Development 143: doi:10.1242/dev.130369: Supplementary information
Supplemental information for: Deborah J. Stumpo, Carol S. Trempus, Charles J. Tucker,
Weichun Huang, Leping Li, Kimberly Kluckman, Donna M. Bortner and Perry J. Blackshear.
Deficiency of the placenta- and yolk sac-specific tristetraprolin family member ZFP36L3
identifies likely mRNA targets and an unexpected link to placental iron metabolism.
Supplemental Data
Development • Supplementary information
Supplemental Fig. S1. Confocal evaluation of placental ZFP36L3 expression at E15.5.
Shown are confocal images of placental sections from WT and KO mice, as indicated, stained
with ZFP36L3 antibody (red). The nuclei were counterstained with DAPI (blue). The sections
were imaged in parallel at the same optical settings. The main anatomical regions of the
placenta are indicated in the WT section, and the numbered boxed areas are indicated for
higher power magnification of single optical sections, as depicted in numerical order on the right
side of the figure. Box 1 represents the junction of the allantois (unstained) with the labyrinth,
with the ZFP36L3-expressing cells representing the syncytiotrophoblast cells (also in box 2).
Box three represents the boundary of the labyrinth region with the spongiotrophoblast region,
with the latter cell type expressing less ZFP36L3 than the former. Box 5 represents the border
between the spongiotrophoblast layer and the maternal decidua, and 6 is the maternal decidua
alone. Boxes 4 and 7-9 demonstrate ZFP36L3 expression in the trophoblast giant cell layer.
Development 143: doi:10.1242/dev.130369: Supplementary information
Development • Supplementary information
Supplemental Fig. S2. Placental ZFP36L3 expression during development. Shown are
placental sections stained with ZFP36L3 antibody as described in the legend to Fig. 1. The
developmental stages for each section are indicated. As before, the brown staining indicates
ZFP36L3 staining, and the orientation of the tissues is roughly the same as that shown in Fig. 1.
Development 143: doi:10.1242/dev.130369: Supplementary information
Development • Supplementary information
Supplemental Fig. S3. ZFP36L3 expression in visceral yolk sac at E15.5. Shown are
sections of the visceral yolk sac stained with ZFP36L3 antibody as described in the legend to
Fig. 1. The brown staining indicates ZFP36L3 staining. In A are shown sections of the highly
folded yolk sac facing the placenta, and in B is shown a section of the non-folded yolk sac
facing the embryo. C shows sections from an umbilical blood vessel and the amnion, showing
lack of staining in those tissues.
Development 143: doi:10.1242/dev.130369: Supplementary information
Development • Supplementary information
Supplemental Figure S4. ZFP36L3 staining in WT and KO placentas at E15.5. Staining
and orientation are as described in the legend to Fig. 1. This experiment shows the specificity
of the antibody by comparing the staining with the immune serum (Imm) to that achieved with
pre-immune serum under the same conditions (Pre-Imm), and also demonstrates the total lack
of expression in the KO placenta. The images on the left are low power (1.25X) views of the
whole placenta, and those on the right are higher power (20X) views of the labyrinth region from
the same sections. Note that the morphology of the KO placenta is very similar to that of the
WT placenta.
Development 143: doi:10.1242/dev.130369: Supplementary information
Development • Supplementary information
Supplemental Figure S5. Zfp36l3 KO locus, vector, and expression. (A) Schematic
representation of the normal genomic locus on the X chromosome for Zfp36l3 as well as the
targeting vector and the resulting disrupted allele generated by homologous recombination. The
5’ flanking region is shown in blue, the uninterrupted open reading frame (ORF) in red, and the
positions of the start and stop codons shown are indicated by arrows. The single intron occurs
after the stop codon and is shown in light green, and the 3’UTR is in dark green. The flanking
regions are shown as black lines, and the positions of various restriction enzyme sites are
indicated above the diagram: H, HindIII; K, KpnI; S, SstI; EV,EcoRV; A, AscI; Nr, NruI. Genomic
5' and 3' probes, located outside the targeting vector, are indicated in purple and were used for
Southern analysis to confirm homologous recombination. The targeting vector contained a
Neomycin selection cassette (PGKNeo), resulting in deletion of the open reading frame. A
diptheria toxin resistance cassette was also inserted in the targeting vector (DTA). (B) PCR
analysis of genomic DNA isolated from Zfp36l3 WT, HET, and KO mice. The WT allele
produced a fragment of 1.1 kb and the KO allele produced a fragment of 0.54 kb. (C) Northern
blot analysis of total RNA isolated from E15.5 male WT and KO placenta (5 µg per lane)
hybridized with a 32P-labeled 1.74-kb Zfp36l3 cDNA probe, demonstrating the presence of
Zfp36l3 RNA in the WT sample (about 2.5 kb) but nothing in the KO lane. The right panel
demonstrates roughly equivalent loading of the gel (ethidium bromide staining), and shows the
positions of the predominant ribosomal RNA species.
Development 143: doi:10.1242/dev.130369: Supplementary information
Development • Supplementary information
Supplemental Figure S6. Potential TTP family member binding sites in Hbegf and Lipg
mRNAs. Shown are sequences from the 3’UTRs of Hbegf (A) and Lipg (B) mRNAs that were
conserved in many mammalian species. The sequences in A were not contiguous, but
represented separated regions of the 3’UTR; the position of the polyadenylation signal is
indicated with an underline. The sequences in B are contiguous. Red color indicates the
potential TTP family member binding site; an asterisk represents base identity at that site.
Development 143: doi:10.1242/dev.130369: Supplementary information
Development • Supplementary information
Supplemental Figure S7. Expression of TFRC protein and Tfrc mRNA. In A is shown a
western blot of TFRC from placental extracts from WT and KO mice (left panel); the expression
of α-actin as a loading control is shown in the right panel. In B are shown NanoString values for
the Tfrc transcript in WT and KO TSC, before (Un) and after (D) differentiation in culture; the WT
and KO mean values were statistically different (P < 0.01; **) both in the undifferentiated and
differentiated states, and there was a large increase in expression upon differentiation. In D is
shown an alignment of potential ZFP36L3 binding sites in the 3’UTR of Tfrc transcripts from
several mammalian species. Asterisks indicate sequence identity at that site; bases in red
indicate potential TTP family member binding sites.
Development 143: doi:10.1242/dev.130369: Supplementary information
Supplemental Table S1. Actual vs. expected rates of live births from two types of matings.
Shown are the numbers of litters evaluated, the numbers and sexes of live weaned offspring, and
the statistical comparisons between the actual percentage of weaned offspring of a given
genotype compared to the expected percentage of surviving weaned offspring for that genotype.
A. Offspring from WT male x Zfp36l3 Het female matings.
Male
Litters
born
103
Animals
born
Male Female WT Het KO
548
273
275
368 105 75
Actual
%
Expected
%
50
50
67
19
14
50
50
50
25
25
WT
196
Female
KO
75
WT
172
Het
103
36**** 14**** 31** 19****
25
25
25
25
B. Offspring from Zfp36l3 KO male x Zfp36l3 Het female matings.
Male
Litters
born
50
Actual
%
Expected
%
Animals
born
247
Male
118
Female
129
48
50
Female
WT Het KO
78 84 85
WT
78
KO
40
Het
84
KO
45
52
32
34
34
32*
16***
34**
18**
50
50
25
25
25
25
25
25
Development • Supplementary information
****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; Wilcoxon signed ranks test
GenBank Acc. #
Gene
WT
SD
KO
SD
fold
KO/WT
p value
q value
7,8 or 9mers
# cons.
NM_009756
Bmp10
72
11.5
337.6
65.7
4.64
7.23E-08
1.72E-05
3*
3
NM_011719
Wnt9b
46.8
12.4
185.8
49.9
3.91
6.49E-06
1.06E-03
1 8, 1 7
2
NM_011215
Ptprn2
68.4
14.0
251.6
36.7
3.64
1.92E-06
3.60E-04
NM_010415
Hbegf
1574.6
262.9
4962.4
383.7
3.15
9.60E-09
2.74E-06
1 7, 1 8
2
NM_013657
Sema3c
136
29.9
400.2
163.8
2.93
8.49E-05
7.72E-03
18
1
NM_153166
Cpne5
182.2
18.3
516.2
62.8
2.82
1.81E-16
1.44E-13
NM_008706
Nqo1
73.4
15.1
203
22.1
2.74
4.34E-06
7.70E-04
NM_010720
Lipg
7923
1008.9
20504
1665.4
2.59
4.94e-324
7.89e-320
27
2
NM_018827
Crlf1
216
42.1
544.8
161.1
2.52
1.81E-04
1.19E-02
NM_001122993
B3galt5
154.8
27.2
387.6
47.9
2.49
4.56E-07
9.59E-05
27
2
NM_009250
Serpini1
88
19.9
217.2
26.3
2.45
5.07E-05
5.26E-03
19
rat only
NM_011391
Slc16a7
55.2
14.8
124.6
24.0
2.23
1.16E-03
3.55E-02
NR_028425
D7Ertd143e
69.4
17.5
149.2
20.7
2.13
6.30E-08
1.53E-05
NM_001013751
Syna
3786.6
583.3
7990.8
789.1
2.11
9.41E-83
3.76E-79
18
0
NM_023755
Tfcp2l1
183.8
42.1
388.4
26.7
2.11
5.70E-06
9.66E-04
1 8, 1 7
rat only
NM_177900
Hapln4
336
42.8
701.4
37.0
2.08
6.05E-12
3.12E-09
29
0
NM_027482
5730508B09Rik
139.8
17.6
288.8
23.9
2.06
3.99E-07
8.50E-05
18
1
NM_011725
Xlr
63.8
14.2
132.2
20.7
2.06
1.35E-06
2.66E-04
NM_011867
Slc26a4
139.6
29.7
285
32.8
2.03
1.74E-04
1.18E-02
NM_178143
Prkaa2
384.4
58.5
770.2
131.5
2
2.42E-04
1.45E-02
3 7, 3 8,
19
some
NM_001281965
Itk
53.6
11.7
107.8
18.0
1.99
9.95E-04
3.29E-02
1 8, 1 7
2
NM_028894
Lonrf3
4584.6
878.0
8952.8
796.3
1.95
1.11E-102
5.93E-99
1 9, 1 8
1
NM_144827
Spata20
73
10.4
143.2
11.9
1.95
2.05E-05
2.60E-03
NM_133730
Krt25
176.6
22.0
343
52.4
1.94
3.79E-04
1.90E-02
NM_172471
Itih5
2323.4
159.1
4439.8
817.1
1.91
7.56E-04
2.86E-02
1 7, 1 8
2
NM_011741
Zan
82
10.3
157
22.7
1.9
1.40E-06
2.73E-04
18
?
NM_008478
L1cam
108.2
9.8
204
36.4
1.88
9.52E-04
3.22E-02
Development • Supplementary information
Development 143: doi:10.1242/dev.130369: Supplementary information
Supplemental Table S2. Average reads from the mRNA-Seq analysis for up- and down-regulated transcripts in WT and Zfp36l3 KO
placenta. The table lists GenBank accession numbers, gene symbols, average reads (n = 5), SD of average, fold increase or decrease of Zfp36l3 KO
means over WT means, and p and q values of the comparisons. Only transcripts whose fold changes were 1.5 fold or greater, and with FDR < 0.05,
are listed; transcripts were also excluded if there was no detectable expression in any of the WT samples. Shown also are the number of potential 7mer (UAUUUAU), 8-mer (UAUUUAUU or UUAUUUAU) or 9-mer (UUAUUUAUU) TTP family member binding sites in the 3’UTRs of each
transcript, with the notation 1 8 meaning that one 8-mer was present. Also shown is an indication of whether or not these potential binding sites were
conserved in other mammals (#cons.).
