S1 Table. List of reagents and equipment used in this study.
REAGENT or RESOURCESOURCEIDENTIFIER

Anti-human prosurfactant protein CAbcam

ab90716

Cas9 protein

Integrated DNA Technologies

Cat# 1074181

Dako REAL Target Retrieval Solution (10x)Agilent Technologies

S2031

Di-N Butyl-Phthalate in xylene, DPX

Merck

Cat# 1.01979.0500

Eosin

Merck

Cat# 1.09844.1000

EtOH, General alcohol 99 degree, synthetic

Japan Alcohol Trading Company Limited

EtOH, Special grade, 99.5

SIGMA-ALDRICH

Code No. 09-0770-4

Hematoxylin Solution, Mayer's

Merck

Cat# 1.09249.0500

Paraffin

Thermo Fisher Scientific

Item Number: 6774060

PBS (Phosphate Buffered Salts) Tablet

Takara Bio

Cat# T900

XyleneNacalai tesque

Product code: 36611-45

BigDye Terminator v1.1 Cycle Sequencing Kit

Thermo Fisher Scientific

Product No.: 4337450

BrightGreen 2X qPCR MasterMix-No Dye

Applied Biological Materials

MasterMix-S

DNeasy Blood & Tissue Kit

QIAGEN

Cat# 69504

FastGene cDNA Synthesis 5x ReadyMix

Nippon Genetics

NE-LS64

Guide-it Mutation Detection Kit

Takara Bio

Product code: 631448

ImmPRESS Polymer Detection Kit for IHCVector Labs

Cat# MP-7401

NucleoZOL

Macherey-Nagel

REF 740404.200

RNAlater

Thermo Fisher ScientificAM7020

Takara Bio

Product code: R045A

Fgf10 Ex3F (5’-TGACTCTTCT GTTGTTAGCGT TG-3’)

Eurofins Genomics

Yasue et al. 2014 [14]Fgf10 Ex3R (5’-ACATCCAAAG CCTTCCTTCC-3’)
Fgf10 primer for MiSeq Fw (5'-GGATACAACACCTATGCATC T-3')
Fgf10 primer for MiSeq Rv (5'-GTCTTTGCCTTTTGAGCTAC-3')

Eurofins Genomics

Yasue et al. 2014 [14]Eurofins Genomics

This paper

Eurofins Genomics

This paper

Digital camera system

NikonDS-Fi1

Digital camera system

Leica Microsystems

DFC310FXDNA sequencer

Thermo Fisher Scientific

ABI PRISM 3100

DNA sequencer

Illumina

MiSeq

Electrophoresis

Agilent Technologies

TAPEStation 4200

Industrial blade

FEATHER Safety Razor

Item No.: 990077

Micro-scissors

Natsume Seisakusho

MB-54-2

Microscope

Leica Microsystems

DM5000B

Microscope

Leica Microsystems

M165FC

NanoDrop

Thermo Fisher Scientific

ND-1000

Paraffin extender

Sakura Seiki

PS-52

Paraffin extender

Sakura Finetek Japan

PS-53

Rotary microtome

Leica Biosystems

RM-2145

Superfrost Plus Microscope Slides

Thermo Fisher Scientific

Cat# 12-550-15

Thermal cycler

BIO-RAD

T100

Thermal cycler

BiometraTgradient

Thermal cycler

RocheLightCycler Nano system

Tweezers

FONTAX

KN3345108

Chemicals

Critical Commercial Assays

DNA polymerase
PrimeSTAR Max DNA polymerase
Oligonucleotides

Machines or Instruments

S2 Table. Summary of deep sequencing data on DNA from different tissuesof E18.5 embryos.
#2 Wild type
Neck Lung Limb
Wt
99.6 100 100
In (with Lys196, His201)000
In (without Lys196 or His201)
000Out000.37

N.A.

Limb
Lung
Neck0%Wt

20%0%

20%

60%

80%

In (without Lys196 or His201)

100%
Out

p -value#12 Type III

0.751529

Limb
Lung
Neck
0%

20%

40%

In (with Lys196, His201)

60%80%

In (without Lys196 or His201)

100%
Out

p -value#13 Type III

4.02E-05

Limb
Lung
Neck
0%

20%

40%

In (with Lys196, His201)

60%

80%

In (without Lys196 or His201)

100%
Out

p -value#14 Type II

N.A.

Limb
Lung
Neck
0%
Wt

20%

40%

In (with Lys196, His201)#15 Type III

60%80%

In (without Lys196 or His201)

100%
Out*

p -value#15 Type III

2.90E-11

Limb
Lung
Neck0%
Wt

20%

40%

In (with Lys196, His201)#16 Type III

60%80%

In (without Lys196 or His201)

100%Out

p -value#16 Type III

N.A.

