- Title
- Role of FAT1 cadherin in neuronal differentiation
- Creator
- Ahmed, Abdulrzag Faraj
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2015
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Fat cadherins comprise the largest of all known members of cadherin superfamily. They are present in all multicellular organisms and retain a high degree of structural conservation. In Drosophila there are two Fat genes: Fat and Fat-like, whilst in vertebrates there are four members called Fat1, Fat2, Fat3 and Fat4. In Drosophila, the archetype Fat (Ft) cadherin is upstream of a discrete branch Hippo signalling where it also functionally intersects with planar cell polarity (PCP), the process which organizes cells within the plane of an epithelial sheet. A second Drosophila Fat gene, Ft2, is only involved in PCP. Evolutionary divergence has seen expansion to four vertebrate members (Fat1, Fat2, Fat3 and Fat4) with most literature addressing their roles in PCP. In contrast to Drosophila, connections between the vertebrate Fat cadherins and Hippo signalling are not completely established nor fully explored. Neurogenesis describes the cellular processes required for the development and maintenance of the central nervous system. Here the Hippo pathway is emerging as a critical nexus that balances self-renewal of neural progenitors against differentiation. However, while neurogenesis in Drosophila involves Fat-Hippo signalling, the upstream elements in vertebrate Hippo signalling are poorly understood. Prominent expression of Fat1 cadherin is evident within the developing vertebrate neuroepithelium and the manifestation of severe neurological phenotypes in Fat1-knockout mice suggests Fat1 may play a critical role in differentiation. Based on these findings, my overarching hypothesis is that the primary function of Fat1 cadherin in the nervous system is to drive neuronal differentiation. This fundamentally occurs through inhibiting self-renewal of neuronal stem cells and/or promoting neuronal differentiation. This function may potentially be driven through one or more cell signalling pathways known to be involved in neurogenesis. The emerging role of the Hippo signalling pathway in stem cell compartments is of particular interest to this notion, along with overlapping effects of Hippo on other major signalling pathways, particularly Shh, BMP and TGF-β pathways. To explore this hypothesis, Chapter 3 investigated the possible roles of FAT1 during neuronal differentiation of SH-SY5Y cells in vitro. These results showed that FAT1 but not other FAT cadherins was induced by neuronal differentiation of SH-SY5Y cells. Using gene-silencing techniques employing shRNA it was then established that FAT1 depletion reduced both neurite initiation and elongation. Moreover, FAT1 knockdown cells displayed comparatively higher rates of proliferation and survival at higher densities. Changes in proliferation were confirmed by altered levels of cell cycle regulators. This indicated that FAT1 was involved in the neuronal differentiation process where it is required for neuritogenesis as well as inhibiting proliferation in a density-dependent manner. Chapter 4 then investigated the possible signalling pathways that are governed by FAT1 during neuronal differentiation. As cell density effects are the hallmark of the Hippo pathway, the involvement of FAT1 engaging Hippo signalling during SH-SY5Y differentiation was examined. Using shRNA-mediated depletion of FAT1 it was inferred that FAT1 served to activate core Hippo kinase components and affected the activities of the Hippo effector TAZ. Suppression of FAT1 promoted the nucleocytoplasmic shuttling of TAZ leading to enhanced transcription of the Hippo target genes ANKRD1 and CTGF. Further investigations indicated that FAT1-signalling did not involve Shh or BMP signalling pathways, however there was crosstalk shown between FAT1-Hippo and elements of the TGF-β pathway. The increase in nuclear TAZ was accompanied with increased nuclear levels of the TGF-β effector Smad3. While FAT1 expression did not influence the levels of TAZ itself, inhibiting FAT1 expression did increase cellular levels of Smad3. Silencing of TAZ reversed the effects of FAT1 depletion thus connecting inactivation of TAZ/TGF-β signalling with Hippo signalling mediated through FAT1. Chapter 5 then sought to substantiate the results obtained with SH-SY5Y cells in an alternate in vitro model of neuronal differentiation involving NTera2 cells. The results obtained were entirely in concordance with findings of the SH-SY5Y model. Of all four FAT cadherins, FAT1 was selectively induced. Moreover, FAT1 depletion using siRNA inhibited the initiation of neurites and increased transcription of the Hippo target genes ANKRD1 and CTGF. Therefore these data support the conclusion that FAT1 is involved in neuritogenesis and affects Hippo signalling during neuronal differentiation. As confirmation of these experimental findings, Chapter 6 then investigated the expression and regulation of the Fat cadherin in a number of different physiological models of neuronal differentiation. In silico analyses were undertaken against a number of publically available microarray datasets. These analyses involved differentiation studies of human stem cells in vitro together with in vitro and in vivo and mouse models. In all cases it was shown that Fat1 expression was increased during differentiation thereby validating the findings in the SH-SY5Y and NTera2 differentiation models. Collectively the results from this thesis establish that FAT1 regulates the neuronal differentiation process through governing two key aspects of neurogenesis; these being neuritogenesis and the suppression of cell proliferation. Moreover, establishing that FAT1 acts new upstream Hippo element, it is shown that FAT1 mediates these functions through the Hippo effector TAZ. Furthermore, this serves to inhibit the crosstalk between TAZ and TGF-β signalling during the early stages of neuronal differentiation.
- Subject
- fat cadherins; neuronal differentiation; Hippo pathway; TGF-beta pathway; TAZ; SH-SY5Y; NT-2 neurons; Fat1; neurite outgrowth
- Identifier
- http://hdl.handle.net/1959.13/1305645
- Identifier
- uon:21081
- Rights
- Copyright 2015 Abdulrzag Faraj Ahmed
- Language
- eng
- Full Text
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| View Details Download | ATTACHMENT02 | Thesis | 6 MB | Adobe Acrobat PDF | View Details Download |