Molecular mechanisms of neuronal circuit formation in the developing nervous system.
Zusammenfassung der Projektergebnisse
During development, axons project to their targets in a step-wise manner in response to attractive and repulsive cues. Their trajectories are broken into short segments, which terminate in specialized regions that form choice points for growing axons. Typically, there is a tight regulation of spatial and temporal patterns of guidance cue expression along axon paths, as well as of the expression of corresponding neuronal receptors. Even though many guidance cues have been identified, we are still far from a comprehensive understanding how complex and accurate neuronal circuitry is established. Major obstacles are presented by the fact that (i) the cues mediating specific growth decisions are still unknown for many choice points and (ii) the fiber projections are often evaluated in isolation without taking neighboring tracts into consideration. However, in vivo, growth cone decisions depend on the interactions of a complex network of neuronal and nonneuronal cells, a multitude of simultaneously detected guidance cues, and the instantaneous integration of all of these inputs. We addressed these challenges by a genome-wide screen to identify differentially expressed factors in sensory neurons that are controlling the dorsal-ventral choice in the limb (Specific Aim 1). In a second project, we used mouse genetic tools to selectively ablate either sensory or motor neurons or remove specific guidance cue receptors in either neuronal subtype and then studied the formation of axon trajectories (Specific Aim 2). The genome-wide expression screen revealed a number of interesting candidate genes whose expression patterns were then studied by in situ hybridization. Overall, the differential expression levels seen by the chip analyses were confirmed for a number of brachial genes. However, the specificity and the differences in dorsal/ventral expression patterns were not as prominent as initially hoped for. As both, the construction of vectors for electroporation and the generation of transgenic mice had at this time not yielded the required tools for a functional analysis, it was decided to not pursue this further at this point, but rather concentrate the available resources on the projects associated with Specific Aim 2. We set out to clarify the respective influence and contribution of sensory and motor axons to the correct establishment of both type of projections and to characterize the molecular nature of the communication between these two axonal populations. The axon guidance receptor neuropilin- 1 (Npn-1) is expressed on all motoneurons and in sensory neurons of the DRG and is therefore in a position to mediate axon-axon interactions. Using cell-type specific removal of Npn-1 from motoneurons, we found a dramatic defasciculation of motor projections to the limb and a significant reduction in the distal advancement. Contradicting earlier chick experiments, the formation of the sensory trajectory appeared completely normal. The disorganized motor projections also caused defects in the dorsal/ventral pathfinding decision. Deletion of Npn-1 from sensory neurons caused a severe defasciculation of the sensory innervation of fore- and hindlimbs. Surprisingly, motor projections were also defasciculated. Our analyses revealed a probable mechanism for the sensory influence on motor axons: Motoneuron-specific deletion of Npn-1 resulted in pronounced defasciculation of motor axons within the plexus region. In contrast, elimination of Npn-1 from sensory neurons caused pronounced defasciculation and spreading over a wide area of sensory and motor projections before these axons reach the plexus as well as in the plexus region. These data suggest that the state of sensory axon fasciculation before entering the plexus region influences motor axon fasciculation after the plexus. We corroborated these data with experiments where sensory or motoneurons were completely removed. Further, our data indicate that the fasciculation and growth patterns of sensory projections are independent of motor axons in the plexus region and distal limb, provided that these axons reach the plexus in a correctly fasciculated manner. These results also underscore the crucial role of Npn-1-signaling for the sorting and selective fasciculation of sensory and motor axons of the vertebrate limb prior to these projections arriving at the important early choice point, the plexus region.
Projektbezogene Publikationen (Auswahl)
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(2011). Cranial nerve fasciculation and Schwann cell migration are impaired after loss of Npn-1. Developmental Biology 15:230-41
Huettl, R.E., Huber, A.B.
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(2011). Npn-1 contributes to axonaxon interactions that differentially control sensory and motor innervation of the limb. PLoS Biology 9(2):e1001020
Huettl, R, Bianchi, E., Soellner, H., Novitch, B., Huber, A.B.
