Fig 4

Fig 4

AB. In situ hybridization on E9.5 embryos, using Hoxa2 (A) and Hoxa3 (B) probes. A. Hoxa2 is highly expressed in the neural crest migrating from rhombomere 4 (asterisk) to the BA2 (arrow). The portion of neural crest migrating just below the otic vesicle (OV) into the BA3 (arrowhead) is also Hoxa2-positive. B. Hoxa3 is expressed in the BA3 (arrowhead). C. Distribution of HOXA2 peaks FE in BA2 and PBA. HOXA2 peaks in BA2 and PBA are ordered based on log2 FE and divided into quartiles. The central quartiles (50% of the data) are shown in the box, while top and bottom whiskers represent the top 25% and bottom 25% (the top and the bottom quartiles) of the data, respectively. HOXA2 peaks display a significantly higher FE in BA2 compared to PBA (p < 2.2e-16; one sided Welsh two sample t-test). D. Comparison of HOXA2 binding in BA2 (red bars) and PBA (green bars) by ChIP-qPCR. Enrichment of each region following immunoprecipitation with HOXA2 and IgG negative control antibody (Neg Ab) is calculated as percentage input; numbers indicate the corresponding FE values in HOXA2 ChIP-seq (BA2 and PBA). Peaks are labelled by their closest genes. Itih4 is a negative control (unbound region). Values represent the average of duplicate samples, and error bars indicate the SEM. E. HOX binding to Meis2 probe, alone and in combination. Co-translation of HOXA2 with PBX/MEIS and with or without HOXA3; the same applies to HOXA3. The highest HOX dose corresponds to 100%; HOXA2 (red) is translated at progressively decreasing levels, either alone, or co-translated with progressively increasing levels of HOXA3 (green) (e.g. 75% HOXA2; 75% HOXA2 + 25% HOXA3; 50% HOXA2; 50%HOXA2 + 50% HOXA3; 50% HOXA3 as indicated below the gel). At 50% and 25% levels, HOXA2 forms a significantly stronger trimeric complex (red arrow) on its own than when HOXA3 is added. PBX/MEIS dimeric complex, black arrow; HOXA3/PBX/MEIS trimeric, green arrow (see also S4C Fig). F. Luciferase activity driven by Meis2 and Zfp703 enhancers co-transfected with expression vector for Hoxa2 or Hoxa3, alone, or at diverse ratio of Hoxa2 to Hoxa3 (3:1; 2:2; 1:3) as indicated. All samples contain Meis2 and Pbx1a expression vectors; Hoxa2 and Hoxa3 are added as indicated. For both enhancers, luciferase activity decreases as Hoxa2 is progressively replaced by Hoxa3 (asterisks indicate significant difference relative to full dose of Hoxa2); for Meis2, enhancer activation is significantly higher with half dose of Hoxa2 alone, relative to Hoxa2 and Hoxa3 together. Values represent fold activation over basal enhancer activity and are presented as the average of at least two independent experiments, each performed in triplicate. Error bars represent the SEM. One-way ANOVA with post-hoc Tukey HSD test: * = p<0.01; ** = p<0.001.