Development 143: doi:10.1242/dev.130369: Supplementary information
Lzts1
56.8
6.9
107
12.1
1.87
9.00E-05
7.93E-03
NM_198423
Bahcc1
552
82.3
1030
193.7
1.86
1.89E-03
4.60E-02
18
1
NM_031199
Tgfa
1279.4
323.0
2378.4
358.3
1.86
1.49E-03
4.08E-02
1 9, 1 8
2
NM_021528
Chst12
481
87.0
889.4
177.0
1.85
1.93E-06
3.70E-04
NM_194061
D630045J12Rik
461.4
62.6
846.2
98.2
1.83
1.12E-05
1.55E-03
1 9, 1 8
2
NM_001080926
Lrp8
2963.4
475.5
5409.8
363.0
1.83
3.52E-33
5.11E-30
1 9, 1 8
2
NM_172992
Phtf2
442.2
86.1
806.8
61.4
1.82
5.90E-05
6.01E-03
28
1
NM_145947
Slc26a7
777.6
122.8
1416.4
133.8
1.82
5.25E-13
2.89E-10
NM_178608
Reep1
239.6
23.3
431.2
35.8
1.8
1.89E-05
2.43E-03
NM_011976
Sema4g
699.6
129.4
1244.4
153.1
1.78
6.19E-04
2.57E-02
18
1
NM_011825
Grem2
720.2
117.7
1277
156.6
1.77
7.47E-06
1.18E-03
NM_001289505
Pex5l
58.4
6.7
104.4
7.7
1.77
1.83E-05
2.37E-03
18
1
NM_172710
Sel1l3
964.6
126.9
1694
139.8
1.76
1.29E-11
6.22E-09
NM_198301
Fam105a
119
19.6
208.4
20.9
1.75
2.47E-04
1.47E-02
NM_033174
Snurf
556.6
126.4
966.2
81.7
1.73
6.13E-04
2.58E-02
18
1
NM_021281
Ctss
71.2
17.9
123
14.7
1.72
2.32E-04
1.42E-02
NM_033521
Laptm4b
1103.4
120.1
1893.8
254.7
1.72
5.02E-04
2.26E-02
NM_172821
Map3k13
84.6
4.7
146.6
8.8
1.72
1.65E-06
3.17E-04
19
1
NM_011973
Mok
88.4
13.2
152.4
21.0
1.72
8.69E-04
3.07E-02
NM_009721
Atp1b1
3815
422.0
6508.8
242.4
1.71
1.80E-82
5.75E-79
1 7, 1 8
2
NM_001286729
Glcci1
135.2
10.3
231.4
9.3
1.71
7.00E-07
1.43E-04
17
1
NM_001253679
Slc7a7
289.8
37.3
497
60.5
1.71
3.90E-04
1.92E-02
NM_001004173
Sgpp2
557.4
67.5
949
48.3
1.7
1.12E-09
3.88E-07
NR_027934
Sgsm1
75.8
13.4
129.6
7.0
1.7
9.88E-05
8.44E-03
1 8, 1 9
2
NM_001163640
Chn2
157.4
10.4
265.2
19.0
1.68
8.93E-06
1.35E-03
17
1
NM_011345
Sele
666
96.2
1117.8
96.8
1.68
6.41E-10
2.27E-07
17
rat only
NM_009331
Tcf7
116.4
19.1
195.8
25.1
1.68
9.98E-04
3.29E-02
NM_001082961
Snrpn
274.8
37.1
458.4
33.5
1.67
8.02E-05
7.41E-03
NM_001252515
Ehbp1
867.4
52.4
1439
69.1
1.66
3.50E-16
2.67E-13
2 9, 1 8
2
NM_011037
Pax2
201
35.4
334
35.7
1.66
7.31E-04
2.81E-02
NM_019926
Mtm1
74.8
9.7
124.4
17.3
1.65
1.04E-03
3.36E-02
18
1
NM_178728
Napepld
269.4
24.2
445.6
30.4
1.65
1.76E-05
2.30E-03
17
0
NM_001033289
Slc9a2
465
25.8
767.6
82.4
1.65
1.12E-04
9.00E-03
18
1
Development • Supplementary information
NM_199364
Development 143: doi:10.1242/dev.130369: Supplementary information
Star
1728.6
309.2
2848.2
432.9
1.65
7.96E-07
1.59E-04
2 8, 1 9
2
NM_028651
Tmtc4
146
14.7
241.2
29.3
1.65
4.01E-04
1.95E-02
1 7, 1 9
2
NM_145977
Slc45a3
502.2
65.7
825.6
108.2
1.64
4.87E-04
2.23E-02
29
1
NM_001195265
Pdzd7
73.2
9.9
119.8
22.2
1.63
8.82E-04
3.10E-02
NR_110489
AW822252
64
11.4
104.6
10.6
1.62
8.05E-04
2.97E-02
NM_001136484
Gcnt1
451
72.7
729.2
37.9
1.62
6.54E-06
1.07E-03
18
rat only
NM_015730
Chrna4
314.6
63.4
503
55.9
1.6
2.12E-03
4.89E-02
NM_028375
Cxx1c
280
36.7
448.2
61.4
1.6
1.41E-03
3.95E-02
NM_010280
Gfra3
91
20.4
146.6
13.1
1.6
1.79E-03
4.52E-02
NM_011165
Prl4a1
1525.8
178.1
2441.8
218.0
1.6
4.31E-08
1.08E-05
NM_015736
Galnt3
531.4
29.3
845.2
101.3
1.59
3.41E-04
1.78E-02
1 7, 1 8
2
NM_001081230
Map9
1059.8
115.9
1690
156.4
1.59
1.44E-04
1.06E-02
5 9, 1 7
most
NM_001039126
Asb1
556.6
36.6
880
77.0
1.58
6.36E-05
6.27E-03
2 7, 2 8,
29
all
NM_001190371
Ankrd29
81.6
14.0
128.4
18.2
1.57
1.83E-03
4.57E-02
NM_145962
Pank3
3850.2
497.1
6042.2
672.4
1.57
7.81E-04
2.91E-02
18
1
NM_027897
Rhpn2
1296.2
102.5
2034.8
123.6
1.57
1.39E-13
8.91E-11
19
1
NM_001134457
Nxpe3
177
25.4
277
28.7
1.56
7.98E-04
2.95E-02
29
1
NM_201361
Rmdn2
230.6
32.0
358.6
14.2
1.55
8.31E-05
7.63E-03
NM_001111304
Tbc1d9
351.4
34.4
545.6
45.9
1.55
1.42E-04
1.06E-02
27
2
NM_001252659
Ldlr
1759.4
281.1
2703.4
251.2
1.54
1.61E-05
2.15E-03
1 7, 2 8
3
NM_176940
Nwd1
347.8
44.8
533.4
66.5
1.53
1.69E-03
4.38E-02
NM_172645
Suco
5850.2
749.5
8934
746.6
1.53
2.09E-06
3.85E-04
27
2
NM_172856
Cers6
1614.6
187.8
2451.6
144.0
1.52
1.34E-10
5.35E-08
19
1
NM_138587
Fam3c
2729.2
283.3
4145.8
275.3
1.52
1.81E-17
1.53E-14
1 7, 2 9
2
NM_133911
Gpr125
5648.8
325.9
8598.2
586.2
1.52
2.35E-45
4.17E-42
NM_175184
Mvb12b
810.8
122.3
1234.8
111.2
1.52
8.95E-04
3.10E-02
18
1
NM_001081035
Nav3
693.2
34.5
1054.4
97.0
1.52
4.25E-05
4.52E-03
NM_001100116
1700047I17Rik2
683.2
78.4
1032.4
110.7
1.51
8.77E-04
3.09E-02
17
1
NM_173752
Lgalsl
4291.8
626.9
6435
541.5
1.5
7.14E-06
1.14E-03
2 7, 1 8
3
NM_030110
Micu3
194.2
27.7
292
24.3
1.5
7.15E-04
2.79E-02
2 8, 3 9
most
NM_001277982
Snap91
471.8
65.3
708
47.6
1.5
3.83E-04
1.92E-02
1 7, 1 8
2
NM_172256
Dync2li1
240
21.3
158.8
14.4
-1.51
2.30E-04
1.42E-02
Development • Supplementary information
NM_011485
Development 143: doi:10.1242/dev.130369: Supplementary information
Slc7a2
2169
194.2
1432.8
127.4
-1.51
1.66E-08
4.50E-06
NM_025748
Adat2
158.4
10.9
104.2
14.7
-1.52
3.51E-04
1.80E-02
NM_027123
Fastkd3
474
35.9
311
28.9
-1.52
1.04E-04
8.53E-03
NM_133727
Kptn
163.6
19.1
107
16.6
-1.52
2.04E-03
4.80E-02
NM_133962
Arhgef18
5393.2
481.3
3528.8
221.9
-1.53
1.40E-13
1.00E-05
NM_207202
Ccdc120
741.4
89.4
484.6
43.9
-1.53
8.66E-04
3.07E-02
NM_001289777
Rufy3
133.6
11.8
87
9.2
-1.53
2.54E-04
1.48E-02
NM_153541
Zbtb8b
744.8
30.2
485.8
60.6
-1.53
1.09E-05
1.53E-03
NM_172133
Adap2
646.2
39.8
419
32.0
-1.54
2.02E-05
2.58E-03
NM_001286529
Fbxl12
158.4
12.3
102.4
16.9
-1.54
6.79E-04
2.70E-02
NM_009201
Slc1a5
2029.8
131.6
1318.8
92.2
-1.54
1.94E-10
7.38E-08
NM_001286948
Pla2g12a
102
12.2
65.4
7.5
-1.55
9.16E-04
3.13E-02
NM_001039194
Aifm2
479.4
45.4
304.2
43.8
-1.57
5.39E-04
2.38E-02
NM_009864
Cdh1
8002.4
696.4
5094.2
248.9
-1.57
1.05E-36
1.68E-33
NM_009903
Cldn4
2692.2
347.9
1709
255.5
-1.57
1.86E-03
4.59E-02
NM_028443
Fam101a
442.2
61.2
280.8
35.2
-1.57
1.82E-03
4.55E-02
NR_110477
Gm12338
312.4
45.9
198.4
10.1
-1.57
1.27E-03
3.74E-02
NM_007912
Egfr
271.6
23.1
171.6
25.3
-1.58
3.84E-04
1.91E-02
NM_173420
Synb
7380.2
1090.4
4681.4
308.4
-1.58
6.93E-20
6.92E-17
NM_001290800
Tyro3
412.4
48.5
258.4
37.7
-1.59
1.03E-03
3.35E-02
NM_001113353
Synj2
110.4
11.5
68.4
4.5
-1.61
1.41E-04
1.06E-02
NR_015582
C330024D21Rik
107.8
13.7
66
11.1
-1.62
1.47E-03
4.06E-02
NM_011514
Suv39h1
656.8
65.0
404.8
28.9
-1.62
1.03E-04
8.54E-03
NM_011786
Aloxe3
141.2
20.4
85.6
9.2
-1.64
3.18E-04
1.69E-02
NM_028775
Cyp2s1
1652.4
145.7
1006.6
105.6
-1.64
1.47E-09
4.89E-07
NM_008103
Gcm1
1154
193.5
690.6
39.3
-1.67
4.91E-04
2.24E-02
NM_172134
Pdxk
657.6
65.1
394.2
26.0
-1.67
6.81E-05
6.63E-03
NM_001098233
Purg
356.8
45.8
213.8
25.5
-1.67
6.08E-04
2.58E-02
NM_008969
Ptgs1
1009.4
133.5
597.6
54.0
-1.69
1.30E-04
9.90E-03
NM_008168
Grik5
266.6
45.6
154.2
21.5
-1.72
2.11E-03
4.89E-02
NM_001252638
Rhobtb1
132.6
26.6
76.6
10.7
-1.72
1.36E-04
1.02E-02
NM_028048
Slc25a35
107.6
16.4
61.2
7.8
-1.75
9.17E-04
3.14E-02
NM_020270
Scamp5
1641.2
98.2
934.6
122.7
-1.76
1.97E-13
1.16E-10
17
1
19
1
Development • Supplementary information
NM_007514
Development 143: doi:10.1242/dev.130369: Supplementary information
Ctsh
1231.6
172.0
693.8
71.8
-1.77
1.63E-11
7.64E-09
NM_019935
Ovol1
159.6
9.2
89.8
16.6
-1.77
7.81E-05
7.26E-03
NM_153168
Lars2
2983
485.8
1642.4
94.1
-1.82
9.76E-27
1.30E-23
NM_028902
Hsf2bp
121.4
15.0
65.4
7.6
-1.84
4.88E-05
5.13E-03
NM_011395
Slc22a3
3582.2
374.3
1948
205.6
-1.84
6.22E-21
7.09E-18
NM_028060
Slc35f2
1566.6
83.0
833.8
162.0
-1.88
1.84E-11
8.41E-09
NM_007559
Bmp8b
486.4
63.2
257.4
19.1
-1.89
6.06E-05
6.05E-03
NR_033228
E030024N20Rik
381.2
66.6
201.2
19.9
-1.89
3.73E-04
1.90E-02
NM_020568
Plin4
579.4
60.5
305.4
30.4
-1.89
1.52E-08
1.42E-05
NM_172737
Shisa7
241.4
28.0
126.2
19.2
-1.91
1.41E-04
1.06E-02
NM_144512
Slc6a13
403.4
50.9
211.2
48.9
-1.91
6.14E-04
2.57E-02
NM_183263
Rnmtl1
213.4
18.4
106.2
18.8
-2
3.86E-05
4.35E-03
NM_008089
Gata1
304
31.0
149.2
22.4
-2.03
4.02E-05
4.38E-03
NM_001109972
Sec61g
122.8
16.9
59.2
10.0
-2.06
1.92E-04
1.23E-02
NM_027360
2010107E04Rik
163
30.3
78
16.4
-2.08
1.15E-03
3.52E-02
NM_028064
Slc39a4
1292.4
261.9
619.6
86.5
-2.08
7.40E-04
2.84E-02
NM_026728
Echdc2
375
43.7
176.6
25.2
-2.12
2.08E-05
2.61E-03
NM_019443
Ndufa1
348.2
71.7
163.6
20.4
-2.12
1.11E-03
3.48E-02
NM_028664
Ankrd45
144
24.1
67
25.5
-2.13
1.23E-07
2.76E-05
NM_026506
Snrpg
446.6
70.4
209.2
32.2
-2.13
6.18E-05
6.13E-03
NM_001024717
Gal3st3
109.4
15.8
50.6
14.8
-2.14
6.21E-04
2.57E-02
NM_138750
Prom2
110.4
17.1
50.2
13.4
-2.18
5.46E-04
2.40E-02
NM_026919
Tmem258
275.2
65.8
125.2
14.7
-2.19
3.29E-04
1.74E-02
NM_175511
Fam78a
549
74.1
243.8
56.8
-2.25
1.82E-04
1.19E-02
NM_010739
Muc13
968.4
219.9
412.8
100.6
-2.34
2.15E-04
1.36E-02
NM_001164406
Sra1
111.2
24.0
46.4
10.1
-2.37
1.08E-03
3.42E-02
NM_001173500
G630090E17Rik
134
13.3
55.2
2.1
-2.4
8.56E-09
2.53E-06
NM_008630
Mt2
2724.6
833.1
1111.4
381.2
-2.45
4.43E-04
2.10E-02
NM_146257
Slc29a4
180
44.5
73
16.2
-2.45
1.94E-03
4.67E-02
NM_016669
Crym
125.4
14.8
50.4
11.0
-2.46
3.87E-05
4.32E-03
NM_013650
S100a8
443
113.4
175.8
40.2
-2.51
3.95E-04
1.94E-02
NM_026547
Lurap1
106.8
24.2
41
5.8
-2.57
7.47E-04
2.85E-02
NM_028877
Palm3
768.8
222.8
292
34.3
-2.63
5.97E-05
6.00E-03
17
1
17
0
Development • Supplementary information
NM_007801
Development 143: doi:10.1242/dev.130369: Supplementary information
Slc27a3
968
187.5
366.2
87.6
-2.64
3.02E-05
3.52E-03
NM_053248
Slc5a5
178.2
36.5
60.2
8.4
-2.93
9.26E-15
6.16E-12
NM_026251
Patl2
236.8
27.0
65.2
16.0
-3.59
1.77E-07
4.87E-05
NM_008653
Mybpc3
113.2
39.1
21.6
7.5
-5.05
2.86E-05
3.38E-03
NM_011638
Tfrc
77504
17724.4
12157
8390.9
-6.37
9.03E-19
8.48E-16
NM_001009549
Zfp36l3
5715.4
399.6
0
0.0
-5716
4.94e-324
7.89e-320
28
2
Development • Supplementary information
NM_011988
Gene
Prob.
