1

1

Fgf10-CRISPR mosaic mutants demonstrate the gene dose-related

2

loss of the accessory lobe and decrease in the number of alveolar

3

type 2 epithelial cells in mouse lung

4
5

Munenori Habuta1 ¶, Akihiro Yasue2 ¶*, Ken-ichi T Suzuki3,4, Hirofumi Fujita1, Keita

6

Sato1, Hitomi Kono1, Ayuko Takayama4, Tetsuya Bando1, Satoru Miyaishi5, Seiichi

7

Oyadomari6, Eiji Tanaka2, Hideyo Ohuchi1*891Department of Cytology and Histology, Okayama University Graduate School of

10

Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan

11

2

12

Tokushima University Graduate School, Tokushima, Japan

13

3

14

Japan

15

4

16

Biology, Okazaki, Aichi, Japan

Department of Orthodontics Dentofacial Orthopedics, Institute of Biomedical Sciences,

Department of Mathematical and Life Sciences, Hiroshima University, Hiroshima,

Center for the Development of New Model Organisms, National Institute for Basic

2

17

5

18

Dentistry and Pharmaceutical Sciences, Okayama, Japan

19

6

20

Tokushima, Japan

Department of Legal Medicine, Okayama University Graduate School of Medicine,

Division of Molecular Biology, Institute for Genome Research, Tokushima University,

21
22* Corresponding authors

23

E-mail: hohuchi@okayama-u.ac.jp (HO); yasuea@tokushima-u.ac.jp (AY)

24
25
26
27

¶

These authors contributed equally to this work.

3

28

Abstract

29

CRISPR/Cas9-mediated gene editing often generates founder generation (F0) mice that30

exhibit somatic mosaicism in the targeted gene(s). It has been known that Fibroblast

31

growth factor 10 (Fgf10)-null mice exhibit limbless and lungless phenotypes, while

32

intermediate limb phenotypes (variable defective limbs) are observed in the Fgf10-

33

CRISPR F0 mice. However, how the lung phenotype in the Fgf10-mosaic mutants is34

related to the limb phenotype and genotype has not been investigated. In this study, we

35

examined variable lung phenotypes in the Fgf10-targeted F0 mice to determine if the lung36

phenotype was correlated with percentage of functional Fgf10 genotypes. Firstly,

37

according to a previous report, Fgf10-CRISPR F0 embryos on embryonic day 16.5

38

(E16.5) were classified into three types: type I, no limb; type II, limb defect; and type III,39

normal limbs. Cartilage and bone staining showed that limb truncations were observed in40

the girdle, (type I), stylopodial, or zeugopodial region (type II). Deep sequencing of the41

Fgf10-mutant genomes revealed that the mean proportion of codons that encode putative42

functional FGF10 was 8.3 ± 6.2% in type I, 25.3 ± 2.7% in type II, and 54.3 ± 9.5% in

43type III (mean ± standard error of the mean) mutants at E16.5. Histological studies

4

44

showed that almost all lung lobes were absent in type I embryos. The accessory lung lobe

45

was often absent in type II embryos with other lobes dysplastic. All lung lobes formed in

46

type III embryos. The number of terminal tubules was significantly lower in type I and II

47

embryos, but unchanged in type III embryos. To identify alveolar type 2 epithelial

48(AECII) cells, known to be reduced in the Fgf10-heterozygous mutant, immunostaining

49

using anti-surfactant protein C (SPC) antibody was performed: In the E18.5 lungs, the50number of AECII was correlated to the percentage of functional Fgf10 genotypes. These51

data suggest the Fgf10 gene dose-related loss of the accessory lobe and decrease in the52

number of alveolar type 2 epithelial cells in mouse lung. Since dysfunction of AECII cells

53

has been implicated in the pathogenesis of parenchymal lung diseases, the Fgf10-CRISPR

54

F0 mouse would present an ideal experimental system to explore it.

55
56

KEYWORDS: Genome editing, Fibroblast growth factor 10, Mouse, Lung, Somatic57

mosaicism

58

5

59

Introduction

60

The recently developed CRISPR/Cas9 system provides a highly efficient means for61

editing the genomes of model and non-model organisms. This powerful tool can help

62

elucidate pathophysiological mechanisms underlying various genetic diseases (reviewed

63

in [1,2]). CRISPR/Cas9 promises the possibility of an ultimate cure for genetic diseases

64

by enabling replacement of mutated genes with normal alleles (reviewed in [3]); however,65

genome-edited founder mice often exhibit somatic mosaicism in the targeted gene,66

meaning more than two mutated alleles for that gene are mixed in the same mouse [4-6].

67

Such mosaicism may be undesirable when it complicates phenotypic analysis [7].

68

However, mosaic mice may help the study of genes whose constitutive mutations are69

lethal, or the study of interactions between mutant and normal cells in the same individual70[8]. Furthermore, recent DNA sequencing advances have enabled us to identify genetic71

mosaicism even in phenotypically normal individuals, the pathological significance of

72

which is still unclear (reviewed in [9,10]).

73

Previously, we reported that somatic mosaicism of a homeobox gene Pax6 mutation74

causes variable eye phenotypes correlated with its gene dosage [11]. In this study, we675

focused on the variable phenotypes of genome-edited Fgf10 founder generation (F0) mice.

76

The Fgf10 gene is required for limb and lung formation; Fgf10 null mutants suffer

77embryonic lethality at birth due to lung agenesis [12,13]. Recent studies have shown that78

a fraction of Fgf10 genome-edited F0 mice exhibits typical limbless and lungless79

phenotypes, and that the severity of the limbless phenotype depends on the Fgf10

80mutation rate [7,14,15]. However, the lung phenotype has not been described. In this81

study, we sought to determine the relationship between lung and limb phenotypes, and82

correlation of these phenotypes to putative functional FGF10 dosage.

83
84

7

85

Materials and methods

86

Reagents and equipment used in this study are listed in S1 Table.

87
88

Mice and ethical statement

89

The animal experimental design was approved by the Committee of Animal Experiments

90of Tokushima University, Tokushima, Japan, and by the Animal Care and Use Committee,

91

Okayama University, Okayama, Japan (Permit numbers: T27-16, T30-8, OKU-2017404,

92

OKU2018605). All surgeries were performed under sodium pentobarbital anesthesia, and93

all efforts were made to minimize suffering. In vitro fertilized (IVF) eggs were prepared94

from male and female BDF1 (C57BL/6 x DBA2 F1) mice as previously reported [14].

95

CRISPR/Cas9 genome editing was performed on one-cell stage zygotes by

96

microinjection (analysis on E16.5 embryos) with Cas9 mRNA and sgRNA targeted at97

exon 3 of the Fgf10 gene according to Yasue et al. (2014) [14] (S1 Fig). After culturing

98

genome-edited zygotes until the two-cell stage, embryos were transferred to the uterine

99

tube of foster mice (Jcl: MCH (ICR) strain developed by CLEA Japan, Inc. (Tokyo,

100

Japan); Jcl for Japan clea, MCH for Multi-Cross Hybrid, and ICR for Institute of Cancer

8

101

Research) and allowed to develop until E16.5. To obtain E18.5 F0 embryos, Cas9 mRNA

102

(100 ng/µL) and synthesized guide RNA (50 ng/µL) were electroporated into one-cell103stage IVF eggs and further manipulation of embryos was performed according to104

Hashimoto et al. (2016) [7]. CRISPR-control embryos were obtained by electroporation

105

of medium only into IVF eggs before processing them for sequencing and histological106

examination. To know developmental variations, the weight of E18.5 embryos was

107

measured with a standard electronic scale.

108

109

Enzyme mismatch cleavage assay

110

Approximately 25 µg genomic DNA were extracted from neck skin tissue (epidermis and111

dermis) of collected embryos using a Qiagen DNeasy Blood & Tissue Kit. The genomic

112

region encompassing the CRISPR/Cas9 target site was amplified by polymerase chain

113

reaction (PCR) using a primer set specific for the Fgf10 gene (S3A Fig, S1 Table) [14].

114

The amplicons were processed for a mismatch cleavage assay using the Guide-it Mutation

9

115

Detection Kit and the products were analyzed by 2% agarose gel electrophoresis (S3B

116

Fig).

117
118

DNA sequencing

119

To verify the presence of the target site mutation, single bands of PCR amplicons of120

approximately 500 bp derived from the collected embryos at E16.5 (neck skin tissues)

121

were subjected to Sanger sequencing. The resulting DNA sequence waves were122

dissociated using a web tool for TIDER data analysis available at https://tide.nki.nl [16].

