Engrailed-1 and Netrin-1 regulate axon pathfinding by association interneurons that project to motor neurons

During early development, multiple classes of interneurons are generated in the spinal cord including association interneurons that synapse with motor neurons and regulate their activity. Very little is known about the molecu lar mechanisms that generate these interneuron cell types, nor is it known how axons from association interneurons are guided toward somatic motor neu rons. By targeting the axonal reporter gene tau-lacZ to the En1 locus, we s how the cell-type-specific transcription factor Engrailed-1 (EN1) defines a population of association neurons that project locally to somatic motor ne urons. These EN1 interneurons are born early and their axons pioneer an ips ilateral longitudinal projection in the ventral spinal cord. The EN1 intern eurons extend axons in a stereotypic manner, first ventrally, then rostrall y for one to two segments where their axons terminate close to motor neuron s. We show that the growth of EN1 axons along a ventrolateral pathway towar d motor neurons is dependent on netrin-1 signaling. In addition, we demonst rate that En1 regulates pathfinding and fasciculation during the second pha se of EN1 axon growth in the ventrolateral funiculus (VLF); however, En1 is not required for the early specification of ventral interneuron cell types in the embryonic spinal cord.