Limb
Lung
Neck0%
Wt

20%40%

In (with Lys196, His201)#17 Type III

60%80%

In (without Lys196 or His201)

100%
Out

p -value#17 Type III

3.38E-50

Limb
Lung
Neck0%
Wt

20%

40%

In (with Lys196, His201)#18 Type I
Neck Lung Limb
Wt
9.27
In (with Lys196, His201)
0N.D. N.D.
In (without Lys196 or His201)
0
Out
90.7

40%

In (with Lys196, His201)#14 Type II

Neck Lung Limb
Wt
3839
In (with Lys196, His201)
3225N.D.
In (without Lys196 or His201)00
Out3036p -value

Limb

Wt

Neck Lung Limb
Wt
100 100
In (with Lys196, His201)
00N.D.
In (without Lys196 or His201)
00
Out
00

OutLung#13 Type III

Neck Lung Limb
Wt
59.8
5756
In (with Lys196, His201)000
In (without Lys196 or His201) 0.39
00
Out
43
4439.8100%

NeckWtNeck Lung Limb
Wt
33
In (with Lys196, His201)
0N.D. N.D.
In (without Lys196 or His201) 64.7
Out*2.3*includes a large deletion80%

3.34E-29#12 Type IIINeck Lung Limb
Wt
8385
In (with Lys196, His201)
00
N.D.
In (without Lys196 or His201)00Out
171560%In (without Lys196 or His201)#11 Type III

Wt

Neck Lung Limb
Wt
6666
In (with Lys196, His201)
00
N.D.
In (without Lys196 or His201)
00
Out
34
34

40%

In (with Lys196, His201)#11 Type III
Neck Lung Limb
Wt
67.86164
In (with Lys196, His201)000
In (without Lys196 or His201)
000Out393632.2

p -value

#2 Wt

60%

80%

In (without Lys196 or His201)

100%Out

p -value#18 Type IN.A.

Limb
Lung
Neck0%
Wt

20%40%

In (with Lys196, His201)

60%80%

In (without Lys196 or His201)

100%OutS2 Table (continued)#19 Type I
Neck Lung Limb
Wt
0.5
In (with Lys196, His201)
0N.D. N.D.
In (without Lys196 or His201) 31.2
Out
68.3

N.A.

Limb
Lung
Neck0%
Wt

20%0%20%40%

In (with Lys196, His201)

60%80%

In (without Lys196 or His201)

100%Out

p -value#21 Type III

0.17603

Limb
Lung
Neck
0%

20%40%

In (with Lys196, His201)

60%

80%

In (without Lys196 or His201)

100%Outp -value#22 Type III

0.271069

Limb
Lung
Neck0%20%40%

In (with Lys196, His201)#23 Type III

60%80%

In (without Lys196 or His201)

100%
Outp -value#23 Type III

N.A.

Limb
Lung
Neck0%
Wt

20%

40%

In (with Lys196, His201)#24 Type III

60%80%

In (without Lys196 or His201)

100%
Out*

p -value#24 Type III

1.18E-26

Limb
Lung
Neck0%
Wt

20%

40%

In (with Lys196, His201)#25 Type III

60%80%

In (without Lys196 or His201)

100%
Outp -value#25 Type III

0.139527

Limb
Lung
Neck0%
Wt

20%40%

In (with Lys196, His201)#26 Type I
Neck Lung Limb
Wt
0.45
In (with Lys196, His201)
0N.D. N.D.
In (without Lys196 or His201)
0Out
99.5

p -value

Limb

Wt

Neck Lung Limb
Wt
00
In (with Lys196, His201)
40.7
40.2
N.D.
In (without Lys196 or His201)
00
Out**
59.8
59.3
**including large insertion

OutLung#22 Type III

Neck Lung Limb
Wt
73.1
82.3
In (with Lys196, His201)
00
N.D.
In (without Lys196 or His201)
00Out
17.7
26.9

100%

NeckWtNeck Lung Limb
Wt
100
97.2
In (with Lys196, His201)
00N.D.
In (without Lys196 or His201)
00
Out*
2.8
0*including large deletion80%

1.89E-09#21 Type III

Neck Lung Limb
Wt
28.9
29.9
In (with Lys196, His201)
00N.D.
In (without Lys196 or His201)
00
Out
71.1
70.1

60%

In (without Lys196 or His201)#20 Type III

Wt

Neck Lung Limb
Wt
56.9
57.5
In (with Lys196, His201)
00
N.D.
In (without Lys196 or His201)
00Out
42.5
43.1

40%

In (with Lys196, His201)#20 Type III
Neck Lung Limb
Wt
71.1
67.7
In (with Lys196, His201)
28.9
32.3
N.D.
In (without Lys196 or His201)00Out00p -value#19 Type I

60%

80%

In (without Lys196 or His201)

100%
Out**p -value#26 Type I

N.A.