123Deep sequencing analysis using DNA extracted from neck skin, lung, and/or limb

124

dermal tissues (Table 1 for E18.5; neck skin only for E16.5 embryos in Table 3; neck skin125

only for additional E18.5 embryos in S3 Table) for the Fgf10 target site was performed

126

according to the previous report on Illumina MiSeq system [17]. Briefly, the Fgf10 on-

127

target site from each individual embryo was amplified using custom barcode primers

128

shown in S1 Table. Library construction and sequencing were performed at the National

129

Institute for Basic Biology (NIBB, Japan) and Bioengineering Lab (Kanagawa, Japan).

130

The pooled sequence data was demultiplexed into each sample and analyzed subsequently

10

131

using a web-based tool, CLiCKAR [17].

132
133

Correction and calculation of putative somatic mutation rate134

in the case of large insertions/deletions (indels)135

PCR amplicons for deep sequencing were analyzed using TAPEStation 4200 system.

136

When larger or smaller DNA bands were detected above or below the main bands, their137

approximate molecular size was recorded. “Peak Molarity” value for each DNA band was

138

documented and the percentage of large indel was estimated by calculating the total value.

139

The putative somatic mutation rate for large indels was calculated proportionally. In

140embryos #3 (type II, E16.5), #23 (type III, E18.5), #25 (type III, E18.5), and #11_2 (type141

II, E18.5), and #22_2 (type III, E18.5), large indels were detected and corrected

142

accordingly.

143
144

Cartilage and bone staining

11

145

After Cesarean dissection, E18.5 embryos, removed their skin and internal organs, were146

fixed in 95% ethanol and processed for whole mount skeletal staining with Alcian blue

147

8GX and Alizarin red S according to standard procedures [18].

148
149

Histology and analysis of terminal tubules

150

Hematoxylin-eosin (HE) staining was performed on mouse sections according to standard

151

procedures. Briefly, sections of 5 µm-thickness were made of the pectoral region of152

paraffin-embedded embryos and mounted on microscope slides. After deparaffinization

153

and rehydration, sections were stained with Mayer’s hematoxylin solution and 0.125%

154

eosin. Following staining, sections were washed, dehydrated, and mounted with DPX.

155

After HE staining, photos were taken with a 10x objective lens for each embryonic lung156

(n = 3 for each type, at E16.5) (Fig 5E-H) and tiled using ImageJ

157(https://imagej.nih.gov/ij/index.html) (RRID:SCR_003070). Terminal tubules were

158

scored in the tiled full-field image (n = 1 for each embryo). The area for the full-field was

159

calculated by ImageJ and the number of terminal tubules per mm2 were obtained and160

further analyzed statistically.

12

161
162

Immunohistochemistry and analysis of SPC-positive cells

163

Immunohistochemistry was performed on the 5 µm-thick deparaffinized sections using

164

ImmPRESS Polymer Detection Kit for IHC. A rabbit polyclonal antibody against a165

synthetic peptide fragment of human prosurfactant protein C (SPC) (within residues 1-

166100) was used at 1:5,000 dilution. The specificity of the antibody for AECIIs in the mouse167

lung has been validated [19]. The public identifier from the Antibody Registry is RRID:

168

AB_10674024. After immunohistochemistry, SPC-positive cells were scored in random169

portions of a section in eight photomicrographs using a 40x objective lens. The total

170

number of 6627 cells on average was counted per sample. The results were presented as

171

a ratio of the number of positive cells per total number of cells [20]. This method has a172

limitation, in which as Fgf10 mutations have been known to affect epithelial tube

173

formation and branching [21] and therefore the number of distal airway epithelial cells in

174

total may be impacted, the accuracy of the data would be ensured by normalizing AECII

175

cell number to total epithelial cell number.

176

13

177

Microscopy and image processing

178

Images for sections were collected with a Nikon DS-Fi1 camera on a Leica DM5000B

179

microscope. Embryos, skeletons, and intestines were observed and imaged using a Leica

180

DFC310FX camera on a Leica M165FC stereomicroscope. Image manipulation such as

181

levels and color balance adjustments, made to some images, and assembly of figures were182

performed with Adobe Photoshop CS6 Extended (RRID:SCR_014199).

183
184

Reverse transcription quantitative polymerase chain reaction

185(RT-qPCR)

186

Dissected embryonic lungs (E18.5) were immersed in RNAlater at 4 °C overnight and187

stored at -80 °C until further use. Total RNA was extracted using NucleoZOL. Five188

micrograms of RNA were reverse-transcribed to cDNA with FastGene cDNA Synthesis

189

5x ReadyMix. RT-qPCR was performed in duplicate wells with BrightGreen 2X qPCR

190

MasterMix-No Dye and LightCycler Nano System using primers as shown in S4 Table.

191

The PCR conditions employed were according to the manufacturer’s protocol: 10 min at

192

95°C for enzyme activation, 40 cycles of 15 sec at 95°C (denaturation) and 60 sec at 60°C

14

193

(annealing and extension). We performed no template negative control experiments, as

194

well as melting curve analysis according to manufacturer’s instructions. Gene expression

195

was normalized to a housekeeping gene, Glyceraldehyde-3-phosophate dehydrogenase

196(Gapdh). Relative mRNA levels were determined by the comparative Ct method (Winer197

et al. 1999). Error bars in S6 Fig show standard deviation and p values were calculated

198

by Microsoft Excel (RRID:SCR_016137) and confirmed by WaveMetrics Igor Pro

199(RRID:SCR_000325).

200
201

Morphometric analysis of the cecum

202

Wild type (n = 3), type I (n = 4), and type II (n = 2) dissected intestines containing the203

cecum, colons, and part of small intestines at E18.5 were photographed at the same204

magnification (S7 Fig). The length of each cecum was measured using ImageJ. Briefly,

205

multi-points were set from the most proximal point abutting the small intestine and colon206

to the most distal cecum, approximately 16 to 18 points in the case of wild type cecum.

207

The sum of distance between each point was regarded as the length of the cecum.

208

15

209

Statistical analysis

210

Significance was determined by one-way analysis of variance (ANOVA) (Fig 4B),

211

Dunnett’s test (Fig 5I), or two-tailed unpaired Student’s t-test (S5L Fig, S7J Fig).

212

Correlation analysis was done by calculating correlation coefficient (Fig 6J-L, S5M Fig).

213

Chi-square test was used for the comparison of deep sequencing data on DNA from two

214

or three different tissues (Table 4). Data were presented as mean ± standard error of the215

mean (SEM). Values of p<0.05 were considered significant otherwise stated. Data

216

analysis was essentially performed using Microsoft Excel except for Dunnett’s test,

217

which was done using R (https://www.r-project.org) (RRID:SCR_001905).

16

218

Results

219

Generation of Fgf10-CRISPR founder embryos and their limb

220skeletal structures

221

To generate Fgf10-CRISPR mice, we used a single guide RNA (sgRNA) to target exon

222

3, as previously described (S1 Fig) [14]. According to Hashimoto and Takemoto (2015)

223[15], the resultant Fgf10-CRISPR F0 embryos were classified by the limb phenotype into

224

three types: type I, no limb; type II, limb defect; and type III, normal limbs. Since skeletal

225

pattern of these three limb phenotypes has not been revealed, cartilage and bone staining226

was performed on E18.5 Fgf10-crispants (n = 4 for wild type; n = 11 for mutant embryos)

227(Table 2, Fig 1, S2 Fig). Normally, the appendicular skeleton consists of girdle (“pectoral”

228

for forelimb and “pelvic” for hindlimb) and limb elements. The skeletons of the fore- and229

hindlimbs consist of stylopod for humerus/femur, zeugopod for the radius/ulna and230

tibia/fibula, and autopod for hand/foot (S2I-K Fig). In type I embryos (n = 6), all limb

231bones were lost and remaining girdle bones were affected to varying degrees (Fig 1A-D;

232

S2A-D Fig). The pectoral girdle consists of the scapula and clavicle, while the pelvic

233

girdle consists of the ilium, pubis, and ischium (S2I-K Fig) (for review [22]). In four out

17

234

of six type I embryos, the size of scapula was reduced and its spine was not formed,

235

indicating that the posterior blade was lost (Fig 1A). As for the pelvic girdle, the ischial236

and pubic bones were lost, while iliac bones reduced bilaterally (Table 2 and Fig 1B). In

237

the remaining two embryos, the scapula was intact with a spine, while the pubis and238

ischium were lost or reduced with the ilium intact bilaterally (Table 2 and Fig 1C, D; S2C,

239

D Fig). Thus, we regarded the latter phenotype as a type between I and II (type I/II). In

240

type II embryos (n = 2), all limb elements were lost in one or two appendages and in the241remaining appendages, limb truncations were observed at the stylopod or zeugopod level242(Table 2 and Fig 1E-O; S2E, F Fig). In type III embryos examined (n = 3), all limb and243

girdle bone structures were normal (Fig 1P-R). These limb skeletal patterns indicated that244

limb truncations were observed in the stylopodial, zeugopodial (type II), or pelvic region

245(type I). Formation of the clavicle, and the superior part of the scapula and pelvic girdle

246

were not affected by lack of Fgf10 gene products.

