The role of Patched1 during development of the mouse cerebellum and regulation of neural stem cells in medulloblastoma

Jonathan Robson (2010). The role of Patched1 during development of the mouse cerebellum and regulation of neural stem cells in medulloblastoma PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

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Author Jonathan Robson
Thesis Title The role of Patched1 during development of the mouse cerebellum and regulation of neural stem cells in medulloblastoma
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2010-04
Thesis type PhD Thesis
Supervisor Professor Brandon Wainwright
Professor Brent Reynolds
Dr Tammy Ellis
Total pages 241
Total colour pages 62
Total black and white pages 179
Subjects 06 Biological Sciences
Abstract/Summary The Sonic Hedgehog (Shh) signalling pathway plays a critical role in the development of many organs including the brain, lungs and skin. Of particular interest to this project, aberrant activity of the Shh pathway has been shown to be involved in the development of the paediatric cerebellar tumour medulloblastoma, typically through loss of function of the Shh receptor Patched 1 (Ptc1). Binding of Shh to Ptc1, or ablation of Ptc1 function, results in Smoothened (Smo) activation resulting in the activation of a number of downstream transcription factors responsible for cell proliferation and migration. In addition to its role in cerebellar development the Shh/Ptc1 pathway has also been associated with neural stem cell regulation although direct assessment of this relationship is difficult given the lack of in vivo neural stem cell markers. The aim of this project was to quantitatively assess the effect increased Shh/Ptc1 pathway activity has on neural stem cells via in vitro stem and progenitor cell assays, and determine if neural stem cells are the cell of origin of medulloblastoma. In order to determine how the Shh/Ptc1 pathway regulates neural stem cell fates Ptc1 function was ablated in the neocortex and subventricular zones of the developing mouse embryo through the use of an inducible Nestin Cre recombinase/Ptc1 conditional transgenic mouse line. Using in vitro neural stem cell assays it was identified that constitutive activation of the Shh/Ptc1 pathway in the neocortex and subventricular zone of the developing brain resulted in increased stem and progenitor cell numbers in vivo. Concomitant with an increase in Gli1 expression in the neocortex, an increase in the rate of symmetric neural stem cell division was observed. While sufficient to increase stem cell and progenitor cell numbers, increased activity of the Shh/Ptc1 pathway was not sufficient to rescue the loss of stem cells observed in neocortices devoid of Notch1 signalling. Although studies are beginning to elucidate the role of the Shh/Ptc1 signalling pathway in medulloblastoma formation little is known about the primary cell type responsible for tumour initiation. The observation that Shh/Ptc1 constitutive activation increased both stem and progenitor cell numbers and increased the rate of cell division prompted us to investigate the possibility that Ptc1 ablated neural stem cells may be a cellular origin of medulloblastoma, whereby the Shh/Ptc1 pathway maintains neural cells in a stem like fate. To identify the relationship between neural stem cells and medulloblastoma formation Ptc1 function was ablated in the developing cerebellum under the control of GFAP Cre recombinase which resulted in constitutive activation of the Shh/Ptc1 pathway in the cerebellar stem cell compartment, leading to medulloblastoma. It was shown that homozygous loss of Ptc1 in the cerebellar ventricular zone and developing external granule layer resulted in increased cell proliferation concomitant with increased Gli1 expression. Neural stem-like cells were isolated from the adult Ptc1 deleted medulloblastoma but not from equivalently aged wild type mice. Unlike primary Ptc1 deleted cells, isolated neurospheres from Ptc1 deleted medulloblastomas failed to recapitulate tumour formation in vivo. In contrast to primary Ptc1 deleted cells, Ptc1 deleted neurospheres showed significantly decreased Shh/Ptc1 pathway expression concomitant with a loss of primary cilia in vitro. To further investigate the possibility of a cancer stem cell as a cell of origin of medulloblastoma the primary Ptc1 deleted cell population was assayed for cancer stem cell markers through Fluorescent Activated Cell Sorting (FACS) of a panel of CD antibodies. This project identified 5 antibodies, CD38, CD44, CD90.2, CD117 and CD121b to have lower binding detection in Ptc1 deleted medulloblastoma compared to wild type cerebella and 3 antibodies, CD1d, CD24 and CD81 which have greater binding detection in Ptc1 deleted medulloblastomas compared to wild type cerebella. Of these, CD24 positive cells were identified to have increased tumour forming potential compared to CD24 negative cells identifying a possible medulloblastoma initiation marker in Ptc1 deleted stem/progenitor cells. Co-localised with CD15, a recently hypothesised neural stem cell marker, the CD24 positive population was found to be split into two distinct cell populations which led to the hypothesis that CD24/CD15 co-expression may identify the tumour forming cell population in medulloblastoma. Collectively these data suggest that medulloblastoma resulting from aberrant Shh/Ptc1 signalling may be due to a persistent stem-like cell whose tumorigenic potential depends on the presence of primary cilia and may be detected in vivo through specific expression profiles of cell surface markers.
Keyword medulloblastoma
brain tumour
primary cilium
stem cell
Additional Notes Coloured pages: 27, 30, 35, 37, 39, 40, 43, 46, 49, 52, 56, 63, 64, 65, 67, 68, 71, 72, 78, 79, 80, 81, 86, 89, 93, 98, 100, 104, 106, 109, 111, 118, 122, 124, 126, 128, 131, 133, 136, 138, 141, 145, 147, 149, 151, 154, 160, 162, 164, 165, 166, 167, 170, 172, 174, 176, 178, 181, 183, 194, 204, 206.

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Created: Fri, 24 Dec 2010, 12:34:41 EST by Jonathan Robson on behalf of Library - Information Access Service