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Lysosomal signaling is required for embryonic stem cell differentiation and human development

Villegas, Florian. Lysosomal signaling is required for embryonic stem cell differentiation and human development. 2017, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_12635

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Abstract

Self-renewal and differentiation are the two key properties that characterize stem cells. To initiate differentiation, pluripotent embryonic stem cells (ESC) must be forced out of self-renewal, the transcriptional networks conferring stem cell identity need to be dissolved and lineages must be chosen. Compared to acquisition and maintenance of ESCs pluripotency, the mechanisms driving exit from this cell state are ill defined. Previous work has identified the tumor suppressor Folliculin (Flcn) as a novel gene required for exit from the ESC state during differentiation (Betschinger et al., 2013a). Flcn loss of function in mice is early embryonic lethal consistent with being required for exit from pluripotency in vivo. It has been shown in ESCs that Flcn and two associated proteins, Fnip1 and Fnip2, regulate the subcellular localization of the basic helix-loop-helix transcription factor Tfe3. In wildtype ESCs, Tfe3 is ubiquitously detected in the nucleus and cytoplasm. Upon ESC differentiation, Tfe3 is excluded from the nucleus whereas Flcn loss of function ESCs harbor constitutive nuclear Tfe3 localization and are, consequentially, impaired in differentiation. Consistently, ectopic nuclear Tfe3 similarly inhibits ESC differentiation. Recently, Flcn has been identified in a metabolic pathway leading to the activation of the mTORC1 complex in an amino-acid sensing dependent manner after nutrient starvation (Tsun et al., 2013). However, mTORC1 activity is not required for ESC differentiation.
To gain insights into the regulation of this process, we conducted a genome-wide CRISPR/Cas9 screen in ESCs that aimed to identify genes that, similar to Flcn, are required for the maintenance of stem cell identity upon long term exposure to differentiation stimuli. Although molecularly related to amino-acid sensing dependent mTORC1 regulation, our findings suggest an alternate mode of action that may be founded in the non-starving nature of ESC differentiation. In addition, we show in ESC that the lysosome regulates the assembly of a Flcn-Tfe3 axis, whose impairment induces an embryonic developmental arrest.
Additionally, in a collaborative work with human geneticists, we identified five Tfe3 point mutations in a human syndrome called hypomelanosis of Ito. Symptoms associated with this developmental disorder include severe intellectual disability, coarse facial features, frontonasal dysplasia, obesity, epilepsy and growth retardation, suggesting a pleiotropic developmental disorder. To understand the developmental nature of the disease, we tested these mutations in ESCs and we found them causing defects in exit from self-renewal and nuclear Tfe3 accumulation.
Taken together, the work presented in this thesis has allowed the identification of the lysosome as a critical organelle required for the control of the exit from pluripotency and indicates that lysosomal recruitment of Tfe3 is required for its inactivation, which is a crucial step to enable normal developmental progression.
Advisors:Gasser, Susan M. and Betschinger, Joerg and Teis, David
Faculties and Departments:05 Faculty of Science
09 Associated Institutions > Friedrich Miescher Institut FMI > Quantitative Biology > Nuclear organization in development and genome stability (Gasser)
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:12635
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:1 Online-Ressource (iv, 103 Seiten)
Language:English
Identification Number:
Last Modified:25 Jun 2018 14:03
Deposited On:25 Jun 2018 14:02

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