Figure  S1:  Deletion  of  Dlx3  in  the  NC  A)   LacZ   staining   of   Dlx3LacZ/WT  mice   (a)   and  Wnt1-­‐cre:R26RLacZ  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:  Dlx3F/WT  (1),  Dlx3F/LacZ   (2),  Wnt1-­‐cre:Dlx3F/WT  (3),  Wnt1-­‐cre:Dlx3F/LacZ   (4).  Four  Dlx3  alleles  are   indicated:   Dlx3WT,   Dlx3LacZ,   Dlx3F   and   the   recombined   allele   Dlx3ΔF.   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:Dlx3F/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 -7.72Dspp NM_010080 0.001147 -7.34Dlx3 NM_010055 0.000002 -7.30Sprr1b NM_009265 0.035011 -6.43Lce1f NM_026394 0.000310 -6.24Krt1 NM_008473 0.022765 -5.26Lce1d NM_027137 0.007985 -4.78Hrnr NM_133698 /// XM_001000796 /// XM_984724 0.035920 -4.44Krt10 NM_010660 0.004838 -4.22Lce1b NM_026822 0.009005 -4.08Mmp20 NM_013903 0.004371 -4.03Lce1a1 NM_025984 0.010656 -3.99Gm94 NM_001033280 0.038235 -3.73Mpzl2 NM_007962 0.000961 -3.60Lyg1 NM_027111 0.034709 -3.41Lce1a2 NM_028625 0.004032 -3.09Lce1a1 NM_025984 0.024455 -2.93Flg XM_001000758 /// XM_001481265 /// XM_485270 0.026064 -2.89Il1f5 NM_001146087 /// NM_001146088 /// NM_019451 0.015698 -2.81Lce1i NM_029667 0.012667 -2.622310002L13Rik XM_128949 /// XM_915395 0.039570 -2.60Tmf1 NM_001081111 0.005235 -2.44Sost NM_024449 0.012198 -2.36Calb1 NM_009788 0.010896 -2.33Klk4 NM_019928 0.001776 -2.31Sprr2h NM_011474 0.041030 -2.29Mepe NM_053172 0.002882 -2.25Enam NM_017468 0.036129 -2.14Krt27 NM_010666 0.005842 -2.13Ptprr NM_001161838 /// NM_001161839 /// NM_001161840 /// NM_011217 0.005624 -2.13Calb1 NM_009788 0.045299 -2.09Serpina9 NM_027997 0.029029 -2.09Acpp NM_019807 /// NM_207668 0.009995 -2.08Calb1 NM_009788 0.018902 -2.08Steap1 NM_027399 0.008801 -2.06Cfd NM_013459 0.009094 -2.04Krt16 NM_008470 0.027709 -2.04Kprp NM_028629 0.012107 -2.02Gng4 NM_010317 0.006333 -2.01Sox21 NM_177753 0.012771 -2.01Krt24 NM_029393 0.019577 -2.00Ckmt1 NM_009897 0.000570 -1.98Hal NM_010401 0.001750 -1.95Aifm3 NM_175178 0.001571 -1.95Clu /// LOC100046120 NM_013492 /// XM_001475611 0.024306 -1.94Mpzl2 NM_007962 0.001446 -1.92Lama3 NM_010680 /// XM_140451 /// XM_981828 0.000270 -1.90Lamb3 NM_008484 0.000258 -1.90Gchfr NM_177157 0.000275 -1.90Gal3st4 NM_001033416 0.032670 -1.89Tspan8 NM_001168679 /// NM_001168680 /// NM_146010 0.007306 -1.86Papln NM_130887 0.008531 -1.86Msi2 NM_054043 /// XM_001471946 0.004237 -1.85Calb1 NM_009788 0.028094 -1.81Thy1 NM_009382 0.005229 -1.81Acpp NM_019807 /// NM_207668 0.007314 -1.80Slurp1 NM_020519 0.008174 -1.79Lce1c /// Lce1f /// Lce1g NM_025413 /// NM_026394 /// NM_028622 0.002190 -1.79Gng4 NM_010317 0.029845 -1.78Aplp1 NM_007467 0.006357 -1.78Ckmt1 NM_009897 