The Role of Dicer in Cerebellar Development and Hedgehog-Mediated Medulloblastoma

Lena Constantin (2011). The Role of Dicer in Cerebellar Development and Hedgehog-Mediated Medulloblastoma PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

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Author Lena Constantin
Thesis Title The Role of Dicer in Cerebellar Development and Hedgehog-Mediated Medulloblastoma
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2011
Thesis type PhD Thesis
Supervisor Professor Brandon Wainwright
Dr Nicole Cloonan
Total pages 243
Total colour pages 29
Total black and white pages 214
Language eng
Subjects 060410 Neurogenetics
060403 Developmental Genetics (incl. Sex Determination)
060405 Gene Expression (incl. Microarray and other genome-wide approaches)
Abstract/Summary Background. MicroRNAs (miRNAs) are a class of short non-coding RNA molecules that guide the RNA-induced silencing protein (RISC) complex to downregulate transcripts with complementary miRNA-matching sequence in their 3’ untranslated region. Approximately 700 miRNAs are predicted in the mouse genome. MicroRNA precursors are cleaved into mature double-stranded RNA duplexes by the RNase III enzyme, Dicer, with the exception of miR-451. To date, multiple studies have implicated miRNAs as important regulators of cerebellar function. For example, miRNAs modulate the pathogenesis of neurodegenerative diseases such as spinocerebellar ataxia type 1, their loss in Purkinje cells leads to neuronal degeneration, and multiple studies have demonstrated that the global repression of miRNAs promotes cellular transformation and tumorigenesis in vivo. Malignancies influenced by miRNA function include the cerebellar tumor, medulloblastoma. Medulloblastoma is the most common pediatric solid brain tumor of the central nervous system. A subset of medulloblastoma arises from cerebellar granule cell precursors that continue to proliferate in response to Sonic Hedgehog (Shh) ligand. The Shh signaling cascade is mediated by the binding of Shh to the transmembrane protein, Patched (Ptch). This binding relieves the inhibitory influence of Ptch on the transmembrane protein, Smoothened (Smo), such that Smo can activate downstream transcription. Therefore, the removal of Ptch from the plasma membrane, or the binding of Ptch to Shh, activates the signaling cascade. At least three miRNAs have been identified that repress medulloblastoma cell proliferation by suppressing central components of the Shh pathway, such as Smo. Results. To address the role of miRNAs in cerebellar granule cell precursor development, exon 24 of Dicer1 was conditionally inactivated via the granule cell precursor-specific Math1-Cre-recombinase transgene using the Cre/loxP recombination system. The site-specific recombination of Dicer1 generated a nonsense transcript with a premature termination codon within exon 25. The loss of exon 24 of Dicer1 resulted in a cortical layering defect in the anterior zone of the cerebellum, due to an increase in granule cell precursor differentiation during postnatal development. The increase in the proportion of differentiating granule cell precursors led to a reduction in mature granule neurons in the anterior zone of the cerebellum only, and mice presented with a high-frequency tremor. The premature differentiation defect was rescued by the constitutive activation of Shh signaling, by inactivation of the Ptch1 ligand receptor, which presumably restored GLI-Kruppel family member (Gli) 2-mediated function. To determine the effect of miRNA loss in Shh-induced medulloblastoma, the Ptch1-conditional mouse model was used to constitutively activate Shh signaling in Dicer1-inactivated granule cell precursors by, again, using the Math1-Cre/loxP recombination system. Mice with granule cell precursor-specific Ptch1-inactivation developed tumors resembling human medulloblastoma with complete penetrance and were therefore used as a background model for tumor studies. Tumor-free survival analysis demonstrated that Dicer1-inactivation alone could not initiate tumorigenesis. However, Dicer1 in combination with Ptch1-inactivation was identified to function as a haploinsufficient tumor suppressor. The expression profile of Dicer1-inactivated tumors uncovered the dysregulation of Wnt and p38 Mapk signaling, and actin-mediated cytoskeletal remodeling. Furthermore, histological analyses showed that exon 24 Dicer1-inactivated tumors displayed increased invasion properties in vivo. Fifteen miRNAs were significantly downregulated in exon 24 Dicer1-inactivated tumors and are hypothesized to mediate some of the aforementioned phenotypes. Intriguingly, the vast majority of miRNAs were maintained at wild-type levels in samples with a high level of genomic Dicer1 inactivation, suggesting that loss of exon 24 of Dicer1 was functionally compensated for. A splice variant of Dicer1 (exon ∆25) was identified at very low levels in granule cell precursors and medulloblastoma. Interestingly, the exon 25 splice variant was significantly upregulated upon recombination of the loxP-flanked exon 24 of Dicer1, to generate a prominent in-frame mRNA product (exon ∆24 ∆25) that was missing the majority of the RNase IIIb domain in recombined cells. Conclusions. The loss of exon 24 and 25 of Dicer1 affected the processing of very few miRNAs. This was surprising considering that Dicer is indispensible for miRNA processing, with the exception of miR-451. The Cre recombinase-mediated deletion of exon 24 of Dicer1 upregulated the splicing of exon 25 to generate an in-frame Dicer1 exon ∆24 ∆25 splice variant. Here, it is hypothesized the Dicer exon ∆24 ∆25 splice variant is capable of processing miRNAs, possibly by forming an inter-molecular dimer to compensate for the loss of the critical RNase IIIb domain. The loss of exon 24 and/or exon 25 of Dicer1 generated reproducible and specific defects in the cerebellum, and likewise in medulloblastoma, indicating that Dicer and/or a subset of Dicer-dependent miRNAs execute important functions in cerebellar development by mediating Shh signaling through Gli2 expression, and in tumors, by decreasing latency and increasing tumor cell invasion.
Keyword cerebellar compartments
granule cell precursor
haploinsufficient tumor suppressor
nonsense-mediated altered splicing
sonic hedgehog pathway
Additional Notes 33, 34, 37, 38, 39, 40, 43, 46, 51, 59, 68, 106, 108, 109, 110, 111, 113, 114, 116, 118, 127, 129, 130, 132, 134, 135, 138, 139, 139

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Created: Thu, 28 Jun 2012, 12:15:39 EST by Ms Lena Constantin on behalf of Library - Information Access Service