Tadmiri R. Venkatesh - US grants

The major focus of our research is to understand the genetic and molecular basis of pattern formation in the nervous system. The nervous system of multicellular organisms is a complex network of diverse cell types with unique positions and patterns of connectivity. Understanding how these intricate patterns develop is a central problem in cell and developmental biology. During the development of the nervous systems of both invertebrates and vertebrates environmental cues and cellular interactions play important roles in the determination of cellular phenotypes and in pattern formation. The compound eye of Drosophila is well suited for studying the cellular and molecular basis of pattern formation. Molecular genetic analysis of the Drosophila eye will contribute to a more complete understanding of what these developmental cues are and how they direct development. Our genetic screens for mutation affecting pattern formation in the developing eye have led us to the discovery of a new mutation. rap (retina aberrant in pattern). Our analysis demonstrates that rap gene function is critical for normal eye pattern formation. Several lines of evidence suggest that rap acts early to regulate the initial steps in ommatidial formation. First, experiments with a temperature sensitive alleleindicate that rap function is required during the third larval instar, a stage during which ommatidial pattern formation in rap mutants is abnormal immediately behind the morphogenetic furrow in the developing eye disc indicating that rap gene functions are important during initial steps. And finally, genetic mosaic studies demonstrate that rap + function is required only in R8 for normal ommatidial pattern formation consistent with the notion that rap acts at the beginning of pattern formation. These results led us to two possible models. In the first model the rap gene product is a signal made by R8 for proper patterning of other R cells. Our recent studies indicate that rap+ gene product is necessary for the proper differentiation of the R8 cell. The research proposed here is aimed at a genetic and molecular analysis of the rap gene with a view to understand the molecular nature of the rap gene product, its expression pattern and identify other gene products that with the rap gene.

Protease inhibitors have been shown to possess a broad range of transformation-suppressing effects including reduction of tumor formation and blockage of transformation in cell culture systems. In previous work our laboratory has described the stage-specific nature of the process of transformation of human epidermal keratinocytes by the oncogenic virus, SV40. The overall goal of this proposal is to use the viral transformed epithelial cells as a model system to study the mechanism of action of protease inhibitors in antioncogenesis. The studies described in this proposal are divided into two parts. In the first part we will characterize the effects of protease inhibitors on transformation in the viral transformed epithelial cells using four types of parameters of growth and differentiation as stage-specific markers of transformation in vitro: a) clonal growth, b) growth dependence on serum and growth factors, c) anchorage independent growth, d) density dependent regulation of growth and differentiation. These studies will determine aspects of growth control and critical stages in the transformation process which are targets of the transformation suppressing effects of protease inhibitors. In the second part we will investigate the role of gene expression as a molecular mechanism of action of protease inhibitors. These studies will have three components: 1) DNA and RNA blot hybridization analyses to determine whether protease inhibitor treatment can prevent two types of SV40-induced changes in the myc and ras protooncogenes: a) structural polymorphisms and b) altered levels of transcription, 2) Immunochemical analysis and RNA blot hybridization to determine whether protease inhibitor treatment can reverse the transformation-related induction of simple epithelial/fetal keratin genes and/or block the expression of a newly characterized cytoskeletal cDNA marker of transformation and, 3) Isolation of transformation-related sequences from cDNA libraries whose expression is either blocked or induced by protease inhibitors.

DESCRIPTION (provided by applicant): This proposal requests funds for a confocal microscope to be used as shared imaging instrument by the faculty in the department of biology at the City College of New York. The confocal microscope will facilitate the research activities of eight NIH-funded investigators some of whom already use confocal microscopy in their research, and will enable others to initiate a new dimension to their ongoing research activities. At the present time there is no confocal microscope available in any of the departments of the Division of science at City College. The current users of the confocal microscopy are using for-fee facilities at other research institutions in New York and Boston. A total of nine principal investigators and over 40 research personnel including postdoctoral fellows, graduate students, undergraduates, and research assistants will use the requested microscope on a daily basis. The research projects of the users cover a diverse variety of confocal applications that include: cellular basis of song learning in birds, neurotransmitter function in nematodes, neuronal development in Drosophila and higher vertebrates, studies on Foraminifera, thymic nurse cell function and Drosophila hematopoiesis and immune response. The use of a confocal microscope is critical for these studies. Funds are requested for a Nikon C1 confocal microscope. This microscope has high quality optics, resolution and excellent image quality and is versatile and compatible to most imaging software. In addition, the low cost of the microscope and quality of service as compared to other manufacturers were important points of concern. The confocal microscope is a key essential instrument and relatively standard in most departments. Acquisition of the confocal microscope will greatly enhance the quality and the productivity of the NIH funded investigators at City College.

