Figure S1. Palatal shelf elevation is affected along the anterior-posterior axis in Wnt1Cre;Erk2fl/fl mice. A-J. MicroCT scan sections of anterior, medial, and posterior regions of
newborn control and Wnt1-Cre;Erk2fl/fl mice. Arrow indicates the most affected side.
Arrowheads indicate the unelevated palatal shelf.
Development • Supplementary information
Development 142: doi:10.1242/dev.125328: Supplementary information
Development 142: doi:10.1242/dev.125328: Supplementary information
Figure S2. NCC migration, cell survival, and proliferation are unaffected in the palatal
mesenchyme of Wnt1-Cre;Erk2fl/fl mice. A-D. LacZ staining (blue) of coronal sections of
control and Wnt1-Cre;Erk2fl/fl;R26Rfl/fl embryos at E10.5 and E11.5. E-H. Active Caspase-3
(green) and BrdU immunostaining (brown) of E12.5 control and Wnt1-Cre;Erk2fl/fl palates.
Development • Supplementary information
Scale bars: 100 m. n = 3 for each stage and analysis.
Figure S3. Midline epithelium cell fate and anterior-posterior patterning are unaffected
in the palatal epithelium of Wnt1-Cre;Erk2fl/fl mice. A-B. Ki67 (green) and K14 (red)
double immunostaining of coronal sections of palatal explants from control and Wnt1Cre;Erk2fl/fl embryos after a 3-day incubation period. n = 4. C-D. Shh in situ hybridization in
the palatal shelves of E14.5 control and Wnt1-Cre;Erk2fl/fl embryos. n = 3.
Development • Supplementary information
Development 142: doi:10.1242/dev.125328: Supplementary information
Development 142: doi:10.1242/dev.125328: Supplementary information
Cre;Erk2fl/fl mice. A-F. Active Caspase-3 immunostaining (green) of coronal sections of
control and Wnt1-Cre;Erk2fl/fl tongues from E12.5 to E14.5. G. Quantification of apoptotic
cells from A-F. Scale bars: 100 m. n = 3 for each stage.
Development • Supplementary information
Figure S4. Apoptosis and proliferation are unaffected in the tongues of Wnt1-
Development 142: doi:10.1242/dev.125328: Supplementary information
Figure S5. Differentiation is unaffected in Wnt1-Cre;Erk2fl/fl tongues but muscle pattern
and fiber organization are altered. A-B. MHC immunostaining (green) of coronal sections
of E13.5 control and Wnt1-Cre;Erk2fl/fl tongues. C-D. MHC immunostaining of coronal
sections of E14.5 control and Wnt1-Cre;Erk2fl/fl tongues. A’-D’ and A”-D” are magnified
misorientation of the intrinsic muscles of the tongue.
Development • Supplementary information
images of the intrinsic muscles of the tongue at both stages. Arrow points to severe
Development 142: doi:10.1242/dev.125328: Supplementary information
Figure S6. Chondrogenesis is unaffected in the Meckel’s cartilage of Wnt1-Cre;Erk2fl/fl
mice. Immunostaining of Link protein (green; A-B, E-F) and in situ hybridization of Ctgf
(C-D, G-H) in the Meckel’s cartilage of E13.5 and E14.5 control and Wnt1-Cre;Erk2fl/fl
Development • Supplementary information
mice. n = 3 for each stage and analysis.
Development 142: doi:10.1242/dev.125328: Supplementary information
Figure S7. Population of osteogenic progenitors in Wnt1-Cre;Erk2fl/fl mandibles is
reduced. A-B. SP7 immunostaining (red) of mandibles in control and Wnt1-Cre;Erk2fl/fl mice
at E12.5. C-F. SP7 immunostaining (red) of mandibles in control and Wnt1-Cre;Erk2fl/fl mice
at E14.5 and E15.5. G-J. In situ hybridization of Type 1 Collagen in mandibles of control and
Wnt1-Cre;Erk2fl/fl mice at E14.5 and E15.5. Insets show low magnification. Scale bars: 200
Development • Supplementary information
m. n = 3 for each stage and analysis.
