Publicação
Monitoring the activity of the Notch pathway in neural progenitor cells
| Resumo: | Neural progenitor (NP) cells proliferate in the ventricular zone (VZ) of the neural tube and migrate towards the mantle layer (ML) upon neural differentiation. In order to produce the correct type and number of neurons at the right time, a precise control of the proliferation of neural progenitor (NP) cells and their differentiation is required. The Notch signaling pathway, through lateral inhibition, is involved in this balance, restraining NP differentiation. However, not much is known about Notch activity in single NPs, mainly regarding possible variations on Notch activity in each NP, and how this putative dynamic activity contributes to the maintenance of these cells in the undifferentiated state. In order to answer these questions, I used several ES cell lines expressing different reporters of Notch activity driven by the promoter of the Hes5 gene. These ES cells can be directed to neural differentiation in adherent monolayer cultures, resulting in the production of neuroepithelial rosettes that mimic their in vivo counterpart, the neural tube. Here, I show that an already described Hes5::GFP reporter ES cell line is not suitable to be used as a reporter of Notch activity since the half-life of the reporter protein is much longer than that of the HES5 protein, not allowing the detection of the termination of Notch activity. I also test other ES cell lines, Hes5::VNP, that express an unstable reporter protein, which might allow a more precise and accurate monitoring of Notch activity dynamics. Using this cell lines, I could observe that not all cells in neuroepithelial rosettes express the reporter protein, and that the levels of Notch activity are variable between NPs. Further engineering of these cell lines needs to be performed in order to be able to construct a double reporter cell line carrying a reporter of Notch activity together with a reporter of neuronal differentiation to allow visualization of differentiated neurons. |
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| Autores principais: | Guedes, Ana Marisa Mendes Gonçalves Vinhais, 1987- |
| Assunto: | Biologia molecular Células estaminais Tubo neural Neurogénese Proteínas Teses de mestrado - 2011 |
| Ano: | 2011 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Neural progenitor (NP) cells proliferate in the ventricular zone (VZ) of the neural tube and migrate towards the mantle layer (ML) upon neural differentiation. In order to produce the correct type and number of neurons at the right time, a precise control of the proliferation of neural progenitor (NP) cells and their differentiation is required. The Notch signaling pathway, through lateral inhibition, is involved in this balance, restraining NP differentiation. However, not much is known about Notch activity in single NPs, mainly regarding possible variations on Notch activity in each NP, and how this putative dynamic activity contributes to the maintenance of these cells in the undifferentiated state. In order to answer these questions, I used several ES cell lines expressing different reporters of Notch activity driven by the promoter of the Hes5 gene. These ES cells can be directed to neural differentiation in adherent monolayer cultures, resulting in the production of neuroepithelial rosettes that mimic their in vivo counterpart, the neural tube. Here, I show that an already described Hes5::GFP reporter ES cell line is not suitable to be used as a reporter of Notch activity since the half-life of the reporter protein is much longer than that of the HES5 protein, not allowing the detection of the termination of Notch activity. I also test other ES cell lines, Hes5::VNP, that express an unstable reporter protein, which might allow a more precise and accurate monitoring of Notch activity dynamics. Using this cell lines, I could observe that not all cells in neuroepithelial rosettes express the reporter protein, and that the levels of Notch activity are variable between NPs. Further engineering of these cell lines needs to be performed in order to be able to construct a double reporter cell line carrying a reporter of Notch activity together with a reporter of neuronal differentiation to allow visualization of differentiated neurons. |
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