Development 142: doi:10.1242/dev.121939: Supplementary Material
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Development 142: doi:10.1242/dev.121939: Supplementary Material
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Development 142: doi:10.1242/dev.121939: Supplementary Material
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Table S1A,B: Statistics determined for total area of hypoxic regions in normal and Hif1a
conditional knock-out (CKO) forebrain after different oxygen exposures (Figure 1D). Twoway ANOVA with post-hoc t-test and Bonferroni adjustment with atmospheric oxygen
concentrations and Hif1a genotype as fixed factors revealed that atmospheric oxygen
concentration and Hif1a genotype have a significant interaction effect on hypoxic area size
(p=0.014, F-value=5.4) and significant differences among atmospheric oxygen concentrations
(p<0.001, F-value=212.8) and Hif1a genotypes (p<0.001, F-value=1599.2). Displayed are
Bonferroni-adjusted p-values. (A) Significances among the different genotypes. (B)
Significances among the different atmospheric oxygen concentrations. Bold values indicate
significant differences.
A
normal vs. Hif1a CKO
10% O2
21% O2
75% O2
< 0.001
< 0.001
< 0.001
B
normal
Hif1a CKO
10% O2 vs. 21% O2
< 0.001
< 0.001
10% O2 vs. 75% O2
< 0.001
< 0.001
21% O2 vs. 75% O2
0.018
0.071
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Table S2A,B: Statistics determined for vessel density in normal and Hif1a conditional knockout (CKO) forebrain after different oxygen exposures (Figure 1E). Two-way ANOVA with
post-hoc t-test and Bonferroni adjustment with atmospheric oxygen concentrations and Hif1a
genotype as fixed factors revealed that atmospheric oxygen concentration and Hif1a genotype
have a significant interaction effect on vessel density (p=0.04, F-value=3.9) and significant
differences among atmospheric oxygen concentrations (p<0.001, F-value=24.5) and Hif1a
genotypes (p<0.001, F-value=1092.9). Displayed are the Bonferroni-adjusted p-values. (A)
Significances among the different genotypes. (B) Significances among the different
atmospheric oxygen concentrations. Bold values indicate significant differences.
A
normal vs. Hif1a CKO
10% O2
21% O2
75% O2
< 0.001
< 0.001
< 0.001
B
normal
Hif1a CKO
10% O2 vs. 21% O2
0.035
< 0.001
10% O2 vs. 75% O2
0.005
0.002
21% O2 vs. 75% O2
1.000
0.102
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Table S3A,B: Statistics determined for brain volume of normal and Hif1a conditional knockout (CKO) forebrain after different oxygen exposures (Figure 1G). Two-way ANOVA with
post-hoc t-test and Bonferroni adjustment with atmospheric oxygen concentrations and Hif1a
genotype as fixed factors revealed that atmospheric oxygen concentration and Hif1a genotype
have no significant interaction effect on brain volume (p=0.75, F-value=0.3) and significant
differences among atmospheric oxygen concentrations (p<0.001, F-value=72.2) and Hif1a
genotypes (p=0.003, F-value=10.7). Displayed are the Bonferroni-adjusted p-values. (A)
Significances among the different genotypes. (B) Significances among the different
atmospheric oxygen concentrations. Bold values indicate significant differences.
A
normal vs. Hif1a CKO
10% O2
21% O2
75% O2
0.07
0.187
0.023
B
normal
Hif1a CKO
10% O2 vs. 21% O2
< 0.001
< 0.001
10% O2 vs. 75% O2
< 0.001
< 0.001
21% O2 vs. 75% O2
0.028
0.297
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Table S4: Statistics determined for the amount of Sox2+ cells in the VZ of normal and Hif1a
conditional knock-out (CKO) forebrain after different oxygen exposures (Figure 2B). Twoway ANOVA with atmospheric oxygen concentrations and Hif1a genotype as fixed factors
revealed that atmospheric oxygen concentration and Hif1a genotype have no significant
interaction effects on the amount of Sox2+ cells in the VZ (p=0.863, F-value=0.1) and no
significant differences among atmospheric oxygen concentrations (p=0.06, F-value=3.7), and
Hif1a genotypes (p=0.375, F-value=0.8). (A) Significances among the different genotypes. (B)
Significances among the different atmospheric oxygen concentrations. Bold values indicate
significant differences.
