Early Motoneuron Growth Outgrowth

During development, chick lumbosacral motoneurons have been reported to form precise topographic projections within the limb from the time of initial outgrowth. This observation implies, first, that motoneurons select the appropriate muscle nerve pathway and, second, that they restrict their ramification within the primary uncleaved muscle masses to appropriate regions. Several reports based on electrophysiology and otthograde horseradish peroxidase (HRP) labeling have shown muscle nerve pathway selection to be fairly precise. However, studies based on retrograde labeling with HRP have produced conflicting reports on the extent to which vertebrate motoneurons make projection errors. Since it is difficult to distinguish between true projection errors and HRP leakage when using retrograde labeling, we decided to assess the distribution of labeled growth cones in 25pm serial plastic sections, following orthograde labeling of identifiable subpopulations of motoneurons during the period of initial axon outgrowth. Examination of a large number of muscle nerves revealed no segmentally inappropriate axons, confirming earlier reports that muscle nerve pathway selection is very accurate. In addition, we observed that growth cones take widely divergent trajectories into the same muscle nerve, suggesting that growth cones are responding independently to some specific environmental cue rather than being passively channeled at this point. The distribution of labeled growth cones within the muscle masses provided direct evidence that motoneurons did not at any time project to obviously inappropriate muscle regions. In fact, motoneuron growth cones remained together as a compact nerve in the central region of their target muscle and only ramified widely within the muscle primordium after the completion of muscle cleavage and the onset of motoneuron cell death, a delay of several days. We conclude that if projection errors occur, they must be of a minor spatial extent. Earlier studies on the development of motor projections in the Received August 22, 1984; Revised January 7, 1985; Accepted January 29, 1985 ’ We thank L. Dahm and H. Tanaka for critical evaluation of the manuscript, and Sara Putnam for her secretarial and editorial assistance. This work was supported by National institutes of Health Grant NS 19640 and by Muscular Dystrophy Assocration and Natronal Institutes of Health postdoctoral fellowships. *To whom correspondence should be sent, at her present address: Natural Science Butlding, Divisron of Biological Sciences, The University of Michigan, Ann Arbor, Ml 48109. chick hindlimb showed that, although axons destined for particular muscles are widely dispersed within individual spinal nerves, they (7) sort out in the plexus region to take up characteristic positions (Lance-Jones and Landmesser, 1981 a) and (2) are found, with only minor exceptions, within segmentally appropriate muscle nerves from the onset of muscle nerve formation (stage (st) 26) (Landmesser and Morris, 1975; Landmesser, 1978; Lance-Jones and Landmesser, 1981 a). Since this is prior to normally occurring motoneuron cell death (Hamburger, 1975) and muscle cleavage (Romer, 1927) this was strong suggestive evidence that the basic motor pattern in this system is first laid down by motoneuron growth cones choosing the appropriate projection pathways, rather than by regressive events involving retraction of erroneously projecting axon collaterals and/or cell death. Generally similar conclusions have been made for motoneurons innervating the chick wing (Hollyday, i983a, b), mouse facial (Ashwell and Watson, 1983) and limb (Lance-Jones, 1982a) muscles, and bullfrog hindlimbs (Fare1 and Bemelmans, 1985). In contrast to these studies, several laboratories have reported substantial initial projection errors that appear to be removed by motoneuron cell death. These range from Lamb’s (1976) observation that a moderate number of the earliest outgrowing Xenopus motoneurons project to a clearly inappropriate site in the limb to the description of Pettigrew et al. (1979) of widespread projection errors in the chick wing. Since these observations were based primarily on the retrograde labeling of motoneurons following horseradish peroxidase (HRP) injections into the uncleaved primary muscle masses, it was not possible to determine whether they resulted from errors in pathway selection or from growth of motoneurons over potential boundaries between muscles within the limb. In addition, they are subject to interpretive difficulties, since it is difficult to rule out leakage of HRP within the small (several hundreds of micrometers) uncleaved primary muscle masses of the vertebrate limb (Landmesser, 1980). It is important to resolve these apparently contradictory observations, since they bear directly upon the mechanisms by which specific projections are formed in this system. We therefore decided to investigate the behavior of segmentally identified populations of chick lumbosacral motoneurons within the primary muscle masses from their time of entry (st 26) until the completion of muscle cleavage and the onset of motoneuron cell death (st 30). By injecting HRP into single segments of the lateral motor column (Lance-Jones and Landmesser, 198la), it is possible to completely label a number of motoneurons in which the segment of origin can be unambiguously identified and in which growth cones can be clearly visualized within the limb. It is thus possible to characterize in some detail the behavior of segmentally identified growth cones at different stages of outgrowth into the limb. Our observations relating to the specific projection of motoneuron growth cones within muscle nerves and their target muscles are presented here. The subsequent paper (Tosney and Landmesser, 1985a) presents more detailed quantitative observations on the morphology of growth cones during their outgrowth into the limb.