of 17
Sertoli
anti-FIS1 | Dn | DAPI
Dn | DAPI
anti-FIS1 | DAPI
0
2
4
6
8
1 0
1 2
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
Fluor. Intensity (au)
Distance (μm)
Sertoli
Germ cell
Sertoli
1 2
0
2
4
6
8
1 0
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
Fluor. Intensity (au)
Distance (μm)
Dn
anti-FIS1
Sertoli
Germ cell
anti-FIS1
| Dn | DAPI
Dn | DAPI
anti-FIS1 | DAPI
P56 S8/
Fis1
S8/Cntrl
Inset
Inset
A
C
B
Fis1
conditional
allele
1
43
2
5
loxP
loxP
TM
Fis1
excised
allele
Stra8-Cre
Recombination
1
43
5
Dn
anti-FIS1
c
Fis1
: 268 bp
ex
Fis1
: 278 bp
WT: 176 bp
Fis1
genomic
locus
1
43
2
5
TM
exFis1/cFis1
+/cFis1
+/cFis1
+/exFis1
+/exFis1
exFis1/cFis1
exFis1/cFis1
+/+
+/+
Development: doi:10.1242/dev.199686: Supplementary information
Development • Supplementary information
Fig. S1. Related to Fig. 1
(A) Schematic
of the conditional (floxed) and
excised
Fis1
alleles.
In the conditional
Fis1
allele,
loxP
sites flank exon
2. After
Cre
-mediated recombination, deletion
of exon 2 causes
a
frameshift in the
Fis1
open reading frame. Genotyping oligos are indicated.
(B) Genotyping
from
mouse tails. Genotyping oligos and
the predicted fragment sizes
are indicated. See also Fig.
S1A.
(C) Immunostaining
of testis sections with an antibody against FIS1. Note that Dendra2-
positive germ cell mitochondria in the
Fis1
mutants (right panel) are negative
for FIS1
immunostaining. Line
scans
of the indicated regions are shown at the bottom. The regions
corresponding to germ and Sertoli cell mitochondria are indicated. Scale bars, 20 μm.
Development: doi:10.1242/dev.199686: Supplementary information
Development • Supplementary information
Na/K-ATPase|DAPI
S8/Cntrl
S8/
Fis1
Na/K-ATPase
DAPI
Fig. S
2. Related to Fig. 2
Immunostaining germ cell boundarie
s us ing an antibody
against t he pl asma m embrane ma
rker
N
a/K-ATPase. The bottom panel (mutant)
shows that the nuc
lei wit hin GCs are not s
eparated
by
pl
asma membrane. Sca
le bar, 20 μ
m.
Development: doi:10.1242/dev.199686: Supplementary information
Development • Supplementary information
γ
H2AX |TUNEL|DAPI
DAPI
Inset
XY
ST
XY
ST
GC
S8/Cntrl
S8/
Fis1
Fig. S3
. Related to Fig. 3
A
nalysis of apoptosis in
Fis1
GCs. TUNEL a ssay was perform
ed in P35 testis se ctions c o-stained
with an antibody
against γ H2AX a
nd DAPI. Note tha t t he aberra
nt
Fis1
GC is not T
UNEL-
positive. ST, spermatid; GC, giant cell. Scale bar, 20 μm.
Development: doi:10.1242/dev.199686: Supplementary information
Development • Supplementary information
Rosa26
COXIV-mCh-EGFP
Stop
Stra8
-Cre
Recombination
Rosa26
COXIV-mCh-EGFP
loxP
loxP
Rosa26
mChEG
conditional
allele
mChEG
excised
allele
Rosa26
genomic
locus
+/+
mChEG/+
H20
mChEG: 358 bp
WT: 500 bp
B
A
SG
SC
SG
SC
S8/Cntrl
ST
PEX14 | DAPI
S8/
Fis1
SC
SG
C
Inset
Respiratory Chain CII
GC
GC
P56 S8/Cntrl
P56
S8/
Fis1
Respiratory Chain CIV
Inset
GC
GC
GC
E
Fluorescence intensity (au)
Distance (
μ
m)
Merge
SP10 | Dn
SP10 | MTR
S8/
Fis1
S8/Cntrl
Line Scan
0.0
0.2
0.4
0.6
0.8
1.0
0 1 2 3
4
0.0
0.2
0.4
0.6
0.8
1.0
0 1 2 3 4
Dn
MTR
G
Distance (
μ
m)
Fluor. intensity (au)
Merge
Dn | DAPI
MTR | DAPI
S8/
Fis1
Line Scan
0 1 2 3
0.0
0.2
0.4
0.6
0.8
1.0
4
F
Dn
MTR
D
P56 S8/Cntrl
P56
S8/
Fis1
GC
Acr
Step 9
Acr
Steps 5-8
Pre-spermatid
Development: doi:10.1242/dev.199686: Supplementary information
Development • Supplementary information
Fig. S4. Related to Fig. 5
(A) Schem
atic show
ing the ge neration of germ c
ell-specific mitophagy
reporter
mice. Genotyping
ol
igos are i ndicated.
(B) G
enotyping from mouse ta
ils. Genotyping oligos a nd predicte
d band sizes a re i ndicated. See
also Fig. S5A.
(C) CO
X (Com
plex IV) enzyme histoche
mistry in P56 testis se ctions.
(D) SDH (Com
plex II) enz
yme histochemistry in P56 testis sections.
(E) MitoTra
cker Red Staining in dissoc
iated spermatids. SP10 marks the acrosome
, which is
uni
que to spermatids. Scale bar, 5 μm
(F) MitoTracker Red Staining in pre-spermatid S8/
Fis1
germ cells. Scale bar, 5 μm
(G) Immunostaining of testis sections with the
PEX14 peroxisome marker. SC, spermatocyte; SG,
spermatogonia. Scale bar, 20 μm.
Development: doi:10.1242/dev.199686: Supplementary information
Development • Supplementary information
S8/
Fis1
Tomogram
Tomogram
S8/
Fis1
S8/
Fis1
ST nuc
ST nuc
AS
AS
AS
AS
AS
AS
B
A
PAG
AS
Tomogram
S8/
Fis1
Development: doi:10.1242/dev.199686: Supplementary information
Development • Supplementary information