Fig. 5: LIN-39 and MAB-5 control unc-3 expression levels.
From: Maintenance of neurotransmitter identity by Hox proteins through a homeostatic mechanism
a ChIP-seq tracks for LIN-39 and MAB-5 on unc-3 locus. Molecular nature of endogenous unc-3 reporters (UNC-3::GFP and UNC-3::GFP LIN-39 site MUT) and transgenic unc-3 reporters (unc-3_223bp::mChe, unc-3_ΔExon3-11_mChe, unc-3_ΔExon7-11_mChe). b Representative images of L4 animals carrying the endogenous unc-3::gfp reporter in WT, lin-39(n1760), and lin-39(n1760); mab-5(e1239) backgrounds. c Quantification of MN number (left graph, n = 19–22 animals) or fluorescence intensity (middle graph, n = 595 neurons) of UNC-3::GFP in WT, lin-39(n1760), and lin-39(n1760); mab-5(e1239) backgrounds. Right graph: Quantification of fluorescent GFP intensity in MNs of animals carrying the UNC-3::GFP and UNC-3::GFP LIN-39 site MUT endogenous reporters. n = 682 neurons. d Quantification of the number of unc-3 mRNA fluorescence aggregates in individual cholinergic MNs. Single-molecule mRNA FISH for unc-3 was performed in WT and lin-39(n1760); mab-5(e1239) animals at L1. For all quantifications, box and whisker plots were used; all data points shown. Unpaired t-test with Welch’s correction was performed and p-values were annotated. n = 22 animals. Representative images of DB5, DA5, DB7, and DA7 neurons. WT (colored red) versus mutant (in blue) for quantification. Magenta: signal from Quasar 670 probe for unc-3 mRNA, Green: unc-17::gfp reporter for cholinergic motor neurons. e Representative images of unc-3_223bp::mChe expression in L4 hermaphrodites in WT, lin-39(n1760), and lin-39(n1760); mab-5(e1239). f and g Quantification of MN numbers (f, n = 13–17 animals) or fluorescence intensity (g, n = 176 neurons) of unc-3_223bp::mChe in panel (e). For fluorescence intensity quantifications, single neurons expressing the mCherry reporters are plotted as individual data points. n > 600. In panel g, n > 200 neurons. h and i Quantification of the number of MNs expressing the unc-3_ΔExon3-11_mChe (h), and unc-3_ΔExon7-11_mChe (i) in WT and lin-39(n1760); mab-5(e1239) mutant animals. n = 10 animals. For all quantifications, box and whisker plots were used; all data points were presented. Box boundaries indicate the 25th and 75th percentile. The limits indicate minima and maxima values, whereas center values (mean) are highlighted in red. Unpaired t-test (two-sided) with Welch’s correction was performed. j Schematic model of a coherent feed-forward loop (FFL) for the control of cholinergic identity. Source data are provided as a Source data file. Scale bars: 5 μm.
