(a) GO analysis of all Pet1- and/or Lmx1b-maintained TACs identified from E14.5 DKO ATAC-seq datasets, depicted as volcano plot of enrichment (x-axis) vs. FDR statistical significance (y-axis), showing many synapse and axon biological processes among the top enriched annotations. (b) Percent of Pet1- and/or Lmx1b-regulated TACs that gain or lose accessibility (blue) or gain accessibility (red) in wildtype Pet1 neurons from E11.5 to E17.5. For example, 33% of all TACs are dynamic and 22% gain accessibility. In contrast, 62% of Lmx1b-regulated TACs are dynamic and 50% gain accessibility. (c) Genome browser tracks of gene-enhancer pairings, ATAC-seq, Pet1 and Lmx1b CUT&RUN, and histone modification ChIPmentation signals at synaptic genes Syt1 and Slc17a8. Tracks show the changes in chromatin accessibility between wildtype (red), Pet1-cKO (yellow), Lmx1b-cKO (yellow), and DKO (yellow) Pet1 neurons within the highlighted (blue) regions at E14.5. Display data ranges for the zoomed-out panels are optimized based on the blue highlighted regions. (d) Relative expression (FPKMs) of Syt1 and Slc17a8 in rostral Pet1-cKO, Lmx1b-cKO, and DKO neurons at E17.5. Data presented as mean ± SEM. *, FDR<.05. (e) Differential expression (from E11.5 to E17.5) of the genes associated with Pet1-occupied TACs in Pet1-cKO vs. wildtype 5-HT neurons (left), genes associated with Pet1-occupied TACs in Lmx1b-cKO vs. wildtype 5-HT neurons (middle), and genes associated with Pet1 and Lmx1b co-occupied TACs in DKO vs. wildtype 5-HT neurons (right). (f) TdTomato (red) and DAPI (blue) co-staining of pericellular baskets in the medial septum of P15 Pet1-Cre; Rosa26Ai9 wildtype, Pet1-cKO, Lmx1b-cKO, and DKO mice. (g) Diagram showing the analyzed brain area and quantification of TdTomato axons (pixels/μm2) (two-way ANOVA; n = 3 animals per genotype and 2 quantified brain sections per animal) (bottom panels). Images were generated through automatic stitching of individual 20× images. (h) Representative immunohistochemistry images of Syt1, TdTomato, and DAPI in the dorsolateral septum of P15 Pet1-Cre; Rosa26Ai9 mice showing the decreased overlap of Syt1 (green) and TdTomato (red) signals in KO animals compared to wildtype. Images were generated through automatic stitching of individual 63× images. (i) Representative immunohistochemistry images of vGlut3 (Slc17a8), TdTomato, and DAPI in the medial septum of adult Pet1-Cre; Rosa26Ai9 mice showing the decreased overlap of vGlut3 (green) and TdTomato (red) signals in KO animals compared to wildtype. Images were generated through automatic stitching of individual 63× images. (j) Quantification of Syt1 (left) and vGlut3 (right) co-localization with TdTomato in the medial septum as a percentage of the total TdTomato signal (two-way ANOVA; n = 2 animals per genotype and 3–4 quantified images per animal). *, P<.05; **, P<.01; ***, P<.001; ****, P<.0001 in panels e, g, j.