Fig. 4

Mouse Ebf1 and Ebf2 rescue MN differentiation and locomotory defects in nematodes lacking unc-3. a Phylogenetic tree generated using Phylogeny.fr [54,55,56]. b Schematic showing the cell body position of ventral nerve cord MNs (labeled with green) in C. elegans. Forty [40] of these MNs are cholinergic and express UNC-3. The expression of two cholinergic MN markers (acr-2/AChR, unc-17/VAChT) is dramatically reduced in unc-3 mutant animals carrying loss-of-function alleles (e151, n3435). Transgenic expression of mEbf1 [Punc-3::mEbf1::unc-54 3’ UTR] or mEbf2 [Punc-3::mEbf2::unc-54 3’ UTR] significantly restores expression of acr-2/AChR and unc-17/VAChT in MNs of unc-3 mutants. Two transgenic lines per construct were used. A representative image of one rescuing line is shown. The pharynx is gfp positive because a pharyngeal promoter (myo-2) fused to gfp was used as a co-injection marker to distinguish transgenic from non-transgenic worms (see Methods). Fifteen animals at larval stage 4 (L4) were imaged for each genotype. Note that the fragment of unc-3 promoter (558 bp) used for rescue drives expression of mEbf1 or Ebf2 in ~ 30 MNs of the C. elegans ventral nerve cord. Expression of acr-2/AChR or unc-17/VAChT in these 30 MNs is significantly affected in unc-3 (−) mutant nematodes, and partially restored when either mEbf1 or mEbf2 is provided using the unc-3558 bp MN-specific promoter. c Quantification of the results shown in panel B. Two transgenic lines (indicated with a and b) per rescuing construct were quantified. Student’s t-test was performed. N = 15. *: p < 0.05, **: p < 0.01, ***: p < 0.001. Statistical comparison between WT and MN > mEbf1/2 lines indicates partial rescue. d Thrashing assay shows that the movement defects of unc-3 mutant nematodes can be significantly restored upon MN-specific expression of mEbf1 or mEbf2. Statistical comparison between WT and MN > mEbf1/2 lines indicates partial rescue. Two day-old adult nematodes were used. N = 10. Student’s t-test was performed. *: p < 0.05, **: p < 0.01, ***: p < 0.001