The developmental regulation of laminin and fibronectin expression during pathfinding by pioneer motoneurons in embryonic zebrafish

The generation of neuromuscular specificity is required for the proper expression of a vertebrate's behavioral repertoire. The pattern of this connectivity depends, in part, on the molecular mechanisms which direct axonal outgrowth. Cues in the embryonic environment are sensed by growth cones where the information is transduced into an appropriate motility response. Laminin and fibronectin, adhesive components of the extracellular matrix, have been implicated in directing the pattern of neurite outgrowth in developing and regenerating vertebrate nervous systems. I have immunochemically identified laminin- and fibronectin-like proteins in the zebrafish. Using antibodies, I have localized these molecules in zebrafish embryos during specific phases of axonal initiation and arborization of individual primary motoneurons. Initially, fibronectin is concentrated at the borders of each myotome but not within them, and laminin is absent from the border, but is present within the myotome. At this developmental time, motoneuronal growth cones navigate within the myotome and do not extend into the border regions. Later, laminin appears and fibronectin diminishes in the borders, coincident with the extension of motoneuron growth cones into these regions. I compared neurite outgrowth in vitro on laminin and fibronectin substrata. Primary motoneurons were putatively identified in dissociated embryonic cell cultures by morphological criteria and by staining for acetylcholinesterase activity. Primary neurons had more robust growth on laminin substrata than on fibronectin. Primary sensory neurons, identified immunohistochemically, morphologically, and by absence of acetylcholinesterase activity, preferred to extend on other cells, but were able to grow on either laminin or fibronectin. The dynamic regulation of fibronectin and laminin during early embryonic development in the zebrafish, and the specific interaction of primary motoneuronal growth cones with these potential substrata, may thus guide the axonal outgrowth of the pioneering motoneurons and pattern the segmental organization of peripheral nerves. In the discussion, I describe the results of collaborative experiments which demonstrate that ectopically localized fibronectin can inhibit axonal outgrowth of identified pioneer motoneurons in vivo and thus, support the model that it may play an important role in patterning axonal outgrowth.