LimbLung
Neck
0%
Wt

20%

40%

In (with Lys196, His201)

60%

80%

In (without Lys196 or His201)

100%
Out

The percentage of sequence reads for each genotype category is shown in contingency
tables. Wt, wild type Fgf10 genotype; In, in-frame mutations in the Fgf10 gene; Out,frameshift mutations in the Fgf10 gene; N.D., not done; N.A., not applicable. p-values of Chisquare test (see Table 4) are shown for reference.

S3 Table. Summary of type II and type III embryos examined for E18.5 lungs
by immunohistochemistry, shown in Fig 6 and Fig S5.
TypeIIIII
WT

EmbryoNo.

Forelimb
Left

Right

Hindlimb
Left

Right

#11_2* TruncatedNormal Truncated Truncated#24_2 Only 3 digits Normal Only 3 digits Normal#25_2 Only 2 digits Truncated Truncated Truncated#8_2
NormalNormalNormalNormal#21_2#22_2*#2_18
NormalNormalNormalNormalSPC (+) cells/totalcells (mean±SEM %)Wt genotype (%) inneck DNA

10.5 ± 1.1
14.8 ± 0.4
10.9 ± 0.7
17.0 ± 0.8
19.5 ± 0.8
14.9 ± 0.8
N.D.

27.78
9.75
9.53
62.40
54.17
38.80
N.D.

Wt genotype (mean±SEM %) in neck DNA

15.7 ± 6.0

51.8 ± 6.9
N.D.

FigureFig S5; Fig 6A,D,G
Fig S5
Fig S5
Fig S5
Fig S5
Fig S5
Fig 6C,F,I*Approximately 295- (#11_2) and 79- (#22_2) bp insertion were detected by microchip
electrophoresis and the number of sequence reads in deep sequencing was correctedaccordingly (see Materials and methods). N.D., not determined.

S4 Table. Primers used for quantitative PCR (qPCR) analysis.
Gene name
Fgf10Hopx
Sftpc
VegfaPdgfa
Plin2Pdgfra
Acta2Gapdh

Primer namemmu_fgf10_qPCR_Fw
mmu_fgf10_qPCR_Rv
mmu_hopx_qPCR_Fw
mmu_hopx_qPCR_Rv
mmu_sftpc_qPCR_Fw
mmu_sftpc_qPCR_Rv
mmu_vegfa_qPCR_Fw
mmu_vegfa_qPCR_Rv
mmu_pdgfa_qPCR_Fw
mmu_pdgfa_qPCR_Rv
mmu_plin2_qPCR_Fw
mmu_plin2_qPCR_Rv
mmu_pdgfra_qPCR_Fw
mmu_pdgfra_qPCR_Rv
mmu_acta2_qPCR_Fw
mmu_acta2_qPCR_Rv
GAPDH_qPCRFw
GAPDH_qPCRRv

Nucleotide sequence of PCR primers
AGCGGGACCAAGAATGAAGACTTGCTGTTGATGGCTTTGACGGTTCAACAAGGTCAACAAGCACC
CCAGGCGCTGCTTAAACCATCACCTCAAACGCCTTCTCATCG
TTTCTGAGTTTCCGGTGCTCC
GCTGTACCTCCACCATGCCA
CCACCAGGGTCTCAATCGGACGTCAAGTGCCAGCCTTCACTGGGTTCAGGTTGGAGGTCG
AAGAGAAGCATCGGCTACGAGGCGATAGCCAGAGTACGTG
TCCTTCTACCACCTCAGCGAG
CCGGATGGTCACTCTTTAGGAAGGTCCCAGACATCAGGGAGTAATCGGATACTTCAGCGTCAGGA
AGGTTGTCTCCTGCGACTTCATGGTCCAGGGTTTCTTACTCC

Amplicon size (bp) GC content (%)

References

91

49.5

This study

106

61.3

This study

147

52.4

This study

131

56.5

This study

148

54.7

This study

78

55.1

Shaul et al. 2010 [1]

103

47.6

Awuah et al. 2013 [2]

102

52.9

Hecker et al. 2009 [3]

184

52.2

Ning et al. 2013 [4]References
1. Shaul ME, Bennett G, Strissel KJ, Greenberg AS, Obin MS. Dynamic, M2-like remodeling phenotypes of CD11c+ adipose tissue
macrophages during high-fat diet-induced obesity in mice. Diabetes. 2010; 59(5): 1171-81. doi: 10.2337/db09-1402.
2. Awuah PK, Nejak-Bowen KN, Monga SPS. Role and regulation of PDGFRα signaling in liver development and regeneration. Am J Pathol.
2013; 182(5):1648-58. doi: 10.1016/j.ajpath.2013.01.047.
3. Hecker L, Vittal R, Jones T, Jagirdar R, Luckhardt TR, Horowitz JC, et al. NADPH oxidase-4 mediates myofibroblast activation andfibrogenic responses to lung injury. Nat Med. 2009;15(9):1077-81. doi: 10.1038/nm.2005.
4. Ning YL, Yang N, Chen X, Xiong RP, Zhang XZ, Li P, et al. Adenosine A2A receptor deficiency alleviates blast-induced cognitive
dysfunction. J Cereb Blood Flow Metab. 2013;33(11):1789-98. doi: 10.1038/jcbfm.2013.127.

