Figure S1: Deletion of Dlx3 in the NC A) LacZ staining of Dlx3 LacZ/WT mice (a) and Wnt1- cre:r26r LacZ mice (b) at E11.5. The arrowhead points at Dlx3 expression in the branchial arches. B) PCR and Southern blot analysis of genomic DNA from the head of E11.5 embryos: Dlx3 F/WT (1), Dlx3 F/LacZ (2), Wnt1- cre:dlx3 F/WT (3), Wnt1- cre:dlx3 F/LacZ (4). Four Dlx3 alleles are indicated: Dlx3 WT, Dlx3 LacZ, Dlx3 F and the recombined allele Dlx3 Δ F. C) qpcr analysis of Dlx3 mrna levels in the head of E11.5 embryos. T- test, n=3.
Figure S2: Dlx3 expression in the developing root Dlx3 expression during radiculogenesis of the first molar at P15. Immunohistochemical analysis using anti- Dlx3 antibody on molar section. Black arrows indicate odontoblasts. Black arrowheads indicate cementoblasts. White arrowheads indicate the HERS. DP, dental pulp; D, dentin; B, bone; MC, Meckel s cartilage. Scale bar 100 µm.
Figure S3: Ameloblast differentiation and enamel formation in Wnt1- cre:dlx3 F/LacZ mice A) Immunohistochemical analysis of Dlx3 expression in mandibular incisors (cross sections) from WT and cko mice at P5, showing that Dlx3 remains expressed in the ameloblasts of cko mice. B) Frontal section of incisor from cko mouse at P15. Ameloblast (Am) followed their normal life- cycle in cko mice, from presecretion to secretion to maturation stage shown here. The structure of ameloblasts was normal, evidencing a ruffled border (arrowheads). The respective morphology of external aprismatic (eae), external prismatic (epe) and internal prismatic (ipe) enamel was not affected. Prism decussation was not altered (horizontal arrows). Scale 100 µm. C) Scanning electron microscopy analysis of the enamel of mandibular incisors (maturation zone) from WT and cko mice at 8 weeks. B, bone; D, dentin; E, enamel. D) Scanning electron microscopy of first mandibular molars from WT and cko mice at 8 weeks.
Figure S4: Primary mandibular cell culture and Dlx3 expression A) Phase contrast observation of cells isolated from mandibles at P0 used for ChIP- seq analysis. The mixed population of cells includes: osteoblasts (a), cells from the dental epithelium (b), and cells from the dental mesenchyme (c). B) RT- PCR analysis of the expression of Rs15 and Dlx3 in primary cells isolated from mouse mandibles at P0.
Table S1: Microarray analysis of mandibles from Wnt1-cre:Dlx3F/LacZ (cko) and WT mice Gene Symbol RefSeq Transcript ID p-value(cko vs. WT) Fold-Change(cKO vs. WT) Lce1c NM_028622 0.001433-7.72 Dspp NM_010080 0.001147-7.34 Dlx3 NM_010055 0.000002-7.30 Sprr1b NM_009265 0.035011-6.43 Lce1f NM_026394 0.000310-6.24 Krt1 NM_008473 0.022765-5.26 Lce1d NM_027137 0.007985-4.78 Hrnr NM_133698 /// XM_001000796 /// XM_984724 0.035920-4.44 Krt10 NM_010660 0.004838-4.22 Lce1b NM_026822 0.009005-4.08 Mmp20 NM_013903 0.004371-4.03 Lce1a1 NM_025984 0.010656-3.99 Gm94 NM_001033280 0.038235-3.73 Mpzl2 NM_007962 0.000961-3.60 Lyg1 NM_027111 0.034709-3.41 Lce1a2 NM_028625 0.004032-3.09 Lce1a1 NM_025984 0.024455-2.93 Flg XM_001000758 /// XM_001481265 /// XM_485270 0.026064-2.89 Il1f5 NM_001146087 /// NM_001146088 /// NM_019451 0.015698-2.81 Lce1i NM_029667 0.012667-2.62 2310002L13Rik XM_128949 /// XM_915395 0.039570-2.60 Tmf1 NM_001081111 0.005235-2.44 Sost NM_024449 0.012198-2.36 Calb1 NM_009788 0.010896-2.33 Klk4 NM_019928 