development | supplementary...

Development 142: doi:10.1242/dev.121939: Supplementary Material

Development | Supplementary Material


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). 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 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

10% O2 21% O2 75% O2

normal vs. Hif1a CKO < 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



Table S2A,B: Statistics determined for vessel density in normal and Hif1a conditional knock-

out (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

10% O2 21% O2 75% O2

normal vs. Hif1a CKO < 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



Table S3A,B: Statistics determined for brain volume of normal and Hif1a conditional knock-

out (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


genotypes (p=0.003, F-value=10.7). Displayed are the Bonferroni-adjusted p-values. (A)



A

10% O2 21% O2 75% O2

normal vs. Hif1a CKO 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



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). 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 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

10% O2 21% O2 75% O2


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



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


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)



A

10% O2 21% O2 75% O2


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



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


genotypes (p<0.001, F-value=37.1). Displayed are the Bonferroni-adjusted p-values. (A)



A

10% O2 21% O2 75% O2

normal vs. Hif1a CKO 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



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

10% O2 21% O2 75% O2

normal vs. Hif1a CKO < 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



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). 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 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

10% O2 21% O2 75% O2


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



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).



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



A

10% O2 21% O2 75% O2


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



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).



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



A

10% O2 21% O2 75% O2


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



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


A

10% O2 21% O2 75% O2


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



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


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

21% O2 75% O2

normal vs. Hif1a CKO 0.906 0.088

B

normal Hif1a CKO

21% O2 vs. 75% O2 0.682 0.06



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


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

21% O2 75% O2

normal vs. Hif1a CKO 0.319 0.109

B

normal Hif1a CKO

21% O2 vs. 75% O2 1.000 0.502



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.



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 anti-

BrdU (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, sc-

13024). Afterwards sections were washed, blocked and incubated for 1 h in secondary

antibodies blocking solution at room temperature. Secondary antibodies were Alexa488-

conjugated donkey anti-rat IgG, Alexa488-conjugated donkey anti-rabbit IgG, Alexa555-

conjugated donkey anti-rabbit IgG, Alexa555-conjugated donkey anti-goat IgG, Alexa647-

conjugated 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



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



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-plug-

in “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.



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.


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