of 3
SI Appendix
Materials and Methods
Animals
The Iberian ribbed newts (
P
leurodeles waltl
) used in this study were obtained from a breeding colony
(1)
at Tottori University
, Hiroshima University, and National Institute
for
Basic Biology.
For anesthesia
before limb amputation and samplings, MS
-
222 (Sigma, St. Louis, MO, USA) was used at a final
concentration
of 0.02%. The developmental stages were defined according to previous methods
(2)
. The
forelimb was amputated in the middle of the zeugopod at 1
-
month post
-
fertilization (mpf). The hindlimb
was amputated at 2
-
2.5 mpf in the middle of the zeugopod or stylopod if its zeugopod
was
severely
truncated. Blastemas were collected at 10 days post
-
amputation (dpa) and allowed to regenerate for 1
-
2
month
s
before observation. Animal rearing and treatments were performed and approved in accordance
with the Guidelines for the Use and Care of E
xperimental Animals and the Institutional Animal Care and
Use Committee of
Hiroshima University,
National Institute for Basic Biology
,
and California Institute
of
Technology.
I
n situ
hybridization chain reaction (HCR)
Li
m
b
b
ud
s
(st. 3
4
for forelimb, st.
40
for hindlimb) and
15
dpa
blastema
s
(
late bud stage
,
(3)
)
were
collected under anesthesia
and then
tissue
s
were
embedded in low melt agarose and/or Tissue
-
Tek OCT
compound (SAKURA, Nagano, Japan) and immediately frozen in liquid nitrogen. Tissues were sectioned
at 14μm. HCR probes (
P63
,
Fgf8
,
and
Fgf10
) were
purchased
from Molecular Technologies and
Molecular Instruments (Los Angeles, CA, USA).
HCR was performed following the manufacturer's
instruction
s
with minor modifications.
RNA sequencing and differential gene expression analysis
The fore
-
and hindlimb buds were collected at st.
33
-
34 and st.
39
-
40 and pooled separately
(n=3
biological replicates)
. For differential gene expression analysis in limb development, the hindlimb bud of
wild type or
Fgf10
mutants were collected individually (n=6, each) at st. 39
-
40. For limb regeneration,
late bud stage blastemas (10 dpa) were collected individually (n=3 for wild type, n=6 for
Fgf10
mutant).
Each limb bud
or blastema sample was soaked in RNAlater
(Invitrogen, Waltham, MA, USA) at 4
o
C
overnight, and stored at
-
80
o
C until extraction. Total RNA was extracted and purified using NucleoSpin
(Takara Bio, Shiga, Japan), following the manufacturer's instructions.
Paired
-
end sequencing of each 150
-
bp end of cDNA libraries was performed with the Hiseq2000 sequencing system (Illumina, San Diego,
CA, USA).
The RNA
-
Seq of normal fore
-
and hind
-
limb bud was conducted by Macrogen Japan (Kyoto,
Japan) and the RNA
-
Seq libraries of blastemas in wild type and
Fgf10
mutants were generated using the
Illumina stranded mRNA Prep kit (San Diego, CA, USA) and sequenced in GENEWIZ (South Plainfield,
NJ, USA).
The
reads were mapped to the reference transcript database (PLEWA04_ORF.cds.fa
(4)
) using
Salmon
(5)
and differential expression analysis was performed by edgeR GLM
(6)
. The volcano plot and
box plot were generated by ggVolcanoR
(7)
and BoxPlotR
(8)
. The data have been deposited with links to
BioProject accession number PRJDB15083 in the NCBI BioProject database
(https://www.ncbi.nlm.nih.gov/bioproject/).
sgRNA design and Cas9 injection
Pw
Fgf10
sequence was obtained from a previous report (M0204228_PLEWA04
(4)
) and compared with
other species to define exon
-
intron boundary. Three sgRNAs (
gRNA1;
CATACGCATCCTACAAATGGAGG, gRNA2; GTTTGTGGCCTTAAATGGGAAGG, gRNA3;
GGGAAGGGAAAACCCAAGCGAGG
) were designed at exon 3 in the same location as used for the
Fgf10
crispant mouse
(9, 10)
, and one sgRNA was designed at exon 1 downstream of the ATG site
(gRNA4; CCGCCTTGTCCCAGCTGCCCTGC)
.
In vitro
transcription of sgRNA and Cas9
ribonucleocomplex injection
into one
-
cell
-
stage embryos
was performed as previously described
(11)
.
Genotyping
For
Fgf10
crispant mutation analysis, both fore
-
and hindlimbs were collected individually for amplicon
sequencing analysis as previously described
(12)
. For the
Fgf10
F2 mutant, genomic DNA was extracted
from the tail tip or limb using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany), and sequenced
by the Sanger sequencing.
MicroCT
i
maging
MicroCT image of
Fgf10
F1 and F2 mutant
limb
(1
-
year
-
old adult) was obtained using R_mCT2 (Rigaku,
Tokyo, Japan).
SI
Reference
s
1.
T. Hayashi
et al.
, Molecular genetic system for regenerative studies using newts.
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55
, 229
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236 (2013).
2.
D. L. Shi, J. C. Boucaut, The chronological development of the urodele amphibian Pleurodeles waltl (Michah).
Int J Dev Biol
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3.
L. E. Iten, S. V. Bryant, Forelimb regeneration from different levels of amputation in the newt,Notophthalmus
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M. Matsunami
et al.
, A comprehensive reference transcriptome resource for the Iberian ribbed newt
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