Publicação
Investigating cell fate decisions of the intestinal stem cell
| Resumo: | The development of multicellular organisms, as well as the maintenance of adult tissue homeostasis, requires the production of new cell types that will build up the new organism and maintain it throughout its entire life. Cell proliferation, differentiation and cell death have to be carefully orchestrated during development, requiring cell-cell communication. This is carried out by a small number of cell signaling pathways that are reiteratively employed both during development and during tissue homeostasis in adults. One of these pathways, which plays many critical roles, both during development and in adult stem cells, is the Notch signaling pathway. During my PhD, I become interested in understanding how different cell types can be specified during development, and how do cells ensure that this occurs properly. During the first part of my thesis, I investigated one mechanism of cell type specification – asymmetric cell division. During the second part of my PhD, I studied the maintenance of tissue homeostasis by the adult stem cells of the Drosophila ntestine. During development, multipotent cells divide and give rise to cells with more restricted fates, in the process of building differentiated adult tissues. One mechanism employed to specify different cell fates is asymmetric cell division: during mitosis, cell fate determinants are asymmetrically localized to one of the future daughter cells, and will determine the fate of that daughter cell (Knoblich, 2001). A good example of this occurs during the development of the sensory organs in Drosophila, where precursor cells, the sensory organ precursors (SOPs), divide asymmetrically to produce two distinct cells, a pIIa cell and a pIIb cell. These will again divide asymmetrically to give rise to the four different cell types of the sensory organs. In this process, the Par6-aPKC complex localizes at the posterior cortex of the SOPs and promotes the actin-dependent localization of the cell fate determinants Numb, Partner of Numb (Pon) and Neuralized to the opposite anterior pole. Both Numb and Neuralized regulate Notch signaling, promoting its activation only in the posterior cell, which will become the pIIa cell (Bardin et al., 2004). The plasma membrane lipid phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the plasma vi membrane localization and activity of various proteins, including several actin regulators, thereby modulating actin-based processes (Di Paolo and De Camilli, 2006). I examined the distribution of PIP2 and of the PIP2-producing kinase Skittles (Sktl) in mitotic SOPs, and found that both Sktl and PIP2 reporters were uniformly distributed in dividing SOPs. However, in the course of this study, I unexpectedly observed that overexpression of full-length Pon or its localization domain (LD) fused to the Red Fluorescent Protein (RFP:PonLD) resulted in asymmetric distribution of Sktl and PIP2 reporters in dividing SOPs. This finding that Pon overexpression alters polar protein distribution is important because RFP::PonLD is often used as a polarity marker of dividing progenitors (Perdigoto et al., 2008). However, since Sktl and PIP2 do not play any detectable role in the asymmetric localization of the cell fate determinants, I decided to not further pursue this project. The second part of my PhD focused on how adult stem cells maintain tissue homeostasis. The maintenance of adult tissue homeostasis during the whole life span of the organism (which in some vertebrates can be more 200 years) requires that stem cells divide to replace the differentiated cell types of the organ, as well as to maintain the stem cell pool. In order to do so, adult stem cells utilize many of the same signaling pathways that are employed during embryonic development to regulate cell growth, proliferation, differentiation, death and morphogenesis. The adult Drosophila Intestinal Stem Cells (ISC) divide throughout the lifetime of the adult fly to replenish gut tissue by producing two differentiated cell types. The ISC division is thought to be asymmetric concerning the fate of the two daughter cells: one remains an ISC and the other becomes a progenitor cell, termed the enteroblast (EB). The ISC is the only dividing cell in the adult gut, and the EBs go on to differentiate directly, either into an enterocyte or an enteroendocrine cell. Enterocytes are polyploid absorptive epithelial cells, while the enteroendocrine cells are diploid cells that express peptide hormones. Lineage labeling analysis demonstrated that ISCs are multipotent stem cells (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006) and Notch signaling has been shown to play a role in regulating cell fate decision of the ISC lineage. The Notch ligand Delta is expressed in the ISCs and Notch signaling is activated in the EB to promote EB, enteroendocrine and vii enterocyte fates (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006; Ohlstein and Spradling, 