Apple us22617 Supplemental Information An Ensemble of Regulatory Elements Controls Runx3 Spatiotemporal Expression in Subsets of Dorsal Root Ganglia Proprioceptive Neurons Supplemental _Fig _ S1 is related to Fig. 1 Supplemental _Fig _ S2 is related to Fig. 1 Supplemental _Fig _ S3 is related to Fig. 1 Supplemental _Fig _ S4 is related to Fig. 2 Supplemental _Fig _ S5 is related to Figs. 3, 5 Supplemental _Fig _ S6 is related to Figs. 3, 5 Supplemental _Fig _ S7 is related to Fig. 3 Supplemental _Fig _ S8 is related to Fig. 6 Supplemental _Fig _ S9 is related to Fig. 7 Supplemental _ Table_ S1 is related to Fig. 1 Supplemental _ Table_ S2 is related to Fig. 1 Supplemental _ Table_ S3 is related to Fig. 1 Supplemental _ Table_ S4 is related to Fig. 1 Supplemental _ Table_ S5 is related to Material and methods Supplemental _ Table_ S6 is related to Fig. 7 Supplemental _ Table_ S7 is related to Fig. 7 Supplemental_ Movie_ S1.mp4 is related to Fig. 3 Supplemental_ Methods.pdf 1 Supplemental Figures 2 Supplemental Fig. S1. The overlapping BACs –C, -E and -A encompass the genomic region required for the established Runx3 expression Sagittal sections of E14.5 BAC transgenic and Runx3LacZ/LacZ knock-in embryos depicting LacZ expression. BAC-B, -D and -F, completely lacked expression in hematopoietic tissues and in DRG and exhibited faint expression at all other sites (Supplemental Table S1). Despite the fact that BAC-B contains both P1 and P2, and BACs -D and -F contain only P2, the expression pattern of the three BACs was highly similar. BACs -A, -C and -E that extend 5’upstream of Runx3 exhibited a more intense LacZ expression in the same organs as BACs -B, -D and -F (Supplemental Table S1), indicating that the region upstream of P1 contains REs that intensify Runx3 expression in these tissues. These BACs also exhibited LacZ expression in hematopoietic organs, with BAC-A conferring the highest reporter expressing in the liver and thymus. Upper panels show embryos outer side and lower panels show inner side. AFP GFP Supplemental Fig. S2. Schematic representation of the Runx3 gene with P1 and P2 specific KI alleles 3 Runx3 gene (upper line) was modified to generate the two KI alleles; P1-driven AFP KI allele (P1AFP, middle line) and P2-driven GFP KI allele (P2GFP, lower line). 4 Supplemental Fig. S3. Strategy for generation of compound heterozygote mutant mice (A) CRISPR-mediated deletion of R1,2,3 REs. Blue arrows underneath the line indicate position of sgRNAs while the shears mark Cas9 cutting sites (see also Supplemental Table S4). (B) Diagnostic PCR for detection of R1,2,3 REs homozygous deletion. PCR primers were designed to detect WT internal R1 and R3 fragments (lanes 1 and 3) and their absence in the mutant (lanes 2 and 4). R3/R1 PCR reaction detecting the deleted allele is seen in mutant (lane 7) but not in WT (lane 6). Lane 5 shows MW markers (see PCR oligos in Supplemental Table S5). (C) Schematic representation of compound heterozygote mice carrying one Runx3 allele bearing CRISPR-deleted REs (CRΔR1,2,3) and a second P2GFP Runx3 null allele. 5 Supplemental Fig. S4. Differential REs Activity in BAC transgenics at E12.5 (A) GFP expression in TrkC/Runx3 neurons: shown are intact BAC-C and various BAC-C RE-deleted brachial DRG. (B) Histograms showing percentage of TrkC/Runx3 neurons expressing BAC-derived GFP. BAC-C-GFP (Ct) and its indicated deleted versions. *p<0.005, **p<0.0001. 