247
248

Genomic analysis of Fgf10-CRISPR founder embryos

18

249

To prescreen mutations at the Fgf10 locus, we performed an enzyme mismatch cleavage

250

assay (S3A, B Fig) and Sanger sequencing with the use of DNA from neck skin tissues

251of the E16.5 embryos. We estimated mutation frequencies based on band intensities [23],252

and found an embryo (type II, #4) with obvious limb defects but showed faint band

253

intensities for mutated DNA after endonuclease treatment (S3B Fig). Sanger sequencing

254

indicated a mixed genotype of wild type and a single nucleotide insertion in this embryo

255(S3C Fig).

256

To get more accurate mutation frequencies, we performed deep sequencing of PCR

257

amplicons containing the Fgf10 target site by use of embryonic (E16.5) neck DNA. By

258

multiple amplicon analysis, we could distinguish between wild type, in-frame and259

frameshift mutations (Fig 2). We also deduced amino acid sequences encoded by the260

targeted Fgf10 locus (Fig 3). Among in-frame mutations, we found that codons that261

encode Lys196 and His201 were preserved or those that encode His201 alone were262

preserved as in #3, #5, and #9 embryos; both residues are known to be indispensable for263

FGF receptor dimerization [24]. We calculated the percentage of the gene sequence that264

still encodes putative functional FGF10 proteins with both residues in each type of mutant

19

265

at E16.5, finding 8.3 ± 6.2% for type I, 25.3 ± 2.7% for type II, and 54.3 ± 9.5% for type

266

III, n = 3 for each type, mean ± SEM) (Table 3) (Fig 4A, B). In the case of E18.5 neck

267

DNA, it was 3.4 ± 2.9% for type I (n = 3), 33% for type II (n = 1), and 66.5 ± 8.8% for

268

type III, respectively (n = 8) (Table 1).

269

To know whether genotyping results using DNA from different tissues were similar,270

we performed deep sequencing on DNA extracted from lung and limb dermal tissues as

271

well as neck skin tissues of the E18.5 embryos (Table 1; S2 Table). We evaluated deep272

sequencing results on type III embryos (n = 10) with Chi-square test (Table 4). We found273

that in 6 embryos there was a significant difference in the frequencies of the functional274

Fgf10 genotypes from the different tissues and in 4 embryos there was no significant

275

difference in the frequencies of the functional Fgf10 genotypes from the different tissues

276(Table 4).

277
278

Analysis of lung phenotypes

279

We next examined the lung histology of Fgf10-mosaic F0 embryos on E16.5 (Table 3,

280

Fig 5, S4 Fig). In the normal mouse lung, there are five lung lobes, four on the right

20

281

(cranial, middle, caudal, and accessory) and one on the left (Fig 5A). The lung histology

282

of type I embryos demonstrated either the absence of all lobes (two of three embryos) or

283

only the presence of a residual lobe (one of three) (Fig 5B). We found that the accessory

284

lobe was absent in two out of three type II embryos, and that either middle or caudal lobe

285

was additionally absent or hypoplastic (Fig 5C). In type III embryos, all five lung lobes

286

were present (Fig 5D). In E18.5 type II embryos, the accessory lung lobe was absent from287

all three embryos examined (embryo #11_2, 24_2, 25_2; S5I-K Fig).

288

Mouse lung development is divided into five stages, namely embryonic,

289

pseudoglandular (E9.5 to 16.5), canalicular (E16.5 to 17.5), saccular (E17.5 to P5), and290

alveolar (P5 to P28) [25]. At E16.5, after branching morphogenesis the lung is in the291transition of pseudoglandular to canalicular stage. We found that the number of terminal

292

tubules was significantly decreased in type I and II residual lungs (Fig 5F, G, I), but not293

significantly altered in type III lungs (Fig 5H, I), compared with wild type (Fig 5E, I). By

294

E18.5, the lung enters the saccular stage and cuboidal cells that produce surfactant protein

295

C (SPC), i.e. alveolar type 2 epithelial (AECII) cells, differentiate on the wall of the296

developing airway space. Since AECII cell differentiation is perturbed in the Fgf10-

21

297

heterozygous lung [26,27], we performed immunohistochemistry on E18.5 lung sections

298

to detect AECII. We counted the number of SPC-positive cells in lungs of type III

299

embryos and examined whether it was correlated to the percentage of functional Fgf10

300

genotype. Representative data of immunostaining for type III lungs are shown (Fig 6B,

301E) along with those for type II lungs (Fig 6A, D) and positive/negative controls (Fig. 6C,

302

F, G-I). We found that the number of SPC-positive cells in lungs of type III embryos was303

correlated to the percentage of functional Fgf10 genotype (Fig 6J, correlation coefficient304

is 0.909 for limb DNA; Fig. 6K, 0.924 for neck DNA). Since the delay in development305

can be caused by variation between embryos even from a same litter due to crowding in306

the uterus, we also examined whether the number of SPC-positive cells was correlated to307

the weight of each embryo, as an index for developmental stage (Table 1). It seemed that308

there was no correlation between them (Fig 6L; correlation coefficient is 0.301).

309

On the other hand, since it was reported that the mesenchymal FGF10 is also important

310

for proper mesenchymal lineage formation during lung development as revealed by

311

Fgf10-hypomorphic lungs [20,28], we examined expression of mesenchymal and its

312

related marker genes [29] in type III lungs at E18.5 by quantitative PCR (Table 1, S4

22

313

Table). However, we could not detect a significant decrease in expression of these genes

314(S6 Fig), possibly because the percentage for functional Fgf10 genotypes was too high to315

mimic a Fgf10-hypomorphic phenotype in lung mesenchymal lineage formation (61.19%

316

in #11, 65.98% in #12, and 70.11% in #17 embryos). This expression analysis should be317

further clarified on earlier stage lungs, and in those which have more loss-of-function

318

mutations in the Fgf10 gene.

319

23

320

Discussion

321

Fgf10 genome edited F0 mice are classified into three limb phenotypes: no limb (type I),

322

limb defect (type II), and normal limbs (type III). In this study, we first examined

323

appendicular skeletal patterns of the Fgf10 mosaic mutant mice by cartilage and bone324

staining. We found that varying degrees of limb and girdle bone truncations were325

observed in type I and type II embryos. We next estimated the rate of putative functional326

Fgf10 genotypes in each mutant type and the mean percentage for type II embryos is 25.3

327

± 2.7 by deep sequencing of neck DNA. Comparison of deep sequencing results on DNA

328

from different tissues (neck, lung, and limb dermis) revealed that in half of the embryos329

the rate of putative functional Fgf10 genotypes were significantly varied between the330

three DNA. Lung phenotypes were examined on two embryonic stages, E16.5 and E18.5.

331

In type II embryos, the accessory lobe was lost and the number of terminal tubules was

332

significantly reduced. In type II and “normal limb (type III)” embryos, the number of333

alveolar type 2 cells, immunostained by anti-surfactant C protein, was correlated with the334

rate of putative functional Fgf10 genotypes.

335

24

336

Truncation patterns of girdle and limb bones in Fgf10-mosaic

337

mutants

338

Skeletal staining in this study revealed that in type II embryos, limb bone structures are339

truncated in more distal structures rather than forming a miniature of whole limb and340

girdle structures. Also, in the mutants, the zeugopodial element consists of one ulna- or

341

tibia-like bone: This pattern is different from those after extirpation of apical ectodermal342

ridge in chicken limb buds, where both zeugopod bones are truncated [30]. Although the343

number of type II embryos examined in this study is only two and therefore further studies344

are needed, a reduced number of Fgf10-expressing cells in the mosaic mutants does not345

seem to know the morphological pattern to be formed. Looking the expression of346

zeugopod patterning genes could help to elucidate the underlying mechanisms.

