Figure 2. The terminal selector UNC-3 is required for MN subclass diversification.
(A) Schematic showing subclass-specific gene expression in posterior MNs. (B–C) The expression of multiple subclass-specific markers (DA9: itr-1, glr-4, mig-13; VA12: mig-13, flp-18; DA1-7/DB: unc-129; VA1-11/VB: del-1) is dramatically affected in unc-3(e151) null mutant animals. Note that flp-18 expression in VA11 is unaffected in unc-3 null animals. Translational fusion reporters were used for glr-4 (glr-4 transl::gfp) and mig-13 (mig-13 transl::gfp), while promoter fragments were fused to gfp for itr-1, flp-18, unc-129, and del-1. Asterisks in B mark autofluorescence from the C. elegans intestinal cells. Numbers at top right of each image indicate the fraction of animals that showed marker expression in the respective MNs. Arrowheads in B and C mark MN subclasses (DA9, VA12, DA1-7 and VA1-11). In schematic shown in C, only DA, DB, VA, and VB MNs are color-coded. (D–E) Cis-regulatory mutational analysis is shown for glr-4 (expressed in DA9 and SAB neurons) and mig-13 (expressed in DA9 and VA12) genes. The glr-4 and mig-13 loci are schematized. Lines indicate genomic region fused to gfp (green) or tagrfp (red). (+) indicates robust reporter gene expression in the respective neurons. (±) indicates significant reduction in the number of neurons expressing the reporter when compared to transgenic animals carrying longer genomic fragments of the cis-regulatory region. (–) indicates complete loss or very faint expression in the respective neurons. Multiple transgenic lines were analyzed for each construct. At least twenty animals were analyzed per transgenic line. Detailed quantification of this analysis is provided on the right. Location of each COE motif (blue vertical lines) is presented as distance from ATG (+1). MUT indicates that COE motif has been mutated (shown as empty blue box) through substitution of 2 nucleotides in the core sequence (for example, COE wild-type site: TCCCNNGGGA >> COE MUT site: TGGCNNGGGA). ***p value < 0.001. Asterisk (*) on VNC in E indicates additional mig-13 expression in VNC MNs anterior to the vulva.