Netrin signal is produced in leech embryos by segmentally iterated sets of central neurons and longitudinal muscle cells

Netrins are secreted molecules capable of attracting or repelling growing axons. They and their receptors, along with other netrin-interacting proteins, are widely conserved among animals from a broad range of phyla. We have raised and purified an antibody against a recently cloned leech netrin, which has allowed us to characterize embryonic netrin expression by cells in peripheral tissues and in the central nervous system. During early gangliogenesis, netrin expression was detected at particularly high levels in five bilateral pairs of central neurons. Towards the end of the period of axonal outgrowth, netrin expression was observed to be restricted to only six central neurons, comprising two bilateral pairs and two impaired cells. A pair of netrin-producing central neurons, the bipolar cells, was identified by their expression of the antigen recognized by the monoclonal antibody Laz1-1. Double staining of sensory afferents from segmental sensilla with the monoclonal antibody Lan3-2 and the bipolar cells with the netrin antibody revealed that the terminals of these afferents grow up to the bipolar cells and turn anteriorly or posteriorly, without extending any further medially. Peripheral netrin expression was found to be restricted to longitudinal muscle cells in the ventral half of the body wall. Extracellular, secreted netrin was detected in a broad longitudinal stripe located symmetrically with respect to the ventral midline. The pattern of expression of netrin in leech embryos is consistent with observed expression patterns in other animals, suggesting that developmental netrin functions are conserved among all bilateral animals.