347

Regarding girdle bones, it has been known that the posterior (inferior) half of the348

scapula is absent and most of the pelvic structures is lacking in Fgf10-null embryos

349[12,13]. This study showed that in type I/II Fgf10-mosaic mutants the posterior scapula

350

with the spine retained. In the mouse, while the pelvic girdle and most of the scapular

351

blade derives solely from the lateral plate mesoderm (LPM), the medial edge of the blade

25

352

arises from dermomyotome cells and the neural crest cells contribute to the scapular spine353[31-33]. Thus, it is likely that a reduced number of Fgf10-expressing cells in the LPM

354

and neural crest cells [34] contributed to the formation of the posterior (inferior) scapula355

in type I/II embryos. In the case of pelvic girdle, a reduced number of Fgf10-expressing

356

cells in the LPM contributed to the formation of the inferior pelvic structures in type I/II

357

embryos.

358
359

Fgf10 mutation rate may not be identical in different tissues

360

Here we showed that there is a correlation between limb phenotypes, lung phenotypes,

361

and overall defective mutation rate in Fgf10-CRISPR knockout F0 mice. It has been

362

shown that even if CRISPR-genome editing materials are introduced into fertilized eggs363

at the one-cell stage, mosaicism in the target genotype is caused under certain

364

experimental conditions until the target sequences are mutated [6,7]. During the early365

embryonic stage, mutated cells and non-mutated cells are intermingled and distributed to366lung and limb primordia proportionally [35], which we believe contributes to the367

correlation between limb and lung phenotypes. However, statistical analysis on deep

26

368

sequencing data of the genotypes shows in more than half cases there is a significant

369

difference of the defective mutation rate in DNA from different tissues. In this study, we

370

could not identify the reason why there were differences in the rate of the functional Fgf10

371

genotypes. In some developmental settings, Fgf10-expressing normal cells might have a372

growth advantage than Fgf10-deficient cells, which should be further explored in the next373

studies [10].

374

Fgf10-CRISPR F0 mice possess degrees of lung dysgenesis that375

correlate with putative functional FGF10 dosage

376

Accessory lobe formation was firstly impaired due to reduction in putative functional

377

FGF10 dosage. Ramasamy et al. (2007) reported that Fgf10-hypomorphic mice, in which

378

Fgf10 expression is reduced by 27%, compared with Fgf10-heterozygous mutants, lack379

accessory lobe formation [20]. Since Fgf10 expression is highest in the mesenchyme of380

the accessory lobe on E11.5 and persists as such until at least E18.5 [21,29], high Fgf10

381

dosage was believed to be required for accessory lobe formation. Our result shows that382

more than 25.3 ± 2.7% (from the analysis of E16.5 embryos) of functional Fgf10 gene383

product is required for accessory lobe formation. Since reduction or loss of accessory lobe

27

384

formation has been observed in loss-of-function mutants of Gli3, Fog2, and Gata4 [36-38538], FGF10 signaling may be related to the expression of these transcription factors

386

during accessory lobe formation.

387

Lineage-tracing analysis of Fgf10-expressing cells during mouse lung development has

388

revealed that there are two waves of Fgf10 expression; the first begins from E11.5 and389

the second from E15.5 [29]. We think that a reduction in the latter cells would cause the390

decrease in the number of terminal tubules in type I and type II Fgf10-CRISPR F0391

embryos. A recent study has shown that AECII as well as AECI independent progenitors392

are present at E13.5 as revealed by single cell RNA sequencing analysis [39]. By the393

saccular stage, the lung mesenchyme surrounding the epithelium becomes thinner and394

cuboidal AECII cells, characterized by the production of surfactant proteins, begin to395

differentiate (summarized in [40]). The number of AECII cells is found to be lower in the396

Fgf10-heterozygous lung [26,27], which is similar to our result in type III Fgf10-mosaic

397

mice. Taken together, Fgf10 expression levels regulate the number of alveolar type 2398epithelial cells in mouse lung in somewhat dose-dependent manner.

28

399

Regarding FGF10 dosage, the Fgf10-hypomorphic mouse aforementioned [20,28] has

400

also clarified that higher FGF10 dosage is required for full development of colonic crypts

401[41]. Our preliminary observation (S7 Fig) showed that in type I embryos the length of402

the cecum was significantly shorter than the wild type and there was an atresia of the403

colon as reported in the Fgf10-null mutants [41,42]. The length of the blind colon varied404

depending upon the embryos (S7D-G Fig), suggesting a correlation with their FGF10

405

dosage.

406
407

Fgf10-CRISPR F0 mice can serve as a series of lung disease408

models409

Volckaert et al. (2013) reported that lung branching morphogenesis does not require

410

localized Fgf10 expression in the distal mesenchyme, because ubiquitous Fgf10

411

overexpression can induce lung formation in Fgf10 knockout mice [43]. Furthermore,

412

Fgf10 expression after lung initiation is required for branching and proximal-distal413

differentiation by regulating Sox2/9 expression in the epithelium [43]. Our result also

29

414

supports this property of Fgf10 as the number of terminal tubules decreased in the Fgf10-415

mosaic mutants where functional FGF10 dosage is reduced.

416

Recent papers have reported that mutations and single nucleotide polymorphisms

417(SNPs) in the Fgf10 gene are correlated with human lung disease (reviewed in [44]). For

418

example, the absence of the right medial-basal airway is associated with a type of chronic

419

obstructive pulmonary diseases and two types of SNPs are found within the same intron

420

of Fgf10 in those cohorts [45]. Although some molecular and cellular differences have421

been identified between mouse and human lungs (reviewed in [46]), the F0 mice422

generated by CRISPR/Cas9-mediated Fgf10 gene editing can become a model animal to423

study the pathophysiology of human pulmonary hypoplasia and related chronic lung424

diseases that may be rooted in the developmental stage as recently postulated [47]).

425
426

Acknowledgements

427

We are grateful for assistance in deep sequencing analysis given from Prof. Shuji

428

Shigenobu, Functional Genomics Facility, NIBB Core Research Facilities, and the NIBB

429

Collaborative Research Program (18-202 to T.H).

30

430

31

431

References

432

1.

433
434

engineering with CRISPR-Cas9. Science. 2014;346(6213):1258096.
2.

435
436

Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9
for genome engineering. Cell. 2014;157(6):1262-78.

3.

437
438

Doudna JA, Charpentier E. Genome editing. The new frontier of genome

Cornu TI, Mussolino C, Cathomen T. Refining strategies to translate genome
editing to the clinic. Nat Med. 2017;23(4):415-23.

4.

Sung YH, Kim JM, Kim HT, Lee J, Jeon J, Jin Y, et al. Highly efficient gene439

knockout in mice and zebrafish with RNA-guided endonucleases. Genome Res.

440

2014;24(1):125-31.

441

5.

Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R. One-step442

generation of mice carrying reporter and conditional alleles by CRISPR/Cas-443

mediated genome engineering. Cell. 2013;154(6):1370-9.

444

6.

Yen ST, Zhang M, Deng JM, Usman SJ, Smith CN, Parker-Thornburg J, et al.

445Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA

446

injections in mouse zygotes. Dev Biol. 2014;393(1):3-9.

32

447

7.

Hashimoto M, Yamashita Y, Takemoto T. Electroporation of Cas9448

protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in449

the mouse. Dev Biol. 2016;418(1):1-9.

450

8.

Zhong H, Chen Y, Li Y, Chen R, Mardon G. CRISPR-engineered mosaicism

451

rapidly reveals that loss of Kcnj13 function in mice mimics human disease452

phenotypes. Sci Rep. 2015;5:8366.

453

9.

454
455

Nat Rev Genet. 2013;14(5):307-20.
10.

456
457

Biesecker LG, Spinner NB. A genomic view of mosaicism and human disease.

Gajecka M. Unrevealed mosaicism in the next-generation sequencing era. Mol
Genet Genomics. 2016;291(2):513-30.

11.