A
normal vs. Hif1a CKO
10% O2
21% O2
75% O2
0.804
0.349
0.718
B
normal
Hif1a CKO
10% O2 vs. 21% O2
1.000
0.689
10% O2 vs. 75% O2
0.916
1.000
21% O2 vs. 75% O2
0.396
0.139
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Table S5A,B: Statistics determined for the amount of Tbr2+ cells in the SVZ of normal and
Hif1a conditional knock-out (CKO) forebrain after different oxygen exposures (Figure 2C).
Two-way ANOVA with post-hoc t-test and Bonferroni adjustment with atmospheric oxygen
concentrations and Hif1a genotype as fixed factors revealed that atmospheric oxygen
concentration and Hif1a genotype have no significant interaction effect on amount of Tbr2+
cells in the SVZ (p=0.24, F-value=1.6) and significant differences among atmospheric oxygen
concentrations (p<0.001, F-value=31.0) and no significant differences among Hif1a
genotypes (p=0.774, F-value=0.1). Displayed are the Bonferroni-adjusted p-values. (A)
Significances among the different genotypes. (B) Significances among the different
atmospheric oxygen concentrations. Bold values indicate significant differences.
A
normal vs. Hif1a CKO
10% O2
21% O2
75% O2
0.114
0.375
0.897
B
normal
Hif1a CKO
10% O2 vs. 21% O2
0.130
1.000
10% O2 vs. 75% O2
< 0.001
0.003
21% O2 vs. 75% O2
0.007
0.002
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Table S6A,B: Statistics determined for the amount of Sox2+ cells in regions basal of the VZ
(SVZ/IZ) of normal and Hif1a conditional knock-out (CKO) forebrain after different oxygen
exposures (Figure 2D). Two-way ANOVA with post-hoc t-test and Bonferroni adjustment
with atmospheric oxygen concentrations and Hif1a genotype as fixed factors revealed that
atmospheric oxygen concentration and Hif1a genotype have no significant interaction effect
on amount of Sox2+ cells in regions basal of the VZ (p=0.118, F-value=2.6) and significant
differences among atmospheric oxygen concentrations (p<0.001, F-value=16.5) and Hif1a
genotypes (p<0.001, F-value=37.1). Displayed are the Bonferroni-adjusted p-values. (A)
Significances among the different genotypes. (B) Significances among the different
atmospheric oxygen concentrations. Bold values indicate significant differences.
A
normal vs. Hif1a CKO
10% O2
21% O2
75% O2
0.002
1.07
< 0.001
B
normal
Hif1a CKO
10% O2 vs. 21% O2
1.000
0.49
10% O2 vs. 75% O2
0.004
0.021
21% O2 vs. 75% O2
0.001
0.31
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Table S7A,B: Statistics determined for the amount of Sox2+/Tbr2+ cells in the IZ of normal
and Hif1a conditional knock-out (CKO) forebrain after different oxygen exposures (Figure
2E). Two-way ANOVA with post-hoc t-test and Bonferroni adjustment with atmospheric
oxygen concentrations and Hif1a genotype as fixed factors revealed that atmospheric oxygen
concentration and Hif1a genotype have significant interaction effect on amount of
Sox2+/Tbr2+ cells in the IZ (p=0.002, F-value=10.4) and significant differences among
atmospheric oxygen concentrations (p<0.001, F-value=49.9) and Hif1a genotypes (p<0.001,
F-value=45.3). Displayed are the Bonferroni-adjusted P-values. (A) Significances among the
different genotypes. (B) Significances among the different atmospheric oxygen concentrations.
Bold values indicate significant differences.