Cat.
1
Avg.
WT
308.7
SD
Wt
34.9
Avg.
KO
472.1
SD
KO
38.2
p WT
vs. KO
2.30E-04
Ratio
KO/WT
1.53
Avg.
Het1
518.8
SD
Het1
24.5
Avg.
Het2
321.6
SD
Het 2
6.4
p Het1
vs. Het2
3.87E-04
Ratio
Het1/Het2
1.61
1
249.0
28.9
369.2
37.6
9.63E-04
1.48
363.0
13.7
265.4
3.4
6.23E-04
1.37
1
322.1
26.7
502.5
17.5
3.36E-06
1.56
529.6
24.6
386.8
64.3
4.27E-02
1.37
1
444.2
20.9
536.1
20.9
2.55E-04
1.21
548.6
16.5
475.0
14.5
9.06E-03
1.16
Cpeb2
1
708.6
55.9
862.8
46.2
2.78E-03
1.22
864.6
34.2
742.1
45.3
3.79E-02
1.17
Dos
1
70.1
16.2
112.0
10.9
2.66E-03
1.60
107.4
8.5
76.0
3.3
8.31E-03
1.41
Elavl1
1
212.4
21.1
355.8
15.5
4.28E-06
1.68
356.2
10.1
200.5
16.3
3.29E-04
1.78
Hbegf
1
694.4
128.8
1820.9
182
7.82E-06
2.62
1769
32.8
713.7
56.2
2.14E-05
2.48
Irx2
1
98.4
12.4
143.9
6.4
1.85E-04
1.46
140.3
12.2
98.7
7.6
1.50E-02
1.42
Ldlr
1
703.9
44.4
1107.5
83.1
2.65E-05
1.57
992.0
87.0
626.1
126.4
2.80E-02
1.58
Lipg
1
3267.4
501.1
7222.6
355
1.24E-06
2.21
6558
182.0
3412.0
607.6
2.18E-03
1.92
Lonrf3
1
1164.7
186.6
1986.4
98.2
5.27E-05
1.71
1838
66.4
1237.4
129.1
4.26E-03
1.49
Mtm1
1
142.4
6.7
169.2
10.0
2.18E-03
1.19
168.3
6.3
137.3
2.1
2.72E-03
1.23
Pank3
1
1391.3
68.5
1927.2
63.4
2.98E-06
1.39
1904
74.9
1490.8
27.7
1.85E-03
1.28
Pdxp
1
62.0
4.1
86.5
10.3
2.27E-03
1.39
102.7
11.8
70.0
6.6
2.70E-02
1.47
Slc38a3
1
42.5
10.5
86.0
12.1
6.25E-04
2.02
115.0
7.3
44.0
5.7
4.15E-04
2.61
Snurf
1
1580.0
270.2
2499.4
277
1.44E-03
1.58
2286
130.9
1378.2
164.2
3.63E-03
1.66
Star
1
149.3
24.7
245.1
20.7
3.42E-04
1.64
222.9
13.8
155.2
28.5
3.91E-02
1.44
Suco
1
1050.5
76.7
1434.2
90.3
1.93E-04
1.37
1437
55.3
1100.6
26.9
1.50E-03
1.31
Tcfcp2l1
1
36.8
5.1
52.8
4.9
1.97E-03
1.43
50.1
9.1
28.1
1.4
2.79E-02
1.78
Tfrc
1
25571.6
4068
4330.7
2556
2.11E-05
0.17
2982
192.7
27004
5116
2.68E-03
0.11
Zfp36l2
1
917.4
73.7
1073.5
81.7
2.19E-02
1.17
1104
69.9
859.4
43.1
1.35E-02
1.28
1110067D
22Rik
1700047I1
7Rik1
Acvr1
2
888.1
124.2
1254.1
100
1.79E-03
1.41
1252
25.6
936.9
56.7
2.02E-03
1.34
2
303.8
16.0
387.4
10.5
2.35E-05
1.28
408.4
25.9
330.5
4.9
1.40E-02
1.24
2
393.4
36.1
504.7
18.8
5.96E-04
1.28
516.6
56.5
383.2
19.7
3.44E-02
1.35
Atp1b1
2
1302.3
159.3
2104.3
59.6
1.31E-05
1.62
2100
25.1
1329.9
160.9
2.59E-03
1.58
Atp6v1b1
2
10.4
5.0
20.5
4.3
1.53E-02
1.97
29.1
1.8
14.7
3.5
6.75E-03
1.98
Fam3c
2
707.9
75.7
1103.0
92.4
1.67E-04
1.56
1067
86.2
808.7
91.8
4.42E-02
1.32
Gcnt1
2
442.2
74.0
591.7
37.7
6.99E-03
1.34
618.7
25.0
463.9
45.5
1.35E-02
1.33
Hapln4
2
75.6
14.5
155.5
18.8
1.48E-04
2.06
149.1
13.3
69.3
4.4
1.29E-03
2.15
Itih5
2
370.6
20.3
672.6
96.6
2.83E-04
1.81
611.1
62.3
352.7
42.6
8.39E-03
1.73
1600002H
07Rik
4921524J1
7Rik
5730508B0
9Rik
B3gnt2
Development • Supplementary information
Development 143: doi:10.1242/dev.130369: Supplementary information
Supplemental Table S3. NanoString quantitation of transcript expression in placenta. From the original list of assayed transcripts, several
(Cpne5 , Mak, Dlx2 , Accn1, Csf2 , Cxcl2 , Cyp3a25, Il10, Il6, Lipm, Sypl2, and Zan mRNAs) were removed because their average expression in
WT placenta was less than 10 normalized reads; the average of the NanoString negative internal controls plus 2 SD was 9.4 normalized reads. Data
from the 139 remaining transcripts are listed here as normalized reads, in order of the indicated probability category. Probability categories (Prob.
Cat.) were determined from the data in this table and from Supplemental Table S5. Shown are average values for WT, KO, Het1 and Het2 (n = 5 in
each case), along with the ratios of KO average over WT average, and Het1 average over Het2 average, and p values from comparing members of
each pair (WT vs. KO; Het1 vs. Het2; unpaired two-tailed t tests). Probability category 1: Significant differences between both pairs of placenta
averages and both times of the TSC time course (n = 22, excluding Zfp36l3 mRNA); category 2: Both placenta comparisons and one time point in
the time course were significantly different (n = 17); category 3: only the WT vs. KO placenta comparison was significantly different, along with
both time points of the time course (n = 5); category 4: only the WT vs. KO placenta comparison was significantly different, plus one time point of
the time course (n = 6); category 5: Both WT vs. KO and Het1 vs. Het2 comparisons were significantly different, but there were no significant
differences in the time course (n = 26); category 5B: Both WT vs. KO and Het1 vs. Het2 were significantly different, but the time course values
were below the limit of detection (n = 6); category 6: only WT vs. KO were significantly different, and there were no significant differences in the
time course (n = 15); category 7: both time points in the time courses were significantly different, but there were no significant differences in the WT
vs. KO placenta comparison (n = 6). An additional 36 transcripts in this assay were unrated and considered low probability. See the text for further
details.
Development 143: doi:10.1242/dev.130369: Supplementary information
2
67.2
8.3
118.8
21.9
2.30E-03 1.77
122.7
15.7
57.4
14.9
1.30E-02
2.14
Phtf2
2
64.0
7.8
97.8
9.1
4.82E-04
1.53
112.1
10.4
70.6
5.7
7.85E-03
1.59
Rhpn2
2
395.4
66.5
558.1
31.5
2.21E-03
1.41
532.5
26.0
381.3
28.1
5.02E-03
1.40
Slc45a3
2
337.6
40.2
599.6
28.0
5.10E-06
1.78
623.4
30.1
380.8
38.4
2.15E-03
1.64
Tbc1d9
2
160.8
7.6
231.0
11.6
7.63E-06
1.44
244.9
17.1
165.8
17.6
1.04E-02
1.48
Tcf7
2
93.9
17.5
134.1
29.3
4.60E-02
1.43
132.2
3.0
88.4
11.2
5.86E-03
1.50
Tpd52l1
2
17.3
3.4
31.8
4.3
8.00E-04
1.83
32.3
3.7
16.3
4.8
2.07E-02
1.98
2310045A
20Rik
Cldn23
2
269.2
50.5
397.7
22.1
1.63E-03
1.48
373.5
22.0
273.2
22.1
1.04E-02
1.37
3
102.1
10.3
134.1
14.4
6.81E-03
1.31
120.5
20.9
119.2
17.0
9.48E-01
1.01
Grem2
3
133.5
21.5
203.4
29.1
4.77E-03
1.52
191.5
30.3
130.8
32.3
1.25E-01
1.46
Phyhipl
3
377.1
74.7
482.2
37.8
3.63E-02
1.28
523.8
44.9
413.1
40.0
6.00E-02
1.27
Sema4g
3
65.6
19.2
98.5
9.5
1.55E-02
1.50
79.4
12.2
56.2
4.3
6.37E-02
1.41
9030409G
11Rik
Jag1
3
792.1
69.8
914.5
42.1
1.70E-02
1.15
924.3
73.0
831.9
111.2
3.82E-01
1.11
4
118.3
20.0
184.5
17.9
1.13E-03
1.56
167.5
12.9
133.5
11.9
5.25E-02
1.25
Mtap9
4
134.6
7.1
193.8
12.3
3.35E-05
1.44
157.1
13.3
125.7
15.5
9.56E-02
1.25
Nexn
4
221.0
23.1
277.9
25.7
1.09E-02
1.26
297.1
24.5
243.7
13.0
5.27E-02
1.22
Slc16a7
4
35.1
10.4
61.5
3.8
1.45E-03
1.75
58.0
12.2
36.7
10.0
1.28E-01
1.58
St3gal6
4
992.8
99.4
1189.7
60.4
9.58E-03
1.20
1251
16.9
1055.7
131.2
1.05E-01
1.18
Ept1
4
725.4
54.8
860.2
23.4
1.94E-03
1.19
807.5
9.8
833.0
27.5
2.85E-01
0.97
Ankrd13d
5
31.1
8.1
52.2
5.1
2.25E-03
1.68
47.6
6.7
27.5
5.1
2.78E-02
1.73
Asb1
5
328.4
13.9
520.1
30.0
2.75E-06
1.58
531.7
22.3
379.3
9.8
8.99E-04
1.40
Bahcc1
5
62.5
3.2
116.0
13.3
5.07E-05
1.86
103.1
11.7
53.9
12.9
1.62E-02
1.91
Chn2
5
99.4
12.9
164.6
19.4
5.07E-04
1.66
175.6
3.8
109.5
4.7
9.98E-05
1.60
D630045J1
2Rik
Efha2
5
48.2
11.2
71.4
8.7
1.12E-02
1.48
80.1
6.2
58.1
4.3
1.45E-02
1.38
5
69.7
9.0
96.4
9.6
3.76E-03
1.38
101.4
9.2
76.3
6.7
3.56E-02
1.33
Ehbp1
5
197.5
15.4
252.0
26.4
7.41E-03
1.28
275.3
14.2
172.5
5.9
6.97E-04
1.60
Fam125b
5
140.3
10.4
197.0
19.6
9.23E-04
1.40
196.7
12.7
152.6
9.1
1.64E-02
1.29
Fam55c
5
60.5
12.7
89.3
9.4
6.66E-03
1.48
87.0
9.4
64.5
5.1
4.10E-02
1.35
Glcci1
5
149.9
14.0
206.5
15.6
6.39E-04
1.38
219.0
19.2
165.4
4.9
1.88E-02
1.32
Hectd2
5
53.7
9.1
81.7
10.0
3.26E-03
1.52
80.4
4.1
60.7
6.3
2.07E-02
1.32
Krt25
5
76.8
18.9
152.1
22.9
9.61E-04
1.98
147.3
8.0
88.8
6.6
1.36E-03
1.66
Laptm4b
5
731.8
97.3
1085.7
90.5
7.04E-04
1.48
1189
106.6
931.5
66.5
4.40E-02
1.28
Map3k13
5
21.1
4.4
29.2
2.5
1.31E-02
1.38
30.2
1.2
19.8
1.8
2.69E-03
1.52
Mfap3l
5
59.8
7.1
71.3
4.3
2.46E-02
1.19
69.1
7.6
51.4
4.6
4.85E-02
1.34
Napepld
5
104.7
6.6
158.2
14.7
1.66E-04
1.51
160.9
8.7
107.0
4.3
1.42E-03
1.50
Prkaa2
5
94.7
6.9
161.0
10.5
5.65E-06
1.70
164.4
11.2
99.7
18.7
1.37E-02
1.65
Reep1
5
83.4
10.4
145.2
6.9
9.13E-06
1.74
164.2
24.8
84.0
14.2
1.66E-02
1.95
Serpini1
5
13.8
2.0
34.4
3.6
8.36E-06
2.49
31.7
1.2
11.5
3.6
1.65E-03
2.76
Sgpp2
5
242.2
24.4
402.1
19.4
7.05E-06
1.66
422.7
16.6
268.5
16.9
7.72E-04
1.57
Slc26a4
5
59.1
11.4
103.6
9.2
2.93E-04
1.75
91.3
3.0
58.4
8.1
5.69E-03
1.56
Slc26a7
5
335.3
52.0
568.5
47.6
1.66E-04
1.70
519.7
39.9
328.8
45.5
1.12E-02
1.58
Slc7a7
5
316.0
14.9
475.9
34.9
2.97E-05
1.51
449.3
38.6
282.5
20.4
5.69E-03
1.59
Spata20
5
34.4
9.1
58.3
7.7
3.83E-03
1.70
67.9
8.0
38.7
3.1
8.43E-03
1.76
Syna
5
1696.5
252.2
3299.9
299
3.64E-05
1.95
2968
30.4
1632.6
