Lili Jing’s research while affiliated with University of Pennsylvania and other places

One of the earliest events in neuromuscular junction (NMJ) development is the accumulation of acetylcholine receptor (AChR) at the center of muscle cells. The unplugged/MuSK (muscle specific tyrosine kinase) gene is essential to initiate AChR clustering but also to restrict approaching growth cones to the muscle center, thereby coordinating pre- and postsynaptic development. To determine how unplugged/MuSK signaling coordinates these two processes, we examined the temporal and spatial requirements of unplugged/MuSK in zebrafish embryos using heat-shock inducible transgenes. Here, we show that despite its expression in muscle cells from the time they differentiate, unplugged/MuSK activity is first required just prior to the appearance of AChR clusters to simultaneously induce AChR accumulation and to guide motor axons. Furthermore, we demonstrate that ectopic expression of unplugged/MuSK throughout the muscle membrane results in wildtype-like AChR prepattern and neuromuscular synapses in the central region of muscle cells. We propose that AChR prepatterning and axonal guidance are spatio-temporally coordinated through common unplugged/MuSK signals, and that additional factor(s) restrict unplugged/MuSK signaling to a central muscle zone critical for establishing mid-muscle synaptogenesis.

(A) Lateral views of 27-hpf wildtype, unplugged and Tg(hsp70l:unplugged SV1-myc); unpluggedbr307/br307 embryos stained for motor neurons (znp-1). Motor axons in wildtype embryos extend into the ventral myotome after the choice point (arrowheads). unplugged motor axons form lateral branches (arrows), or stall (data now shown) at the choice point. Axonal pathfinding defects were rescued in Tg(hsp70l:unplugged SV1-myc); unpluggedbr307/br307 embryos after the appropriate HS treatment. (B) Quantification of motor axonal phenotypes. Embryos were heat-shocked from the 10-somite stage to 27 hpf. HS treatment did not have obvious effect on motor axon pathfinding in wildtype and unplugged embryos (columns 1–3). In the absence of HS treatment, motor axons remain disrupted in Tg(hsp70l:unplugged FL-myc); unpluggedbr307/br307 (column 4) and Tg(hsp70l:unplugged SV1-myc); unpluggedbr307/br307 (data not shown) embryos. After HS treatment, motor axons were significantly rescued in transgenic embryos (columns 5–6). 20 hemisegments in each embryo were scored. Results are expressed as the average of multiple embryos (n≥20). (0.54 MB TIF)

(A-D) Representative images for the AChR prepattern rescue in 21-somite Tg(hsp70l:unplugged SV1-myc); unpluggedbr307/br307 embryos after HS treatments. Embryos were stained for motor neurons (znp-1, green) and AChRs (α-BTX, red). Segments 11-15 were imaged in each embryo. (E-H) Tg(hsp70l:unplugged SV1-myc); unpluggedbr307/br307 embryos were heat-shocked for 40 minutes at the 20-somite stage and examined at the 21-somite stage for motor neurons and AChRs. AChR prepatterning was not well rescued in segments 11-15 (E and G). Posterior segments 16-20 from the same embryo display better rescue of the prepatterning (F and H). (G and H) Enlarged images of the highlighted segments in E and F. Arrow in (H) indicate the large wildtype-like prepatterned clusters. Arrowheads in (G) mark the small punctate clusters. Scale bars: 20 µM. (I-L) Rescue of neural synapses at 48hpf by Tg(hsp70l:unplugged FL-myc). Embryos were heat-shocked for 40 minutes at 48hpf, fixed at 51hpf and stained for motor axons (znp-1, green) and AChR clusters (α-BTX, red). (I and J) Neural synapses induced by heat shock treatment of Tg(hsp70l:unplugged FL-myc); unpluggedbr307/br307 embryos persisted for 3 hours following the heat shock. (K and L) No neural synapses were induced in unpluggedbr307/br307 embryos lacking the transgene. Arrows in I and K point to rescued and non-rescued synapses respectively with insets showing enlarged views. (1.07 MB TIF)

Tg(hsp70l:unplugged SV1-myc); unpluggedbr307/br307 embryos were heat-shocked starting at the indicated times points, and examined at 27 hpf for motor axon pathfinding. Results were summarized from one experiment, analyzed as in Figure 3A, and represented as mean±SEM. 20 hemisegments were scored in each embryo (n = 180−400, average = 328, hemisegments per time point). (0.14 MB TIF)

Early during neuromuscular development, acetylcholine receptors (AChRs) accumulate at the center of muscle fibers, precisely where motor growth cones navigate and synapses eventually form. Here, we show that Wnt11r binds to the zebrafish unplugged/MuSK ectodomain to organize this central muscle zone. In the absence of such a zone, prepatterned AChRs fail to aggregate and, as visualized by live-cell imaging, growth cones stray from their central path. Using inducible unplugged/MuSK transgenes, we show that organization of the central muscle zone is dispensable for the formation of neural synapses, but essential for AChR prepattern and motor growth cone guidance. Finally, we show that blocking noncanonical dishevelled signaling in muscle fibers disrupts AChR prepatterning and growth cone guidance. We propose that Wnt ligands activate unplugged/MuSK signaling in muscle fibers to restrict growth cone guidance and AChR prepatterns to the muscle center through a mechanism reminiscent of the planar cell polarity pathway.

Vertebrates display diverse patterns of neuromuscular innervation, but little is known about how such diversity is generated. In mammals, neuromuscular junctions form predominantly at equatorial locations, giving rise to a focal innervation pattern along a central endplate band. In addition, vertebrate striated muscles exhibit two nonfocal neuromuscular patterns, myoseptal and distributed innervation. Although agrin-MuSK-rapsyn signaling is essential for the focal innervation pattern, it is unknown whether the same genetic program also controls synaptogenesis at nonfocal innervation sites. Here we show that one of three transcripts generated by the zebrafish unplugged locus, unplugged FL, encodes the zebrafish MuSK ortholog. We demonstrate that UnpFL/MuSK is critical for the assembly of focal synapses in zebrafish and that it cooperates with dystroglycan in the formation of nonfocal myoseptal and distributed synapses. Our results provide the first genetic evidence that neuromuscular synapse formation can occur in the absence of MuSK and that the combinatorial function of UnpFL/MuSK and dystroglycan generates diverse patterns of vertebrate neuromuscular innervation. • muscle-specific kinase • neuromuscular junction • synaptogenesis