Yasue A, Kono H, Habuta M, Bando T, Sato K, Inoue J, et al. Relationship458

between somatic mosaicism of Pax6 mutation and variable developmental eye

459

abnormalities-an analysis of CRISPR genome-edited mouse embryos. Sci Rep.

460

2017;7(1):53. Epub 2017/03/02. doi: 10.1038/s41598-017-00088-w

461
462

12.

Min H, Danilenko DM, Scully SA, Bolon B, Ring BD, Tarpley JE, et al. Fgf-10
is required for both limb and lung development and exhibits striking functional

33

463
464

similarity to Drosophila branchless. Genes Dev. 1998;12(20):3156-61.
13.

465
466

Sekine K, Ohuchi H, Fujiwara M, Yamasaki M, Yoshizawa T, Sato T, et al.
Fgf10 is essential for limb and lung formation. Nat Genet. 1999;21(1):138-41.

14.

Yasue A, Mitsui SN, Watanabe T, Sakuma T, Oyadomari S, Yamamoto T, et al.

467

Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by468

the TALEN and CRISPR/Cas systems. Sci Rep. 2014;4:5705.

469

15.

Hashimoto M, Takemoto T. Electroporation enables the efficient mRNA

470

delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome471

editing. Sci Rep. 2015;5:11315.

472

16.

Brinkman EK, Kousholt AN, Harmsen T, Leemans C, Chen T, Jonkers J, et al.

473

Easy quantification of template-directed CRISPR/Cas9 editing. Nucleic Acids

474

Res. 2018;46(10):e58.

475

17.

Iida M, Suzuki M, Sakane Y, Nishide H, Uchiyama I, Yamamoto T, et al. A

476

simple and practical workflow for genotyping of CRISPR-Cas9-based knockout

477

phenotypes using multiplexed amplicon sequencing. Genes Cells. 2020;00:1-12.

478

doi: 10.1111/gtc.12775

34

479

18.

Hogan BLMB, R.; Costantini, F.; Lacy, E. . Manipulating the mouse embryo: a480

laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory

481

Press; 1994.

482

19.

Stachtea XN, Tykesson E, van Kuppevelt TH, Feinstein R, Malmstrom A,

483

Reijmers RM, et al. Dermatan Sulfate-Free Mice Display Embryological484

Defects and Are Neonatal Lethal Despite Normal Lymphoid and Non-

485

Lymphoid Organogenesis. PLoS One. 2015;10(10):e0140279.

486

20.

Ramasamy SK, Mailleux AA, Gupte VV, Mata F, Sala FG, Veltmaat JM, et al.

487

Fgf10 dosage is critical for the amplification of epithelial cell progenitors and488

for the formation of multiple mesenchymal lineages during lung development.

489

Dev Biol. 2007;307(2):237-47.

490

21.

Bellusci S, Grindley J, Emoto H, Itoh N, Hogan BL. Fibroblast growth factor

491

10 (FGF10) and branching morphogenesis in the embryonic mouse lung.

492

Development. 1997;124(23):4867-78.

493
494

22.

Sears KE, Capellini TD, Diogo R. On the serial homology of the pectoral andpelvicgirdlesoftetrapods.

Evolution.

2015;69(10):2543-55.

doi:

35

495
496

10.1111/evo.12773
23.

Abu-Bonsrah KD, Zhang D, Newgreen DF. CRISPR/Cas9 Targets Chicken

497

Embryonic Somatic Cells In Vitro and In Vivo and generates Phenotypic

498

Abnormalities. Sci Rep. 2016;6:34524.

499

24.

Ibrahimi OA, Yeh BK, Eliseenkova AV, Zhang F, Olsen SK, Igarashi M, et al.

500

Analysis of mutations in fibroblast growth factor (FGF) and a pathogenic

501

mutation in FGF receptor (FGFR) provides direct evidence for the symmetric502

two-end model for FGFR dimerization. Mol Cell Biol. 2005;25(2):671-84.

503

25.

Gao X, Hogan BL. Development of the Respiratory System. Kaufman's Atlas

504

of Mouse Development Supplement: With Coronal Sections: Academic Press;

505

2016.

506

26.

Chao CM, Yahya F, Moiseenko A, Tiozzo C, Shrestha A, Ahmadvand N, et al.

507

Fgf10 deficiency is causative for lethality in a mouse model of508

bronchopulmonary dysplasia. J Pathol. 2017;241(1):91-103.

509
510

27.

Li J, Wang Z, Chu Q, Jiang K, Li J, Tang N. The Strength of Mechanical Forces
Determines the Differentiation of Alveolar Epithelial Cells. Dev Cell.

36

511
512

2018;44(3):297-312 e5.
28.

Mailleux AA, Kelly R, Veltmaat JM, De Langhe SP, Zaffran S, Thiery JP, et al.

513

Fgf10 expression identifies parabronchial smooth muscle cell progenitors and514

is required for their entry into the smooth muscle cell lineage. Development.

515

2005; 132(9):2157-66. doi: 10.1242/dev.01795

516

29.

El Agha E, Herold S, Al Alam D, Quantius J, MacKenzie B, Carraro G, et al.

517

Fgf10-positive cells represent a progenitor cell population during lung518

development and postnatally. Development. 2014;141(2):296-306.

519

30.

520
521

Summerbell D. A quantitative analysis of the effect of excision of the AER fromthe chick limb-bud. J Embryol Exp Morphol. 1974;32(3):651-60.

31.

Durland JL, Sferlazzo M, Logan M, Burke AC. Visualizing the lateral somitic

522

frontier in the Prx1Cre transgenic mouse. J Anat. 2008;212(5):590-602. doi:523

10.1111/j.1469-7580.2008.00879.x

524

32.

Valasek P, Theis S, Krejci E, Grim M, Maina F, Shwartz Y, et al. Somitic origin

525

of the medial border of the mammalian scapula and its homology to the avian526

scapula blade. J Anat. 2010;216(4):482-8. doi: 10.1111/j

37

527

33.

Matsuoka T, Ahlberg PE, Kessaris N, Iannarelli P, Dennehy U, Richardson WD,

528

et

529

2005;436(7049):347-55. doi: 10.1038/nature03837

530

34.

al.

Neuralcrest

origins

of

the

neck

and

shoulder.

Nature.

Teshima TH, Lourenco SV, Tucker AS. Multiple Cranial Organ Defects after

531

Conditionally Knocking Out Fgf10 in the Neural Crest. Front Physiol.

532

2016;7:488. doi: 10.3389/fphys.2016.00488

533

35.

534
535

Soriano P, Jaenisch R. Retroviruses as probes for mammalian development:
allocation of cells to the somatic and germ cell lineages. Cell. 1986;46(1):19-29.

36.

Grindley JC, Bellusci S, Perkins D, Hogan BL. Evidence for the involvement536

of the Gli gene family in embryonic mouse lung development. Dev Biol.

537

1997;188(2):337-48.

538

37.

Ackerman KG, Herron BJ, Vargas SO, Huang H, Tevosian SG, Kochilas L, et539

al. Fog2 is required for normal diaphragm and lung development in mice and540

humans. PLoS Genet. 2005;1(1):58-65.

541
542

38.

Ackerman KG, Wang J, Luo L, Fujiwara Y, Orkin SH, Beier DR. Gata4 isnecessary for normal pulmonary lobar development. Am J Respir Cell Mol Biol.

38

543
544

2007;36(4):391-7.
39.

Frank DB, Penkala IJ, Zepp JA, Sivakumar A, Linares-Saldana R, Zacharias

545

WJ, et al. Early lineage specification defines alveolar epithelial ontogeny in the546

murine lung. Proc Natl Acad Sci U S A. 2019;116(10):4362-71. doi:

547

10.1073/pnas.1813952116

548

40.

549
550

Ornitz DM, Yin Y. Signaling networks regulating development of the lower
respiratory tract. Cold Spring Harb Perspect Biol. 2012;4(5).

41.

Sala FG, Curtis JL, Veltmaat JM, Del Moral PM, Le LT, Fairbanks TJ, et al.

551

Fibroblast growth factor 10 is required for survival and proliferation but not

552

differentiation of intestinal epithelial progenitor cells during murine colon553

development. Dev Biol. 2006;299(2):373-85. doi: 10.1016/j.ydbio.2006.08.001

554

42.

Burns RC, Fairbanks TJ, Sala F, De Langhe S, Mailleux A, Thiery JP, et al.

555

Requirement for fibroblast growth factor 10 or fibroblast growth factor receptor

556

2-IIIb signaling for cecal development in mouse. Dev Biol. 2004;265(1):61-74.

