Growth cones navigate along relatively stereotyped pathways both vertebrates and invertebrates, and such stereotyped growth patterns ultimately contribute to the specificity of synaptic connections. This proposal is part of a general objective to determine how growth cones utilize a number of qualitatively different guidance mechanisms to navigate in the complex embryonic environment, and what the underlying bases of these mechanisms are,. The studies proposed here will examine growth cone navigation and guidance in a recently described identified cell in the medicinal leech, Hirudo medicinalis. Each of these Comb- or C-cells projects an array of about 70 individual, parallel growth cones along an oblique trajectory in the body wall, and each of the 21 midbody segments contains a bilateral, mirror-image pair of C-cells. The experiments in this proposal will examine three hypotheses about C-cell growth cone guidance and navigation. 1) Growth cones use guidance cues in the environment to navigate. Possible guidance cells will be identified and their relationship to growth cones determined by light and electron microscopy. The importance of such cells will be tested by ablating the early in development. To examine the influence of more diffuse guidance cues, pathways used by C-cell growth cones will be surgically rotated to challenge their sub-sequent navigation. 2) Growth cones interact among themselves to orient and align. Whole C-cells will be ablated to create vacant territories to then determine how the remaining growth cones of adjacent C-cells respond. Furthermore, a small number of growth cones on a single cell will be ablated to test whether the remaining adjacent ones can converge to occupy the vacancy. 3) Growth cones use several different guidance mechanisms but the relative importance of any single mechanism changes with time and development. Ablations of guidance cells and ablations of growth cones on a single cell (as above) will be performed at different developmental stages. the influence of these manipulations should vary in a predictable way, depending upon the stage initiated. It will also be possible to specifically delay the outgrowth of a single C-cell by injecting cytochalasin intracellularly. By delaying growth cone extension at different stages it will be possible to determine the stages at which guidance by factors extrinsic to the cell are more, or less important than factors intrinsic to the cell.