2007). In my work, I have shown that the transcriptional repression of Notch target genes is required to maintain the ISC identity (Bardin et al., 2010). However, how Notch signaling is modulated to control the acquisition of the EB fate and two distinct differentiated cell fates is not understood. It has been proposed that the specification of the differentiated fates could be related to the level of Notch signaling (Ohlstein and Spradling, 2007), a mechanism that has also been proposed to be employed by other stem cell lineages (Mazzone et al., 2010). Given the role of Notch signaling in many stem cell fate decisions, it is important to understand how it can promote alternative fates in a single precursor cell. To answer this question and to identify novel regulators of the cell fate decisions in the ISC lineage, we carried out a chemical mutagenesis screen in Drosophila. During this screen, random mutations were induced and mitotic clones, in the F2 generation, were screened for mutants in which intestinal cell type specification was affected. Several complementation groups with defects in cell type specification were identified, which can be divided into three groups: mutants in which the stem cell is lost, mutants with overproduction of enteroendocrine like cells and mutants with excess of stem cell-like cells. I characterized briefly and mapped some of these mutations. I further investigated the role of one of the genes identified in this screen. One complementation group was found to specifically affect the self-renewing of ISCs without affecting the production of differentiated cells. I mapped this complementation group to the Gmd (GDP-mannose 4,6-dehydratase) gene. Clones of Gmd mutant cells have an overproduction of Delta positive, proliferative, multipotent stem cell-like cells in the intestine. However, unlike the loss of Notch signaling, loss of Gmd does not affect differentiation, as shown by the finding that enterocytes and enteroendocrine cells are normally specified in Gmd mutant clones. This suggests that in Gmd mutants ISCs, the ISC can divide asymmetrically, in relation to the fate of the daughter cells, to self-renew and produce EBs, or symmetrically to give rise to two ISCs. Gmd is required for the production of fucose and has been shown to be required for the O-fucosyltransferase 1 (Ofut1)-dependent fucosylation of the Notch viii receptor (Okajima and Irvine, 2002; Okajima et al., 2003; Sasamura et al., 2003). It is thought that O-fucosylated Notch protein serves only as a subsequent substrate for further glycosylation by the glycosyltransferase Fringe (Fng), since the loss of fucosylation results in defects only in developmental contexts that require Fngdependent modification of Notch (Okajima et al., 2008; Okajima et al., 2005). Interestingly, I found that Gmd plays a fringe-independent, but Ofut1-dependent, role to limit symmetric ISC divisions. Furthermore, the Gmd phenotype can be suppressed by the expression of active nuclear Notch. In addition, I found that Notch signaling could still be activated in Gmd mutant clones, but that precise asymmetric fate decisions in the daughter cells do not occur. Studies in mammalian cells found that O-fucosylated Notch receptor is more strongly activated by its ligands (Chen et al., 2001; Moloney et al., 2000; Stahl et al., 2008). My data are consistent with Gmd being required for high levels of Notch activation in the ISC lineage, which is necessary to limit symmetric division of the ISC. To test how the levels of Notch signaling affect the ISC lineage, I analyzed the temperature-dependent effect of rumi mutations in the intestine. Rumi is a component of the Notch signaling pathway but the requirement for Rumi is temperature-dependent: at 18ºC rumi mutants have a very mild loss of Notch signaling phenotype while at 28ºC they have a stronger phenotype (Acar et al., 2008). I found that rumi mutant clones in the intestine are wild-type at 18ºC but have a stronger phenotype at 28ºC. At 21ºC and 22ºC, rumi mutant clones have an overabundance of ISCs but differentiation is essentially unaffected, similar to Gmd mutants. This data support a model in which different cell fate decisions require different levels of Notch signaling: while the asymmetric decision between ISC and EB requires high levels of Notch signaling, further differentiation of the EB into enterocyte or enteroendocrine cells can occur with lower levels of Notch activity. Such requirement of high levels of Notch signaling for daughter cell commitment, that is, exit from self-renewal, could be a mechanism to prevent loss of the ISC. I propose that this could be a general mechanism utilized by stem cells to ensure their maintenance, in which high threshold signaling prevents noisy, low levels of signaling that could lead to the loss of the stem cell. |
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| Autores principais: | Perdigoto, Carolina Noiva Leiras Rodrigues, 1982- |