87.9
3.48E-05
1.82
Xlr
5
159.3
20.5
247.2
25.4
6.65E-04
1.55
235.7
15.2
151.4
2.8
1.53E-03
1.56
B3galt5
5B
174.7
14.9
272.6
30.4
4.14E-04
1.56
260.7
11.8
199.4
18.7
1.71E-02
1.31
Development • Supplementary information
Itk
Development 143: doi:10.1242/dev.130369: Supplementary information
5B
85.0
12.5
277.3
43.7
2.91E-05 3.26
333.2
48.5
122.3
47.5
1.17E-02
2.73
Plxna4
5B
146.5
28.8
196.7
22.3
2.47E-02
1.34
201.8
7.0
141.8
16.3
8.68E-03
1.42
Ptprn2
5B
15.1
5.5
60.6
11.8
1.18E-04
4.00
67.1
4.5
24.3
6.3
1.45E-03
2.76
Slc18a2
5B
29.6
2.4
39.4
3.1
9.85E-04
1.33
51.5
2.6
35.2
6.6
3.16E-02
1.46
Wnt9b
5B
20.1
2.5
45.0
10.4
1.66E-03
2.24
54.6
5.3
20.8
5.4
3.16E-03
2.63
Bicd1
6
15.2
2.3
23.0
3.0
3.12E-03
1.51
19.2
2.4
12.2
5.8
1.85E-01
1.58
Brsk1
6
10.8
2.2
23.3
3.5
3.07E-04
2.16
16.9
4.0
9.6
3.6
1.25E-01
1.77
Cxcl1
6
148.5
22.9
248.8
51.6
7.46E-03
1.68
192.7
26.9
171.9
41.8
5.85E-01
1.12
Fam105a
6
311.2
35.3
438.7
34.9
8.91E-04
1.41
465.8
39.7
379.9
38.0
9.18E-02
1.23
Lass6
6
302.0
39.6
416.7
21.6
9.47E-04
1.38
436.1
37.6
374.8
12.1
9.32E-02
1.16
Lpl
6
215.0
18.1
281.4
26.7
3.35E-03
1.31
307.1
53.8
254.9
41.3
3.37E-01
1.20
Lrp8
6
214.0
23.6
321.3
46.0
3.19E-03
1.50
324.9
15.4
244.5
55.5
1.20E-01
1.33
Mmp9
6
11.3
3.1
17.4
2.7
1.59E-02
1.55
16.0
4.7
13.1
3.9
5.30E-01
1.23
Pex5l
6
103.8
10.0
177.9
9.4
4.65E-06
1.71
180.2
2.5
140.8
25.5
9.50E-02
1.28
Prl4a1
6
2078.6
253.0
3390.0
161
2.30E-05
1.63
2960
332.3
2417.5
518.1
2.80E-01
1.22
Sele
6
284.8
38.2
425.4
42.3
1.15E-03
1.49
430.9
39.0
316.1
45.5
5.34E-02
1.36
Slc9a2
6
95.0
7.4
150.9
17.2
3.29E-04
1.59
143.0
33.4
108.2
5.3
2.20E-01
1.32
Sostdc1
6
20.3
4.1
40.6
9.7
4.93E-03
2.00
51.8
12.1
30.6
3.2
7.46E-02
1.69
Sox8
6
18.2
3.4
27.4
5.7
2.39E-02
1.51
24.3
4.2
23.6
1.9
8.55E-01
1.03
Tbc1d10a
6
209.8
25.2
252.5
18.1
2.49E-02
1.20
286.0
2.7
228.8
34.9
8.17E-02
1.25
Pex2
7
882.4
54.1
811.6
38.7
6.59E-02
0.92
863.8
27.6
918.0
20.7
9.02E-02
0.94
Socs3
7
151.1
18.9
175.0
10.6
5.80E-02
1.16
166.3
22.4
161.3
6.4
7.78E-01
1.03
Srprb
7
1934.7
166.5
1973.8
45.9
6.62E-01
1.02
1936
64.7
1989.9
19.5
3.24E-01
0.97
Tead1
7
716.8
61.1
780.2
66.6
1.98E-01
1.09
769.4
30.7
717.9
57.6
3.27E-01
1.07
Tgfa
7
165.5
47.4
230.9
41.2
7.07E-02
1.40
232.9
33.5
166.3
32.0
1.12E-01
1.40
Zfp36
7
2301.6
107.7
2360.4
68.7
3.84E-01
1.03
2333
101.0
2412.4
109.8
4.92E-01
0.97
Sema3c
72.2
22.8
141.1
67.0
8.73E-02
1.95
122.4
16.1
56.8
18.3
1.90E-02
2.15
1600014C10Rik
654.2
20.8
666.6
29.5
5.10E-01
1.02
621.4
10.7
676.5
36.5
1.10E-01
0.92
A2bp1
12.8
3.1
18.4
4.3
6.53E-02
1.44
16.4
1.6
15.0
3.9
6.61E-01
1.09
Actb
73632.0
1358
74466
2278
5.47E-01
1.01
74308
329.7
73857
690.9
4.52E-01
1.01
AI506816
26.4
9.3
27.2
3.8
8.78E-01
1.03
24.7
4.1
31.6
5.9
2.48E-01
0.78
Cd300a
39.2
8.2
45.1
6.4
2.90E-01
1.15
44.2
5.1
36.7
6.9
2.81E-01
1.21
Cxcl10
36.1
7.8
39.6
7.5
5.37E-01
1.10
40.1
7.3
38.0
6.6
7.78E-01
1.06
Cyp2a4
12.1
2.4
11.3
2.5
6.56E-01
0.93
13.5
1.9
11.5
5.6
6.63E-01
1.17
Dusp2
182.8
28.1
198.4
23.9
4.22E-01
1.09
195.9
39.6
176.0
57.9
7.09E-01
1.11
Egr1
83.5
16.4
75.5
9.7
4.29E-01
0.90
86.9
7.6
90.4
8.1
6.81E-01
0.96
Etnk1
603.5
77.1
652.4
66.5
3.65E-01
1.08
644.7
46.8
594.1
30.6
2.70E-01
1.09
Fos
607.2
44.1
612.1
60.0
9.00E-01
1.01
643.8
16.7
793.3
12.8
5.54E-04
0.81
Galnt3
62.5
8.8
72.7
4.8
7.39E-02
1.16
71.5
9.3
54.9
3.9
8.05E-02
1.30
Gapdh
18133.1
335.5
17940
541
5.62E-01
0.99
17963
79.7
18073
168.4
4.47E-01
0.99
Il1a
52.5
22.8
44.6
5.2
5.21E-01
0.85
38.6
4.0
40.6
4.6
6.73E-01
0.95
Il1b
13.1
1.4
16.2
4.3
2.04E-01
1.24
21.6
4.0
11.2
2.9
4.00E-02
1.93
Il23a
24.6
6.6
32.7
6.5
1.20E-01
1.33
45.0
14.4
28.5
6.6
2.13E-01
1.58
Kcnq5
12.9
4.4
17.3
5.0
2.25E-01
1.34
17.9
4.1
20.2
5.0
6.52E-01
0.89
Kirrel3
18.7
2.7
28.3
9.9
9.99E-02
1.51
28.7
3.5
24.0
3.0
2.16E-01
1.20
Myd116
1463.6
122.6
1459.2
99.3
9.57E-01
1.00
1408.8
90.4
1450.8
69.3
6.29E-01
0.97
Myl12b
2051.5
110.8
1981.7
68.7
3.16E-01
0.97
2037.9
46.0
2192.9
140.7
2.13E-01
0.93
Development • Supplementary information
Bmp10
Development 143: doi:10.1242/dev.130369: Supplementary information
19.6
2.7
22.8
5.8
3.51E-01 1.16
22.4
0.2
26.2
2.2
7.05E-02
0.86
Ppp1r9a
266.9
46.6
291.4
38.8
4.41E-01
1.09
317.8
26.2
245.4
16.6
3.00E-02
1.30
Ptger4
95.8
6.6
99.0
10.1
6.14E-01
1.03
110.3
3.7
100.6
10.1
2.72E-01
1.10
Ptgs2
242.8
62.0
281.4
66.5
4.21E-01
1.16
252.0
21.4
323.2
12.0
1.48E-02
0.78
Ptpn20
28.5
8.9
40.0
8.6
9.99E-02
1.40
34.7
2.4
26.9
8.1
2.65E-01
1.29
Sgsm1
26.2
2.9
30.0
2.8
9.33E-02
1.15
30.0
2.1
21.1
5.3
9.22E-02
1.42
Sgtb
36.8
9.1
51.3
9.5
5.99E-02
1.39
57.2
6.3
40.5
5.7
5.03E-02
1.41
Tiprl
10.6
2.1
10.2
2.7
8.13E-01
0.96
9.2
3.6
9.3
2.8
9.74E-01
0.99
Tmtc4
21.6
6.0
28.4
3.5
8.30E-02
1.32
34.9
2.9
24.3
4.0
3.92E-02
1.44
Tnf
20.5
9.9
19.8
4.2
9.07E-01
0.97
21.4
3.1
20.1
4.8
7.60E-01
1.07
Tpbpa
823119
5138
1
5.5
0.93
1.30
9523
3
1.7
85086
3
22.0
3478
7
2.4
0.87
5.42E-02
74356
4
33.7
2.09E-01
22.5
76349
3
29.3
3.23E-01
Wnt7a
1009
38
2.5
4.83E-03
1.53
Zfp36l1
1168.8
193.3
1253.0
4.83E-01
1.07
1310.8
247.3
1231.3
34.3
6.76E-01
1.06
Zfp36l3
476.4
65.7
3.8
122.
8
0.7
5.33E-07
0.01
9.4
0.7
534.6
58.4
2.20E-04
0.02
Development • Supplementary information
Pilra
Development 143: doi:10.1242/dev.130369: Supplementary information
Supplemental Table S4. NanoString quantitation of transcript expression in undifferentiated and differentiated TSC. Shown here are the
normalized reads from the NanoString assays of undifferentiated (Un) and differentiated (Dif) WT and KO TSC (n=4 in each group). Also shown
are the ratios of KO/WT in each group, and the p values from the comparison of WT and KO averages in each group (unpaired, two-tailed t tests).
Probability categories and other information are described in the legend to Supplemental Table S3.
Prob.
cat.
WT Un
avg
WT
Un
SD
KO
Un
avg
KO
Un
SD
Ratio
KO/
WT Un
p WT
vs. KO
Un
WT Dif
avg
WT
Dif
SD
KO Dif
avg
KO
Dif
SD
p WT vs
KO Dif
35.7
Ratio
KO/
WT
Dif
1.68
1600002H
07Rik
4921524J1
7Rik
5730508B
09Rik
B3gnt2
1
426.1
12.0
727.3
19.0
1.71
4.24E-07
865.1
35.3
1455.3
1
255.9
15.9
318.8
14.2
1.25
2.22E-03
814.8
89.7
510.0
16.6
0.63
1.16E-03
1
70.3
10.0
102.3
16.4
1.45
2.82E-02
94.8
15.1
75.7
6.9
0.80
9.34E-02
1
859.8
71.4
1288.8
62.1
1.50
2.25E-04
838.3
76.4
539.5
31.0
0.64
7.63E-04
Cldn23
1
25.1
3.0
62.0
11.1
2.47
1.45E-03
85.0
1.5
181.2
12.9
2.13
1.39E-05
Cpeb2
1
657.8
43.5
834.0
93.5
Dos
1
171.3
13.4
298.2
22.5
1.27
2.53E-02
1052.3
58.5
1489.6
46.0
1.42
5.25E-05
1.74
1.56E-04
254.7
19.3
418.7
20.1
1.64
5.26E-05
Elavl1
1
373.2
39.1
358.3
12.7
0.96
5.53E-01
696.4
30.4
762.3
17.5
1.09
1.73E-02
Hbegf
1
748.5
23.3
1169.5
65.7
1.56
4.48E-05
3210.4
121.9
6669.5
242.3
2.08
5.63E-07
Irx2
1
495.9
Ldlr
1
1437.0
28.7
940.1
14.4
1.90
3.47E-07
1660.3
146.6
1181.3
9.6
0.71
1.32E-03
55.7
1714.1
79.5
1.19
2.58E-03
3725.8
95.0
4565.4
131.5
1.23
1.08E-04
Lipg
1
260.8
15.4
204.6
13.6
0.78
3.17E-03
1899.3
285.8
463.4
24.8
0.24
1.30E-04
Lonrf3
1
26.3
3.7
37.1
5.9
1.41
3.75E-02
853.3
46.4
1600.9
52.3
1.88
1.60E-06
Mtm1
1
146.9
11.3
285.7
24.1
1.95
1.03E-04
95.2
4.9
102.5
7.2
1.08
2.00E-01
Pank3
1
1285.3
93.0
1149.8
48.1
0.89
6.63E-02
3572.7
49.7
3391.3
49.4
0.95
4.19E-03
Pdxp
1
401.9
30.3
371.6
27.9
0.92
2.49E-01
632.0
45.0
470.8
16.5
0.74
1.13E-03
Slc38a3
1
91.4
10.7
382.9
29.6
4.19
3.72E-06
119.7
6.6
123.6
7.6
1.03
5.25E-01
Snurf
1
535.0
28.1
515.1
35.4
0.96
4.74E-01
1161.4
44.5
1189.4
31.0
1.02
4.06E-01
Star
1
9.3
2.8
12.8
3.7
1.38
2.42E-01
26.0
5.1
25.0
0.5
0.96
7.37E-01
Suco
1
556.6
38.2
957.0
45.1
1.72
2.32E-05
1598.6
204.5
1917.6
36.6
1.20
3.76E-02
Tcfcp2l1
1
126.3
15.6
489.6
20.3
3.88
3.00E-07
215.0
13.4
858.9
27.7
4.00
2.91E-08
Tfrc
1
5887.5
230.5
2921.9
89.6
0.50
8.10E-07
40152.1
1530.0
29664.5
1115.7
0.74
7.34E-05
Zfp36l2
1
711.9
30.7
663.3
70.0
0.93
3.13E-01
1267.2
47.3
1096.0
41.2
0.86
3.23E-03
1110067D
22Rik
1700047I1
7Rik1
Acvr1
2
236.6
14.5
404.6
10.6
1.71
3.54E-06
1790.5
83.0
1894.2
31.6
1.06
8.94E-02
2
488.6
37.7
963.0
39.2
1.97
5.31E-06
768.0
19.9
1156.4
52.9
1.51
2.13E-05
2
457.7
37.3
610.6
21.0
1.33
8.25E-04
383.0
70.5
249.4
12.1
0.65
1.78E-02
Atp1b1
2
920.5
53.0
955.8
59.9
1.04
4.74E-01
1500.3
50.9
1577.0
32.6
1.05
7.04E-02
Atp6v1b1
2
5.5
2.2
3.5
1.2
0.64
2.16E-01
5.7
1.1
5.3
1.2
0.93
7.07E-01
Fam3c
2
669.4
58.7
434.3
24.5
0.65
6.86E-04
932.0
57.8
934.6
33.6
1.00
9.49E-01
Gcnt1
2
621.3
31.5
809.7
81.2
1.30
9.55E-03
481.1
26.6
798.8
35.6
1.66
1.69E-05
Hapln4
2
89.7
2.8
175.9
19.9
1.96
3.07E-04
35.0
12.5
54.9
3.4
1.57
3.80E-02
Itih5
2
3.9
1.5
8.9
2.8
2.26
3.36E-02
20.7
3.1
14.4
4.9
0.69
1.08E-01
