Fig. 2

Muscle pioneers express netrin 1a along the HMS prior to and during PAC formation and are required for PAC formation. (A-B2) mRNA in situ hybridization (ISH) shows netrin 1a expression in MPs and neural tube (NT) at 22 and 32 hpf. (B2) Transverse section of zebrafish embryo in B at the level indicated. (C-H) The muscle pioneers (MPs) are the critical source of Netrin 1a for PAC formation. Treatment with 50 µM cyclopamine prevents nearly all MP formation and leads to failure of PAC formation. 48 hpf fli1a:egfp^y1 embryos antibody stained for vessels (GFP, green) and MPs (4D9 anti-engrailed, magenta). Confocal projections. (C-E) In DMSO-treated control embryos, MPs are present at the HMS (D) and the PAC forms normally (C, arrowheads). (F-H) In cyclopamine-treated embryos, very few MPs form (G) and the PAC is missing (F, arrowheads). (I) PAC formation was assessed in hemisegments 7-11 in DMSO-treated (97±2% complete; 3±2% partial; 0±0% absent) and cyclopamine-treated (21±6% complete; 15±4% partial; 64±7% absent) embryos. All values are mean±s.e.m.; P-value determined by Mann-Whitney U test comparing absent PACs. n, number of hemisegments (number of embryos). A, anterior; D, dorsal; NT, neural tube. Scale bars: 200 μm in A-B2 50 μm in C-H.