0.000990 -1.78Mpzl2 NM_007962 0.001377 -1.78Fam84a NM_029007 0.011010 -1.77Fetub NM_001083904 /// NM_001083905 /// NM_021564 0.000247 -1.77Grik1 NM_010348 /// NM_146072 0.000185 -1.76H2-K1 NM_001001892 0.021524 -1.76Rsad1 NM_001013381 0.000585 -1.75H2afv NM_029938 /// XM_001476068 /// XM_907680 0.000727 -1.75Bglap /// Bglap-rs1 /// Bglap2 NM_001032298 /// NM_001037939 /// NM_007541 /// NM_031368 0.003405 -1.74Ildr2 NM_001164528 /// XM_001473525 /// XM_001479680 0.010476 -1.74Magi2 NM_001170745 /// NM_001170746 /// NM_015823 0.000033 -1.71Fgf1 NM_010197 0.004570 -1.71Pmch NM_029971 0.007400 -1.71H2afv NM_029938 /// XM_001476068 /// XM_907680 0.001879 -1.71Clu /// LOC100046120 NM_013492 /// XM_001475611 0.026030 -1.71Taf15 NM_027427 0.000697 -1.71Rgs4 NM_009062 0.000735 -1.71Ctsw NM_009985 0.000669 -1.70Acpp NM_019807 /// NM_207668 0.003879 -1.70Myo5b NM_201600 0.025634 -1.69Gabrb2 NM_008070 0.017538 -1.69Gabrb2 NM_008070 0.016715 -1.68Clu /// LOC100046120 NM_013492 /// XM_001475611 0.034895 -1.68Lipc NM_008280 0.000936 -1.68Plekhh1 NM_181073 /// XM_126961 /// XM_904513 0.002187 -1.68Nebl NM_028757 0.001861 -1.67Galnt12 NM_172693 0.010051 -1.66Lrrc8e NM_028175 0.014589 -1.66Bnc1 NM_007562 0.036475 -1.65Aplp1 NM_007467 0.032885 -1.65Cxcl14 NM_019568 0.041994 -1.65Pla2g5 NM_001122954 /// NM_011110 0.000591 -1.64

Sfn NM_018754 0.049839 -1.64LOC100044106 /// Plxnc1 NM_018797 /// XM_001472518 0.029062 -1.63Dtna NM_010087 /// NM_207650 0.036984 -1.63Ildr2 NM_001164528 /// XM_001473525 /// XM_001479680 0.000674 -1.63Cxadr NM_001025192 /// NM_009988 0.001416 -1.63Frmd5 NM_172673 0.001229 -1.62Acer1 NM_175731 0.018036 -1.61Sp6 NM_031183 0.041276 -1.61Tulp2 NM_001045555 /// NM_008807 0.000031 -1.61Msx2 NM_013601 0.000166 -1.61Cadps2 NM_153163 0.004795 -1.60Celsr1 NM_009886 0.029236 -1.60Acpl2 NM_153420 0.001383 -1.59Ankrd6 NM_001012450 /// NM_001012451 /// NM_080471 0.003669 -1.59Slc20a2 NM_011394 0.000180 -1.59LOC100047138 /// Tesc NM_021344 /// XM_001477488 0.009799 -1.59Sp6 NM_031183 0.045256 -1.58Cyp26a1 NM_007811 0.007596 -1.58D3Bwg0562e NM_177664 0.000397 -1.58Chdh NM_001136240 /// NM_172264 /// NM_175343 0.017189 -1.58Chst1 NM_023850 0.018296 -1.57Sema3e NM_011348 0.020516 -1.57Ovol1 NM_019935 0.043176 -1.57Dhcr24 NM_053272 0.016472 -1.56H2-D1 /// H2-K1 /// LOC100044874 NM_001001892 /// NM_010380 /// XM_001473540 /// XM_001473561 0.024496 -1.56Fbln7 NM_024237 0.006508 -1.56Ctnnd2 /// LOC100045979 NM_008729 /// XR_032172 0.040127 -1.56St8sia1 NM_011374 0.025821 -1.55St8sia1 NM_011374 0.037737 -1.55Satb1 NM_001163630 /// NM_001163631 /// NM_001163632 /// NM_009122 0.042313 -1.54Amtn NM_027793 /// XM_284166 /// XM_904684 0.021539 -1.54Ddx6 NM_001110826 /// NM_007841 /// NM_181324 0.000792 -1.54Ccdc109b NM_025779 0.008994 -1.545430407P10Rik NM_001159657 /// NM_144883 0.001612 -1.54Vangl2 NM_033509 0.021963 -1.54Bmp7 NM_007557 0.018306 -1.53Ndrg1 NM_008681 0.008842 -1.53AI504432 NR_033498 /// XR_035122 /// XR_035125 0.001009 -1.53Ahnak2 NM_001033476 /// XM_001475946 0.048309 -1.53Sft2d3 NM_026006 /// XM_484715 /// XM_907118 0.025226 -1.52LOC100047579 /// Tmem20 NM_175507 /// XM_001478437 0.002710 -1.52Fgfr1 NM_001079908 /// NM_001079909 /// NM_010206 0.004277 -1.52Gpr115 NM_030067 /// XM_001476141 /// XM_894986 0.012733 -1.52Dsc3 NM_007882 0.025952 -1.51Kcnk2 NM_001159850 /// NM_010607 /// NR_027627 0.025121 -1.51Dner NM_152915 0.007201 -1.51Tspan7 NM_019634 0.011323 -1.50Col17a1 NM_007732 0.013710 -1.50Itgb8 NM_177290 0.005183 1.51Cdc42ep3 NM_026514 0.000991 1.51Rps25 NM_024266 0.004597 1.52Mbp NM_001025245 /// NM_001025251 /// NM_001025254 /// NM_001025255 0.017621 1.53Cuedc1 NM_001172099 /// NM_198013 0.001388 1.53Galnt3 NM_015736 /// XM_885274 /// XM_980400 0.024536 1.53Kcnq1ot1 NR_001461 0.027648 1.53Rasgrp2 NM_011242 0.012318 1.53Fzd8 NM_008058 0.015919 1.53Insig2 NM_133748 /// NM_178082 0.044400 1.54Gas5 NR_002840 0.036613 1.55Mia1 NM_019394 0.024064 1.55LOC100046044 /// Nr2f1 NM_010151 /// XM_001475459 0.021194 1.57Sorbs1 NM_001034962 /// NM_001034963 /// NM_001034964 /// NM_009166 0.005546 1.59Mbp NM_001025245 /// NM_001025251 /// NM_001025254 /// NM_001025255 0.009162 1.59Agt NM_007428 0.009661 1.61Tm6sf1 NM_145375 0.045524 1.61Slc25a34 NM_001013780 0.044000 1.62Matn3 NM_010770 0.005050 1.62Cytl1 NM_001081106 0.043218 1.63Hapln1 NM_013500 0.025343 1.67Ihh NM_010544 0.002968 1.69F5 NM_007976 0.020700 1.69Tpm3 NM_022314 0.048015 1.70Adamts18 NM_172466 0.003134 1.72Lgals2 NM_025622 0.031390 1.72Comp NM_016685 0.011603 1.75Kif5b NM_008448 0.010564 1.77Myh7 NM_080728 0.009848 1.78Tpm3 NM_022314 0.048843 1.78Kcnma1 NM_010610 0.004060 1.84Myl3 NM_010859 0.015917 1.87Slc30a2 NM_001039677 0.002411 1.89Mbp NM_001025245 /// NM_001025251 /// NM_001025254 /// NM_001025255 0.003170 2.14Myl3 NM_010859 0.006910 2.16Chad NM_007689 0.005633 2.27Tns3 NM_001083587 0.000420 2.27Myl3 NM_010859 0.006451 2.50Expi NM_007969 0.036893 2.51Hba-a1 /// Hba-a2 NM_001083955 /// NM_008218 0.000041 2.53Mbp NM_001025245 /// NM_001025251 /// NM_001025254 /// NM_001025255 0.001805 2.66Gm6293 /// Uqcr10 NM_197979 /// XM_886587 0.000086 2.81Dcpp1 NM_019910 0.021356 3.17Gm6293 /// Uqcr10 NM_197979 /// XM_886587 0.000045 7.99Dcpp1 /// Dcpp2 /// Dcpp3 /// LOC100046800 NM_001039238 /// NM_001077633 /// NM_019910 /// XM_001475821 0.005344 16.68