Itk
2
7.5
0.7
7.2
3.0
0.96
8.73E-01
20.0
3.2
11.0
1.3
0.55
3.91E-03
Phtf2
2
170.8
9.5
195.8
16.5
1.15
6.25E-02
156.6
3.7
166.9
3.7
1.07
1.43E-02
Rhpn2
2
37.7
11.2
35.7
8.6
0.95
8.18E-01
122.2
12.6
330.4
19.8
2.70
4.78E-06
Sema3c
2
329.6
29.6
187.3
15.5
0.57
3.17E-04
102.4
6.3
85.8
5.8
0.84
1.56E-02
Slc45a3
2
95.1
7.5
110.6
13.8
1.16
1.38E-01
50.9
1.6
105.0
5.9
2.06
4.73E-06
Tbc1d9
2
71.1
6.3
73.0
17.6
1.03
8.67E-01
129.5
5.2
245.1
13.1
1.89
7.59E-06
Tcf7
2
1067.0
42.9
3465.7
81.8
3.25
8.09E-09
694.7
23.3
645.4
13.8
0.93
1.98E-02
9.15E-07
Development • Supplementary information
Gene
Tpd52l1
2
90.6
11.8
121.5
9.0
1.34
1.13E-02
75.4
9.1
80.5
3.1
1.07
3.97E-01
Grem2
3
141.1
21.1
116.3
11.0
0.82
1.20E-01
74.2
13.2
56.8
3.6
0.76
6.93E-02
Phyhipl
3
41.8
2.5
51.1
8.7
1.22
1.25E-01
417.1
27.3
429.7
20.9
1.03
5.48E-01
Sema4g
3
36.6
5.3
41.5
5.3
1.13
3.02E-01
90.7
7.8
122.3
3.2
1.35
6.35E-04
Jag1
4
68.0
6.0
30.0
3.0
0.44
6.36E-05
253.4
24.3
153.2
2.4
0.60
3.88E-04
Mtap9
4
149.1
6.6
121.2
2.0
0.81
4.08E-04
237.5
17.7
241.7
7.0
1.02
7.18E-01
Nexn
4
127.2
6.1
200.6
15.9
1.58
2.97E-04
48.1
3.5
185.3
9.9
3.85
4.98E-07
Slc16a7
4
6.9
3.2
21.9
4.5
3.17
3.50E-03
5.8
1.9
10.7
1.4
1.84
1.08E-02
St3gal6
4
149.7
8.3
68.7
12.8
0.46
9.28E-05
84.6
10.1
75.6
1.8
0.89
1.78E-01
2310045A
20Rik
Ankrd13d
5
18.7
7.0
37.9
8.6
2.02
2.42E-02
51.6
3.4
165.5
8.1
3.21
4.94E-07
5
37.3
9.4
62.0
6.5
1.66
9.86E-03
23.8
5.6
48.2
5.3
2.02
1.57E-03
Asb1
5
145.9
12.5
282.5
21.2
1.94
7.21E-05
170.9
9.6
171.7
13.3
1.00
9.33E-01
Bahcc1
5
24.9
4.8
15.9
4.6
0.64
5.63E-02
11.4
3.4
11.6
3.5
1.02
9.46E-01
Chn2
5
58.2
4.4
32.5
5.3
0.56
6.80E-04
20.1
3.4
11.4
1.0
0.57
5.33E-03
D630045J
12Rik
Efha2
5
190.5
14.3
176.5
20.8
0.93
3.74E-01
74.2
9.3
98.7
6.3
1.33
9.29E-03
5
106.7
11.6
174.0
9.5
1.63
2.36E-04
70.7
6.0
56.1
7.4
0.79
3.68E-02
Ehbp1
5
109.1
15.0
145.2
24.9
1.33
7.49E-02
112.3
11.5
115.6
8.6
1.03
6.98E-01
Fam125b
5
115.6
6.1
294.8
10.5
2.55
2.32E-07
186.3
9.9
169.0
6.9
0.91
4.64E-02
Fam55c
5
429.8
34.5
783.0
36.9
1.82
1.92E-05
94.1
17.5
184.3
11.6
1.96
3.00E-04
Glcci1
5
150.4
14.1
86.2
7.3
0.57
4.20E-04
30.7
6.2
43.7
5.6
1.42
3.64E-02
Hectd2
5
82.7
12.7
103.2
5.5
1.25
4.23E-02
114.6
9.8
70.0
2.6
0.61
2.67E-04
Krt25
5
9.3
2.4
11.2
5.1
1.20
5.88E-01
15.3
2.4
11.7
2.3
0.77
1.10E-01
Laptm4b
5
1017.0
59.4
2019.9
91.8
1.99
3.94E-06
266.1
17.3
276.6
7.3
1.04
3.69E-01
Map3k13
5
28.1
3.7
11.0
4.0
0.39
1.65E-03
22.2
5.9
18.5
5.1
0.83
4.39E-01
Mfap3l
5
82.2
11.5
82.5
6.3
1.00
9.74E-01
47.4
4.7
32.1
4.7
0.68
7.03E-03
Napepld
5
15.1
3.4
14.9
2.9
0.99
9.48E-01
71.2
7.7
159.5
20.7
2.24
4.49E-04
Prkaa2
5
96.5
9.8
206.5
14.1
2.14
3.14E-05
30.1
6.1
91.0
8.0
3.02
4.57E-05
Reep1
5
125.9
10.3
160.4
14.1
1.27
1.40E-02
58.0
13.9
204.0
17.0
3.52
2.61E-05
Serpini1
5
13.8
2.4
13.7
3.9
1.00
9.91E-01
14.3
3.3
10.5
1.5
0.73
1.18E-01
Sgpp2
5
20.1
2.9
12.1
1.8
0.60
6.50E-03
15.8
3.9
18.4
3.7
1.17
4.27E-01
Slc26a4
5
3.3
1.4
4.7
3.6
1.42
5.59E-01
7.8
0.6
10.3
1.5
1.31
3.79E-02
Slc26a7
5
3.8
0.6
5.1
1.9
1.35
3.06E-01
11.7
2.3
12.8
1.7
1.09
5.30E-01
Slc7a7
5
59.7
2.7
356.8
33.9
5.98
5.24E-06
23.3
5.4
46.8
6.9
2.01
3.62E-03
Spata20
5
8.2
2.9
8.9
1.3
1.09
7.14E-01
11.1
1.2
11.3
1.9
1.02
8.98E-01
Syna
5
10.0
2.6
19.6
0.8
1.95
8.12E-04
335.4
32.3
1266.3
38.9
3.78
6.33E-08
Xlr
5
17.2
5.2
288.1
21.9
16.74
7.90E-07
80.8
7.5
437.3
38.8
5.41
4.34E-06
Bicd1
6
6.2
1.7
7.2
4.0
1.17
6.94E-01
4.8
2.0
6.4
2.5
1.32
4.28E-01
Brsk1
6
77.8
4.1
131.2
5.4
1.69
9.62E-06
30.3
2.8
64.6
7.1
2.13
2.37E-04
Cxcl1
6
13.2
2.5
10.9
2.4
0.82
2.77E-01
5.4
0.7
9.2
0.6
1.70
3.39E-04
Ept1
6
1178.2
88.4
1542.1
37.9
1.31
6.05E-04
1367.1
44.6
1251.8
30.1
0.92
9.96E-03
Fam105a
6
21.9
5.2
37.5
6.3
1.72
1.65E-02
19.3
4.4
37.4
2.0
1.93
6.27E-04
Lass6
6
731.1
104.4
743.1
95.4
1.02
8.88E-01
73.8
18.8
106.3
5.6
1.44
2.82E-02
Lpl
6
9.7
4.0
7.6
2.7
0.79
4.88E-01
62.6
4.2
203.8
15.3
3.26
4.73E-06
Lrp8
6
53.9
4.9
51.5
8.6
0.95
6.79E-01
66.5
9.1
27.1
6.1
0.41
7.78E-04
Mmp9
6
1819.7
31.5
1244.0
72.0
0.68
1.47E-05
31.3
6.2
7.5
2.6
0.24
8.97E-04
Pex5l
6
7.0
2.7
11.1
1.7
1.59
6.47E-02
23.8
6.7
10.8
2.3
0.45
1.89E-02
Prl4a1
6
7.5
2.2
6.4
4.1
0.86
7.07E-01
8.8
3.9
9.3
1.7
1.05
8.52E-01
Development • Supplementary information
Development 143: doi:10.1242/dev.130369: Supplementary information
Sele
6
5.7
1.8
7.0
2.7
1.22
5.33E-01
7.0
2.7
6.0
4.0
0.87
7.51E-01
Slc9a2
6
4.9
1.2
7.9
1.7
1.63
4.36E-02
2.4
1.2
2.4
1.5
1.03
9.45E-01
Sostdc1
6
4.5
1.0
5.4
1.3
1.20
3.89E-01
4.4
1.1
7.4
3.9
1.69
2.45E-01
Sox8
6
13.0
3.6
19.9
3.7
1.53
5.88E-02
27.6
6.6
17.7
3.8
0.64
6.23E-02
Tbc1d10a
6
117.7
6.9
204.9
9.2
1.74
1.21E-05
242.6
12.1
295.9
7.9
1.22
7.10E-04
9030409G
11Rik
Pex2
7
188.6
3.8
201.2
14.9
1.07
2.05E-01
456.9
50.2
628.3
20.9
1.38
1.57E-03
7
933.5
55.5
1156.2
59.1
1.24
3.13E-03
1137.1
134.2
750.5
11.6
0.66
2.53E-03
Socs3
7
161.7
10.9
414.1
24.1
2.56
3.15E-06
321.5
27.5
274.7
11.0
0.85
3.36E-02
Srprb
7
2234.0
143.7
3464.9
127.2
1.55
3.17E-05
3210.1
89.5
2732.6
33.1
0.85
1.30E-04
Tead1
7
1600.9
59.5
2353.5
136.8
1.47
1.24E-04
964.9
28.7
1124.1
61.7
1.17
6.70E-03
Tgfa
7
16.8
3.4
23.1
4.4
1.37
9.91E-02
299.6
21.8
261.4
23.2
0.87
8.32E-02
Zfp36
7
3517.3
60.6
4158.9
183.2
1.18
1.20E-03
4613.4
319.1
3547.9
97.2
0.77
1.47E-03
B3galt5
5B
5.9
0.8
11.0
4.6
1.85
1.09E-01
6.3
1.3
6.8
2.3
1.08
7.62E-01
Bmp10
5B
4.2
0.8
6.8
1.3
1.64
2.22E-02
4.7
2.1
4.6
2.3
0.97
9.40E-01
Plxna4
5B
5.8
1.3
6.5
3.1
1.12
7.29E-01
7.9
3.4
8.0
4.2
1.02
9.68E-01
Ptprn2
5B
10.8
2.5
17.2
2.2
1.59
1.73E-02
6.1
1.3
9.8
1.4
1.60
1.48E-02
Slc18a2
5B
3.7
1.5
4.9
0.9
1.35
2.54E-01
3.6
2.8
4.7
0.6
1.33
5.07E-01
Wnt9b
5B
4.93E-01
5.0
3.1
8.1
1.5
1.62
1.66E-01
6.6
3.8
8.5
2.2
1.28
1600014C10
Rik
A2bp1
194.0
9.4
298.5
14.2
1.54
4.05E-05
546.9
34.6
555.0
9.5
1.01
4.0
2.3
8.6
2.9
2.15
7.87E-02
4.4
1.8
4.3
2.7
0.99
9.78E-01
Actb
150297
1497
4088
0.98
2.58E-01
140991
2578
121401
1465
0.86
2.67E-05
AI506816
8.2
2.7
14715
4
10.6
2.6
1.30
2.99E-01
6.1
2.1
5.7
1.6
0.93
7.89E-01
Cd300a
150.6
9.7
143.2
10.1
0.95
4.00E-01
10.7
2.9
14.1
2.5
1.32
1.76E-01
Csf2
4.4
1.6
5.7
1.3
1.31
3.11E-01
7.4
2.6
5.7
0.7
0.76
3.00E-01
Cxcl10
38.8
7.0
23.8
6.4
0.61
3.47E-02
18.5
2.5
34.5
3.3
1.86
5.34E-04
Cyp2a4
3.8
1.1
6.4
1.4
1.68
4.06E-02
5.7
2.8
6.5
2.8
1.14
7.42E-01
Dusp2
72.8
4.8
127.7
18.1
1.75
2.27E-03
86.0
15.5
46.7
5.7
0.54
6.21E-03
Egr1
532.2
15.5
1949.3
179.7
3.66
9.78E-06
75.8
7.2
61.4
8.9
0.81
7.16E-02
Etnk1
452.2
29.8
591.9
41.5
1.31
3.20E-03
449.9
24.7
555.9
24.9
1.24
1.93E-03
Fos
517.6
10.6
2127.1
91.1
4.11
8.41E-08
511.4
41.5
1065.4
40.5
2.08
3.12E-06
Galnt3
106.7
3.6
48.6
8.5
0.45
3.43E-05
127.6
15.1
75.6
8.0
0.59
1.89E-03
Gapdh
54316.5
546.9
55513
1521
1.02
2.47E-01
57914.9
1041.3
67248.2
819.4
1.16
1.84E-05
Il1a
7.1
4.1
6.1
4.4
0.85
7.69E-01
41.3
6.2
18.3
2.2
0.44
9.45E-04
Il1b
4.0
0.5
3.6
0.6
0.90
4.21E-01
4.5
2.4
4.1
1.9
0.92
8.37E-01
Il23a
149.5
5.6
158.4
24.2
1.06
5.56E-01
32.8
4.1
40.0
4.1
1.22
7.80E-02
Kcnq5
13.0
2.6
15.3
3.2
1.17
3.85E-01
16.1
2.1
28.8
3.6
1.79
1.93E-03
Kirrel3
6.3
2.2
3.4
0.8
0.55
7.95E-02
3.3
0.8
6.2
2.2
1.90
7.15E-02
Myd116
558.1
30.6
2814.3
115.2
5.04
5.35E-08
844.2
39.1
1113.6
16.9
1.32
3.46E-05
Myl12b
2254.5
87.2
3039.8
84.7
1.35
3.04E-05
2191.9
65.7
3812.7
33.1
1.74
2.16E-08
Pilra
2.8
1.2
6.3
2.0
2.21
4.19E-02
4.6
2.3
4.2
2.4
0.90
8.09E-01
Ppp1r9a
120.6
4.6
168.2
6.9
1.40
5.98E-05
140.9
8.2
142.1
12.4
1.01
8.92E-01
Ptger4
28.8
1.1
38.9
5.5
1.35
2.07E-02
19.8
1.6
12.0
2.1
0.61
2.37E-03
Ptgs2
1165.2
67.3
601.0
38.8
0.52
1.55E-05
88.8
4.8
205.7
23.0
2.32
1.35E-04
Ptpn20
4.2
0.6
4.0
3.5
0.96
9.38E-01
4.5
2.1
3.8
0.6
0.84
5.78E-01
Sgsm1
87.0
12.2
180.8
19.6
2.08
4.07E-04
65.0
19.2
58.6
5.2
0.90
5.97E-01
Sgtb
58.2
4.3
141.4
8.6
2.43
5.50E-06
41.2
6.6
75.5
9.6
1.83
2.20E-03
Development • Supplementary information
Development 143: doi:10.1242/dev.130369: Supplementary information
Tiprl
9.9
2.0
8.8
0.3
0.89
3.67E-01
17.4
0.5
12.5
3.2
0.72
3.67E-02
Tmtc4
33.1
4.4
51.6
6.7
1.56
7.17E-03
35.6
2.5
34.1
5.1
0.96
6.68E-01
Tnf
11.6
3.6
5.7
2.6
0.49
6.35E-02
7.0
1.5
5.3
1.8
0.76
2.60E-01
Tpbpa
7.3
2.6
8.2
2.6
1.13
6.74E-01
443.8
48.7
14.6
2.9
0.03
5.04E-06
Wnt7a
149.7
3.1
253.5
10.5
1.69
3.29E-06
35.7
8.7
64.9
5.3
1.82
2.58E-03
Zfp36l1
825.3
49.5
511.9
25.5
0.62
6.68E-05
837.9
21.9
1129.3
35.1
1.35
1.85E-05
Zfp36l3
7.0
3.0
2.5
1.4
0.35
5.55E-02
143.4
4.2
4.3
1.1
0.03
2.23E-09
Development • Supplementary information
Development 143: doi:10.1242/dev.130369: Supplementary information
Development 143: doi:10.1242/dev.130369: Supplementary information
Gene
Prob.
