Patterns of cell-cell coupling in embryonic spinal cord studied via ballistic delivery of gap-junction-permeable dyes

Intercellular communication mediated by gap junctions is developmentally regulated in many tissues, including the nervous system. In rodent lumbar spinal cord, extensive gap junctional coupling among motor neurons innervating the same muscle is present at birth but is no longer present 1 week later. Little is known about how this motor-pool-specific coupling arises during embryonic development. To address this question, we developed a novel method of visualizing patterns of cell-cell coupling that can be applied to a wide range of tissues. Gap-junction-permeable dyes adsorbed to metal beads were delivered into individual cells in embryonic cerebral cortex or spinal cord using pressure. Dye diffused off of the bead surface into the cytoplasm, crossed gap junctions, and labeled clusters of coupled cells. For embryonic cerebral cortex, this method revealed patterns of cell-cell coupling similar to those reported with other techniques. In embryonic lumbar spinal cord, cell-cell coupling is widespread in the ventricular zone at E11, and the extent of coupling decreases until birth. In the ventral horn, motor neurons are coupled into clusters at E14, with little change in the extent of coupling at E16, and a similar extent of coupling is present at birth. The cell types within clusters, identified by using antibodies against homeodomain transcription factors, were surprisingly heterogeneous in both the ventricular zone and the motor columns. Taken together, these data suggest that the spatial and temporal patterns of cell-cell coupling are dynamic and that cell-type-specific gap junctional coupling arises gradually during spinal cord development.