Summary of the gene profiles examined by qPCR
Gene name Expressed by/inRole
Hopx
AECI
Sftpc
AECII
Vegfa
Distal lung epithelium
Stimulates endothelial proliferation
Pdgfa
Distal lung epithelium
Stimulates proliferation of alveolar smooth muscle cell progenitors
Plin2
Lipofibroblast
PdgfraAlveolar smooth muscle cell progenitors
Acta2Smooth muscle cell

S1 Fig. Structure of the mouse Fgf10 gene indicating the target sites for
CRISPR/Cas9 system.

S2 Fig. Cartilage and bone staining to reveal skeletal structures.

A-H, whole mount staining. Left (A, C, E, G) and right (B, D, F, H) lateral views are shown.
Arrows show truncated limb and girdle bones. I-K, wild type skeletal structures, showing
scapula and forelimb (I), hindlimb (J), and pelvic girdle (K). au, autopod; f, femur; fi, fibula;h, humerus; il, ilium; is, ischium; p, pubis; r, radius; sc, scapula; sp, spine; st, stylopod; t,tibia; u, ulna; ze, zeugopod. Scale bars: 1 mm.

S3 Fig. Mismatch cleavage assay.

A, PCR primers were designed in the upstream region of the exon 3 and in the exon 3,giving rise to a PCR amplicon size of 501 bp. DNA fragments of 309 bp and 192 bp aregenerated by the Resolvase when the Fgf10 genome has been cleaved by Cas9 and nonhomologous end joining has been achieved. B, Electrophoresis of the enzyme- treatedmouse genomic DNA from the Fgf10-CRISPR F0 embryonic necks. The DNA ladder for
DNA size reference and a result of DNA from a wild type (WT) mouse are shown on the left.
Three DNA fragments of approximately 500 bp (▼), 300 bp (5 in gray), and 200 bp (5) areseen in all the lanes except for the wild type and #4 lanes. In embryo #3, an extra band forlarge insertion (328 base) is shown (see Fig 2). C, Genomic analysis of the #4 embryo as
revealed by Sanger sequencing. Deduced amino acid sequences are also shown. Lys-196
and His-201 are highlighted in yellow and green, respectively. Altered amino acids areindicated in red. Asterisks indicate stop codons.

S4 Fig. Limb phenotypes and lung histology of all embryos examined atE16.5 as summa- rized in Table 3.

Scale bars: 500 μm.

S5 Fig. Limb phenotypes, lung histology, and the number of SPC-positive
cells per total cell number of type II and type III embryos at E18.5.

Source data for (L, M) are available in S3 Table. Lateral views of type II (A-F), and type III(G, H) embryos at E18.5. Arrowheads show limb defects. (I-K) In all three type II embryos,
the accessory lobe was lost. In embryo #24_2, (J), the middle lobe (Mi) was also
undetectable. Data in (L) are presented as means ± SEM. (M) In these embryos, thenumber of SPC-positive cells was more correlated to the percentage of wild type Fgf10
genotype (correlation coefficient [R] was 0.728 for neck DNA) than that including in-frame
mutations with Lys196 and His201 retained (R = 0.334). Scale bar: 2 mm (A-H).

S6 Fig. Quantitative PCR analysis for mRNA expression of mesenchymal
and its related marker genes in wild type and type III lungs at E18.5.

S7 Fig. Cecum and colons of the wild type and Fgf10-CRISPR F0 (type Iand type II) embryos at E18.5.

A-C, wild type (Wt) cecum (c), colon, and small intestine (si) are shown from three embryos
examined. Ileum and colon were cut at dissection. D-G, type I cecum is reduced comparedwith the wild type. Whether the cecum epithelium is absent or not cannot be identified fromthese photos. Type I embryos show an atresia of the colon, but the length varies depending
upon the embryos. H-I, type II embryos examined (n = 2) do not exhibit a reduced cecum or
an atresia of the colon. The colons presented here were cut as distally as possible. J, theapproximate length of the cecum. The length of type I cecum is significantly decreased
compared with the wild type. The length of two type II embryos examined is also shown forreference. Scale bar: 1 mm (in A for all to scale).