Development 142: doi:10.1242/dev.125328: Supplementary information
Movie 1. Comparison of control and Wnt1-Cre;Erk2fl/fl mice from E12.5 to E16.5 showing an
Development • Supplementary information
interconnection between the palate, tongue, and mandible.
Development 142: doi:10.1242/dev.125328: Supplementary information
Table S1. Quantification of the mandibular phenotype in Wnt1-Cre;Erk2fl/fl mice at different
stages. 72% of the cases were asymmetric. The presence, size, and continuity of the Meckel’s
cartilage and the osteogenic front were taken into account to determine the most affected
side.
Phenotype
E14.5
E15.5
Symmetric
6
3
Asymmetric
7
3
7
4
E16.6
2
E18.5
P0
Total
2
11 (27.5%)
1
3
16 (40%)
1
1
13 (32.5%)
(left)
Asymmetric
Development • Supplementary information
(right)
Development 142: doi:10.1242/dev.125328: Supplementary information
Table S2. Differentially expressed transcripts in the palate of Wnt1-Cre;Erk2fl/fl mice at
E13.5. Transcripts were selected by identifying those having a fold change > 1.5 and FDR <
0.05 in Wnt1-Cre;Erk2fl/fl (mutant) versus Erk2fl/fl (control) mice, as described in the Methods
section. Geometric means are given for the expression values over all samples in either
Affymetrix ID
1435239_at
1424609_a_at
1448972_at
1436596_at
1419728_at
1428029_a_at
1440495_at
1429685_at
1439566_at
1416298_at
1436294_at
1438278_a_at
1424208_at
1449393_at
1438644_x_at
1444596_at
1432393_a_at
1436279_at
1419391_at
1456242_at
1438799_at
1450708_at
1456498_at
Gene Symbol
Gria1
Gm4354
Gria1
H2afv
Cxcl5
H2afv
Rmst
Gabrb2
Gprin3
Mmp9
Ankrd29
Cwc22
Ptger4
Sh2d1a
Commd9
Pax7
Thg1l
Slc26a7
Myog
Gm7325
Dlx6os1
Scg2
Itga4
Mutant Geo.
Mean
15.399
114.374
20.390
53.789
37.211
1201.342
6.998
26.636
6.314
46.968
17.983
84.357
67.176
5.947
154.752
12.423
55.881
27.772
267.428
70.584
16.296
16.200
12.810
Wild-Type
Geo. Mean
37.352
253.077
45.025
112.328
72.088
2072.950
11.450
43.574
10.176
75.632
28.240
131.007
103.484
8.962
101.786
8.097
34.495
16.842
159.295
37.144
8.440
7.235
5.209
Fold Change
-2.426
-2.213
-2.208
-2.088
-1.937
-1.726
-1.636
-1.636
-1.612
-1.610
-1.570
-1.553
-1.540
-1.507
1.520
1.534
1.620
1.649
1.679
1.900
1.931
2.239
2.459
Adjusted pvalue
0.005
0.018
0.004
0.000
0.000
0.001
0.044
0.036
0.044
0.003
0.004
0.014
0.020
0.047
0.012
0.012
0.029
0.002
0.047
0.018
0.007
0.009
0.011
Development • Supplementary information
group.
