on the difficulty of increasing dental complexity

W W W. N A T U R E . C O M / N A T U R E | 1

SUPPLEMENTARY INFORMATIONdoi:10.1038/nature10876

Supplementary Figures

Supplementary Figure 1. Examples of single treatment concentrations tested to increase cusp number. Day 13+8 for ShhGFP and day 13+6 for ShhGFP;Eda-null images of cultured molars. Extra cusps indicated with arrowheads and fused second molar indicated with dashed line. Eda added for two days onto day 13 NF-kappaB-beta-galactosidase reporter molars15 show activation of Eda/Edar/NF-kappaB signalling. Anterior towards the left, buccal towards the top. Scale bars, 0.5 mm.

Supplementary Figure 2. Shh signalling inhibits cusp formation. a, Enamel knots are delayed and smaller in Shh-treated molars and the later developing posterior part, the talonid is inhibited. Cyclopamine-treated molars show new and closely packed cusps (arrowheads), and the fusion of the first and second molars (dashed line). Samples are ShhGFP (heterozygous for Shh) visible light photographs above, fluorescent light photographs below. b, The effects of Shh and cyclopamine are similar in wild type molars. Anterior towards the left, buccal towards the top. n = 10 in each group, for Shh treatments, the average cusp number is 4.4, s.d. is 0.5. Scale bar, 0.5 mm.

Supplementary Figure 3. Number of cusps as the function of number of adjusted pathways (number of treatments). First molar calculations on the left and combined first and second molar calculations on the right, bubble size represents number of teeth. Linear regressions are y = 2.31x + 4.675, r2 = 0.635, P < 0.001 (left) and y = 3.392x + 5.375, r2 = 0.682, P < 0.001 (right).

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2 | W W W. N A T U R E . C O M / N A T U R E

RESEARCH

Supplementary Figure 4. The effects of overexpression of Eda, Edar and compound Eda; Edar under K14 promoter analyzed using x-ray tomography reconstructions (3 µm voxel resolution) of adult mouse teeth. Conforming to a previous report17, the K14-Eda dentition frequently has an extra tooth (arrow), together with wider bucco-lingual separation of cusps and incipient central crests connecting cusps longitudinally (shown with an asterisk in the first molar). The K14-Edar dentition frequently (57 %) has an extra lingual cusp on the second molar (arrowhead), as reported previously34, but also the incipient development of two longitudinal crests connecting cusps (asterisks). The compound K14-Eda; K14-Edar dentition shows enhanced development of the longitudinal crests (asterisks) and a lingual extra cusp of the second molar is also present (88 %). The hypoconulid of the first molar is reduced and the second molar anteroconid cusps are taller. A unique feature of the compound teeth is the presence of a well-developed buccal cingulid (shown in the close-up, present in 63 % of specimens) with a cingular cusp on the first molar (arrowhead). A cingulid (called cingulum in upper molars) is a characteristic feature of the mammalian dentition and is also present in some murine rodents (e.g., Apodemus). K14-Eda, n = 36 (ref. 17); K14-Edar, n = 14; K14-Eda; K14-Edar, n = 8. All views are occlusal except for the bottom view of K14-Eda;K14-Edar which is obliquely buccal. Cracks caused by preparation have been removed from the reconstructions. Anterior towards the left, buccal towards the bottom. M1, first molar; M2, second molar; M3, third molar. Scale bars, 0.5 mm.

Supplementary Figure 5. An example x-ray microtomography slide before (left) and after (right) segmentation. Segmented stacks had 2 µm voxel resolution and the segmented area corresponds to the dental mesenchyme. Scale bar, 0.5 mm.