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SPECIFIC AIMS A primary goal of this proposal is to understand the molecular genetic mechanisms regulating glia differentiation. Traditionally, glia were thought of mainly as support cells for neuronal function. In recent years, however, it has become increasingly clear that glia are pivotal for proper neuronal development and function. Glia mediate a remarkable array of cellular functions including axon ensheathment, establishment of blood brain barrier, trophic response, ionic equilibrium, synaptogenesis, axon pruning, engulfment and neuronal plasticity. To carry out these important functions, glia must themselves differentiate and function properly. Indeed, glia malfunction often precedes neuronal/axonal degeneration in many human neurodegenerative diseases (BAUMANN and PHAM-DINH 2001;BENARROCH 2005;FREEMAN 2005b;FREEMAN 2005c;KIM and DE VELLIS 2005;SCHWABE et a/. 2005;SHAHAM 2005;WYSS-CORAY and MUCKE 2002). Despite their immense importance in neurobiology, glia are remarkably understudied and the molecular genetic mechanisms that direct the differentiation of glia are poorly understood. The developing nervous system of Drosophila offers a superb experimental system in which to understand these mechanisms. Drosophila glia are remarkably similar to the mammalian glia in their development, structure and function (FREEMAN and DOHERTY 2005). In Drosophila, molecular and genetic manipulations are readily feasible and the cellular signaling pathways that regulate nervous system development are highly conserved between Drosophila and mammalian systems. Thus an understanding of the regulatory mechanisms that direct glia differentiation in Drosophila will provide important insights into mammalian glia differentiation and will be invaluable in our understanding of human neurological disorders. The focus of our proposal is on the role of ubiquitination as a regulatory mechanism during glia differentiation. Tagging of specific proteins for degradation by ubiquitination has emerged as an important regulatory mechanism in nervous system development and disease. We previously identified and molecularly characterized Rap/Fzr, an activator of the multi-subunit ubiquitin ligase, APC (Anaphase promoting complex). Our work has shown that Rap/Fzr regulates cell cycle progression and is required for proper neuronal patterning in the developing eye (JACOBS etal. 2002;KARPILOW et a/. 1989;KARPILOW etal. 1996;PIMENTEL and VENKATESH 2005b). To identify novel cellular functions of Rap/Fzr we carried out genetic studies which showed that Rap/Fzr interacts with several genes required for glia differentiation (Kaplow et al. 2006 submitted). We have recently made the novel observation that Rap/Fzr regulates glia differentiation by interacting with an Ets domain transcription factor, Pointed, already known to be required for glia differentiation and with Apc2. a catalytic subunit of the APC ubiquitin ligase. Our working hypothesis is that glia differentiation is regulated by novel interactions involving Rap/Fzr, Pointed and Apc2. Rap/Fzr binds Pointed and targets it to the ubiquitin ligase complex (APC), where it is ubiquitinated. Pointed is eventually degraded by the 26S proteosome. In our model, the level of Pointed is regulated by Rap/Fzr, and is a key determinant in the regulation of glia differentiation (Figure 1). The specific aims of this proposal are: Specific Aim I. To test whether glia differentiation is negatively regulated by Rap/Fzr. Our preliminary results suggest that Rap/Fzr is a negative regulator of glia differentiation, a) We will test at the cellular level the effects of loss-of-function Rap/Fzr mutations on glia differentiation in the developing larval brain and the eye. We will generate homozygous rap/fzr- mutant clones in a wild type tissue background using the FLP-FRT technique, b) To directly assess the effects of Rap/Fzr loss-of-function on glia differentiation in a spatially and temporally restricted manner, we will use RNAi and UAS-GAL4 techniques to knockdown Rap/Fzr function in specific tissues at specific times, c) To test the effects of gain-of-function of Rap/Fzr on glia differentiation, we will generate random clones of cells expressing Rap/Fzr, using UAS-GAL4 as well as FLP-FRT systems. We will determine the role of Rap/Fzr in regulating the number and position of glia in the developing larval nervous system. These experiments will address whether the Rap/Fzr function is necessary for glia differentiation in a cell autonomous manner. Specific aim II. To test whether Rap/Fzr regulates glia differentiation by direct interaction with Pointed: Our working hypothesis is that Rap/Fzr targets Pointed for ubiquitination by the ubiquitin ligase APC. Pointed is an ETS domain transcription factor which is required for glia differentiation, a) We will test whether Rap/Fzr binds Pointed using in vitro and in vivo biochemical assays. We will use tissue extracts from larval central nervous system (CMS) and tissue culture S2 cell extracts and assay for binding interactions between Rap/Fzr and Pointed by co-immunoprecipitation assays, b) We will test whether Rap/Fzr and Pointed co-localize in glia in vivo using confocal microscopy in combination with immunohistochemistry. c) We will test whether Rap/Fzr and Pointed interact physically using yeast two-hybrid assays, d) We will perform in vitro ubiquitination assays and test whether Pointed can serve as a substrate for ubiquitination by Rap/Fzr and APC. Specific Aim III. To test whether Apc2/Morula regulates glia differentiation: Apc2 is the catalytic subunit of the ubiquitin ligase complex, APC, encoded by the morula gene. Our preliminary studies suggest that Apc2 is a negative regulator of glia differentiation. To directly assess the role of Apc2 in glia differentiation at the cellular level: a) We will test the effects of loss-of-function of APC on glia differentiation. We will induce clones of Apc2-/Apc2- tissue in the developing larval brain and the eye and determine whether Apc2 is required cell autonomously for glia differentiation, b) We will test the effect of loss-of-function of Apc2 on glia differentiation using the RNAi technique, c) We will test the effects of the gain-of-function of Apc2 on glia differentiation by generating random clones of cells expressing Apc2 using the UAS-GAL4 and the FLP-FRT techniques.