durante el desarrollo del esbozo del miembro. Nuestros datos también indican que la expresión de flrt3 no está regulada a través de los FCF en la CEA, sin embargo, la activación ectópica de WNT3A es capaz de inducir la expresión de flrt3. En conjunto, nuestros resultados demuestran que flrt3 es una molécula clave durante el desarrollo de las extremidades de pollo, siendo necesaria, pero no suficiente, para la correcta formación y mantenimiento de la CEA bajo el control de la señalización a través de BMP y WNT. Durante el desarrollo de las extremidades, la estructura de la CEA se mantiene a través de un fino control del balance entre la proliferación y apoptosis. Este equilibrio se encuentra genéticamente controlado aunque se sabe muy poco acerca de las moléculas involucradas en este proceso. En esta tesis presentamos evidencias en las que oct4, molécula necesaria para establecer y mantener la población de células pluripotentes necesarias durante la embriogénesis en ratón y humanos, controla la tasa de proliferación en las células de la CEA. La expresión ectópica de oct4 en el ectodermo del esbozo de la extremidad perturba la razón entre la apoptosis y la proliferación y, además, su expresión está controlada por la actividad de la vía canónica de los Wnt. También describimos en este trabajo la localización y comportamiento especiales de las células de la CEA en proliferación como respuesta a la actividad de oct4. Por consiguiente, podemos inferir que el rol de oct4 será el de un factor necesario para mantener un nicho celular responsable por la renovación de la CEA.