SUPPLEMENTARY INFORMATION RESEARCH

Supplementary Tables

Supplementary Table 1. Majority of single gene mutations affecting teeth cause the loss of teeth or cusps. Change in cusps tabulated for the lower first molar. Genes in bold have both gain- and loss-of-function mutations affecting molar development. Note that mutations can be partial or conditional. Double gene mutations are also known to affect teeth, predominantly causing developmental arrest. For detailed reviews of the genes and references, see refs 5 and 6. Possible increases are based on histological data alone with undetermined change in cusp number. M2, second molar.Gene Direction of mutation Change in cusps Feed-back inhibitorActivin BetaA Loss Loss, early developmental arrestActRIIA & ActRIIB Loss (double mutation) Loss, early developmental arrestBmpr1a Loss Loss, early developmental arrestDkk1 Gain Loss, early developmental arrest YesDlx1 & Dlx2 Loss (double mutation) Loss, early developmental arrestFgf8 Loss Loss, early developmental arrestFgfr2IIIb Loss Loss, early developmental arrestGli2 & Gli3 Loss (double mutation) Loss, early developmental arrestLef1 Loss Loss, early developmental arrestMsx1 Loss Loss, early developmental arrestp63 Loss Loss, early developmental arrestPax9 Loss Loss, early developmental arrestPitx2 Loss Loss, early developmental arrestRunx2 Loss Loss, early developmental arrestShh Loss Loss, early developmental arrestSmad2 Loss Loss, early developmental arrestBeta catenin Gain Reduction (but many teeth)Epiprofin Loss Reduction (but many teeth)Eda Loss ReductionEdar Loss ReductionFollistatin Loss Reduction YesIkappaBalfa Gain Reduction YesMsx2 Loss ReductionFgf3 Loss Pattern change (hypoconulid lost in M2, ref. 7)Pattern change (hypoconulid lost in M2, ref. 7)Follistatin Gain Pattern change YesIkkalpha Loss Pattern changeSmo Loss Pattern changeSpry2 Loss Pattern change YesSpry4 Loss Pattern change YesEda Gain Mild increase (≈1 cusps)Lrp4 Loss Mild increase (≈1 cusps)Sostdc1 Loss Mild increase (≈1 cusps) YesEdar Gain Possible increase (refs 34, 35)Jagged2 Loss Possible increase (ref. 36)



RESEARCH

Supplementary Table 2. Single treatment concentrations tested to increase cusp number. Samples were treated from E13 onwards for four days. All samples are ShhGFP heterozytes, with those so indicated also Eda-null. Molar areas were measured and cusp numbers counted on day eight for ShhGFP and on day six for ShhGFP;Eda-null. M1, first molar; s.d., standard deviation.Molecule Concentration Samples M1 cusps s.d. M1 area s.d.control 0 µg/ml 10 5.60 0.52 638976 119049

Eda 0.5 µg/ml 10 5.40 0.52 657835 83639Eda 0.75 µg/ml 4 5.25 0.50 523348 33473

Eda-null control 0 µg/ml 14 3.14 0.36 264562 51101

Eda on Eda-null 0.01 µg/ml 12 4.50 1.00 427998 78839

Eda on Eda-null 0.1 µg/ml 13 5.77 0.44 505560 80580

Eda on Eda-null 0.4 µg/ml 12 5.67 0.49 538851 104953Eda on Eda-null 1 µg/ml 13 5.62 0.65 525973 105105

Activin-A 0.1 µg/ml 6 6.33 0.52 658280 62201

Activin-A 0.2 µg/ml 6 6.33 0.82 577439 88383

Activin-A 0.5 µg/ml 10 7.80 1.23 531498 45356

Activin-A 0.75 µg/ml 10 7.90 1.37 499427 36701

Activin-A 3 µg/ml 6 8.67 2.80 380333 27748Activin-A 5 µg/ml 4 2.25 0.50 199812 52756

cyclopamine 0.5 µM 3 6.33 0.58 543327 71224

cyclopamine 1 µM 10 7.50 0.97 329637 79383

cyclopamine 2 µM 10 6.80 1.55 360658 67402

cyclopamine 4 µM 3 7.33 0.58 351696 14780

Supplementary Table 3. Summary statistics of treatment cultures. M1, first molar; M2, second molar; Total area, M1 + M2; s.d., standard deviation. Number of samples for each treatment was ten unpaired molars from at least three different litters (see Supplementary Table 4).

ControlControlControlEda

0.5 µg/mlEda

0.5 µg/mlEda

0.5 µg/mlActivin A 0.5 µg/mlActivin A 0.5 µg/mlActivin A 0.5 µg/ml

cyclopamine 2µM

cyclopamine 2µM

cyclopamine 2µMControlControlControl

Eda 0.5 µg/ml

Eda 0.5 µg/ml

Eda 0.5 µg/ml

Activin A 0.5 µg/mlActivin A 0.5 µg/mlActivin A 0.5 µg/ml

cyclopamine 2µM

cyclopamine 2µM

cyclopamine 2µM

Average s.d. Average s.d. Average s.d. Average s.d.

