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.10 RELATIONSHIP TO CYTOSKELETON (PROJECT 2) AND BIOLOGICAL NETWORKS (PROJECT 1)
VI.11 TIMELINE
< Previous | Page 12 of 27 | Next >
VI.8 PRELIMINARY RESULTS:
We describe recent published and unpublished findings by Consortium members as a context for the Specific Subprojects. We present details in the Subproject descriptions.
VI.8.i Experimental Results:
Dr. Newman's laboratory has established that ectopic expression of the amino-terminal heparin-binding domain of fibronectin enhances or inhibits precartilage condensation formation depending on its level of expression and the initial cell density. They also found that ectodermal fibroblast growth factors (FGFs) act via FGF receptor 2 to mediate lateral inhibition of cartilage nodule formation (Moftah et al., 2002).
Dr. Weijer's laboratory has studied the mechanisms that control cell movement during the formation of the primitive streak in the chick embryo, mapping the pattern of cell movement before primitive streak formation and during its elongation using DiI labeling and localized transfection of subpopulations of cells in the epiblast with GFP expression constructs. Postero-lateral cells move toward the posterior marginal zone while the cells anterior to the posterior marginal zone move anteriorly along the midline of blastoderm disk. In loss of function experiments, epiblast cells, which had lost their ability to either divide, by local application of DNA synthesis inhibitors, or move, did not show the same movement pattern. So the formation and elongation of the primitive streak depends both on cell division and cell movement. In another set of experiments they have shown that the movement of mesoderm precursor cells emerging at various anterior posterior positions from the primitive streak is controlled by chemo-repulsion and chemo-attraction. FGF8 produced in the streak was identified as the chemo-repellent that sends cells away from the streak. In the posterior part of the embryo the cells are attracted by a signal coming from the border region of the area opaca and area pellucida and these cells will form extra embryonic structures and the haemapoetic system. Cells emerging more anterior are attracted back in towards the midline by FGF4 released by the forming notochord to form somites and lateral plate mesoderm (Yang et al., 2002). Using expression of dominant active and negative GFP tagged CDC42constructs we have now shown that the FGF4 mediated chemo-attraction requires activation of CDC42, while the chemo-repulsion by FGF8 does not.
Dr. Forgacs' laboratory measures cell-cell adhesion and cell-ECM binding constants in cell aggregates. Intact (not dissociated) tissue fragment from the limb buds of four-day-old chick embryos (pre-cartilage limb bud mesenchyme), under appropriate conditions round up to form spherical aggregates. Mechanical deformation of these aggregates, using a parallel plate compression apparatus, allows the measurement of tissue viscoelastic properties, particularly surface tensions. The quantitative results show that limb bud tissues are more cohesive than the surrounding flank tissue, with leg limb buds being more cohesive than wing in agreement with the qualitative analysis of Heintzelman et al. (1978). The measurement of intracellular viscoelastic properties by a powerful magnetic tweezers connects tissue properties to those of individual cells. They have used phagocytosis to insert magnetic particles into individual cells (verified, using confocal microscopy).
Dr. Forgacs and Dr. Newman, in collaboration with Dr. Sackmann's laboratory in Munich, Germany have measured the viscoelastic properties of model extracellular matrices to gain deeper insight into ECM's role in early morphogenesis.
Dr. Glazier and Dr. Newman's laboratories have experimentally investigated mesenchymal condensation and developed a CPM model based on local cell-cell adhesion and cell-extracellular matrix (ECM) interactions. The simulation reproduces the phenomenology of in vitro chick limb chondrogenic mesenchymal condensation which depends on the initial cell density. Unlike in the Turing mechanism where chemical diffusion drives patterning, the pattern results from cell diffusion biased by direct cell-cell adhesion and cell-ECM binding. The mechanism also differs from chemotaxis which requires long-range cell movement (Brand et al., 1985; Downie and Newman, 1994;.Glazier and Graner, 1993; Gould, et al., 1972; Graner and Glazier, 1992; Hall, and Miyake, 1995; Hamburger and Hamilton, 1951; Leonard et al., 1991; Li and Muneoka, 1999; Miura and Shiota, 2000; Mochizuki et al., 1998; Moftah et al., 2002; Newman, 1988; Newman and Tomasek, 1996; Newman et al., 1981; Oberlender and Tuan, 1994; Ohsugi et al., 1997; Tomasek et al., 1982; Turing, 1952; Widelitz et al., 1993).
Dr. Marrs's laboratory has developed the zebrafish model to study cadherin adhesion molecules. Morpholino knockdown technology has produced zebrafish that lack E-cadherin, N-cadherin and R-cadherin. These experimental models allow examination of defects in cell migration and fasciculation/defasciculation during gastrulation and retinotectal axon migration.
Dr. Shou's laboratory has developed a series mouse models in which genes that regulate cardiac development are either deleted (knockout model) or overexpressed (transgenic model). These models include Bone Morphogenic Protein (BMP-10) knockout and transgenic overexpressor, and FKBP12 knockout and transgenic overexpressor. These models allow examination of abnormal cardiac developmental phenotypes and help to determine the molecular mechanisms of ventricular trabeculation-compaction. We have an excellent mouse core facility to generate additional transgenic and knockout mice at IU Medical.
Dr. Grow's Laboratory has developed a 7200-feature microarray from a Xenopus laevis cDNA embryonic heart library and is currently fabricating a 15000-feature microarray from this library (Project 1, Subproject 7). These arrays allow temporal profiling of gene expression during early heart differentiation to address specific questions regarding cardiovascular development. We will extend this approach to additional species.