(E) Validation of the specific expression of the eight homeodomain TFs (Lab, Dfd, Scr, VVl, Lim1, Hth, Unc4 and Mirr) in MN2c, MN2a, MN4 and MN3 by immunohistochemistry or in situ HCR. For expression analysis, proteins or transcripts were detected with specific antibodies (Lab, Dfd, Scr, Hth, Vvl) or HCR probes (Lim1, unc4) using wild-type embryos. Mirr expression was detected by GFP antibody stainings using a GFP fusion line (mirr::GFP). The stereotypic position of MN2c, MN4, MN2a and MN3 was identified by the two axon projections A and B (labelled by FasII), which invariantly innervate the MHE (projection A) and MHD (projection B) muscles in wild-type embryos. MN2c and MN4 are the two MNs adjacent to MN2a. The four MNs are highlighted by yellow circles. 