M1+M2 cusps 6.30 1.34 7.00 2.00 8.60 1.90 8.40 2.01

Cusps M1 (normals + extras) 5.60 0.52 5.40 0.52 7.80 1.23 6.80 1.55

M1 normal cusps (anteroconids counted as one) 5.60 0.52 5.30 0.48 5.30 0.48 4.70 0.82

Anteroconid duality in % 60.00 - 80.00 - 70.00 - 40.00 -

Hypoconulid presence in % 60.00 - 30.00 - 40.00 - 20.00 -

M1 extra cusps (if present) 0.00 0.00 0.10 0.32 2.40 1.17 2.33 0.87

M2 presence in % 30.00 - 70.00 - 40.00 - 100.00 -

M2 day of appearance (if M2 present) 4.67 - 6.14 - 4.00 - 4.50 -

M2 cusps (if M2 present) 2.33 0.58 2.57 1.51 2.25 0.96 2.30 0.82

M1-M2 fusion in % (if M2 present) 0.00 - 0.00 - 25.00 - 100.00 -

Total molar area (µm2) E13+8 days 669148 141629 741861 131154 575258 72925 443906 114407

M1 area (µm2) e13+8 days 638976 119049 657835 83639 531498 45356 360658 67402

M1 extra cusps area (if present) (µm2) e13+8 days 0 0 44739 0 95954 41364 97669 37357

M2 area (if present) (µm2) e13+8 days 30172 51475 120037 54320 109399 30397 83247 63544

First extra cusp day of appearance - - - - 4.70 0.48 4.22 0.67



Eda + Activin AEda + Activin AEda + Activin AActivin A +

cyclopamineActivin A +


cyclopamine Eda + cyclopamineEda + cyclopamineEda + cyclopamineEda + cyclopamine +

Activin AEda + cyclopamine +


Activin AEda + Activin AEda + Activin AEda + Activin AActivin A +



cyclopamine Eda + cyclopamineEda + cyclopamineEda + cyclopamineEda + cyclopamine +



Activin A

Average s.d. Average s.d. Average s.d. Average s.d.

M1+M2 cusps 11.50 1.51 13.10 1.52 13.50 2.27 15.20 2.10

Cusps M1 (normals + extras) 8.00 1.15 9.00 1.60 10.13 1.13 12.50 1.84

M1 normal cusps (anteroconids counted as one) 4.90 0.32 3.75 0.89 4.38 0.52 3.75 0.71

Anteroconid duality in % 80.00 - 12.50 - 0.00 - 0.00 -

Hypoconulid presence in % 0.00 - 0.00 - 0.00 - 0.00 -

M1 extra cusps (if present) 3.10 0.99 5.25 1.28 6.14 1.35 8.75 2.05

M2 presence in % 90.00 - 100.00 - 100.00 - 100.00 -

M2 day of appearance (if M2 present) 3.33 - 3.38 - 3.63 - 3.40 -

M2 cusps (if M2 present) 3.89 1.17 4.25 0.71 3.00 0.93 2.70 1.16

M1-M2 fusion in % (if M2 present) 100.00 - 100.00 - 100.00 - 100.00 -

Total molar area (µm2) E13+8 days 733707 158289 470364 36043 460225 54412 470239 70005

M1 area (µm2) e13+8 days 549127 86733 339562 33646 390009 29155 357562 52309

M1 extra cusps area (if present) (µm2) e13+8 days 143851 50032 195609 43058 204765 46988 240537 69684

M2 area (if present) (µm2) e13+8 days 205088 73165 132117 28494 96125 30594 110355 46709

First extra cusp day of appearance 4.00 0.47 3.90 0.32 3.50 0.53 3.40 0.52

Supplementary Table 4. Primary data used in cusp number and tooth size tabulations. M1, first molar; M2, second molar.

Measurements at E13 + 8 days in cultureMeasurements at E13 + 8 days in cultureMeasurements at E13 + 8 days in cultureMeasurements at E13 + 8 days in cultureTreatment Sample identity M1 cusps M1+M2 cusps M1 area (µm2) M1+M2 area (µm2)

Control 22.10.2009 A left 6 6 748638 74863822.10.2009 B left 6 8 751943 82889222.10.2009 D left 6 6 726356 72635622.10.2009 E left 6 9 672027 81415222.10.2009 F left 6 6 542837 54283724.03.2010 E left 5 5 564244 56424428.04.2010 C left 5 7 653175 73581828.04.2010 D right 6 6 786194 78619412.05.2010 E right 5 5 443103 44310326.05.2010 D right 5 5 501244 501244

Average 5.60 6.30 638976 669148Standard deviation 0.52 1.34 119049 141629

Eda 0.5 µg/ml 10.12.2008 B right 5 5 512499 51249910.12.2008 C right 5 9 792121 100900210.12.2008 D right 5 6 709417 76135930.3.2009 A right 6 5 638148 71931530.3.2009 D right 5 5 639790 63979030.3.2009 E right 5 6 767602 85414624.6.2009 A right 6 9 623686 75195024.6.2009 B right 5 9 580139 70441224.6.2009 C right 6 10 633198 78438324.6.2009 D right 6 6 681749 681749


