P53-dependent Tumorigenesis And Gene Expression In Mammalian Brain

Astrocytic gliomas are the most common brain cancers of adults. The goal of this research was to examine the molecular genetic determinants of gliomas, with particular emphasis on the tumor suppressor gene P53. First, I studied the frequency, nature and timing of P53 mutations in human gliomas. I observed that glioma-prone families do not have inherited mutations of P53, nor of the Nf1-GRD, Nf2, MTS1, MTS2, or CDK4 genes. However, a high frequency of loss of heterozygosity at chromosome 9p was noted in gliomas that cluster in families. In a second study of low-grade gliomas that progressed to higher grade malignancies, I observed that those with P53 mutations progressed slowly to anaplastic pathology whereas those without mutation progressed directly to glioblastoma omitting the anaplastic stage. Next, I studied the biological consequences of P53 disruption on tumorigenesis and brain development in mice. I introduced a disrupted P53 gene into astrocytoma-susceptible inbred mice to augment astrocytoma formation and generate an experimental model. These P53-null mice did not exhibit enhanced incidence of astrocytic glioma but did develop high grade lymphomas at a significantly younger age than control P53-null mice. Lastly, I examined gene expression in the brains of wild-type and P53-null mouse embryos. Expression of the WAF1/p21 and IGF-BP3 genes in the developing head responded to loss of p53 in a gender-specific manner, suggesting that female-restricted defects of neural tube closure in P53-null mice may be explained by insufficient compensatory gene expression. I also noted that P53-deficiency causes lethal developmental defects other than exencephaly in females. Loss of one copy of P53 caused a moderate decrease in the number of female embryos relative to males early in gestation, while loss of both copies resulted in additional perinatal attrition of females. Together, the experiments described in this thesis contribute to our knowledge of the role of P53 in glioma predisposition and progression in humans, and to tumorigenesis and nervous system development in mice

Identification of genes influencing dendrite morphogenesis in developing peripheral sensory and central motor neurons.

BACKGROUND: Developing neurons form dendritic trees with cell type-specific patterns of growth, branching and targeting. Dendrites of Drosophila peripheral sensory neurons have emerged as a premier genetic model, though the molecular mechanisms that underlie and regulate their morphogenesis remain incompletely understood. Still less is known about this process in central neurons and the extent to which central and peripheral dendrites share common organisational principles and molecular features. To address these issues, we have carried out two comparable gain-of-function screens for genes that influence dendrite morphologies in peripheral dendritic arborisation (da) neurons and central RP2 motor neurons. RESULTS: We found 35 unique loci that influenced da neuron dendrites, including five previously shown as required for da dendrite patterning. Several phenotypes were class-specific and many resembled those of known mutants, suggesting that genes identified in this study may converge with and extend known molecular pathways for dendrite development in da neurons. The second screen used a novel technique for cell-autonomous gene misexpression in RP2 motor neurons. We found 51 unique loci affecting RP2 dendrite morphology, 84% expressed in the central nervous system. The phenotypic classes from both screens demonstrate that gene misexpression can affect specific aspects of dendritic development, such as growth, branching and targeting. We demonstrate that these processes are genetically separable. Targeting phenotypes were specific to the RP2 screen, and we propose that dendrites in the central nervous system are targeted to territories defined by Cartesian co-ordinates along the antero-posterior and the medio-lateral axes of the central neuropile. Comparisons between the screens suggest that the dendrites of peripheral da and central RP2 neurons are shaped by regulatory programs that only partially overlap. We focused on one common candidate pathway controlled by the ecdysone receptor, and found that it promotes branching and growth of developing da neuron dendrites, but a role in RP2 dendrite development during embryonic and early larval stages was not apparent. CONCLUSION: We identified commonalities (for example, growth and branching) and distinctions (for example, targeting and ecdysone response) in the molecular and organizational framework that underlies dendrite development of peripheral and central neurons.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Rab-mediated vesicular transport is required for neuronal positioning in the developing Drosophila visual system