| Assunto: | Células estaminais Células estaminais adultas Intestinos Divisão celular Endocitose Glicosilação Apoptose Ciclo celular Drosophila Teses de doutoramento - 2011 Receptores notch |
| Ano: | 2011 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | The development of multicellular organisms, as well as the maintenance of adult tissue homeostasis, requires the production of new cell types that will build up the new organism and maintain it throughout its entire life. Cell proliferation, differentiation and cell death have to be carefully orchestrated during development, requiring cell-cell communication. This is carried out by a small number of cell signaling pathways that are reiteratively employed both during development and during tissue homeostasis in adults. One of these pathways, which plays many critical roles, both during development and in adult stem cells, is the Notch signaling pathway. During my PhD, I become interested in understanding how different cell types can be specified during development, and how do cells ensure that this occurs properly. During the first part of my thesis, I investigated one mechanism of cell type specification – asymmetric cell division. During the second part of my PhD, I studied the maintenance of tissue homeostasis by the adult stem cells of the Drosophila ntestine. During development, multipotent cells divide and give rise to cells with more restricted fates, in the process of building differentiated adult tissues. One mechanism employed to specify different cell fates is asymmetric cell division: during mitosis, cell fate determinants are asymmetrically localized to one of the future daughter cells, and will determine the fate of that daughter cell (Knoblich, 2001). A good example of this occurs during the development of the sensory organs in Drosophila, where precursor cells, the sensory organ precursors (SOPs), divide asymmetrically to produce two distinct cells, a pIIa cell and a pIIb cell. These will again divide asymmetrically to give rise to the four different cell types of the sensory organs. In this process, the Par6-aPKC complex localizes at the posterior cortex of the SOPs and promotes the actin-dependent localization of the cell fate determinants Numb, Partner of Numb (Pon) and Neuralized to the opposite anterior pole. Both Numb and Neuralized regulate Notch signaling, promoting its activation only in the posterior cell, which will become the pIIa cell (Bardin et al., 2004). The plasma membrane lipid phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the plasma vi membrane localization and activity of various proteins, including several actin regulators, thereby modulating actin-based processes (Di Paolo and De Camilli, 2006). I examined the distribution of PIP2 and of the PIP2-producing kinase Skittles (Sktl) in mitotic SOPs, and found that both Sktl and PIP2 reporters were uniformly distributed in dividing SOPs. However, in the course of this study, I unexpectedly observed that overexpression of full-length Pon or its localization domain (LD) fused to the Red Fluorescent Protein (RFP:PonLD) resulted in asymmetric distribution of Sktl and PIP2 reporters in dividing SOPs. This finding that Pon overexpression alters polar protein distribution is important because RFP::PonLD is often used as a polarity marker of dividing progenitors (Perdigoto et al., 2008). However, since Sktl and PIP2 do not play any detectable role in the asymmetric localization of the cell fate determinants, I decided to not further pursue this project. The second part of my PhD focused on how adult stem cells maintain tissue homeostasis. The maintenance of adult tissue homeostasis during the whole life span of the organism (which in some vertebrates can be more 200 years) requires that stem cells divide to replace the differentiated cell types of the organ, as well as to maintain the stem cell pool. In order to do so, adult stem cells utilize many of the same signaling pathways that are employed during embryonic development to regulate cell growth, proliferation, differentiation, death and morphogenesis. The adult Drosophila Intestinal Stem Cells (ISC) divide throughout the lifetime of the adult fly to replenish gut tissue by producing two differentiated cell types. The ISC division is thought to be asymmetric concerning the fate of the two daughter cells: one remains an ISC and the other becomes a progenitor cell, termed the enteroblast (EB). The ISC is the only dividing cell in the adult gut, and the EBs go on to differentiate directly, either into an enterocyte or an enteroendocrine cell. Enterocytes are polyploid absorptive epithelial cells, while the enteroendocrine cells are diploid cells that express peptide hormones. Lineage labeling analysis demonstrated that ISCs are multipotent stem cells (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006) and Notch signaling has been shown to play a role in regulating cell fate decision of the ISC lineage. The Notch ligand Delta is expressed in the ISCs and Notch signaling is activated in the EB to promote