A
normal vs. Hif1a CKO
10% O2
21% O2
75% O2
< 0.001
0.587
0.001
B
normal
Hif1a CKO
10% O2 vs. 21% O2
1.000
< 0.001
10% O2 vs. 75% O2
< 0.001
< 0.001
21% O2 vs. 75% O2
0.001
0.716
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Table S8: Statistics determined for the amount of mitotic cells in VZ of normal and Hif1a
conditional knock-out (CKO) forebrain after different oxygen exposures (Figure 4B). Twoway ANOVA with atmospheric oxygen concentrations and Hif1a genotype as fixed factors
revealed that atmospheric oxygen concentration and Hif1a genotype have no significant
interaction effects on the amount of apical mitotic cells (p=0.723, F-value=0.3) and no
significant differences among atmospheric oxygen concentrations (p=0.184, F-value=1.8),
and Hif1a genotypes (p=0.725, F-value=0.4). (A) Significances among the different
genotypes. (B) Significances among the different atmospheric oxygen concentrations. Bold
values indicate significant differences.
A
normal vs. Hif1a CKO
10% O2
21% O2
75% O2
0.264
0.899
0.850
B
normal
Hif1a CKO
10% O2 vs. 21% O2
1.000
0.238
10% O2 vs. 75% O2
1.000
0.585
21% O2 vs. 75% O2
1.000
1.000
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Table S9A,B: Statistics determined for the amount of mitotic cells in SVZ of normal and
Hif1a conditional knock-out (CKO) forebrain after different oxygen exposures (Figure 4C).
Two-way ANOVA with post-hoc t-test and Bonferroni adjustment with atmospheric oxygen
concentrations and Hif1a genotype as fixed factors revealed that atmospheric oxygen
concentration and Hif1a genotype have significant interaction effect on the amount of mitotic
cells in SVZ (p=0.001, F-value=9.7) and significant differences among atmospheric oxygen
concentrations (p<0.001, F-value=41.9) and Hif1a genotypes (p=0.013, F-value=7.0).
Displayed are the Bonferroni-adjusted P-values. (A) Significances among the different
genotypes. (B) Significances among the different atmospheric oxygen concentrations. Bold
values indicate significant differences.
A
normal vs. Hif1a CKO
10% O2
21% O2
75% O2
0.160
0.160
< 0.001
B
normal
Hif1a CKO
10% O2 vs. 21% O2
< 0.001
0.001
10% O2 vs. 75% O2
0.005
< 0.001
21% O2 vs. 75% O2
0.005
0.058
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Table S10A,B: Statistics determined for the amount of mitotic cells in IZ of normal and
Hif1a conditional knock-out (CKO) forebrain after different oxygen exposures (Figure 4D).
Two-way ANOVA with post-hoc t-test and Bonferroni adjustment with atmospheric oxygen
concentrations and Hif1a genotype as fixed factors revealed that atmospheric oxygen
concentration and Hif1a genotype have significant interaction effect on the amount of mitotic
cells in IZ (p<0.001, F-value=20.0) and significant differences among atmospheric oxygen
concentrations (p<0.001, F-value=26.1) and Hif1a genotypes (p<0.001, F-value=37.5).
Displayed are the Bonferroni-adjusted P-values. (A) Significances among the different
genotypes. (B) Significances among the different atmospheric oxygen concentrations. Bold
values indicate significant differences.
A
normal vs. Hif1a CKO
10% O2
21% O2
75% O2
0.303
0.407
< 0.001
B
normal
Hif1a CKO
10% O2 vs. 21% O2
0.144
0.094
10% O2 vs. 75% O2
< 0.001
0.658
21% O2 vs. 75% O2
< 0.001
0.909
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Table S11A,B: Statistics determined for the amount of TUNEL+ cells in the VZ/SVZ/IZ of
normal and Hif1a conditional knock-out (CKO) forebrain after different oxygen exposures
(Supplementary Figure S2B). Two-way ANOVA with post-hoc t-test and Bonferroni
adjustment with atmospheric oxygen concentrations and Hif1a genotype as fixed factors
revealed that atmospheric oxygen concentration and Hif1a genotype have significant
interaction effect on amount of TUNEL+ cells in the VZ/SVZ/IZ (p=0.156, F-value=2.1) and
significant differences among atmospheric oxygen concentrations (p<0.001, F-value=345.3)
and Hif1a genotypes (p=0.073, F-value=3.6). Displayed are the Bonferroni-adjusted p-values.