<p>Abstract</p> <p>Background</p> <p>The establishment of tissue architecture in the nervous system requires the proper migration and positioning of newly born neurons during embryonic development. Defects in nuclear translocation, a key process in neuronal positioning, are associated with brain diseases such as lissencephaly in humans. Accumulated evidence suggests that the molecular mechanisms controlling neuronal movement are conserved throughout evolution. While the initial events of neuronal migration have been extensively studied, less is known about the molecular details underlying the establishment of neuronal architecture after initial migration.</p> <p>Results</p> <p>In a search for novel players in the control of photoreceptor (R cell) positioning in the developing fly visual system, we found that misexpression of the RabGAP RN-Tre disrupted the apical localization of R-cell nuclei. RN-Tre interacts with Rab5 and Rab11 in the fly eye. Genetic analysis shows that Rab5, Shi and Rab11 are required for maintaining apical localization of R-cell nuclei.</p> <p>Conclusions</p> <p>We propose that Rab5, Shi and Rab11 function together in a vesicular transport pathway for regulating R-cell positioning in the developing eye.</p

Ihog and Boi are essential for Hedgehog signaling in Drosophila

<p>Abstract</p> <p>Background</p> <p>The Hedgehog (Hh) signaling pathway is important for the development of a variety of tissues in both vertebrates and invertebrates. For example, in developing nervous systems Hh signaling is required for the normal differentiation of neural progenitors into mature neurons. The molecular signaling mechanism underlying the function of Hh is not fully understood. In <it>Drosophila</it>, Ihog (Interference hedgehog) and Boi (Brother of Ihog) are related transmembrane proteins of the immunoglobulin superfamily (IgSF) with orthologs in vertebrates. Members of this IgSF subfamily have been shown to bind Hh and promote pathway activation but their exact role in the Hh signaling pathway has remained elusive. To better understand this role <it>in vivo</it>, we generated loss-of-function mutations of the <it>ihog </it>and <it>boi </it>genes, and investigated their effects in developing eye and wing imaginal discs.</p> <p>Results</p> <p>While mutation of either <it>ihog </it>or <it>boi </it>alone had no discernible effect on imaginal tissues, cells in the developing eye disc that were mutant for both <it>ihog </it>and <it>boi </it>failed to activate the Hh pathway, causing severe disruption of photoreceptor differentiation in the retina. In the anterior compartment of the developing wing disc, where different concentrations of the Hh morphogen elicit distinct cellular responses, cells mutant for both <it>ihog </it>and <it>boi </it>failed to activate responses at either high or low thresholds of Hh signaling. They also lost their affinity for neighboring cells and aberrantly sorted out from the anterior compartment of the wing disc into posterior territory. We found that <it>ihog </it>and <it>boi </it>are required for the accumulation of the essential Hh signaling mediator Smoothened (Smo) in Hh-responsive cells, providing evidence that Ihog and Boi act upstream of Smo in the Hh signaling pathway.</p> <p>Conclusions</p> <p>The consequences of <it>boi;ihog </it>mutations for eye development, neural differentiation and wing patterning phenocopy those of <it>smo </it>mutations and uncover an essential role for Ihog and Boi in the Hh signaling pathway.</p

Proportional Venn diagrams to describe the degree of overlap among genes that emerged from both screens

The total is shown at top left, and then broken down by the predicted site of gene product activity.<p><b>Copyright information:</b></p><p>Taken from "Identification of genes influencing dendrite morphogenesis in developing peripheral sensory and central motor neurons"</p><p>http://www.neuraldevelopment.com/content/3/1/16</p><p>Neural Development 2008;3():16-16.</p><p>Published online 10 Jul 2008</p><p>PMCID:PMC2503983.</p><p></p

Rows show examples representing the main phenotypic categories recovered from the central (RP2) neuron dendrite misexpression screen

