Notch Signaling in Pigment Cells and Generation of Mouse Models for Ocular Diseases

Bhushan Sarode
EPFL, 2013
Thèse EPFL

Résumé

Cellular signaling mediated by Notch receptors results in coordinated regulation of cell growth, survival and differentiation. In melanocytes, Notch signaling is essential for the maintenance of melanocytes and the melanocyte stem cell population. Whereas, in pigmented eye structures, specifically in ciliary body and iris, the role of Notch signaling in their structure and their development is largely unknown. In the first part, I have tried to identify possible Notch target genes in melanocytes which might indicate why the melanocyte stem cell population disappears in the absence of Notch. For that purpose, I carried out microarray analysis on Notch deficient melanoblasts and identified putative Notch targets. I have furthermore established mouse melanocyte cell lines from mice which could be used as a valuable tool to study melanocyte biology. In the second part, I have elucidated the functional significance of the Notch cascade in pigmented eye structures. The ciliary body and iris are pigmented epithelial structures in the anterior eye segment that function to maintain correct intraocular pressure and regulate exposure of the internal eye structures to light respectively. In this part I have analyzed the role of the Notch signaling by performing both loss- and gain-of-function experiments for Notch in the murine ocular pigmented epithelium using Mart-1::Cre mice. Loss of canonical Notch2 signaling results in normal iris development but ciliary body aplasia. In contrast, Notch gain-of-function induces the reciprocal phenotype and causes aniridia and ciliary body hyperplasia. The loss and gain-of-function models I have generated develop ocular hypotony and ocular hypertension respectively, mimicking the human diseases phthisis bulbi and closed angle glaucoma. Importantly, the ocular hypertension that occurs following Notch overexpression is responsive to hypotensive drugs, and thus can serve as a platform to evaluate novel hypotensive agents that may be used to treat closed angle glaucoma. In summary, the present study has identified novel putative Notch target genes which will provide us information to study the pigmentation and related disorder. The data obtained in the second chapter provide novel insight into the role of Notch signaling during development of the ciliary body and iris and thus represent a conceptual advance in our understanding of how pigmented structures of the eye are formed. In addition, the genetic models presented in this study represent robust tools with which to study the etiology, pathology and treatment of diseases related to aberrant intraocular pressure, such as phthisis bulbi and closed angle glaucoma.

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https://infoscience.epfl.ch/record/190833?ln=fr

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Bhushan Sarode
EPFL, 2013
Thèse EPFL

Résumé

Official source

https://infoscience.epfl.ch/record/190833?ln=fr

À propos de ce résultat

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Notch signaling in the pigmented epithelium of the anterior eye segment promotes ciliary body development at the expense of iris formation

Friedrich Beermann, Craig Nowell, Freddy Radtke, Bhushan Sarode

The ciliary body and iris are pigmented epithelial structures in the anterior eye segment that function to maintain correct intra-ocular pressure and regulate exposure of the internal eye structures to light, respectively. The cellular and molecular factors that mediate the development of the ciliary body and iris from the ocular pigmented epithelium remain to be fully elucidated. Here, we have investigated the role of Notch signaling during the development of the anterior pigmented epithelium by using genetic loss- and gain-of-function approaches. Loss of canonical Notch signaling results in normal iris development but absence of the ciliary body. This causes progressive hypotony and over time leads to phthisis bulbi, a condition characterized by shrinkage of the eye and loss of structure/function. Conversely, Notch gain-of-function results in aniridia and profound ciliary body hyperplasia, which causes ocular hypertension and glaucoma-like disease. Collectively, these data indicate that Notch signaling promotes ciliary body development at the expense of iris formation and reveals novel animal models of human ocular pathologies.