6 Supplemental Fig. S5. Histograms showing the results of counting TrkC neurons in serial sections of E15.5 DRG (A) Total number of Runx3 expressing TrkC/GFP cells in C5-T1 DRGs of: WT/P2GFP (marked as Ct), ΔR1= (CRΔR1/P2GFP), ΔR2= (CRΔR2/P2GFP), ΔR3= (CRΔR3/P2GFP), R1= (CRΔR2,3/P2GFP), R2= (CRΔR1,3/P2GFP), R3= (CRΔR1,2/P2GFP), Data were obtained by counting the total number of TrkC/GFP neurons in serial sections of an entire ganglion (n=2-4 embryos of either control or each mutant). *p<0.05, **p<0.001, ***p<0.0002. (B) Total number of TrkC/GFP cells in C5-T1 DRGs acquired by counting the same set of embryos as indicated in (A). 7 Supplemental Fig. S6. Histograms showing the proportion of TrkC neurons in various RE-deleted brachial DRGs (A) Percentage of Runx3 expressing TrkC/GFP cells present in RE-mutant compared to WT/P2GFP embryos. Data was deduced from the cell counting presented in Supplemental Fig. S5A (average mutant cell number/average WT/P2GFPx100). *p<0.05, **p<0.001, ***p<0.0002. (B) Percentage of TrkC/GFP cells present in mutant compared to WT/P2GFP embryos. Data was deduced from the cell counting presented in Supplemental Fig. S5B. 8 Supplemental Fig. S7. The DRG REs also regulate Runx3 expression in the trigeminal ganglion Expression of endogenous Runx3, TrkC and GFP in trigeminal ganglion at E14.5. Panels (from left to right): CRΔR1,2,3/P2GFP, CRΔR1,3/P2GFP, CRΔR1/P2GFP. 9 Supplemental Fig. S8. Deletion of R2 evokes GFP expression in TrkB neurons of BAC transgenics (A) Expression of Runx3, TrkB and GFP in BAC-C and (B) in BAC-C-ΔR2. Panels in (A) or (B) from left to right: transgenic embryos at E11, E11.5, E12.5, and E14.5. (C) Histograms showing percentage of Runx3 expressing TrkB neurons in E11-E14.5 WT embryos. (D) Histograms showing percentage of GFP expressing TrkB neurons in E11-E14.5 BAC-C-ΔR2 transgenic embryos. *p< 0.0001 compared to WT (C) at E11.5, E12.5 and E14.5. (E) Expression of endogenous Runx3, TrkB and GFP in CRΔR2/P2GFP E12.5 embryos. 10 Supplemental Fig. S9. Gene expression analysis discovers TrkC-neuron specific Runx3-responsive genes that were previously presumed to be Brn3a-targets RNA-seq was conducted on TrkC neurons of E11.5 P2GFP/+ and P2GFP/GFP embryos (see Supplemental Materials and methods). This early developmental stage was selected because at this stage the number of TrkC neurons is relatively high. FACS-sorted P2GFP/+ neurons express significantly higher level of Runx3 compared to Runx1 or Runx2 as well as a higher level of Ntrk3 compared to Ntrk1 and Ntrk2 (Supplemental Table S7), thereby providing strong indication for the TrkC identity of the neurons used for RNA-seq analysis. Data analysis revealed pronounced gene expression changes between P2GFP/+ and P2GFP/GFP neurons (Supplemental Table S7), underscoring the central role of Runx3 in TrkC neuron homeostasis. Interestingly, Ingenuity upstream regulator analysis revealed that several previously reported Brn3a (Pou4f1) regulated genes behave as Runx3-responsive genes. Namely, similarly to their response in Brn3a-mutant those genes were either upregulated (marked in pink) or downregulated (marked in green) in P2GFP/GFP neurons (lacking Runx3) compared to P2GFP/+ neurons. These findings are consistent with the observation that in TrkC neurons, Brn3a is an upstream regulator of Runx3. 