557

doi: 10.1016/j.ydbio.2003.09.021.

558

43.

Volckaert T, Campbell A, Dill E, Li C, Minoo P, De Langhe S. Localized Fgf10

39

559

expression is not required for lung branching morphogenesis but prevents

560

differentiation of epithelial progenitors. Development. 2013;140(18):3731-42.

561

44.

562
563

Danopoulos S, Shiosaki J, Al Alam D. FGF Signaling in Lung Development
and Disease: Human Versus Mouse. Front Genet. 2019;10:170.

45.

Smith BM, Traboulsi H, Austin JHM, Manichaikul A, Hoffman EA, Bleecker

564

ER, et al. Human airway branch variation and chronic obstructive pulmonary565

disease. Proc Natl Acad Sci U S A. 2018;115(5):E974-E81.

566

46.

567
568

Nikolic MZ, Sun D, Rawlins EL. Human lung development: recent progress andnew challenges. Development. 2018;145(16).

47.

Hobbs BD, de Jong K, Lamontagne M, Bosse Y, Shrine N, Artigas MS, et al.

569

Genetic loci associated with chronic obstructive pulmonary disease overlap with570

loci for lung function and pulmonary fibrosis. Nat Genet. 2017;49(3):426-32.

571

40

572

Tables573

Table 1. Summary of Fgf10-CRISPR F0 embryos at E18.5. These embryos were574

genotyped by deep sequencing (NGS).

575*SPC (+) cells/total cells (mean ± SEM %) is shown in parenthesis of IHC for Fig 6J and576

K. -, no limb; c-b staining, cartilage and bone staining; N.A., not applicable; N.A.**, not577

applicable due to low expected frequency (<5); N.D., not done; qPCR, quantitative PCR.

578***contains wild type genotype and in-frame genotype retaining nucleotides for Lys196

579

and His201.

580
581

Table 2. Summary of Fgf10-CRISPR F0 embryos (E18.5) processed for cartilage

582

and bone staining.

583-, no bone; ±, bone hypoplasia; +, normal bones; N.A., not applicable; N.D., not584

determined due to separation of proximal parts of the limb from the trunk for lung analysis.

585
586

Table 3. Summary of limb and lung phenotypes of Fgf10-CRISPR F0 embryos at

587

E16.5.

41588-, no limb or no lung lobe; ±, limb defect or small lung lobes; +, normal limbs or normal589

appearance of lung lobes. *a residual lung lobe. Genotypes of crispants (types I-III) were590

prescreened by an enzyme mismatch cleavage assay (S3B Fig) and determined by deep591

sequencing (NGS).

592
593

Table 4. Chi-square test for genotyping results of DNA derived from different tissues.

594

The number of sequence reads in deep sequencing is shown in contingency tables for595

observed frequencies. Wt, wild type Fgf10 genotype; Out, frameshift mutations in the596

Fgf10 gene; W/o & Out, in-frame mutation without nucleotides encoding Lys196 and/or

597

His201 plus frameshift mutations in the Fgf10 gene; In (w), in-frame mutations with598

nucleotides encoding Lys196 and His201; In (w/o), in-frame mutations without

599

nucleotides encoding Lys196 and/or His201. When p-values <0.05, there is statistically600

significant difference in the Fgf10 genotypes of the different tissues.

601

42

Table 1. Summary of Fgf10-CRISPR F0 embryos at E18.5. These embryos were genotyped by deep sequencing (NGS).

Type

I

II

III

Wild type

Hindlimb

Chi-square test of
Functional
NGS results
Fgf10 genotype(p-value)(%) in neck DNA

Functional Fgf10
genotype (%) in limb
DNA

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

N.D.

67.75

61.19

64.23

0.751528903

N.D.

65.98

66.45

Identical

4.01545E-05

N.D.

82.59

84.77

Not identical

2.89945E-11

59.78

56.84

56.22

Not identicalLimb, lung

N.A.**

N.D.

100.00100.00Identical

Limb, lung3.37691E-50

N.D.

70.11

64.00

Not identical

IHC (19.9 ± 0.7)

Limb, neck1.892E-09

100.00***0.7742IHC (16.9 ± 0.8)

Limb, neck0.176029751

56.92

N.D.

57.52

Identical

Fig 6J-M

0.8757IHC (9.8 ± 0.5)

Limb, neck0.271069452

28.92

N.D.

29.94

Identical

Fig 6B,E,H;Fig 6J-M

#23

0.9232IHC (24.2 ± 0.8)

Limb, neckN.A.**

100.00

N.D.

97.20

Identical

Fig 6J-M#24

0.9976IHC (18.8 ± 0.7)

Limb, neck1.18483E-26

73.06

N.D.

82.26

Not identicalFig 6J-M#25

0.9832IHC (15.2 ± 1.4)

Limb, neck

0.139527497

40.75

N.D.

40.16

Identical

Fig 6J-M

0.7472Lung qPCR

Limb, lung, neckN.A.**

99.63

100.00100.00Identical

Right

Weight(gram)

Analysis*

Tissues for NGS

Left

Right

Left

#18

ー

ー

#19

ー

ー

ー

ー

0.6303collected for c-b staining

Neck

N.A.

9.27

ー

ー

0.6899collected for c-b stainig

Neck

N.A.

0.50

#26

ー

ー

ー

ー

0.4682

C-b staining

Neck

N.A.

0.45

ー

Humerus only

ー

Short
femur

0.5784C-b staining

Neck

N.A.

32.96

#11

0.7799Lung qPCR

Limb, lung, neck3.34357E-29#120.9111Lung qPCR

Limb, lung#13

0.9699Lung collected for qPCR

Limb, lung#15

0.9572Lung collected for qPCR

Limb, lung, neck#16

0.9254Lung collected for qPCR

#17

0.7524Lung qPCR

1.0555

#21
#22

#14

#20

#2

Forelimb

NormalNormalNormalNormalNormalNormalNormalNormalFunctional Fgf10
genotype (mean±SEM%) in neck DNA

Genotype
identity between
different DNA

Functional Fgf10
genotype (%) in lung
DNA

Embry
o No.

3.4 ± 2.9

N.A.

65.8 ± 9.0

N.A.

N.D.

FigureFig 1J-O
Not identicalFig 1P-R

100.00*** Not identical***Fig 6J-M*SPC (+) cells/total cells (mean ± SEM %) is shown in parenthesis of IHC for Fig 6J and K. -, no limb; c-b staining, cartilage and bone staining; N.A., not applicable;
N.A.**, not applicable due to low expected frequency (<5); N.D., not done; qPCR, quantitative PCR. ***contains wild type genotype and in-frame genotype retaining
nucleotides for Lys196 and His201.

43

Table 2. Summary of Fgf10-CRISPR F0 embryos (E18.5) processed for cartilage and bone staining.

Type Subtype

Embryo
No.

I
I/II

#26

-

-

±
±

+
+

±
±

#34

-

-

±

+

#35

-

-

±

+

#40

-

-

+

#46

-

-

+

Left

Right

+
+

-

±

+

±

+

+

+

+

+

Scaplua Clavicle Scaplua Clavicle

Pelvic girdle
Left

Right

Functional Fgf10
genotypes (%) in neck
DNA

Functional Fgf10
genotypes (%) in lung
DNA

Functional Fgf10
genotypes (%) in limb
DNA

FigureIlium

Ischium

Pubis

Ilium

Ischium

Pubis

-

±
±

-

-

±
±

-

-

N.A.
0.45

N.A.
N.A.

N.A. Fig 1,Fig S2
N.A.

-

-

±

-

-

±

-

-

N.A.

N.A.

N.A.

-

-

±

-

-

±

-

-

N.A.

N.A.

N.A.

+

-

-

+

±

-

+

±

-

N.A.

N.A.

N.A.

+

-

-

+

±

-

+

-

-

N.A.

N.A.

N.A. Fig 1,Fig S2-

Truncated
at distal
femur

+

±

-

+

-

±

32.96

N.A.

N.A.

+

+

-

+

+

+

N.A.

N.A.

N.A. Fig 1,Fig S2

N.D.

Humerus
only

#33

+

Humerus,radiusonly,noautopod

+

+

+

+

-

Truncated
at distal
tibia, nofibula

#11

+

+

+

N.D.

+

N.D.