(A) Significances among the different genotypes. (B) Significances among the different
atmospheric oxygen concentrations. Bold values indicate significant differences.
A
normal vs. Hif1a CKO
10% O2
21% O2
75% O2
0.063
0.587
0.077
B
normal
Hif1a CKO
10% O2 vs. 21% O2
< 0.001
< 0.001
10% O2 vs. 75% O2
< 0.001
< 0.001
21% O2 vs. 75% O2
0.517
0.97
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Table S12A,B: Statistics determined for the amount of intermediate cells in the SVZ of
normal and Hif1a conditional knock-out (CKO) forebrain at E12 after different oxygen
exposures (Supplementary Figure S4C). Two-way ANOVA with atmospheric oxygen
concentrations and Hif1a genotype as fixed factors revealed that atmospheric oxygen
concentration and Hif1a genotype have no significant interaction effect on the amount of
intermediate cells in the SVZ at E12 (p=0.234, F-value=1.6) and no significant differences
among atmospheric oxygen concentrations (p=0.096, F-value=3.6) and Hif1a genotypes
(p=0.183, F-value=2.1). (A) Significances among the different genotypes. (B) Significances
among the different atmospheric oxygen concentrations. Bold values indicate significant
differences.
A
normal vs. Hif1a CKO
21% O2
75% O2
0.906
0.088
B
21% O2 vs. 75% O2
normal
Hif1a CKO
0.682
0.06
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Table S13A,B: Statistics determined for the amount of mitotic intermediate cells in the SVZ
of normal and Hif1a conditional knock-out (CKO) forebrain at E12 after different oxygen
exposures (Supplementary Figure S4D). Two-way ANOVA with atmospheric oxygen
concentrations and Hif1a genotype as fixed factors revealed that atmospheric oxygen
concentration and Hif1a genotype have no significant interaction effect on the amount of
mitotic intermediate cells in the SVZ (p=0.633, F-value=0.24) and no significant differences
among atmospheric oxygen concentrations (p=0.633, F-value=0.24) and Hif1a genotypes
(p=0.074, F-value=3.8). (A) Significances among the different genotypes. (B) Significances
among the different atmospheric oxygen concentrations. Bold values indicate significant
differences.
A
normal vs. Hif1a CKO
21% O2
75% O2
0.319
0.109
B
21% O2 vs. 75% O2
normal
Hif1a CKO
1.000
0.502
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Supplementary Materials and Methods
Animals and genotyping
For experiments, we crossed Hif1aflox/flox mice with Hif1aflox/flox Nestin-Cre+/- conditional
knockout mice. The embryos were distinguished by genotyping for the corresponding allele
using PCR (Fig. 1B). For identification of the mutation, genomic DNA from the tails of E16
embryos was isolated according to the manual (DNeasy Blood & Tissue Kit, Qiagen). DNA
was then used for genotyping using standard PCR as described (Tomita et al., 2003). Hif1a
αflox/flox mice were a kind gift from Shuhei Tomita, MD, PhD. All adult mice types showed no
identifiable phenotypes and had a normal lifespan.
