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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.

The AER has a vital role in directing embryonic limb development. Several models have been developed that attempt to explain how the AER directs limb development, but none of them are fully supported by existing data. I provide evidence that FGFs secreted from the AER induce a gradient of Wnt5a. I also demonstrate that limb mesenchyme grows toward increasing concentrations of Wnt5a. We hypothesize that the changing shape of the AER is critical for patterning the limb along the proximal to distal axis. To better understand the pathway through which Wnt5a elicits its effects, we have performed various genetic studies. We demonstrate that Wnt5a does not signal via the Wnt/β-catenin pathway. However, we show that Wnt5a mutants share many common defects with Vangl2 mutants suggesting that Wnt5a signals through the Wnt/planar cell polarity (PCP) pathway.

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.