Wiley-Blackwell2014

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Notch signaling is a conserved cell-to-cell communication pathway essential in the development and maintenance of various tissues. Upon ligand binding, Notch receptors on the cell surface are proteolytically cleaved, generating the intracellular domain of Notch (NICD) which acts as a transcriptional regulator. Signaling through Notch1 is essential for normal T cell development, and de-regulated Notch1 signaling leads to the development of acute lymphoblastic T cell leukemia (T-ALL). Mouse models of retroviral overexpression of N1ICD in hematopoietic progenitors in bone-marrow (BM) chimeric mice clearly show that Notch1 in T-ALL acts as an oncogene. MicroRNAs (miRNAs) are small RNA molecules that function as post-transcriptional negative regulators of gene expression. They have been described to play either oncogenic or tumor suppressive roles in Notch1-driven T-ALL. However, it is currently unknown whether miRNAs are essential in Notch1-driven leukemogenesis or in T-ALL maintenance. Dicer1 is an essential enzyme for the generation of mature, functional miRNAs. In this study, the global requirement for miRNAs in T-ALL was assessed via conditional ablation of Dicer1 during early N1ICD-mediated leukemogenesis and in later stages of established T-ALL in vivo. Interestingly, both T-ALL development and maintenance in N1ICD overexpressing BM chimeras were dependent on Dicer1. This was found to be due to induction of apoptosis in Dicer1-deficient T-ALL cells. Microarray-based expression analysis demonstrated that potentially oncogenic miRNAs were up-regulated in both mouse and human T-ALL samples compared with normal T cell progenitors. Among the abundantly expressed miRNAs, candidates previously linked to T-ALL were detected as well as novel candidates. Among them, miR-21, which has previously been linked to tumor biology in a variety of solid tumors, was found to be the most differentially regulated in T-ALL and early and late T cell progenitors. It was also found to be abundantly expressed in primary mouse T-ALL specimens as well as human T-ALL cell lines and primary patient T-ALL samples. Using a novel lentivirus-based miRNA activity reporter system, I demonstrated that miR-21 is active in T-ALL cell lines both in vitro and in vivo. In addition, KD of miR-21 led to apoptosis in T-ALL cells with high miR-21 activity, consistent with miR-21 target gene de-repression. Taken together, these findings demonstrate for the first time that miR-21, in the context of Notch1-driven T-ALL, acts in an oncogenic fashion. It does so by facilitating T-ALL cell survival, which is, at least in part, due to repression of programmed cell-death protein 4 (Pdcd4), a miR-21 target gene and potential novel tumor suppressor in T-ALL. In summary, this study shows that in Notch1-driven T-ALL, miRNAs play an essential role by facilitating T-ALL cell survival, and that miR-21 is a novel oncogenic miRNA in this disease.

EPFL2014

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Notch1 and Notch2 receptors influence progressive hair graying in a dose-dependent manner

Friedrich Beermann, Freddy Radtke

The Notch signaling pathway is involved in diverse biological processes such as cell fate decisions or stem cell maintenance. In this study, we assessed the role of this pathway for melanocyte development and hair pigmentation using RBP-Jkappa, Notch1, and Notch2 conditional knockout mice. Disruption of the Notch pathway by inactivating RBP-Jkappa in the melanocyte lineage using Tyr::Cre mice led to a severe coat color dilution. Similarly, hair graying was observed when Notch1 and/or Notch2 receptors were ablated in melanocytes. This phenotype was proportional to the number of floxed Notch alleles, with the most pronounced effect seen in Tyr::Cre/ degrees ; Notch1(flox/flox); Notch2(flox/flox) mice. Deletion of Notch1 and/or Notch2 in melanoblasts did not induce a congenital defect. The number of Dct-expressing cells at embryonic stages was not affected, but melanocytes located within the hair matrix progressively disappeared during the first regeneration of the hair follicle. In contrast, non-follicular melanocytes and pigmentation in the dermis and in the choroid were not affected. We suggest that both Notch1 and Notch2 receptors contribute to the maintenance of melanoblasts and melanocyte stem cells, and are essential for proper hair pigmentation.

2007

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EPFL2014