EB, enteroendocrine and vii enterocyte fates (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006; Ohlstein and Spradling, 2007). In my work, I have shown that the transcriptional repression of Notch target genes is required to maintain the ISC identity (Bardin et al., 2010). However, how Notch signaling is modulated to control the acquisition of the EB fate and two distinct differentiated cell fates is not understood. It has been proposed that the specification of the differentiated fates could be related to the level of Notch signaling (Ohlstein and Spradling, 2007), a mechanism that has also been proposed to be employed by other stem cell lineages (Mazzone et al., 2010). Given the role of Notch signaling in many stem cell fate decisions, it is important to understand how it can promote alternative fates in a single precursor cell. To answer this question and to identify novel regulators of the cell fate decisions in the ISC lineage, we carried out a chemical mutagenesis screen in Drosophila. During this screen, random mutations were induced and mitotic clones, in the F2 generation, were screened for mutants in which intestinal cell type specification was affected. Several complementation groups with defects in cell type specification were identified, which can be divided into three groups: mutants in which the stem cell is lost, mutants with overproduction of enteroendocrine like cells and mutants with excess of stem cell-like cells. I characterized briefly and mapped some of these mutations. I further investigated the role of one of the genes identified in this screen. One complementation group was found to specifically affect the self-renewing of ISCs without affecting the production of differentiated cells. I mapped this complementation group to the Gmd (GDP-mannose 4,6-dehydratase) gene. Clones of Gmd mutant cells have an overproduction of Delta positive, proliferative, multipotent stem cell-like cells in the intestine. However, unlike the loss of Notch signaling, loss of Gmd does not affect differentiation, as shown by the finding that enterocytes and enteroendocrine cells are normally specified in Gmd mutant clones. This suggests that in Gmd mutants ISCs, the ISC can divide asymmetrically, in relation to the fate of the daughter cells, to self-renew and produce EBs, or symmetrically to give rise to two ISCs. Gmd is required for the production of fucose and has been shown to be required for the O-fucosyltransferase 1 (Ofut1)-dependent fucosylation of the Notch viii receptor (Okajima and Irvine, 2002; Okajima et al., 2003; Sasamura et al., 2003). It is thought that O-fucosylated Notch protein serves only as a subsequent substrate for further glycosylation by the glycosyltransferase Fringe (Fng), since the loss of fucosylation results in defects only in developmental contexts that require Fngdependent modification of Notch (Okajima et al., 2008; Okajima et al., 2005). Interestingly, I found that Gmd plays a fringe-independent, but Ofut1-dependent, role to limit symmetric ISC divisions. Furthermore, the Gmd phenotype can be suppressed by the expression of active nuclear Notch. In addition, I found that Notch signaling could still be activated in Gmd mutant clones, but that precise asymmetric fate decisions in the daughter cells do not occur. Studies in mammalian cells found that O-fucosylated Notch receptor is more strongly activated by its ligands (Chen et al., 2001; Moloney et al., 2000; Stahl et al., 2008). My data are consistent with Gmd being required for high levels of Notch activation in the ISC lineage, which is necessary to limit symmetric division of the ISC. To test how the levels of Notch signaling affect the ISC lineage, I analyzed the temperature-dependent effect of rumi mutations in the intestine. Rumi is a component of the Notch signaling pathway but the requirement for Rumi is temperature-dependent: at 18ºC rumi mutants have a very mild loss of Notch signaling phenotype while at 28ºC they have a stronger phenotype (Acar et al., 2008). I found that rumi mutant clones in the intestine are wild-type at 18ºC but have a stronger phenotype at 28ºC. At 21ºC and 22ºC, rumi mutant clones have an overabundance of ISCs but differentiation is essentially unaffected, similar to Gmd mutants. This data support a model in which different cell fate decisions require different levels of Notch signaling: while the asymmetric decision between ISC and EB requires high levels of Notch signaling, further differentiation of the EB into enterocyte or enteroendocrine cells can occur with lower levels of Notch activity. Such requirement of high levels of Notch signaling for daughter cell commitment, that is, exit from self-renewal, could be a mechanism to prevent loss of the ISC. I propose that this could be a general mechanism utilized by stem cells to ensure their maintenance, in which high threshold signaling prevents noisy, low levels of signaling that could lead to the loss of the stem cell. |
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