+

+

+

+

+

+

+

+

67.75

61.19

64.23

#12

+

+

+

N.D.

+

N.D.

+

+

+

+

+

+

+

+

N.A.

65.98

66.45

#17

+

+

+

N.D.

+

N.D.

+

+

+

+

+

+

+

+

N.A.

70.11

64.00

#1

+

+

+

N.D.

+

N.D.

+

+

+

+

+

+

+

+

N.A.

N.A.

N.A.

#4

+

+

+

N.D.

+

N.D.

+

+

+

+

+

+

+

+

N.A.

N.A.

N.A.

#5

+

+

+

N.D.

+

N.D.

+

+

N.D.

N.D.

N.D.

+

+

+

N.A.

N.A.

N.A.

#7_18

+

+

+

+

+

+

+

+

+

+

+

+

+

+

N.A.

N.A.

N.A. Fig 1,Fig S2

II

Wild type

Hindlimb
Right

Right

#14

III

Pectoral girdle
Left

Left

#4_18
I

Forelimb

N.D.

N.D.

+

+

Fig 1

Fig 1

-, no bone; ±, bone hypoplasia; +, normal bones; N.A., not applicable; N.D., not determined due to separation of proximal parts of the limb from the trunk for lunganalysis.

44

Table 3. Table 3. Summary of limb and lung phenotypes of Fgf10-CRISPR F0 embryos at E16.5.

Forelimb
Hindlimb
Lung
EmbryoType
Cranial Middle Caudal Accessory Left
No.
Left Right Left Right
lobe
lobe
lobe
lobe
lobe
I

II

III

WT

#9
#5
#7
#11
#3
#4
#1
#2
#8
#1
#2
#3

±
±
±
+
+
+
+
+
+

±
±
+
+
+
+
+
+
+

±
±
+
+
+
+
+
+
+

±
±
±
+
+
+
+
+
+

-

-

+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+

-

-

±
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+

±*

+
+
+
+
+
+
+
+
+

# of
terminal
tubules
/mm2

0
0
18.7
87.4
59.0
106.8
113.4
127.1
122.2
131.2
117.9
157.6

# of terminal
Functional Fgf10 Functional Fgf10
tubules/mm2 (mean genotype (%) (neck genotype (mean± SEM %)± SEM)
DNA)

6.2 ± 6.2

84.4 ± 13.9

120.9 ± 4.0

0.00
4.63
20.35
20.60
25.39
29.96
42.83
46.97
73.06

135.6 ± 11.6

8.3 ± 6.2

25.3 ± 2.7

54.3 ± 9.5

Not determined

FigureFig S4
Fig S4
Fig 5,Fig S4
Fig S4
Fig 5,Fig S4
Fig S4
Fig 5,Fig S4
Fig S4
Fig S4
Fig 5,Fig S4
Not shown
Not shown-, no limb or no lung lobe; ±, limb defect or small lung lobes; +, normal limbs or normal appearance of lung lobes. *a residual lung lobe. Genotypes of crispants(types I-III) were prescreened by an enzyme mismatch cleavage assay (Fig S3B) and determined by deep sequencing (NGS).

45

1

Table 4. Chi-square test for genotyping results of DNA derived from different tissues.

2
3
4

Embryo No. Type
#2

Wild
type

5
6

#11

#12

10

#13

13

#15

#16

#17

19
#20

#21

#22

Lung Limb
Total
706 19627 20333
364 9911 10275
1070 29538 30608

Lung
Limb
Total
710.8 19622 20333
359.2 9916 10275
1070 29538 30608

0.752

Wt
Out
Total

Lung Limb
Total
4203 45331 49534
886 8147 9033
5089 53478 58567

Wt
Out
Total

Lung
Limb
Total
4304 45230 49534
784.9 8248
9033
5089 53478 58567

4E-05

Wt
Out
Total

Neck Lung Limb Total
7295 20266 22243 49804
4908 15388 17318 37614
12203 35654 39561 87418

Wt
Out
Total

Neck Lung Limb
Total
6952 20313 22539 49804
5251 15341 17022 37614
12203 35654 39561 87418

3E-11

Wt
W/o & Out
Total

Lung Limb
Total
15698 38223 53921
00015698 38223 53921

Wt
Out
Total

Lung
Limb
Total
15698 38223 53921
00015698 38223 53921
EF: less than 5

N.A.

Wt
Out
Total

Lung Limb
Total
3441 21706 25147
2869 13857 16726
2690 20007 22697
9000 55570 64570

Wt
In (w)
Out
Total

Lung
Limb
Total
3505 21642 25147
2331 14395 16726
3164 19533 22697
9000 55570 64570

3E-50

Wt
In (w)Out
Total

Neck Limb
Total
29282 5549 34831
11875 2653 14528
41157 8202 49359

Wt
In (w)
Total

Neck Limb
Total
29043 5788 34831
12114 2414 14528
41157 8202 49359

2E-10

Wt
In (w)
Total

Neck Limb
Total
23077 10726 33803
17464 7923 25387
40541 18649 59190

Wt
Out
Total

Neck Limb
Total
23153 10650 33803
17388 7999 25387
40541 18649 59190

0.176

Wt
Out
Total

Neck Limb
Total
2653
963 3616
6521 2253 8774
9174 3216 12390

Wt
Out
Total

Neck Limb
Total
2677 938.6
3616
6497 2277
8774
9174 3216 12390

0.271

Wt
Out
Total

Neck Limb
Total
82324 1146 83470
033
33
82324 1179 83503

Wt
Out*
Total

Neck Limb
Total
82291 1179 83470
32.53 0.466
3382324 1179 83503
EF: less than 5

N.A.

Wt
Out*
Total

Neck Limb
Total
8862 2633 11495
3268
568 3836
12130 3201 15331

Wt
Out
Total

Neck Limb
Total
9095 2400 11495
3035 800.9
3836
12130 3201 15331

1E-26

Wt
Out
Total

Neck Limb
Total
12665 10417 23082
18415 15520 33935
31080 25937 57017

In(w)
Out**
Total

Neck Limb
Total
12582 10500 23082
18498 15437 33935
31080 25937 57017

0.155

In(w)
Out**
Total

III

25
#23
*including
large
deletion

III

#24

III

#25
**including
large
insertion

Wt
Out
Total

III

23
24

Neck Lung Limb
Total
6290 13430 18408 38128
3578 7641 10472 21691
9868 21071 28880 59819

3E-29

Wt
Out
Total

III

21
22

Neck Lung Limb Total
6686 12893 18549 38128
3182 8178 10331 21691
9868 21071 28880 59819

III

18
20

Wt
Out
Total

III

16
17

N.A.

III

14
15

Wt
Out
Total

III

11
12

Wt
Out
Total

p- value

Neck Lung Limb
Total
4894 8758 58874 72526
0.067 2.174
15184895 8760 58889 72544
EF: less than 5III9Expected frequencies (EF)Neck Lung Limb Total
4877 8760 58889 72526
18
0
0
18
4895 8760 58889 72544

III

7
8

Observed frequencies

III

46

26

The number of sequence reads in deep sequencing is shown in contingency tables for27

observed frequencies. Wt, wild type Fgf10 genotype; Out, frameshift mutations in the28

Fgf10 gene; W/o & Out, in-frame mutation without nucleotides encoding Lys196 and/or

29

His201 plus frameshift mutations in the Fgf10 gene; In (w), in-frame mutations with30

nucleotides encoding Lys196 and His201; In (w/o), in-frame mutations without

31

nucleotides encoding Lys196 and/or His201. When p-values <0.05, there is statistically32

significant difference in the Fgf10 genotypes of the different tissues.

33
34

47

35

Figure legends36

Fig 1. Cartilage and bone staining to reveal limb skeletal structures.

37

A-I, L-R, left or right lateral views of limb and girdle regions (at E18.5) are shown.

38

Cartilage is stained with Alcian blue and bone is stained with Alizarin red. A, B, type I

39#4_18 embryo. C, D, type I/II #46 embryo. E-I, type II #33 embryo. L-O, type II #14

40

embryo. P-R, type III #12 embryo. J, K, Lateral views of type II #14 embryo are shown.

41

Arrows in (K) show truncated limbs. au, autopod; f, femur; fi, fibula; h, humerus; il,42ilium; is, ischium; p, pubis; r, radius; sc, scapula; sp, spine; st, stylopod t, tibia; u, ulna;

43ze, zeugopod. Scale bars: 1 mm.