0.001776-2.31 Sprr2h NM_011474 0.041030-2.29 Mepe NM_053172 0.002882-2.25 Enam NM_017468 0.036129-2.14 Krt27 NM_010666 0.005842-2.13 Ptprr NM_001161838 /// NM_001161839 /// NM_001161840 /// NM_011217 0.005624-2.13 Calb1 NM_009788 0.045299-2.09 Serpina9 NM_027997 0.029029-2.09 Acpp NM_019807 /// NM_207668 0.009995-2.08 Calb1 NM_009788 0.018902-2.08 Steap1 NM_027399 0.008801-2.06 Cfd NM_013459 0.009094-2.04 Krt16 NM_008470 0.027709-2.04 Kprp NM_028629 0.012107-2.02 Gng4 NM_010317 0.006333-2.01 Sox21 NM_177753 0.012771-2.01 Krt24 NM_029393 0.019577-2.00 Ckmt1 NM_009897 0.000570-1.98 Hal NM_010401 0.001750-1.95 Aifm3 NM_175178 0.001571-1.95 Clu /// LOC100046120 NM_013492 /// XM_001475611 0.024306-1.94 Mpzl2 NM_007962 0.001446-1.92 Lama3 NM_010680 /// XM_140451 /// XM_981828 0.000270-1.90 Lamb3 NM_008484 0.000258-1.90 Gchfr NM_177157 0.000275-1.90 Gal3st4 NM_001033416 0.032670-1.89 Tspan8 NM_001168679 /// NM_001168680 /// NM_146010 0.007306-1.86 Papln NM_130887 0.008531-1.86 Msi2 NM_054043 /// XM_001471946 0.004237-1.85 Calb1 NM_009788 0.028094-1.81 Thy1 NM_009382 0.005229-1.81 Acpp NM_019807 /// NM_207668 0.007314-1.80 Slurp1 NM_020519 0.008174-1.79 Lce1c /// Lce1f /// Lce1g NM_025413 /// NM_026394 /// NM_028622 0.002190-1.79 Gng4 NM_010317 0.029845-1.78 Aplp1 NM_007467 0.006357-1.78 Ckmt1 NM_009897 0.000990-1.78 Mpzl2 NM_007962 0.001377-1.78 Fam84a NM_029007 0.011010-1.77 Fetub NM_001083904 /// NM_001083905 /// NM_021564 0.000247-1.77 Grik1 NM_010348 /// NM_146072 0.000185-1.76 H2-K1 NM_001001892 0.021524-1.76 Rsad1 NM_001013381 0.000585-1.75 H2afv NM_029938 /// XM_001476068 /// XM_907680 0.000727-1.75 Bglap /// Bglap-rs1 /// Bglap2 NM_001032298 /// NM_001037939 /// NM_007541 /// NM_031368 0.003405-1.74 Ildr2 NM_001164528 /// XM_001473525 /// XM_001479680 0.010476-1.74 Magi2 NM_001170745 /// NM_001170746 /// NM_015823 0.000033-1.71 Fgf1 NM_010197 0.004570-1.71 Pmch NM_029971 0.007400-1.71 H2afv NM_029938 /// XM_001476068 /// XM_907680 0.001879-1.71 Clu /// LOC100046120 NM_013492 /// XM_001475611 0.026030-1.71 Taf15 NM_027427 0.000697-1.71 Rgs4 NM_009062 0.000735-1.71 Ctsw NM_009985 0.000669-1.70 Acpp NM_019807 /// NM_207668 0.003879-1.70 Myo5b NM_201600 0.025634-1.69 Gabrb2 NM_008070 0.017538-1.69 Gabrb2 NM_008070 0.016715-1.68 Clu /// LOC100046120 NM_013492 /// XM_001475611 0.034895-1.68 Lipc NM_008280 0.000936-1.68 Plekhh1 NM_181073 /// XM_126961 /// XM_904513 0.002187-1.68 Nebl NM_028757 0.001861-1.67 Galnt12 NM_172693 0.010051-1.66 Lrrc8e NM_028175 0.014589-1.66 Bnc1 NM_007562 0.036475-1.65 Aplp1 NM_007467 0.032885-1.65 Cxcl14 NM_019568 0.041994-1.65 Pla2g5 NM_001122954 /// NM_011110 0.000591-1.64
Sfn NM_018754 0.049839-1.64 LOC100044106 /// Plxnc1 NM_018797 /// XM_001472518 0.029062-1.63 Dtna NM_010087 /// NM_207650 0.036984-1.63 Ildr2 NM_001164528 /// XM_001473525 /// XM_001479680 0.000674-1.63 Cxadr NM_001025192 /// NM_009988 0.001416-1.63 Frmd5 NM_172673 0.001229-1.62 Acer1 NM_175731 0.018036-1.61 Sp6 NM_031183 0.041276-1.61 Tulp2 NM_001045555 /// NM_008807 0.000031-1.61 Msx2 NM_013601 0.000166-1.61 Cadps2 NM_153163 0.004795-1.60 Celsr1 NM_009886 0.029236-1.60 Acpl2 NM_153420 0.001383-1.59 Ankrd6 NM_001012450 /// NM_001012451 /// NM_080471 0.003669-1.59 Slc20a2 NM_011394 0.000180-1.59 LOC100047138 /// Tesc NM_021344 /// XM_001477488 0.009799-1.59 Sp6 NM_031183 