cat.
1
WT 2h
avg
81.32
WT 2h
SD
4.03
WT 4h
avg
72.44
WT 4h
SD
3.80
KO 2h
avg
94.43
KO 2h
SD
3.52
KO 4h
avg
83.54
KO
4h SD
4.70
p 2h
p 4h
0.005400
0.019012
1
55.02
3.14
38.12
0.71
77.77
4.90
59.72
10.94
0.000508
0.014273
1
78.93
12.44
69.73
10.20
103.86
4.33
94.40
9.04
0.016879
0.020185
1
59.10
2.34
32.61
2.86
83.75
4.88
56.01
7.05
0.000220
0.001789
Cldn23
1
69.74
3.49
60.10
5.06
99.42
9.37
75.99
6.20
0.002128
0.013801
Cpeb2
1
71.44
1.60
59.72
2.20
78.94
3.68
76.31
7.09
0.017782
0.008254
Dos
1
66.62
1.91
56.35
2.85
75.56
2.69
65.15
5.19
0.003342
0.042104
Elavl1
1
72.62
2.74
61.70
3.96
82.89
6.43
85.32
5.15
0.043793
0.000750
Hbegf
1
69.49
0.87
56.01
0.70
95.62
3.66
81.62
3.96
0.000020
0.000033
Irx2
1
47.40
2.08
24.18
1.48
54.85
2.56
34.89
3.22
0.007923
0.001954
Ldlr
1
72.86
1.85
67.21
3.28
80.00
2.42
74.21
3.37
0.006655
0.041692
Lipg
1
74.94
3.80
65.31
3.45
92.84
7.22
97.11
12.50
0.008942
0.005406
Lonrf3
1
76.06
2.81
68.35
1.97
94.44
7.87
80.75
7.05
0.008863
0.026225
Mtm1
1
83.61
6.09
62.26
2.33
97.47
7.15
82.73
8.39
0.043014
0.006544
Pank3
1
60.54
2.25
39.04
0.35
74.66
3.98
49.74
2.84
0.001743
0.000645
Pdxp
1
70.53
2.97
60.64
6.13
84.35
8.64
79.88
6.00
0.039550
0.008130
Slc38a3
1
91.64
9.88
84.19
10.00
118.75
10.17
110.99
16.00
0.016192
0.049164
Snurf
1
95.47
2.24
87.82
2.45
106.56
6.27
99.68
3.43
0.027861
0.002796
Star
1
73.42
12.05
86.20
7.03
111.45
22.62
119.87
18.17
0.042297
0.024194
Suco
1
68.09
0.57
48.77
2.41
80.50
3.65
75.66
7.06
0.001127
0.000782
Tcfcp2l1
1
72.73
2.66
67.85
7.50
84.70
5.18
89.23
6.89
0.011971
0.010891
Tfrc
1
86.37
2.59
76.32
3.21
103.38
5.70
91.88
1.09
0.003314
0.000210
Zfp36l2
1
52.82
2.34
30.18
1.86
67.36
2.96
42.92
3.95
0.000546
0.002328
1110067D22
Rik
1700047I17
Rik1
Acvr1
2
79.28
2.76
68.20
1.56
85.90
4.28
73.15
2.23
0.065233
0.019734
2
90.73
3.43
70.57
2.72
97.25
3.76
83.03
4.11
0.068153
0.004645
2
60.27
2.31
47.95
5.41
70.64
3.24
62.39
9.20
0.004033
0.057587
Atp1b1
2
96.11
3.46
78.60
3.50
97.85
4.67
88.81
4.16
0.623904
0.017397
Atp6v1b1
2
67.33
24.78
72.29
12.61
139.72
41.15
73.36
30.76
0.040098
0.957096
Fam3c
2
92.95
1.64
76.51
2.14
87.40
3.05
82.32
4.26
0.032325
0.079616
Gcnt1
2
80.81
3.27
50.87
3.17
84.38
1.80
65.86
3.32
0.148962
0.001306
Hapln4
2
67.37
8.24
66.93
12.78
84.40
19.01
95.54
10.25
0.204415
0.023270
Itih5
2
82.48
27.61
85.31
17.45
157.90
34.18
121.33
24.10
0.024862
0.080860
Itk
2
75.89
18.63
59.50
15.47
104.49
40.64
104.65
21.58
0.310240
0.025774
Phtf2
2
71.88
2.67
78.33
3.39
100.24
10.09
86.13
6.10
0.003299
0.101096
Rhpn2
2
71.47
8.17
64.33
7.49
85.69
5.64
66.60
6.62
0.047799
0.707219
Sema3c
2
98.13
11.83
77.74
11.36
99.45
18.70
102.38
12.36
0.921112
0.044017
Slc45a3
2
58.12
2.73
68.67
9.23
76.06
3.32
60.17
12.40
0.000356
0.377697
Tbc1d9
2
71.88
7.32
60.19
11.69
90.91
4.49
81.51
11.19
0.008544
0.062657
Tcf7
2
83.56
2.02
60.01
3.43
84.20
5.69
68.30
4.26
0.859508
0.039294
Tpd52l1
2
82.55
7.92
85.63
13.76
99.98
7.31
88.40
5.55
0.031045
0.758096
1600002H07
Rik
4921524J17
Rik
5730508B09
Rik
B3gnt2
Development • Supplementary information
Supplemental Table S5. NanoString quantitation of transcript expression after 2 and 4h of actinomycin D in differentiated WT and KO
TSC. Shown here are the percentages of time 0 (set at 100%) for the indicated transcripts assayed after actinomycin D treatment of differentiated
TSC (n=4 in each group). Also shown are the ratios of KO/WT means at each time point, and the p values from the comparison of WT and KO
averages at each time point (unpaired, two-tailed t tests). Probability categories and other information are described in the legend to Supplemental
Table S3.
Grem2
3
76.04
6.76
75.01
4.05
110.97
10.10
112.48
8.48
0.002502
0.000456
Phyhipl
3
79.77
2.79
62.64
6.13
92.93
3.88
75.62
6.37
0.003084
0.043828
Sema4g
3
68.36
11.95
66.70
4.07
90.79
9.32
96.81
5.02
0.042723
0.000194
Jag1
4
69.18
4.30
52.38
5.46
80.50
8.19
68.39
8.32
0.078418
0.031712
Mtap9
4
74.92
7.41
61.51
2.67
87.63
10.00
78.74
4.18
0.127226
0.000947
Nexn
4
84.89
18.41
56.05
3.72
78.06
7.16
76.52
8.30
0.571576
0.008031
Slc16a7
4
52.92
5.94
51.55
28.79
96.74
33.17
109.30
17.89
0.065226
0.025567
St3gal6
4
86.12
12.23
76.78
9.27
109.07
7.63
77.27
10.53
0.032955
0.953647
2310045A20
Rik
Ankrd13d
5
109.30
5.83
103.51
17.36
93.20
8.18
96.22
6.11
0.032114
0.517801
5
107.29
19.17
85.32
14.38
90.41
4.35
77.43
14.51
0.187359
0.528328
Asb1
5
69.49
4.65
49.70
7.89
70.16
3.21
55.28
5.11
0.844109
0.343140
Bahcc1
5
96.10
18.98
85.91
41.43
82.53
44.48
84.13
17.57
0.644197
0.947745
Chn2
5
85.57
11.22
69.53
17.93
71.40
16.91
85.70
35.18
0.272181
0.504593
D630045J12
Rik
Efha2
5
81.55
5.68
75.71
13.36
74.25
4.59
75.09
3.27
0.133821
0.940576
5
103.91
5.04
81.02
11.51
86.53
19.15
81.59
1.43
0.179452
0.935450
Ehbp1
5
89.80
13.48
64.96
9.37
81.10
6.76
75.93
12.19
0.356310
0.262824
Fam125b
5
78.59
6.53
74.49
6.66
72.95
8.83
73.14
9.12
0.407841
0.842777
Fam55c
5
70.35
7.28
54.05
9.78
70.19
3.34
68.27
9.96
0.972296
0.128140
Glcci1
5
75.52
5.80
54.03
5.42
64.71
7.48
44.94
12.80
0.095319
0.300641
Hectd2
5
70.73
4.59
58.00
5.53
64.89
10.40
85.56
19.28
0.407654
0.054739
Krt25
5
84.93
20.19
119.38
6.70
73.34
16.76
93.87
28.65
0.473122
0.183892
Laptm4b
5
98.05
5.69
88.92
2.60
99.74
9.29
98.58
7.05
0.797411
0.067698
Map3k13
5
76.94
6.16
47.06
16.38
78.14
18.61
73.79
24.94
0.918983
0.171632
Mfap3l
5
75.95
15.41
70.49
5.82
73.02
2.87
76.15
15.40
0.756729
0.573502
Napepld
5
81.00
8.27
48.49
7.76
81.76
1.80
51.34
8.41
0.881373
0.680647
Prkaa2
5
92.51
20.32
84.24
16.55
71.87
9.86
66.14
8.59
0.164555
0.143739
Reep1
5
89.28
13.58
83.50
11.07
79.92
8.06
82.75
3.44
0.344145
0.914499
Serpini1
5
118.75
12.11
82.77
19.86
108.25
46.82
76.53
25.69
0.719820
0.750226
Sgpp2
5
104.49
14.98
100.35
15.17
95.85
6.48
81.04
28.64
0.394827
0.341959
Slc26a4
5
84.65
20.72
78.81
47.13
57.32
28.05
61.02
36.88
0.223507
0.625061
Slc26a7
5
77.33
13.58
81.82
9.90
116.90
40.00
85.35
45.01
0.155843
0.898991
Slc7a7
5
99.83
26.76
88.24
10.04
124.88
13.78
102.95
28.00
0.199547
0.424598
Spata20
5
78.04
24.26
74.84
26.15
114.66
12.21
87.74
27.95
0.058267
0.580767
Syna
5
80.91
4.86
73.80
6.41
86.23
6.31
81.95
4.32
0.291220
0.117359
Xlr
5
108.45
7.45
97.01
17.19
117.80
3.26
106.12
6.71
0.093365
0.425220
Bicd1
6
73.63
34.64
81.76
57.53
67.67
19.77
83.07
27.59
0.804615
0.972777
Brsk1
6
108.37
17.67
97.56
12.66
89.48
6.42
98.55
20.49
0.132285
0.945618
Cxcl1
6
107.87
35.44
122.61
15.37
74.84
43.92
71.29
42.98
0.349808
0.099449
Ept1
6
83.52
3.52
69.27
2.91
89.26
3.94
82.97
3.43
0.108847
0.001868
Fam105a
6
90.29
12.48
87.50
15.28
100.94
10.46
67.19
5.73
0.300659
0.074484
Lass6
6
96.18
13.14
74.02
3.93
91.66
6.94
76.14
14.10
0.617373
0.810118
Lpl
6
116.40
6.25
113.67
4.40
110.23
6.00
103.38
12.46
0.263354
0.225910
Lrp8
6
60.17
3.85
62.06
15.91
76.78
12.46
91.67
19.03
0.069502
0.084169
Mmp9
6
91.60
15.00
75.69
21.04
92.10
28.80
95.42
22.48
0.979559
0.309360
Pex5l
6
102.50
10.04
108.08
20.40
80.89
26.78
64.14
35.03
0.238604
0.109586
Prl4a1
6
131.74
30.45
136.80
67.12
76.64
25.21
93.56
29.63
0.052293
0.346770
Sele
6
112.17
28.83
126.31
32.44
145.92
29.18
118.44
59.97
0.204045
0.848178
Development • Supplementary information
Development 143: doi:10.1242/dev.130369: Supplementary information
Slc9a2
6
85.44
40.93
159.09
82.77
122.51
100.73
66.08
27.59
0.576439
0.114362
Sostdc1
6
77.21
35.36
106.70
45.56
58.15
33.56
65.80
23.14
0.523703
0.214957
Sox8
6
69.07
12.36
53.27
5.27
68.25
28.58
68.05
22.55
0.964872
0.311154
Tbc1d10a
6
77.07
6.99
71.79
4.80
84.12
3.19
70.21
4.46
0.163101
0.691570
9030409G11
Rik
Pex2
7
71.99
4.20
52.03
3.45
83.78
6.12
71.68
6.95
0.033260
0.004634
7
62.74
3.72
40.97
1.88
85.28
3.66
64.07
5.46
0.000294
0.000446
Socs3
7
24.92
1.78
17.55
1.43
35.07
2.06
24.43
3.69
0.000655
0.023694
Srprb
7
89.17
1.65
78.97
2.29
105.80
5.14
91.59
3.42
0.001761
0.001799
Tead1
7
75.38
2.73
63.34
3.70
86.25
3.66
82.98
3.36
0.006161
0.000492
Tgfa
7
66.34
2.78
57.28
1.79
89.52
2.97
92.20
4.60
0.000063
0.000018
Zfp36
7
25.50
1.30
15.53
0.88
39.40
1.88
21.92
0.44
0.000043
0.000030
B3galt5
5B
105.53
41.79
96.98
20.81
100.92
13.89
86.33
33.05
0.862171
0.653403
Bmp10
5B
131.17
36.42
89.43
55.94
128.47
63.41
47.47
21.11
0.951137
0.269758
Plxna4
5B
115.40
42.58
103.80
29.63
107.17
16.07
87.16
22.81
0.764687
0.470079
Ptprn2
5B
155.16
30.68
106.10
64.57
106.19
54.76
86.77
54.23
0.225392
0.705061
Slc18a2
5B
79.63
16.93
133.78
66.61
95.12
46.97
54.80
27.73
0.610362
0.106746
Wnt9b
5B
116.55
34.22
122.40
58.36
109.34
25.48
49.70
14.47
0.779442
0.081136
1600014C10
Rik
A2bp1
83.21
3.40
75.11
4.29
94.93
5.56
83.43
6.96
0.020676
0.128577
69.40
30.70
134.42
21.85
101.04
36.16
66.13
24.49
0.291933
0.011314
Actb
100.29
1.25
97.82
1.31
99.15
3.95
98.63
2.51
0.651639
0.635186
AI506816
111.17
15.24
111.63
32.50
