Homeodomain TFs regulate Ig genes expressed in embryonic MNs.Figure 5(A, B) Analysis of gene classes associated with the homeodomain TF clusters. For each gene in the dataset, normalized, scaled expression in single cells was modelled as a function of homeodomain TF cluster identity, and significance of the association was determined using an F-test. Panel (A) depicts the number of genes with significant homeodomain TF cluster association falling into distinct gene classes. p-values were retrieved by a Fisher test. Panel (B) contrasts the p-values for homeodomain TF cluster association between Ig domain genes and other genes using a boxplot. See Methods, section Data visualization for a definition of boxplot elements. P values are from a two-sided Wilcoxon test. ***: p<0.005. (C) Single cells were grouped into 60 clusters according to their expression of genes encoding homeodomain proteins (Fig 2A) and arranged along the inferred AP position (Fig 1D, 1E). For each cluster, the mean expression of Ig encoding genes was computed. Heatmap depicts Ig encoding genes in columns and clusters in rows; mean expression is color-coded and median expression level is visualized by circle size. Gene families are highlighted in color codes. The colored bars highlight the Ig genes expressed in anterior MNs (MN2c, MN2a, MN4, MN3). (D) Quantification of Ig gene expression changes after interference with Dfd expression in neuronal cells (elav>DfdRNAi) by qPCR. Two genes, DIPgamma and DIPkappa, were selected as potential Dfd targets based on their co-expression in Dfd positive MNs. The bar chart shows the fold enrichment after normalization against Act5C of the Dfd and Ig genes in control versus elav>DfdRNAi conditions from three biological replicates, and shows that Dfd and DIP-kappa expression are significantly reduced in elav>DfdRNAi embryos, whereas the expression of the homeodomain TF gene exd is unchanged (internal control). p-values were calculated from a t-test: *<0.05, **<0.01, ns non-significant. Error bar denotes standard deviation. (E) HCR for DIPgamma and DIPkappa transcripts in Drosophila stage 17 embryos. RNA expression of both genes is analyzed in control (elav>GFPRNAi) versus Dfd depleted (elav>DfdRNAi) animals, the ventral nerve cord is highlighted by a white dashed line, the Dfd expressing subesophageal ganglion (SOG) is indicated by a yellow circle. (F) Venn diagram displaying an overlap of Ig domain genes expressed in MNs and genes that are bound by Dfd based on the Dfd ChIP-Seq performed in a whole embryo (Sorge et al, 2012). p-value was calculated using a hypergeometric test using as a reference all protein coding genes n=13920. (G) Venn diagram displaying the overlap of Ig domain genes expressed in MNs that are bound by Ubx identified in a neuronal tissue specific ChIP-Seq (Domsch et al, 2019). p-value was calculated as described in (F). (H) iRegulon analysis was used to identify TF motifs enriched in the vicinity of Ig encoding genes. 3 of the top 15 highest- ranked motifs of TFs are shown; the predicted targets of these motifs are homeodomain TFs. (I) Schematic drawing of the relationship between the homeodomain and the Ig molecule clusters in a single neuron.