Development 142: doi:10.1242/dev.125328: Supplementary information
Table S3. Differentially expressed transcripts in the palate of Wnt1-Cre;Erk2fl/fl mice at
E14.5. Transcripts were selected by identifying those having a fold change > 1.5 and FDR <
0.05 in Wnt1-Cre;Erk2fl/fl (mutant) versus Erk2fl/fl (control) mice, as described in the Methods
section. Geometric means are given for the expression values over all samples in either
Affymetrix ID
Gene Symbol
1449880_s_at
1421979_at
1436528_at
1429379_at
1449417_at
1436521_at
1434990_at
1453128_at
1429029_at
1440216_at
1418425_at
1440962_at
1443921_at
1428663_at
1419295_at
1429571_a_at
1430030_at
1441975_at
1423935_x_at
1418831_at
1418572_x_at
1422324_a_at
1435697_a_at
1448612_at
1424976_at
1449166_at
1421752_a_at
1418449_at
1457044_at
1418608_at
1431786_s_at
1417275_at
1429844_at
1460347_at
Bglap
Phex
Kazald1
Lyve1
Ambn
Slc36a2
Ppm1e
Lyve1
Sgms2
Ifitm5
Sp7
Slc8a3
Ranbp3l
Sgms2
Creb3l1
Spaca1
5330426P16Rik
Acpp
Krt14
Pkp3
Tnfrsf12a
Pthlh
Cytip
Sfn
Rhov
S100a14
Serpinb5
Lad1
Macc1
Calml3
1190003J15Rik
Mal
2310043J07Rik
Krt14
Mutant
Geo. Mean
9.936
61.451
53.605
22.062
20.122
4.074
13.965
4.208
14.481
303.906
87.579
158.301
2.357
414.976
26.854
8.420
57.575
17.176
649.725
21.271
59.125
164.839
6.179
29.001
17.152
77.994
59.531
12.060
44.971
15.315
18.267
47.644
43.274
269.001
Wild-Type
Geo. Mean
28.680
143.278
119.200
46.699
41.673
8.128
23.799
7.071
24.257
498.076
142.708
257.463
3.787
652.834
41.519
12.958
38.333
11.358
422.700
13.792
38.129
106.171
3.977
18.621
10.944
49.566
37.777
7.635
28.427
9.639
11.455
29.591
26.621
165.345
Fold Change
Mutant/WT
-2.887
-2.332
-2.224
-2.117
-2.071
-1.995
-1.704
-1.680
-1.675
-1.639
-1.629
-1.626
-1.607
-1.573
-1.546
-1.539
1.502
1.512
1.537
1.542
1.551
1.553
1.554
1.557
1.567
1.574
1.576
1.580
1.582
1.589
1.595
1.610
1.626
1.627
Adjusted
p-value
0.027
0.038
0.035
0.003
0.045
0.020
0.009
0.004
0.048
0.030
0.026
0.032
0.038
0.008
0.041
0.025
0.000
0.014
0.000
0.031
0.001
0.002
0.041
0.000
0.006
0.002
0.006
0.000
0.003
0.012
0.026
0.034
0.040
0.000
Development • Supplementary information
group.
Development 142: doi:10.1242/dev.125328: Supplementary information
Rspo2
Krt6a
Hbegf
Arg1
Tnfrsf12a
Mtm1
Serhl
Pmaip1
Hspb8
Gjb2
Nppc
Pthlh
Krt6a
Upk1b
Bnc1
Ly6d
Ifi202b
Ifi202b
42.995
270.447
21.971
5.929
37.805
18.767
21.612
25.476
96.335
56.301
5.686
32.426
43.141
22.911
28.514
37.975
21.030
57.494
26.349
164.546
13.365
3.544
22.447
11.116
12.544
14.756
55.741
31.802
3.157
17.384
22.538
11.647
13.625
15.326
5.785
11.433
1.632
1.644
1.644
1.673
1.684
1.688
1.723
1.726
1.728
1.770
1.801
1.865
1.914
1.967
2.093
2.478
3.635
5.029
0.014
0.001
0.000
0.022
0.013
0.014
0.036
0.028
0.000
0.018
0.000
0.027
0.000
0.003
0.006
0.000
0.000
0.000
Development • Supplementary information
1455893_at
1422784_at
1418350_at
1419549_at
1418571_at
1454904_at
1434145_s_at
1418203_at
1417013_at
1423271_at
1422790_at
1427527_a_at
1422783_a_at
1435831_at
1424890_at
1416930_at
1421551_s_at
1457666_s_at