The oblique muscle organizer in Hirudo medicinalis, an identified embryonic cell projecting multiple parallel growth...

The oblique muscle layer in the leech body wall is built upon the processes of a unique identified embryonic cell, the Comb- or C-cell. Each C-cell is composed of a spindle-shaped soma that projects approximately 70 parallel processes through the developing body wall at an angle oblique to the long axis. The morphogenesis of this cell and the navigation of its growth cones were examined by intracellular dye filling and antibody staining. At the earliest stages described each C-cell had about six processes, with those near the center of the cell oriented obliquely. As processes were added at the axial ends of the soma they often projected along previously developed longitudinal or circular muscle founder cells and then secondarily aligned themselves parallel to the older processes from the same C-cell. All growth cones initially extended to a particular location in the body wall, where they ceased growing until all 70 processes had been added (over the course of about 5 days). As adjacent segmental homologs met, their growth cones intermingled, eventually sorting out to align parallel. When one of these cells was ablated early--but not later--in development, the remaining adjacent segmental homologs expanded into the vacant territory, consistent with a hypothesis of mutual avoidance between segmental homologs. Most processes that expanded into the experimentally induced vacancy remained correctly oriented and parallel; the few exceptions projected instead along the mirror-image trajectory. Thus, expression of specific avoidance between adjacent C-cell processes is developmentally regulated and functions as a guidance mechanism in vivo, in that it serves to restrict possible trajectories. After aligning its growth cones, each cell stopped adding processes and the processes rapidly extended in concert along relatively precise trajectories. Processes of contralateral homologs cross to form the orthogonal grid used as a scaffold by myocytes to form the oblique muscles. The advancing fronts of growth cones reached the dorsal midline at about the same time as body closure occurs (at about Embryonic Day 20) at which time the C-cells became granular, lost processes, and presumably died. This sequence of developmental events is consistent with temporal and spatial regulation of different morphogenetic strategies, including--but not limited to--specific avoidance, and further suggests testable hypotheses of mechanisms of growth cone navigation in the intact embryo.