Organogensis

VI.1 COORDINATORS
VI.2 PARTICIPANTS
VI.3 SUMMARY
VI.4 INTRODUCTION
VI.5 SPECIFIC AIMS
VI.6 BACKGROUND AND SIGNIFICANCE
VI.7 THEORETICAL FRAMEWORK
VI.8 PRELIMINARY RESULTS
VI.9 RESEARCH DESIGN AND METHODS

• VI.9.i Gastrulation
  • VI.9.i.a Overview
  • VI.9.i.b Experiments
    • VI.9.i.b.1 The role of chemotaxis in primitive streak formation in chick
      • VI.9.i.b.1.i Hypothesis
      • VI.9.i.b.1.ii Methodology
    • VI.9.i.b.2 Zebrafish gastrulation
      • VI.9.i.b.2.i Overview
      • VI.9.i.b.2.ii Methodology
    • VI.9.i.c Models of Gastrulation
• [ Complete VI.9 Outline ]

VI.10 RELATIONSHIP TO CYTOSKELETON (PROJECT 2) AND BIOLOGICAL NETWORKS (PROJECT 1)
VI.11 TIMELINE

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VI.9 RESEARCH DESIGN AND METHODS:

The descriptions of experimental and computational methods in the following sections are necessarily condensed. Please see the cited literature and the papers listed in the investigators' CVs for additional details on procedures and methods.

VI.9.i Gastrulation:

VI.9.i.a Overview:

The following studies all relate to the developmental effects of cell motility and translocation. They provide a natural connection to the modeling of motility of individual cells described in the cytoskeleton portion of this proposal (Project 2), but demonstrate the effect of context-both neighboring cells and ECM. These phenomena suit modeling using the CPM described in Section VI.7.

VI.9.i.b Experiments:

The precise mechanisms by which wnt and vg1 signals control cell movement is not known (Skromne and Stern, 2001, Heisenberg, 2000). We will continue to investigate the role of chemotaxis in directing movement of mesoderm cells during gastrulation in chick embryo. We will compare normal and mutant zebrafish embryos during gastrulation to investigate the genetic pathways and how they control cell movement.

VI.9.i.b.1 The role of chemotaxis in primitive streak formation in chick:
VI.9.i.b.1.i Hypothesis:

We propose that chemotaxis helps direct cell movement during gastrulation. We first must finish our quantification of cell movement patterns in vivo during early streak formation, extension and regression and then will interfere with these movements by local expression of activating and inhibitory forms of signaling and signal transduction components to investigate their role in gastrulation.

VI.9.i.b.1.ii Methodology:

By labeling groups of cells (normally 20-50 cells) with a fluorescent dye (e.g. DiI) in cultured early stage chick embryo (HH stage X to HH stage 4) and tracking cell movement using fluorescence video-microscopy, we can visualize single cell movements in the area pellucida before and during early streak formation. We have established methods to electroporate early prestreak chick embryos and induce expression of GFP fusion proteins. Embryos survive for up to 36 hours. To trace cell movement quantitatively during streak formation we will combine these observations with positive identification of the moving cells by analyzing their gene expression patterns using antibody staining and in-situ hybridization techniques and by expressing GFP in a cell-type specific manner using heterologous promoters.

We will determine whether chemotaxis, substrate guided movement or differential adhesion between cells dominate these movements. We have shown that movement of the mesoderm cells away from the streak involves both negative chemotaxis to FGF8, as well as attraction by unknown molecules. We are trying to identify the unknown attractants by local application of candidate attractants (other growth factors and molecules like SLIT's. We are in the process of setting up an expression cloning system to express pools of cDNA's in tissue culture cells that will then be implanted as cell pellets in the embryo and could lead to the identification of new attractants and repellents. We will investigate how cells polarize in response to FGF signaling and how this results into directed movement. To visualize polarization we will transfect cells with GFP tagged Pleckstrin Homology (PH) domains specific for particular phosphatidyl-inositol-lipids such as PI[3,4,5]P3. We have used this technique successfully to visualize cell-cell signaling and cell polarization during the movement of individual cells in tissues of Dictyostelium mounds and slugs (Dormann et al.,2002 ) and initial experiments indicate that they can be used in the chick. We are now further dissecting these signaling pathways from FGF detection to cell polarization and cell movement using localized expression of activated and inhibitory GFP tagged signaling components and selective downregulation of some signaling components by expression of small interfering RNA's (siRNA). We concentrate on dissecting the role of the FGF receptors and on the manipulation of phosphatidyl-inositol lipids by over expression of hydrolyzing enzymes such as the 5'phosphatase SHIP2 and the 3' phosphatase PTEN. We express molecules that interfere with the signaling pathways to the actin cytoskeleton such as WASP and CDC42 (see Project 2) in subpopulations of cells in different regions of the embryo. This approach allows selectively immobilization of populations of cells in vivo and study of the consequences for development. FGF signaling and its down stream targets such as CDC42 are also involved in the regulation of the expression of various adhesion molecules, i.e. the down regulation of E-cadherin during entry of epiblast cells into the streak. Therefore we will also assess the effect of the experiments described above on the expression of cell adhesion molecules and its role in the control of movement.