Oxygen treatment
Animal cages were placed into an oxygen chamber (InerTec AG) and oxygen and carbon
dioxide concentrations were measured and controlled using integrated probes in the same
chamber. Both oxygen concentrations were non-lethal for the animals and no behavioural
abnormalities were observed during the intervention. To assess the tissue oxygen tension
within the prenatal brain, we injected the chemical reagent pimonidazole hydrochloride
(hypoxyprobe, hpi) 1h before sacrifice (Fig. 1A). Pimonidiazole is reductively activated in
hypoxic cells with an oxygen level of less than 1.1% O2 and form a stable adduct which is
detectable by a specific antibody (Raleigh et al., 1987). To screen the oxygen supply to the
brains of the offspring, pregnant mice were intravenously injected with hypoxyprobe at 60
mg/kg body weight. After 1 hour incubation, embryonic brains were dissected, fixed
overnight in 4% paraformaldehyde (PFA) in phosphate buffered salin (PBS) and then
appropriate placed in 30% sucrose/1x PBS mix for cryoprotection. Brains were snap-frozen
and sectioned coronally at 20 µm (Leica) and collected on Superfrost Ultra Plus coated glass
slides (Menzel) for immunhistochemical analysis.
To investigate the role of brain tissue oxygen tension on earlier stages of cortical
neurogenesis (SVZ development), brains of normal and Hif1a knockout litters were examined
after exposing pregnant mice to normoxic (21%) or hyperoxic (75%) conditions from E10 to
E12 similar as described above.
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Birth-dating study and immunohistochemistry
For BrdU immunostaining, sections were washed in TBS, permeabilized in 1.5 N HCl at 37°C
for 30 min, washed 3 times with TBS, blocked in 0.2% TritonX-100 (ThermoSCIENTIFIC)
containing 8% donkey serum (Jackson ImmunoResearch) (TBS+), then incubated in rat antiBrdU (1:200, Abcam, ab6326) overnight at 4°C in 3% donkey serum (TBS+). For
immunohistochemistry, staining was performed on 20 µm thick sections. After antigen
retrieval using heating procedure in 0,01M sodium citrate at PH6.0 (Dako REAL) in a
standard microwave, sections were washed in TBS and blocked for 30 minutes at room
temperature. Then they were incubated overnight with primary antibodies at 4°C. The
following primary antibodies were used in this study: rabbit anti-NeuN (Rbfox3) (1:800,
Millipore, ABN78), rabbit anti-Tbr1 (1:100, Millipore, AB10554), rat anti-Ctip2 (1:250,
Abcam, ab18465), goat anti-Sox2 (1:200, Santa Cruz, sc-17319), mouse anti-phospho-H3
(1:150, Cell Signaling TECHNOLOGY, 9706S) and rabbit anti-Tbr2 (1:100, Abcam,
ab23345), p-Vimentin (1:500, Abcam, ab22651), Cux1 (Bcl11b) (1:350, Santa Cruz, sc13024). Afterwards sections were washed, blocked and incubated for 1 h in secondary
antibodies blocking solution at room temperature. Secondary antibodies were Alexa488conjugated donkey anti-rat IgG, Alexa488-conjugated donkey anti-rabbit IgG, Alexa555conjugated donkey anti-rabbit IgG, Alexa555-conjugated donkey anti-goat IgG, Alexa647conjugated donkey anti-mouse IgG, Alexa647-conjugated donkey anti-rat IgG (1:500, all
from Molecular Probes). Sections were then washed again and mounted with Fluoromount G
mounting medium (BIOZOL). Nuclei were stained using bisbenzimide H33258 fluorochrome
trihydrochloride (Hoechst; Invitrogen, H3570).
To identify fragmented DNA of apoptotic cells, embryonic frozen forebrain sections
were labelled using terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick
end labeling (TUNEL) assay (Roche), for 1h at 37°C. TUNEL positive cells were detected by
fluorescence (FITC, 520 nm) and imaged using Spinning disc microscope and ZEN blue
software (Carl Zeiss Microscopy).