11 Supplemental Table S1 Expression pattern of BACs surrounding Runx3 The detailed expression pattern of the six BACs: BAC-A, -B, -C, -D, -E, -F, listed in the Table according to their position in the murine genome starting from the 5’-end, and a Runx3 LacZ knock-in line (Runx3 KI). Original BAC names are indicated in brackets (see UCSC browser; mm9). The intensity of expression was evaluated qualitatively and is marked by + to +++. No expression is marked by a minus (-). The numbers in brackets indicate the number of positively-stained embryos out of the total number of embryos analyzed for each BAC. BAC E BAC A BAC B BAC F BAC D Runx3 BAC C Organ BAC (RP23- (RP24- (RP24- (RP24- (RP23- KI (RP23-394B) 307D6) 118B14) 252E9) 180O8) 57P18) Thymus ++ + (2/9) ++ (1/7) ++ (6/12) - (9/9) - (5/5) - (8/8) Liver ++ +/- (3/9) + (1/7) + (6/12) - (9/9) ++ (1/5) - (8/8) Eyelid mesenchyme + ++ (8/9) ++ (7/7) +++ (12/12) ++ (3/9) + (4/5) + (5/8) Tongue – mesenchymal element ++ ++ (9/9) ++ (7/7) +++ (11/12) + (7/9) + (5/5) + (8/8) of filiform papillae DRG Cervical ++ ++ (8/9) ++ (4/7) + (1/12) - (9/9) - (5/5) +/- (1/8) Brachial ++ ++ (8/9) +/- (5/7) +/- (4/12) - (9/9) - (5/5) +/- (1/8) Thoracic + ++ (8/9) +/- (5/7) +/- (8/12) - (9/9) - (5/5) +/- (2/8) Lumbar ++ ++ (7/9) + (5/7) +/- (6/12) - (9/9) - (5/5) +/- (1/8) Coccygeal (Tail) ++ ++ (9/9) ++ (5/7) + (7/12) - (9/9) - (5/5) +/- (1/8) Hair follicles + ++ (7/9) ++ (7/7) ++ (11/12) + (2/9) + (3/5) +/- (3/8) Whiskers upper ++ + (8/9) ++ (7/7) +++ 10/12) + (2/9) +/- (4/5) - (8/8) Whiskers lower + +++ (9/9) +++ (7/7) +++ 12/12) ++ (6/9) ++ (5/5) ++ (8/8) Inner Epithelium of the ear + + (1/9) - (7/7) + (1/12) + (2/9) +/- (3/5) + (2/8) Teeth mesenchyme + + (4/9) + (6/7) + (9/12) - (9/9) ++ (1/5) +/- (1/8) Nails upper limbs ++ +++ (8/9) +++ (7/7) +++ (11/12) + (3/9) + (4/4) - (8/8) Nails lower limbs ++ +++ (8/9) ++ (7/7) +++ (11/12) + (2/9) + (2/4) - (8/8) Nose mesenchyme ++ +++ (9/9) +++ (7/7) +++ (12/12) ++ (8/9) ++ (5/5) ++ (7/8) Penis ++ +++ (9/9) +++ (7/7) +++ (10/10) + (4/5) ++ (4/5) + (3/5) Upper limb cartilage Scapula ++ +++ (9/9) +++ (7/7) +++ (11/12) + (6/9) ++ (5/5) ++ (8/8) Humerus ++ +++ (9/9) +++ (7/7) +++ (11/12) + (6/9) ++ (5/5) ++ (8/8) Radius ++ +++ (9/9) +++ (7/7) +++ (12/12) + (6/9) ++ (5/5) ++ (8/8) Ulna ++ +++ (9/9) +++ (7/7) +++ (12/12) + (6/9) ++ (5/5) ++ (8/8) Carpus - - (9/9) +/- (1/7) - (12/12) - (9/9) - (5/5) - (8/8) Metacarpus + +++ (7/9) +++ (6/7) +++ (12/12) +/- (4/9) ++ (4/5) ++ (8/8) Digits P1 + +++ (4/9) +++ (5/7) +++ (12/12) +/- (1/9) + (4/5) ++ (8/8) Digits P2 - +++ (3/9) ++ (4/7) +++ (9/12) - (9/9) +/- (3/5) ++ (6/8) Lower limb cartilage Femur ++ +++ (9/9) +++ (7/7) +++ (12/12) + (6/9) ++ (5/5) ++ (8/8) Tibia ++ +++ (9/9) +++ (7/7) +++ (12/12) + (6/9) ++ (5/5) ++ (8/8) Fibula ++ +++ (9/9) +++ (7/7) +++ (12/12) + (6/9) ++ (5/5) ++ (8/8) Tarsus - +/- (2/9) +/- (5/7) + (11/12) - (9/9) + (4/5) + (2/8) Metatarsus + +++ (7/9) +++ (6/7) +++ (12/12) +/- (3/9) ++ (5/5) ++ (8/8) Digits P1 + +++ (3/9) +++ (5/7) +++ (12/12) - (9/9) ++ (4/5) ++ (7/8) Digits P2 - - (9/9) + (2/7) +++ (9/12) - (9/9) - (5/5) +++ (4/8) Ribs ++ +++ (9/9) +++ (7/7) +++ (11/12) + (5/9) ++ (5/5) + (8/8) Vertebra cartilage ++ +++ (9/9) +++ (7/7) +++ (11/12) + (5/9) ++ (5/5) + (8/8) 12 Supplemental Table S2 A list of candidate cis-regulatory elements in the murine Runx3 locus (chr4:134,953,991- 135,208,237; mm10).