Activin A 0.5 µg/ml 20.01.2010 C right 6 7 496553 56871725.05.2010 A left 8 8 575412 57541225.05.2010 B left 9 12 532402 67119225.05.2010 C right 9 11 541269 638925



RESEARCH

25.05.2010 D right 6 6 439480 43948025.05.2010 E left 8 8 486010 48601025.05.2010 F left 9 9 555438 55543826.05.2010 B left 7 10 534355 66334226.05.2010 C right 7 7 578416 57841626.05.2010 D right 9 8 575648 575648


cyclopamine 2 µM 14.7.2009 H left 4 8 499392 73809221.10.2009 A right 7 9 319968 37865624.3.2010 C left 7 5 340206 44213311.5.2010 A left 7 9 335359 38718811.5.2010 B right 6 9 313751 38367811.5.2010 F right 6 5 304015 34523711.5.2010 G left 6 8 300640 3623596.10.2010 C left 7 10 353836 4912856.10.2010 Q left 10 11 457754 4803646.10.2010 R left 8 10 381661 430063


Eda 0.5 µg/ml 22.10.2009 A right 8 13 625490 813372 + Activin A 0.5 µg/ml 22.10.2009 C right 7 13 608385 923142

22.10.2009 D right 8 11 626530 87599322.10.2009 E right 8 13 514997 80568122.10.2009 F right 9 13 704092 95007424.03.2010 D right 7 10 478476 64804624.03.2010 F left 6 9 424254 55719824.03.2010 G right 10 10 514726 51472628.04.2010 A right 9 12 493955 64303128.04.2010 B left 8 11 500365 605803


Activin A 0.5 µg/ml 6.10.2010 B right 11 14 369384 456028 + cyclopamine 2 µM 6.10.2010 G right 8 13 327804 495158

6.10.2010 I right ? 12 x 4759206.10.2010 J left 8 12 349312 4679056.10.2010 L left ? 13 x 4542846.10.2010 O right 8 13 330656 4474286.10.2010 P left 8 12 331278 4917016.10.2010 U left 9 13 357162 4715556.10.2010 V right 8 12 270927 4025876.10.2010 W left 12 17 379974 541073


Eda 0.5 µg/ml 6.10.2010 A left 10 12 346311 419016 + cyclopamine 2 µM 6.10.2010 C right 10 14 430853 544533

6.10.2010 I left 12 15 414079 5232756.10.2010 M right 9 11 373699 4716366.10.2010 N right 9 12 x 4261736.10.2010 O left 11 15 394153 4734206.10.2010 S left ? 18 x 4230586.10.2010 T right ? 12 403776 4037766.10.2010 V left 9 11 355580 396116



6.10.2010 W right 11 15 401624 521244Average 10.13 13.50 390009 460224Standard deviation 1.13 2.27 29154 54412

Eda 0.5 µg/ml 24.3.2010 D left 15 17 409400 471271 + Activin A 0.5 µg/ml 24.3.2010 F right 11 12 312080 378801 + cyclopamine 2 µM 6.10.2010 D left 12 14 312729 377125

6.10.2010 E right 13 17 x 5022816.10.2010 J right 13 16 356638 4734476.10.2010 L right 10 13 312243 4172056.10.2010 M left 11 13 323637 4228686.10.2010 S right 12 17 384140 5611386.10.2010 T left 16 18 449625 5374786.10.2010 U right 12 15 x 560777


Suplementary Table 5. Correlations (Spearman’s Rank Order) between number of molecules used and molar metrics. Note that even with the Bonferroni multiple comparison correction n = 6, the correlations remain significant. Pearson’s correlations provide equivalent results. M1, first molar; M2, second molar.

rs PCusp number M1 0.79 <0.001Cusp number M1+M2 0.85 <0.001

Crown area M1 -0.53 <0.001Crown area M1+M2 -0.33 0.003

Cusp density M1 0.77 <0.001Cusp density M1+M2 0.81 <0.001

References35. Tucker, A. S., Headon, D. J., Courtney, J.-M., Overbeek, P. & Sharpe, P. T. The

activation level of the TNF family receptor, Edar, determines cusp number and tooth number during tooth development. Dev. Biol. 268, 185–194 (2004).

36. Mitsiadis, T. A., Graf, D., Luder, H., Gridley, T. & Bluteau, G. BMPs and FGFs target Notch signalling via jagged 2 to regulate tooth morphogenesis and cytodifferentiation. Development 137, 3025–3035 (2010).

on the difficulty of increasing dental complexity

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