95.33
46.61
108.24
66.15
0.595944
0.939211
Cd300a
107.11
28.33
99.72
31.83
107.32
12.89
88.72
23.84
0.990786
0.648917
Csf2
55.26
17.28
56.97
29.79
128.12
32.61
65.65
39.50
0.014141
0.771478
Cxcl10
98.38
15.09
94.55
22.51
92.71
11.56
75.11
15.97
0.623622
0.268323
Cyp2a4
95.91
21.48
73.48
42.12
98.01
30.62
72.77
32.66
0.925602
0.982427
Dusp2
46.82
5.76
41.20
1.74
65.71
12.31
52.06
21.37
0.052757
0.414303
Egr1
45.76
5.05
31.62
3.08
42.79
11.09
32.83
4.53
0.687563
0.715582
Etnk1
75.23
3.78
77.74
2.33
86.83
4.08
91.98
13.45
0.011245
0.120696
Fos
29.57
0.93
16.77
0.67
27.51
1.59
14.69
1.88
0.100742
0.121001
Galnt3
100.59
3.96
77.11
6.56
97.64
8.55
81.66
5.55
0.606669
0.394309
Gapdh
99.69
1.25
102.22
1.37
101.00
4.11
101.44
2.64
0.616955
0.666753
Il1a
72.06
5.80
55.68
5.98
90.25
27.13
78.63
30.56
0.299389
0.249010
Il1b
68.47
16.74
126.44
73.53
111.35
55.06
156.70
131.92
0.244347
0.740380
Il23a
87.56
20.08
93.75
25.36
114.92
12.57
117.22
19.75
0.092340
0.252802
Kcnq5
72.95
13.30
69.26
16.17
79.04
15.38
49.20
8.42
0.622292
0.105197
Kirrel3
162.61
31.18
177.75
79.17
73.03
34.51
79.67
12.17
0.015683
0.078199
Myd116
30.79
1.28
21.08
1.81
31.18
0.89
19.29
1.07
0.679052
0.191454
Myl12b
102.50
3.35
93.09
2.79
106.11
2.89
100.04
2.53
0.206491
0.018698
Pilra
90.45
37.00
92.61
61.83
68.13
36.95
77.17
49.45
0.487709
0.747058
Ppp1r9a
87.86
12.36
81.12
8.20
100.08
9.28
112.57
10.37
0.220232
0.006210
Ptger4
79.89
13.15
64.69
9.90
46.80
21.63
86.48
26.33
0.064205
0.228175
Ptgs2
87.32
8.23
74.01
17.96
103.95
6.85
96.05
9.64
0.036054
0.110251
Ptpn20
106.94
20.96
101.98
34.21
110.04
61.45
106.76
10.08
0.936758
0.824256
Sgsm1
60.77
15.78
69.71
11.86
75.42
14.28
89.46
9.80
0.278191
0.067811
Sgtb
93.37
15.42
84.82
10.74
79.86
9.24
84.69
16.20
0.240773
0.991136
Tiprl
61.36
20.17
42.29
10.53
72.81
16.82
51.24
21.18
0.478683
0.536178
Tmtc4
101.42
14.15
93.76
22.35
96.99
23.40
78.19
20.07
0.788598
0.404036
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Tnf
68.73
9.20
84.24
12.38
118.59
57.78
65.33
39.32
0.190424
0.457003
Tpbpa
122.30
17.35
77.84
14.89
127.52
19.07
133.37
36.32
0.738073
0.049766
Wnt7a
74.36
10.01
68.90
15.41
87.52
12.47
80.62
9.14
0.203847
0.300379
Zfp36l1
49.68
2.53
33.81
1.10
51.87
2.42
37.51
3.36
0.321163
0.119802
Zfp36l3
81.41
9.03
66.48
7.40
115.63
50.16
123.61
62.82
0.289051
0.168764
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Development 143: doi:10.1242/dev.130369: Supplementary information
Supplemental Materials and Methods
Mice
Zfp36l3 knockout mice (B6-Zfp36l3tm1Pjb) were generated by Caliper Life Sciences
(Hopkinton, MA) using standard techniques. Briefly, the RP23-58C23 BAC clone containing the
mouse chromosome X Zfp36l3 sequence (X chromosome nucleotide # 49,938,675 ~
50,010,021) was used to generate the 5' homology arm (1.2 kb), the 3' homology arm (6.2 kb),
as well as the 5' and 3' probes for screening correctly targeted events. All fragments were
generated by PCR. The final targeting vector contained, in addition to the 5' and 3' homology
arms, the positive selection marker Neomycin (Neo) surrounded by LoxP sites and the negative
selection marker diphtheria toxin (DTA). The targeting vector was linearized with NotI and
electroporated into C57Bl/6 embryonic stem (ES) cells. Genomic DNA from electroporated ES
cells was first screened for homologous recombination by PCR using N38PCRF (5'ACATTTCACTCACTGCTGCACAGAC-3') and NeoPCRF1 (5’CGGAGATGAGGAAGAGGAGAACAGC-3’), which amplified a 1.5 kb product when
homologous recombination occurred. Potential positively recombined ES cell clones were
confirmed by Southern analysis. Genomic DNA was digested with HindIII (5' end), SpeI (3' end)
clones. One clone was subjected to Cre electroporation to remove the Neo cassette. The
deletion of Neo was confirmed by PCR (N38Neodel5F, 5'-CGGCGCAGGACATAGGACAATG3'; N38NeodeZFP36L3R, 5'-CTTCTTCAGAACCACACATTGCTTAACAC-3'). The Neo-deleted
allele was 0.33 kb in size (WT allele, 2.6 kb and mutant allele with neo, 2.2 kb). The Neodeleted clone was injected into C57BL/6J-TyrC-2J blastocysts to generate chimeric mice. Male
chimeras were mated to C57BL/6NTac females and the appropriate offspring were used to
generate the homozygous Zfp36l3 KO mice (-/-) used in this study, which were therefore on a
100% C57BL/6 background.
Development • Supplementary information
and EcoRV (Neo) and 5', 3', and Neo probes were used to identify correctly targeted ES cell
Development 143: doi:10.1242/dev.130369: Supplementary information
Since Zfp36l3 is located on the X chromosome, knockout (KO) males were generated by
breeding WT males with heterozygous (Het) females, or both KO males and females were
generated by breeding KO males with Het females. Genomic DNA was isolated from offspring
as previously described (Stumpo et al., 2004). Approximately 50 ng of genomic DNA was used
for PCR genotyping using the following conditions: 16 mM ammonium sulfate, 67 mM Tris-HCl
(pH 8.8), 0.01% (vol/vol) Tween 20, 5 mM ß-mercaptoethanol, 1.5 mM MgCl2, 0.1 mg/ml bovine
serum albumin, 0.2 mM deoxynucleoside triphosphates, 0.4 µM ZFP36L3NeoDelF (5'GGGCTGGGACTCAACCTTTTAG-3'), 0.4 µM ZFP36L3NeoDelR (5'GTGTCTCACAAAAACGTGTGGGT-3'), 0.4 µM MGZFP36L3R (5'TTGGCTGAGTTGTTGCTCG-3') and 1 U Bioline Taq DNA polymerase (Bioline USA Inc,
Tauton, MA). PCR cycling conditions were 94 oC for 2 min; 30 cycles of 94oC for 30 s, 58oC for
30 s, and 72oC for 2 min; and a final incubation at 72oC for 10 min. These primers amplified a
1.1 kb nucleotide fragment from the endogenous allele and a 0.54 kb fragment from the Neodeleted allele.
All mouse experiments were conducted according to US Public Health Service Policy on
the humane care and use of laboratory animals. The National Institute of Environmental Health
this study.
Immunohistochemistry
For routine paraffin section immunohistochemistry of ZFP36L3 in yolk sac and placenta,
tissues were processed, embedded, sectioned and stained as described previously (Blackshear
et al., 2005; Frederick et al., 2008). For immunostaining of frozen sections for confocal
microscopy, placentas were frozen in OCT, and 8-10 mm sections were cut, placed onto
SuperFrost Plus microscope slides, and stored frozen at -80 oC. Slides were stained by routine
Development • Supplementary information
Sciences Institutional Animal Care and Use Committee approved all animal procedures used in
Development 143: doi:10.1242/dev.130369: Supplementary information
procedures using the ZFP36L3 immune and preimmune antibodies cited above (1:1000 dilution)
and secondary donkey anti-rabbit Alexa Fluor-594 (1:500; Invitrogen), as well as the following
additional antibodies: For the type 1 transferrin receptor, a rat anti-mouse CD71 antiserum, or a
negative control serum (rat IgG isotype control; BD Biosciences 553264 and 559073), were
used at 1:500; these were stained with donkey anti-rat IgG Alexa Fluor 594 (Invitrogen), used at
1:1000. For double staining with anti-ZFP36L3 antibody (1:000), the secondary antibodies used
were donkey anti-rabbit IgG Alexa Fluor-488 (green; for ZFP36L3; 1:500; Invitrogen) and
donkey anti-rat IgG Alexa Fluor-594 (red; for CD71; 1:1000; Invitrogen). Sections were mounted
with Prolong Gold mounting medium plus DAPI (Invitrogen). For HB-EGF staining, the primary
antibody was a goat polyclonal IgG (M-18, Santa Cruz sc-1414), used at 1:50. The secondary
antibody used was donkey anti goat AlexaFluor-488 at 1:100.
Western blotting of TFRC
Mouse placentas from E15.5 WT and KO Zfp36l3 embryos were homogenized in a
buffer containing 50mM Tris-HCl (pH 8), 150 mM NaCl, 1% NP-40, and protease inhibitors
(Complete Mini; Roche, Indianapolis, IN) using a Tekmar Tissumizer, cleared by centrifugation
(12,000 x g, 20 min, 4°C), and quantified using the Bradford Assay (Bio-Rad, Hercules, CA).
precast gels (Bio-Rad) and transferred to nitrocellulose membranes. Blots were incubated (1.25
h at 4°C) with mouse anti-human Transferrin Receptor antibody (1:1000 dilution; 13-6800,
Invitrogen, Carlsbad, CA), followed by incubation (1h at 4°C) with horseradish peroxidaseconjugated goat anti-mouse IgG (1:5000 dilution; Bio-Rad). The blot was stripped with stripping
buffer (Pierce) and re-incubated (2 h at 4°C) with anti-beta Actin antibody (1: 5000 dilution;
mAbcam 8224, Abcam, Cambridge, MA), followed by incubation (1 h at 4°C) with horseradish
Development • Supplementary information
Equivalent amounts of denatured protein (25 µg) were separated on 10% Criterion Tris-HCl
Development 143: doi:10.1242/dev.130369: Supplementary information
peroxidase-conjugated goat anti-mouse IgG (1:25,000 dilution; Bio-Rad). SuperSignal West
Pico chemiluminescent substrate (Pierce, Rockford, IL) was used as a detection agent.
