The development of the caudal nerve cord and muscle innervation in the appendicularian Oikopleura dioica was assessed using differential interference contrast and confocal microscopy, phalloidin staining of actin, and in situ hybridization for the neuronal markers tubulin and choline acetyltransferase (ChAT). The caudal nerve cord first appears as a stream of tubulin mRNA-positive neurons that extends into the tail from the caudal ganglion. By this stage a few actin-rich nerve fibers course longitudinally along the cord. As the tail lengthens, the caudal nerve cord extends and becomes more fasciculated and the neurons cluster at stereotyped longitudinal positions. The number of neurons in the nerve cord reaches a relatively stable maximum of about 29. A subset of neurons in the caudal ganglion and caudal nerve cord expresses ChAT mRNA. These putative motoneurons are distributed along nearly the full extent of the tail in numbers consistent with an independent innervation of each tail muscle cell. The longitudinal series of putative motoneurons is not aligned with the muscle cells, but peripheral nerve fibers extending to the muscle cells are, indicating that motor axons grow along the cord before exiting adjacent to their peripheral target. Muscle innervation occurs roughly coincident with the onset of ChAT mRNA expression. Our results provide the first molecular identification of motoneurons and the first developmental characterization of the motor system in an appendicularian and help set the stage for gene expression studies aimed at understanding the evolution of developmental patterning in this part of the chordate central nervous system.
(c) 2007 Wiley-Liss, Inc.