Tese de mestrado. Biologia (Biologia Evolutiva e do Desenvolvimento). Universidade de Lisboa, Faculdade de Ciências, 2011During limb development, the proximal-distal outgrowth is controlled by a thickening of ectodermal cells at the distal tip of the limb, termed apical ectodermal ridge (AER). This transient embryonic structure is essential for the patterning and limb outgrowth, being a conserved feature in vertebrate development and it is also important to maintain proliferation in adjacent tissues before their differentiation. Despite AER induction and maintenance are orchestrated by complex interactions between the FGF, WNT/β-catenin and BMP signaling pathways, little is known about the molecules involved in the maintenance of the proliferation versus apoptosis and the renewal of its cells during development. In recent studies, it has been showed by our lab that one of these molecules, oct4, could be involved in the control of proliferative balance within the AER cells. In this thesis we present evidences of the involvement of two more molecules in this process, lgr5 and lgr6, which are known as adult stem cells markers. In here, we describe the expression pattern of lgr5 and lgr6 during limb bud development using the chicken embryo as a model and show that their expression patterns in the limb bud are consistent with the areas of cell proliferation within the AER. Moreover, we performed lgr5 gain-of-function experiments through in ovo electroporation and studied the relationship of lgr5 and lgr6 with different signaling pathways known to be involved in the AER induction and maintenance. The phenotypes obtained point to the involvement of lgr5 in the maintenance of a proliferative niche in the AER. We also present here, evidences showing that lgr6 is controlled by WNT signaling. Our results support a model in which lgr5 and lgr6 control the activation and maintenance of a niche of undifferentiated cells that are in continuous proliferation at the base of AER. This niche can be responsible for the renewal of AER until it disappears by massive programmed cell death
