TISSUE SPECIFIC CONTROL OF CELL PROLIFERATION

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DESCRIPTION (Adapted from investigator's abstract): An essential component of tissue morphogenesis, function, and homeostasis is the differential control of cell proliferation in the different cell types that comprise a tissue. The long term goal of the proposed studies is to understand the molecular mechanisms underlying such cell type-specific controls of cell proliferation. As a starting point toward the identification of genes that participate in the cell type-specific regulation of cell proliferation, Gossler has chosen to focus on mouse mutations that affect the proliferation of particular cell types or tissues. In this proposal, the mouse mutation under study is Pintail (Pt). Pintail was isolated from the offspring of mice treated with the carcinogen methylcholantrene. It is a semidominant mutation on chromosome 4 and it specifically affects the proliferation of notochord cells in a dose dependent manner. A descriptive study of Pt, published nearly 40 years ago, showed that the frequency of mitotic cells in the notochord was reduced to approximately 50% and 30%, of normal values in, respectively, heterozygotes and homozygotes. As a result, the notochord is significantly smaller or completely absent in the posterior portion of the embryo; this in turns generates skeletal abnormalities. The proposed experiments are divided into four specific aims. Specific Aim 1 further explores the Pt phenotype. First, an analysis of BrdU incorporation into notochord cells of mutant and wild type embryos will be performed to define the G1, S, G2, and M parameters of the cell cycle in Pt mutant cells. In addition, an expression analysis will be conducted on markers for defined phases of the cell cycle. Second, chimeras will be constructed by the injection of Pt ES cells into wild type blastocysts to assess the cell autonomy of the Pt mutation. Specific Aim 2 proposes to establish a fine scale genetic map of the Pintail mutation using at least 2000 backcross animals generated from two different intersubspecific backcrosses. Using the genetic map and chromosomal walking techniques, a physical map and a BAC contig will be constructed for the Pt critical interval. The Pt gene will be identified in Specific Aim 3 using the candidate gene approach, cDNA selection, and/or sequencing of a <50 kb critical region. The authenticity of the Pt gene will be verified by expression analyses, sequencing, and appropriate transgenic or knockout experiments. In Specific Aim 4, transgenic approaches will be undertaken to functionally analyze the pintail gene and its role in cell type specific control of cell proliferation.