| A properly functioning nervous system requires the establishment of accurate neural connections, which are formed during embryonic and early postnatal development. One of the challenges in understanding nervous system development has been to determine how a limited number of signaling pathways can set up the patterning of a very complex nervous system. Wnts are a family of secreted proteins, initially characterized as morphogens during embryonic patterning (Rijsewijk et al., 1987; McMahon and Moon, 1989). However, they are also highly expressed in the developing and adult nervous system, and recent studies identify surprisingly versatile roles for Wnts in axon pathfinding, axonal branching, and synapse formation (Hall et al., 2000; Krylova et al., 2002; Lyuksyutova et al., 2003; Li et al., 2009). Our laboratory recently found that Wnt5a, expressed in peripheral sympathetic neurons, regulates sympathetic axonal growth, branching, and target innervation (Bodmer et al., 2009). However, Wnt5a is also highly expressed in some peripheral targets, raising the question of the source of Wnt5a involved in target innervation. In addition, previous studies were performed on Wnt5a mutants, which have severe tissue patterning defects that complicate the analysis of innervation patterns. To resolve these issues and to determine whether target derived or neuronal sources of Wnt5a are required for final target innervation, we generated a mouse line in which the Wnt5a gene can be conditionally deleted, by flanking exon 2 with loxP sequences (Wnt5af/f). To enhance the efficiency of excision of Wnt5a gene by Cre recombinase, we mated our Wnt5a conditional knockout mouse to Wnt5a null line so that only one copy of the floxed allele had to be recombined (Wnt5f/–). This line was then further mated to a Cre line. Unlike the global Wnt5a knockout mice, Wntl-Cre;Wnt5af/– mice, where Wnt5a was excised in the progenitors of peripheral neurons, had normal anterior-posterior patterning. In addition, initial axon outgrowth from sympathetic ganglia and axon navigation to the target tissues was normal. However, compared to wild-type littermates, the axons in Wntl-Cre; Wnt5af/– mice failed to undergo secondary and tertiary branching upon reaching their targets. Similar axon branching deficits were observed when a sympathetic neuron-specific Cre line was mated to the Wnt5a conditional mice, eliminating the possibility of contributions from glial Wnt5a in final target innervation. These defects were not due to deficits in proliferation, noradrenergic specification, or survival of sympathetic neurons. Dorsal root ganglia (DRG) sensory neurons exhibited a similar role for nerve-derived Wnt5a in target innervation. Thus, the use of conditional Wnt5a mutant mice reveals an in vivo role for neuron-derived Wnt5a in regulating peripheral nerve projections, in the absence of any overt patterning deficits.;In collaboration with the Greenberg lab, we identified atypical single-pass transmembrane receptor tyrosine kinases, Rors, as the likely Wnt5a receptors that mediate sympathetic axon branching. Rors are expressed in sympathetic neurons during the developmental period when their axons are reaching final targets, and loss of Ror 1 and 2 in sympathetic neurons results in similar arborization deficits as observed in Wnt1-Cre;Wntf/– mice. Using compartmentalized neuronal cultures, we demonstrate that the Ror receptor tyrosine kinases are required locally in sympathetic axons to mediate Wnt5a-dependent branching. Together, our study reveals an autocrine Wnt5a-Ror signaling pathway that directs sympathetic axon branching during target innervation. |