0.045256-1.58 Cyp26a1 NM_007811 0.007596-1.58 D3Bwg0562e NM_177664 0.000397-1.58 Chdh NM_001136240 /// NM_172264 /// NM_175343 0.017189-1.58 Chst1 NM_023850 0.018296-1.57 Sema3e NM_011348 0.020516-1.57 Ovol1 NM_019935 0.043176-1.57 Dhcr24 NM_053272 0.016472-1.56 H2-D1 /// H2-K1 /// LOC100044874 NM_001001892 /// NM_010380 /// XM_001473540 /// XM_001473561 0.024496-1.56 Fbln7 NM_024237 0.006508-1.56 Ctnnd2 /// LOC100045979 NM_008729 /// XR_032172 0.040127-1.56 St8sia1 NM_011374 0.025821-1.55 St8sia1 NM_011374 0.037737-1.55 Satb1 NM_001163630 /// NM_001163631 /// NM_001163632 /// NM_009122 0.042313-1.54 Amtn NM_027793 /// XM_284166 /// XM_904684 0.021539-1.54 Ddx6 NM_001110826 /// NM_007841 /// NM_181324 0.000792-1.54 Ccdc109b NM_025779 0.008994-1.54 5430407P10Rik NM_001159657 /// NM_144883 0.001612-1.54 Vangl2 NM_033509 0.021963-1.54 Bmp7 NM_007557 0.018306-1.53 Ndrg1 NM_008681 0.008842-1.53 AI504432 NR_033498 /// XR_035122 /// XR_035125 0.001009-1.53 Ahnak2 NM_001033476 /// XM_001475946 0.048309-1.53 Sft2d3 NM_026006 /// XM_484715 /// XM_907118 0.025226-1.52 LOC100047579 /// Tmem20 NM_175507 /// XM_001478437 0.002710-1.52 Fgfr1 NM_001079908 /// NM_001079909 /// NM_010206 0.004277-1.52 Gpr115 NM_030067 /// XM_001476141 /// XM_894986 0.012733-1.52 Dsc3 NM_007882 0.025952-1.51 Kcnk2 NM_001159850 /// NM_010607 /// NR_027627 0.025121-1.51 Dner NM_152915 0.007201-1.51 Tspan7 NM_019634 0.011323-1.50 Col17a1 NM_007732 0.013710-1.50 Itgb8 NM_177290 0.005183 1.51 Cdc42ep3 NM_026514 0.000991 1.51 Rps25 NM_024266 0.004597 1.52 Mbp NM_001025245 /// NM_001025251 /// NM_001025254 /// NM_001025255 0.017621 1.53 Cuedc1 NM_001172099 /// NM_198013 0.001388 1.53 Galnt3 NM_015736 /// XM_885274 /// XM_980400 0.024536 1.53 Kcnq1ot1 NR_001461 0.027648 1.53 Rasgrp2 NM_011242 0.012318 1.53 Fzd8 NM_008058 0.015919 1.53 Insig2 NM_133748 /// NM_178082 0.044400 1.54 Gas5 NR_002840 0.036613 1.55 Mia1 NM_019394 0.024064 1.55 LOC100046044 /// Nr2f1 NM_010151 /// XM_001475459 0.021194 1.57 Sorbs1 NM_001034962 /// NM_001034963 /// NM_001034964 /// NM_009166 0.005546 1.59 Mbp NM_001025245 /// NM_001025251 /// NM_001025254 /// NM_001025255 0.009162 1.59 Agt NM_007428 0.009661 1.61 Tm6sf1 NM_145375 0.045524 1.61 Slc25a34 NM_001013780 0.044000 1.62 Matn3 NM_010770 0.005050 1.62 Cytl1 NM_001081106 0.043218 1.63 Hapln1 NM_013500 0.025343 1.67 Ihh NM_010544 0.002968 1.69 F5 NM_007976 0.020700 1.69 Tpm3 NM_022314 0.048015 1.70 Adamts18 NM_172466 0.003134 1.72 Lgals2 NM_025622 0.031390 1.72 Comp NM_016685 0.011603 1.75 Kif5b NM_008448 0.010564 1.77 Myh7 NM_080728 0.009848 1.78 Tpm3 NM_022314 0.048843 1.78 Kcnma1 NM_010610 0.004060 1.84 Myl3 NM_010859 0.015917 1.87 Slc30a2 NM_001039677 0.002411 1.89 Mbp NM_001025245 /// NM_001025251 /// NM_001025254 /// NM_001025255 0.003170 2.14 Myl3 NM_010859 0.006910 2.16 Chad NM_007689 0.005633 2.27 Tns3 NM_001083587 0.000420 2.27 Myl3 NM_010859 0.006451 2.50 Expi NM_007969 0.036893 2.51 Hba-a1 /// Hba-a2 NM_001083955 /// NM_008218 0.000041 2.53 Mbp NM_001025245 /// NM_001025251 /// NM_001025254 /// NM_001025255 0.001805 2.66 Gm6293 /// Uqcr10 NM_197979 /// XM_886587 0.000086 2.81 Dcpp1 NM_019910 0.021356 3.17 Gm6293 /// Uqcr10 NM_197979 /// XM_886587 0.000045 7.99 Dcpp1 /// Dcpp2 /// Dcpp3 /// LOC100046800 NM_001039238 /// NM_001077633 /// NM_019910 /// XM_001475821 0.005344 16.68