Erschienen:
[Erscheinungsort nicht ermittelbar]: [Verlag nicht ermittelbar], 2010
Sprache:
Englisch
Identifikator:
Entstehung:
Hochschulschrift:
Dissertation, 2010
Anmerkungen:
Beschreibung:
Tese de doutoramento em Biologia (Biologia do Desenvolvimento), apresentada à Universidade de Lisboa através da Faculdade de Ciências, 2009 ; The mesoderm of the amphibian embryo forms from the equatorial region of the late blastula in response to signals derived from the vegetal hemisphere of the embryo. These signals include members of the transforming growth factor type ß (TGFß) family, including Vg1, Activin, the nodal-related proteins Xnr1, 2, 4, 5 and 6, and derrière. Two significant characteristics of these signals are that they can act at long range and that they behave as morphogens, eliciting different responses at different concentrations. These properties raise the question of how such molecules exert their long-range effects: what route do they take to traverse a field of responding cells? As a representative of mesoderm inducing TGFß molecules, we have used mainly Activin for our experiments addressing questions about morphogen progression and signalling. Activin is a potent inducer of mesoderm and its patterning. We developed several different assays involving a fluorescently labelled form of the protein to visualize morphogen progression in tissue. In vivo detection of TGFß induced complex formation of signal transducers such as Smad2 and Smad4 by bimolecular fluorescent complementation (BiFC) served as a direct read-out of morphogen function. The combination of both imaging techniques allowed us to observe morphogen passage and direct response in living cells. Our experiments suggest that in contrast to other morphogens, Activin does not pass through neighbouring cells by transcytosis, but appears to travel exclusively through the extracellular space. The number of cognate receptors presented on cell surfaces is crucial for the range of progression and signalling as it blocks ligand passage if presented in excess. Inhibition of endocytosis successfully blocks cellular uptake of Activin, but does not interfere with either signalling capacity or range. Furthermore, during our studies on Smad complex formation we made several important observations. Most interestingly, we found that the nuclear import of the Smad complex is developmentally regulated such that nuclear accumulation in response to a TGF stimulus does not occur before the start of zygotic transcription, indicating the presence of regulatory controls that impose temporal restrictions upon the nuclear import of Smad signalling complexes. Future elucidation of the molecular nature of these import controls may be particularly relevant to our understanding of the mid-blastula transition, as the onset of zygotic gene regulation may require the import of transcriptional regulators. ; Fundação para a Ciência e Tecnologia (FCT), (SRFH/BD/11807/2003)