44
45

Fig 2. Genomic analysis of the Fgf10-CRISPR F0 embryos at E16.5 as revealed by46

deep sequencing.

47

The target nucleotide sequence is highlighted in yellow. Proto-spacer adjacent motif

48

sequence is shown in blue. Insertion and deletion sequences are shown in red.

49
50

Fig 3. Deduced amino acids for in-frame mutations after deep sequencing.

48

51

Wild type (WT) sequences are shown on the top. The embryo (E16.5) number (#),

52

nucleotide number of small insertion (+) or deletion (-) (Indels) are shown on the left.

53

Classified “types” are shown on the right. Amino acids corresponding to the guide RNA

54

sequence are underlined. Deleted and altered amino acids are indicated in red.

55
56

Fig 4. Schematic representation of genomic analysis of Fgf10-CRISPR F0 embryos

57

at E16.5 by deep sequencing.

58

A, percentage of total reads for the Fgf10 crispants. wt, wild type Fgf10 target nucleotide

59

sequence; in, in-frame mutations by small insertion or deletion; out, frameshift mutations.

60

Among in-frame mutations, the percentage of those that preserve the codons for Lys196

61

and His201 is shown in green, while the percentage that eliminate either of them is shown62

in yellow. B, percentage of wild type and in-frame mutations in which nucleotides for63

both Lys196 and His201 are retained: 8.3 ± 6.2% for type I, 25.3 ± 2.7% for type II, and64

54.3 ± 9.5% for type III. Data are presented as means ± SEM. *p<0.01 (p=0.008).

65
66

Fig 5. Limb phenotypes and lung histology of Fgf10-CRISPR F0 embryos at E16.5.

49

67

Representative embryos are shown for wild type (WT), type I (embryo #7), type II (#3),

68

and type III (#1). A–D, transverse section of the embryonic chest region. E–H, close-up

69

of the embryonic lung (boxed area) shown in (A–D), respectively. Asterisks show

70

putative terminal tubules in the lung. I, the number of lung terminal tubules per unit area.

71

In type I and type II embryos, there is a significant decrease in the number compared with

72

that of wild type. Data are presented as means ± SEM. Source data are available in Table73

3. Ac, accessory lobe; Ca, caudal lobe; Cr, cranial lobe; L, left lobe; Mi, middle lobe.

74

Scale bars: 500 µm in (A–D), and 100 µm in (E–H).

75
76

Fig 6. Immunohistochemistry of the lung in type II and type III Fgf10-crispants and77

wild type embryos at E18.5.

78

Representative data are shown. Nuclei are stained with Hematoxylin. A-C, Localization

79

of Surfactant protein C (SPC) (alveolar type 2 epithelial cells) is indicated by brown80

staining. D-F, close-up of the boxed area shown in (A–C), respectively. G-I, negative81

control, using normal rabbit IgG instead of anti-SPC antibody. Scale bars: 100 µm in (A-82C); 50 µm in (D-I). J, K, the number of SPC-positive cells in type III embryos is

50

83

correlated to the percentage of functional Fgf10 genotypes in limb (J) or neck DNA (K).

84

L, the number of SPC-positive cells in type III embryos is not to correlated to their weight

85(K). Source data for (J-L) are available in Table 1. M, Schematic drawings to show

86

percentage of total reads for the type III Fgf10 crispants. wt, wild type Fgf10 target

87

nucleotide sequence; in, in-frame mutations by small insertion or deletion; out, out-of-

88

frame mutations. Among in-frame mutations, the percentage of those that preserve the89codons for Lys196 and His201 is shown in green, while the percentage that eliminate

90

either of them is shown in yellow.

91

51

92

Supporting information

93

Supplementary Tables

94

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

95
96

S2 Table. Summary of deep sequencing data on DNA from different tissues of E18.5

97

embryos.

98

The percentage of sequence reads for each genotype category is shown in contingency

99

tables. Wt, wild type Fgf10 genotype; In, in-frame mutations in the Fgf10 gene; Out,100

frameshift mutations in the Fgf10 gene; N.D., not done; N.A., not applicable. p-values of101

Chi-square test (see Table 4) are shown for reference.

102
103

S3 Table. Summary of type II and type III embryos examined for E18.5 lungs by104

immunohistochemistry, shown in Fig 6 and Fig S5.

105*Approximately 295- (#11_2) and 79- (#22_2) bp insertion were detected by microchip

106electrophoresis and the number of sequence reads in deep sequencing was corrected107

accordingly (see Materials and methods). N.D., not determined.

52

108
109

S4 Table. Primers used for quantitative PCR (qPCR) analysis.

110
111

Supplementary Figures

112

S1 Fig. Structure of the mouse Fgf10 gene indicating the target sites for

113

CRISPR/Cas9 system.

114
115

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

116

A-H, whole mount staining. Left (A, C, E, G) and right (B, D, F, H) lateral views are

117

shown. Arrows show truncated limb and girdle bones. I-K, wild type skeletal structures,118

showing scapula and forelimb (I), hindlimb (J), and pelvic girdle (K). au, autopod; f,119femur; fi, fibula; h, humerus; il, ilium; is, ischium; p, pubis; r, radius; sc, scapula; sp,120

spine; st, stylopod; t, tibia; u, ulna; ze, zeugopod. Scale bars: 1 mm.

121
122

S3 Fig. Mismatch cleavage assay.

53

123

A, PCR primers were designed in the upstream region of the exon 3 and in the exon 3,

124

giving rise to a PCR amplicon size of 501 bp. DNA fragments of 309 bp and 192 bp are

125

generated by the Resolvase when the Fgf10 genome has been cleaved by Cas9 and non-126

homologous end joining has been achieved. B, Electrophoresis of the enzyme-treated127mouse genomic DNA from the Fgf10-CRISPR F0 embryonic necks. The DNA ladder for

128

DNA size reference and a result of DNA from a wild type (WT) mouse are shown on the129

left. Three DNA fragments of approximately 500 bp (▼), 300 bp (▽ in gray), and 200

130

bp (▽) are seen in all the lanes except for the wild type and #4 lanes. In embryo #3, an

131

extra band for large insertion (328 base) is shown (see Fig 2). C, Genomic analysis of the132#4 embryo as revealed by Sanger sequencing. Deduced amino acid sequences are also

133

shown. Lys-196 and His-201 are highlighted in yellow and green, respectively. Altered134

amino acids are indicated in red. Asterisks indicate stop codons.

135
136

S4 Fig. Limb phenotypes and lung histology of all embryos examined at E16.5 as

137

summarized in Table 3.

138

Scale bars: 500 µm.

54

139
140

S5 Fig. Limb phenotypes, lung histology, and the number of SPC-positive cells per

141

total cell number of type II and type III embryos at E18.5.

142

Source data for (L, M) are available in S3 Table. Lateral views of type II (A-F), and type143

III (G, H) embryos at E18.5. Arrowheads show limb defects. (I-K) In all three type II

144embryos, the accessory lobe was lost. In embryo #24_2, (J), the middle lobe (Mi) was145

also undetectable. Data in (L) are presented as means ± SEM. (M) In these embryos, the146number of SPC-positive cells was more correlated to the percentage of wild type Fgf10

147

genotype (correlation coefficient [R] was 0.728 for neck DNA) than that including in-148

frame mutations with Lys196 and His201 retained (R=0.334). Scale bar: 2 mm (A-H).

149
150

S6 Fig. Quantitative PCR analysis for mRNA expression of mesenchymal and its

151

related marker genes in wild type and type III lungs at E18.5.

152
153

S7 Fig. Cecum and colons of the wild type and Fgf10-CRISPR F0 (type I and type154II) embryos at E18.5.

55

155

A-C, wild type (Wt) cecum (c), colon, and small intestine (si) are shown from three156

embryos examined. Ileum and colon were cut at dissection. D-G, type I cecum is reduced

157

compared with the wild type. Whether the cecum epithelium is absent or not cannot be

158

identified from these photos. Type I embryos show an atresia of the colon, but the length

159

varies depending upon the embryos. H-I, type II embryos examined (n = 2) do not exhibit

160

a reduced cecum or an atresia of the colon. The colons presented here were cut as distally161

as possible. J, the approximate length of the cecum. The length of type I cecum is162

significantly decreased compared with the wild type. The length of two type II embryos

163

examined is also shown for reference. Scale bar: 1 mm (in A for all to scale).