RNA Isolation, Northern Analysis and Quantitative PCR
Mouse tissues were collected into RNAlater (Applied Biosystems/Ambion) according to
the manufacturer’s instructions. Total cellular RNA was isolated using the Illustra RNAspin kit
(GE Healthcare). Northern analysis was performed as previously described (Stumpo et al.,
2009) using a mouse Zfp36l3 cDNA, consisting of a 1.74 kb XhoI fragment corresponding to
nucleotides 488 to 2231 of NM_001009549, a mouse transferrin receptor (Tfrc) ~900 bp HindIII
fragment (IMAGE clone ID 6406097, GenBank accession number BC054522), or a
glyceraldehyde-3-phosphate dehydrogenase (Gapdh) cDNA (Carballo et al., 1998) to monitor
gel loading. cDNA was synthesized from RNA using the Superscript III reverse transcription kit
(Invitrogen), and quantitative PCR was performed on an ABI/Prism7900HT Sequence Detector
System using SYBR® Green Master Mix (Applied Biosystems). All reactions were performed in
triplicate in 384-well plates using 1.5 ng of cDNA in a 10 µl reaction volume. Results were
normalized to the endogenous control α-actin and the fold change was calculated using the 2-
were used for quantitative PCR:
Zfp36l3: PCR product 113 bp; sense 5' TGGACTTTCTGCAATAGAGACTTAG 3', antisense 5'
TGTTGAGCTGGAGTTGGAAG 3'
Tfrc: PCR product 132 bp; sense 5' TCCTTGCATATTCTGGAATCCC 3', antisense 5'TGGTTGAGCTGAGGAACTTTC 3'
Development • Supplementary information
ΔΔCt method of relative quantification (Livak and Schmittgen, 2001). The following primers
Development 143: doi:10.1242/dev.130369: Supplementary information
Actb: PCR product 151 bp; sense 5' AGAGGGAAATCGTGCGTGAC 3', antisense 5'
AACCGCTCGTTGCCAATAGT 3'
Hbegf: PCR product 195 bp; sense 5' CCCCTATACACATATGACCACAC 3', antisense 5'
AGGTTCCTATAGCTCAGGTCC 3'
Lipg: PCR product 124 bp; sense 5’ CAGCCCACCCTCTACATTATC 3’, antisense 5’
TTCAAGAGATCGCCCAAGTC 3’.
Ribonucleoprotein immunoprecipitation (RIP) analysis
The RNA precipitation was performed as described (Keene et al., 2006), using freshly
isolated placentas from CRL:CD1 (ICR) mice (Charles River Laboratories) at embryonic (E) day
14.5 of gestation (where E0.5 was the date of detection of the vaginal plug). Briefly, placentas
were homogenized with a Potter-Elvejhem homogenizer (Wheaton Instruments) in ice-cold
polysome lysis buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES (pH 7.0), 0.5% Nonidet-P40,
1 mM dithiothreitol (DTT), 100 U/ml RNase inhibitor (Applied Biosystems), 400 µM vanadyl
ribonucleoside complexes (VRC; New England Biolabs), 1x complete EDTA-free protease
inhibitor cocktail (Roche Applied Science)), incubated on ice for 5 min, and stored at -80°C.
bovine serum albumin (SantaCruz sc-2003) was washed five times with ice-cold NT2 buffer (50
mM Tris-HCL (pH 7.4), 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40), resuspended in 2.5 ml of
ice-cold NT2 buffer and then incubated (rotating) with an L3 antiserum or its corresponding preimmune serum (Frederick et al., 2008) at 1:50 rotating overnight at 4 °C. The placenta lysate
was thawed, centrifuged (15 min, 15,000 x g, 4°C) and then precleared with washed agarose
beads. Thirty mg (1 ml) of the lysate supernatant was rotated at 4 °C for 1 h with washed
Protein A/G PLUS-Agarose immunoprecipitation reagent (0.5 ml bead volume) in 850 µl of icecold NT2 buffer containing 2000 U RNase inhibitor (200 U/ml), 400 µM VRC, and 1mM DTT.
Development • Supplementary information
Protein A/G PLUS-Agarose immunoprecipitation reagent (0.5 ml bead volume) pre-blocked with
Development 143: doi:10.1242/dev.130369: Supplementary information
The antibody-bound agarose beads (after an overnight incubation) and the precleared
lysate/agarose beads were centrifuged at 500 x g, 5 min, 4°C. The pre-cleared lysate from the
1 h incubation was mixed with the pre-immune serum- or antibody-bound beads and rotated
overnight at 4°C. The immunoprecipitation mixture was centrifuged at 500 x g, 5 min, 4 oC and
the beads were washed 5 times with ice-cold NT2 buffer. RNA was released from the beads by
incubating with 1 ml NT2 buffer containing 300 µg Proteinase K at 55°C for 30 min. The beads
were centrifuged at 500 x g, 1 min, 4°C and the supernatant containing the RNA was mixed with
3.5 ml of RNA lysis buffer (GE Healthcare Illustra RNAspin mini) and RNA was isolated
according to the manufacturer’s instructions including the on-column digestion of DNA with
RNase-free DNase (all of the eluted sample from one immunoprecipitation was processed on
one RNA mini column). RNA was eluted in 25 µl of diethylpyrocarbonate (DEPC)-treated water.
The immunoprecipitation was done on 4 different days with different placenta lysates and 9 µl of
the eluted RNA from each immunoprecipitation was analyzed by RT-PCR. Briefly, first strand
cDNAs were synthesized using oligo(dT)12-18 primers and Superscript III reverse transcriptase
(Life Technologies). Real-time PCR was performed with Power SYBR Green PCR mastermix
(Applied Biosystems) and the ABI Prism 7900 Sequence Detection System (Applied
Biosystems). Primers used for real-time PCR were validated for their amplification efficiency
pre-immune serum – CT immune serum)
) of mRNA associated with ZFP36L3 antibody.
Element analysis
Fetal mice were removed at E15.5 from three pregnant dams, quick frozen in liquid
nitrogen and stored at -80°C until genotyping could be performed. At that point, 9 WT and 8 KO
fetuses from three litters were used for total element analysis, using a panel of 20 elements,
performed at the University of Georgia Chemical Analysis Laboratory at the Center for Applied
Isotope Studies. The samples were ashed and then subjected to inductively coupled plasma -
Development • Supplementary information
and specificity prior to use, and are listed above. Results are presented as fold enrichment (2(CT
Development 143: doi:10.1242/dev.130369: Supplementary information
atomic emission spectroscopy, according to EPA method 6010C. See
http://www.cais.uga.edu/analytical_services/chemical_analysis/elements3.htm for further
details.
Steroid hormone analysis
Placentas from E15.5 mice were frozen and used for analysis of progesterone and
corticosterone concentrations at the University of Virginia Ligand Assay and Analysis Core of
the Center for Research in Reproduction (https://med.virginia.edu/research-inreproduction/laboratory-facilities/).
Deep sequencing of mRNA (mRNA-Seq).
For these analyses, we prepared total cellular RNA from 5 placentas from WT mice and
5 KO mice at E15.5 as described above, and treated these as independent biological samples.
The placentas were from female Het and male WT matings, and the WT mice were thus
littermates of the KO mice. Yolk sacs were removed from similar sets of animals; in this case, a
single yolk sac was used to generate a single biological sample, and there were 4 WT and 4 KO
biological samples for sequencing. Library construction and sequencing was performed as
Raw sequencing reads were mapped to the mm10 RefSeq cDNA sequences from the
UCSC Genome Browser using BWA (Li and Durbin, 2009), with the default setting of its pairedend read mapping protocol. This was done separately for each replicate. After read mapping,
we used the improved version of EpiCenter (Huang et al., 2011)
(http://www.niehs.nih.gov/research/resources/soft ware/biostatistics/epicenter) to identify
differentially expressed genes. Specifically, we first filtered out all reads that were not mapped
in pair to their reference cDNA sequences, and then counted reads mapping to each cDNA
sequence. We then normalized read counts in each replicate sample, so that the total read
Development • Supplementary information
described previously (Lai et al., 2013).
Development 143: doi:10.1242/dev.130369: Supplementary information
count in each sample was normalized by the average number of total mapped reads of all
samples from both groups. After normalization, we removed all transcripts that had a
normalized read count of less than 100 in both groups from further analysis, as recommended
by the EpiCenter tool for reducing false positives. From the remaining transcripts, we generated
a list of differentially expressed transcripts at the cutoff of 5% false discovery rate (FDR) as
reported by EpiCenter, which employs whole genome-wide data to estimate variation in read
counts between biological replicates, and uses it to determine statistical significance of gene
expression changes between groups.
Isolation of trophoblast stem cells, differentiation and NanoString analysis of mRNA
decay.
For trophoblast stem cell (TSC) isolation, uteri were collected from 9 superovulated
female mice at E3.5. Eight morulae, 41 blastocysts and 6 hatched blastocysts were recovered
by flushing the uterine horns with Embryomax M2 medium (Millipore). TSC were generated as
described (Tanaka et al 1998). Fifteen TSC lines were generated and cryopreserved. For
routine culture, all TSC lines were maintained on Mitomycin C-treated primary mouse embryonic
fibroblasts (MEF) (Millipore) with TSC medium containing 70% MEF-conditioned medium with a
(Sigma, H3149) (70CM+1.5XF4H medium).
For genotyping, cells were trypsinized, and 10 µl of the trypsin/cell mix was transferred
to PCR wells containing 20 µl of freshly prepared lysis buffer (1.0 ml TE pH 8.0, 20 µl 10%
Triton, and 20 µl Proteinase K (10 mg/ml). The plate was heated at 55 °C for 30 min, 94 °C for
10 min and 20 °C for 1 min. Cell lysates were stored at -20 °C.
Confirmatory genotyping was performed on genomic DNA prepared from the TSC lines
to eliminate potential contamination of TSC samples with primary MEFs. Purified genomic DNA
Development • Supplementary information
final concentration of 37.5 ng/ml FGF4 (R&D Systems, 235-F4/CF) and 1.5 µg/ml heparin
Development 143: doi:10.1242/dev.130369: Supplementary information
was prepared from TS cells cultured on gelatinized plates (0.1% gelatin, Stem Cell
Technologies). The cells were treated with 1.5 ml salt lysis buffer (100 mM NaCl, 100 mM
EDTA, 50 mM Tris/HCl (pH 8.0), 1% SDS) with 17 µl of Proteinase K (10 mg/ml). Plates were
then heated at 55 °C overnight in a humidified chamber.
PCR analysis was performed on lysates or genomic DNA from TSC lines to establish
sex and Zfp36l3 genotypes. Primers used for Zfp36l3 genotyping included MGZFP36L3R,
ZFP36L3NeoDelF and ZFP36L3NeoDelR, as already described. This assay results in a 1.1 kb
WT product and a 543 bp KO product. The Zfp36l3 assays were performed using standard
PCR reaction conditions with EX Taq Buffer (Takara) and the following cycling conditions: 2
min at 94 °C; 30 cycles for 30 sec at 94 °C, 30 seconds at 58 °C, and 1 min at 72 °C; 10 min
at 72 °C; and hold at 10 °C. Genotyping for the Y chromosome was performed by Sry PCR
analysis using the following primers to produce a 480 bp product: Sry2 forward (5'TAGTGTTCAGCCCTACAGCC-3'), and Sry2 reverse (5'CCCGAATTCGAGTACAGGTGTGCAGCTCT-3'). The Sry assay was performed using
standard PCR reaction conditions with LA Taq Buffer (Takara) and the following cycling
conditions: 2 min at 94 °C; 30 cycles for 30 sec at 94 °C, 30 sec at 62 °C, and 1 min at 65
To differentiate TSC, WT (B3) and Zfp36l3 -/- (A2) TSC lines were thawed and
expanded on 10 cm tissue culture plates containing Mitomycin C-treated primary MEFs
(Millipore). The TSC lines were maintained in 70 CM +1.5xF4H culture medium and expanded
1:3 onto 10 cm tissue culture plates containing Mitomycin C-treated MEFs. Cells at ~ 70%
confluence were trypsinized and replated in 70CM+1.5xF4H medium. The plates were
incubated for 30 min in 5% CO2 at 37°C to allow fibroblasts to reattach to the plates.
Nonadherent TSC were removed, counted and plated onto gelatinized (0.1% gelatin, Stem Cell
Technologies) 60 mm plates at 2x10⁵ cells per plate. Eight plates per cell line were placed in
Development • Supplementary information
°C; 10 min at 65 °C; and hold at 10 °C.
Development 143: doi:10.1242/dev.130369: Supplementary information
normal 70CM+1.5xFGFH medium and 24 plates per line were placed in differentiation medium
(TS media without heparin or FGF4).
RNA was prepared from pre-differentiation samples when the plates were ~ 70%
confluent. Eight plates for each cell line were washed 3 times with PBS, and the cells were
lysed by the addition of 500 µl of RNA lysis buffer (RA1 lysis buffer, GEHealthcare Illustra
RNAspin mini kit, containing 1% of freshly added β-mercaptoethanol) per plate. The plates
were scraped, and the contents of two plates were pooled and transferred to cryovials and
stored at -80°C. Differentiated cells were used after 5 days of culture in differentiation medium.
RNA samples for time point 0 in the mRNA decay curves were prepared as above, using 8
plates from each cell line. Thirty min later, 20 µl of actinomycin D (5 µg/ml) was added to each
of the remaining 16 plates per cell line. RNA samples were prepared from 8 plates each, as
described above, at 2 h and 4 h after actinomycin D. RNA was isolated using the Illustra
RNAspin kit (GE Healthcare).
Based on the results of the RNA-Seq experiment, we devised a custom codeset for
NanoString assays for mRNA quantitation in the total cellular RNA samples. This method can
be used to quantitate mRNA concentrations without amplification (Geiss et al, 2008). The
stable and labile in other cell systems), TTP family member mRNAs, internal normalization
controls, and test transcripts. Assays were performed at the NanoString analysis center in
Seattle, WA.
References (all other references referred to in this Supplemental Materials and Methods
section are listed in the main text).
Livak, K.J., and Schmittgen, T.D. (2001). Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408.
Development • Supplementary information
codeset included positive and negative controls for mRNA stability (i.e., transcripts known to be
Development 143: doi:10.1242/dev.130369: Supplementary information
Lai, W.S., Stumpo, D.J., Kennington, E.A., Burkholder, A.B., Ward, J.M., Fargo, D.L., and
Blackshear, P.J. (2013). Life without TTP: apparent absence of an important anti-inflammatory
protein in birds. American journal of physiology. Regulatory, integrative and comparative
physiology 305, R689-700.
Development • Supplementary information
Li, H., and Durbin, R. (2009). Fast and accurate short read alignment with Burrows-Wheeler
transform. Bioinformatics 25, 1754-1760.