Left and centre columns: confocal images (maximal Z-projections) of RP2 neurons at 25–31 hours AEL, visualised with . Control RP2 neuron with brackets indicating the dendritic tree. Control RP2 neuron in the context of a set of axon tracts visualised by anti-FasciclinII staining (magenta), with arrowheads pointing from top to bottom to the lateral, intermediate and medial FasciclinII tracts and the midline indicated by a dotted line. Dendrites between the lateral and central intermediate Fasciclin II fascicle are defined as 'lateral'; dendrites located between the central intermediate fascicle and the midline as 'medial'; the same applies to (o,p). Same neuron as in (b) but with sectors of its dendritic tree pseudo-coloured to highlight branches targeted to anterior lateral (magenta), anterior medial (yellow) and posterior lateral (cyan) regions. Anterior is left and the ventral midline is down. Experimental cells: misexpression lines are indicated in the bottom right-hand corner of each panel. Right column: quantifications of the dendritic phenotypes shown in the left and central columns. As illustrated in (f), both dendritic tree length and number of branching events are reduced in the 'Growth' and 'Branching' categories. 'Branching' phenotypes have trees with an anterior-posterior extent comparable to controls (Additional file ) but have an altered pattern of branching: fewer branching events and more segments that are longer (>5 μm). *< 0.01, **< 0.005, -test, N = 5. Error bars indicate the standard error. Arrows in (b,o,p) point to medial branches present in controls (b) and absent/reduced in experiments (o,p). Black asterisks in (e,p) indicate the cell body of the contralateral RP2 neuron. Scale bar: 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Identification of genes influencing dendrite morphogenesis in developing peripheral sensory and central motor neurons"</p><p>http://www.neuraldevelopment.com/content/3/1/16</p><p>Neural Development 2008;3():16-16.</p><p>Published online 10 Jul 2008</p><p>PMCID:PMC2503983.</p><p></p

RP2 neurons at 25–31 hours AEL and visualised with in the context of FascicilinII positive axon bundles (magenta) demarcating the medial and lateral neuropile (maximal Z-projections of confocal image stacks)

Control. Misexpression of (activated ) leads to a lack of dendritic innervation of the medial neuropile (normally located anterior to the axon (arrowhead in (a)) and a concomitant expansion of dendrites in the lateral neuropile posterior to the axon (arrowhead in (b))). Dendritic extent anterior or posterior to the axon is indicated by brackets. Quantification of anterior, posterior and total (combined) maximal dendritic extent for controls (green, N = 10) and expression RP2 neurons (magenta, N = 8). The significance of pair-wise comparisons using Student's -test is indicated. Anterior is left and the ventral midline is down. Scale bar: 20 μm.<p><b>Copyright information:</b></p><p>Taken from "Identification of genes influencing dendrite morphogenesis in developing peripheral sensory and central motor neurons"</p><p>http://www.neuraldevelopment.com/content/3/1/16</p><p>Neural Development 2008;3():16-16.</p><p>Published online 10 Jul 2008</p><p>PMCID:PMC2503983.</p><p></p

Three-dimensional reconstructions from confocal image stacks of RP2 neurons at 25–31 hours AEL and visualised with generated with AMIRA software

Control. Misexpression of causes aberrant dendritic targeting to the posterior. Brackets in (a) indicate the dendritic tree. Dendrograms derived from the reconstructions with branch points highlighted in magenta and the cell body and axon offset from the dendritic tree by green. Quantification of the dendritic architectures for controls (green, N = 4) and expressing RP2 neurons (magenta, N = 4). The significance of pair-wise comparisons using Student's -test is indicated. Error bars indicate the standard error. Anterior is left and the ventral midline is down. Scale bar: 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Identification of genes influencing dendrite morphogenesis in developing peripheral sensory and central motor neurons"</p><p>http://www.neuraldevelopment.com/content/3/1/16</p><p>Neural Development 2008;3():16-16.</p><p>Published online 10 Jul 2008</p><p>PMCID:PMC2503983.</p><p></p