For Hypoxyprobe/von Willebrand factor (vWF) immunofluorescence, unspecific
binding sites were blocked by 30 min incubation with TBS+. Sections were subsequently
incubated for 2 hours with Hypoxyprobe-FITC (1:150, hpi) and von Willebrand factor (1:100,
Chemicon, AB7356) from rabbit in TBS+. Afterwards, 90 min incubation with the secondary
antibody Alexa Fluor anti-rabbit 555 (Molecular probes, A31572) diluted 1:500 in TBS+ was
performed. Again, cell nuclei were stained with Hoechst staining (Invitrogen, H3570) and
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sections were mounted with Fluoromount mounting medium (BIOZOL) and stored at 4o C
before imaging. For all stainings, a minimum of four control and mutant samples were
analysed for each marker.
Image Acquisition and quantitative measurements
NeuN immunostained images taken with a 20× objective were used for the calculation of the
radial thickness of the cortical plate. To that end, the distance from the subplate/ cortical plate
boundary to pia were measured at approximately 5 positions on the medial sections of the
embryonic telencephalon (100 µm intervals), beginning at the rostral side of the dorsal lateral
ventricles to the caudal side. The percentage of the several cortical layers was determined in a
250 µm-wide columns using specific markers.
Mitotic (pH3+) cells were separately counted at the ventricular surface (apical
progenitors), in the SVZ and OSVZ at 20× magnification. TUNEL+ cells were separately
counted in the neurogenic regions and cortical plate in both Hif1α genotypes in the various
oxygen conditions. Quantification of BrdU+/NeuN+ cells within the cortical plate was
performed on 250 µm wide-fields. Coronal forebrain sections were immunolabeled using
antibodies to BrdU and NeuN. The numbers of BrdU+/Ctip2+ and BrdU+/Ctip2- cells were
determined within the subplate and deeper layers of the cortical plate and BrdU+/Cux1+ cells
in the upper layer on 250 µm wide-columns of the cortical plate. Multiple z-stack images
were acquired from the dorsal telencephalon and analysed using Fiji software (NIH). Cell
counts occurred from three to eight age-matched embryos per oxygen condition. All double
stainings were confirmed by parallel viewing of the two different optic channels and/or using
the orthogonal view plug-in using the Fiji software (NIH).
For analysis of the various progenitor populations, Sox2 and Tbr2 expressing cells in
the VZ and SVZ and more basal of these zones were counted in 250 µm wide-columns. The
quantification of Tbr2+ intermediate progenitor cells and their mitosis in the E12 forebrain
was performed on 200-μm-wide fields. Tbr2/pH3 double-immunofluorescence images were
used to determine the number of mitotic cells in the embryonic telencephalon located at the
basal side of the ventricle.
Vessel density per area was quantified from E12, E14 and E16 embryonic medial
telencephalon sections stained with the vessel marker von Willebrand factor (vWF). For each
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image, the percentage of vWF stained blood vessels was counted and offset to the estimated
area (excluding the ventricular volume) using Fiji software (NIH). For hypoxic tissue
measurement, the area of positive pixels for pimonidazole was quantified using the Fiji-plugin “3D cell counter” and defined threshold criteria.
The whole brain volume of E12, E14 and E16 brains was analysed using a computer
coupled to a Zeiss Axioplan 2 with StereoInvestigator software (NIH). In this study we used
one series of sections and stained with hematoxylin/eosin. For the measurement of the brain
size, every twelfth section was taken, starting from the rostral part of the telencephalon
throughout the beginning of the hindbrain. The outline of every brain section was carried out
at 5× magnification. The volume was estimated by the product of the total volume of all tissue
slice profiles and section thickness.
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Supplementary References
Raleigh, J. A., Miller, G. G., Franko, A. J., Koch, C. J., Fuciarelli, A. F. and Kelly, D. A.
(1987). Fluorescence immunohistochemical detection of hypoxic cells in spheroids
and tumours. British journal of cancer 56, 395-400.
Tomita, S., Ueno, M., Sakamoto, M., Kitahama, Y., Ueki, M., Maekawa, N., Sakamoto,
H., Gassmann, M., Kageyama, R., Ueda, N., et al. (2003). Defective brain
development in mice lacking the Hif-1alpha gene in neural cells. Molecular and
cellular biology 23, 6739-6749.
Development | Supplementary Material