Gap functions are membrane channels that mediate the direct diffusion of small ions and molecular between adjoining cells. Our hypothesis is that gap junctions are one cellular component that responds to morphogenetic signals in the limb bud. Gap junctions could transmit the morphogen or second messenger signals stimulated in the responding cells. Alternatively, gap junctions could create domains of cells that the morphogenetic signal then patterns. This could crete position specific cell growth or differentiation (e.g., cells differentiate into a specific skeletal element). Gap junctions are found in the AER, distal mesenchyme (the progress zone) and condensing mesenchyme of the forming skeletal anlage. Therefore, gap junctions are present in some of the signaling centers when critical decisions are being made about cell fate. My goal is to clarify the roles of gap junction communication in limb patterning and examine limb bud development at the cellular level. Two approaches will be used to examine the role of gap junctions in limb morphogenesis. First we will examine the expression of gap junction genes in two mouse mutants where limb deformities are seen. We will study limb deformity (ld) mice where the AER is altered and disruption of the Shh/FGF-4 feedback loop has been shown. A second mutant mouse line we will examine is brachypodism (bp) where persistent cell proliferation in the progress zone occurs and produces truncated adult limbs. Since gap junctions play a role in cell proliferation, persistent proliferation may be correlated to excess gap junctions expression. A second approach is to use in vitro methods. We will use an organ culture system that allows the maintenance of near normal limb development and the manipulation of the mammalian limbs. We will alter gap junction expression of function and determine if abnormalities or defects results from these perturbations. Gap junction communication can be blocked by treating cell cells with anti-connexin antibodies and allow us to examine if gap junctions are indeed necessary for normal limb development. We will also use micromass cell cultures of mutant mouse mesenchymal cells. Micromass cultures are cells grown at high cell density, which permits the cells to aggregate and differentiate resembling normal bone formation. We can examine whether normal cell growth and differentiation characteristics can be achieved by treatment of the cells with growth factors.
