ESM Methods Animal procedures. Tail-vein glycaemia was measured following a 2h fast. Glucose

tolerance was evaluated by intraperitoneal glucose tolerance test (IPGTT), performed in

overnight fasted mice by injection of glucose (2 g/kg body weight). The trapezoidal

method was used to calculate the area under the curve (AUC) for IPGTT. For organ

sampling, mice were euthanized using 40 µl/g body weight of 10 mg/ml ketamine (Ceva,

Brussels, Belgium) + 0.1% (v/v) xylazine (Bayer, Leverkusen, Germany) diluted in 0.9%

NaCl. Blood cells were flushed from the circulation of anesthetized mice by systemic perfusion with PBS.

Protein analysis. For quantification of beta cell proliferation by Ki67 immune reactivity,

at least 3,000 beta cells were counted per sample from at least 3 representative tissue

sections. Mean beta cell size was determined from the mean cross-sectional area per

beta cell. The latter was determined by dividing the insulin positive area for each islet by

the number of nuclei it contained. For quantification, a mean of 4,624 beta cells were

counted per biological sample. Mouse islets were isolated as previously described in

Coppens et al. (Diabetologia, 2013), for immunoblotting 25µg total protein was loaded

per lane, resolved by SDS-PAGE, transferred to PVDF membrane, blocked with 5%

solution of nonfat powdered milk in Tris-buffered saline and incubated with primary

antibody (ESM Table 2) followed by incubation with HRP-conjugated secondary antibody and visualized by enhanced chemiluminescence.

ESM Table 1. PCR genotyping primer pairs. Target Sequence (5’-3’) Orientation RIPrtTA TAGATGTGCTTTACTAAGTCATCGCG forward

RIPrtTA GAGATCGAGCAGGCCCTCGATGGTAG reverse

tetO-sFLT1 CGACTCACTATAGGGAGACCC forward

tetO-sFLT1 TGGCCTGCTTGCATGATGTGCTGG reverse

hGH CCTAGCTGCAATGGCTACAG forward

hGH GCACTGGAGTGGCAACTTCC reverse

ESM Table 2. Primary antibodies and antigen retrieval.

Antibody Host Source (Cat. #)

Dilution Antigen Retrieval

Ki67 Rat (IgG2a) eBioscience

(14-5698)

IHC 1:1000 HIER, 10mM citrate pH6

Insulin Guinea pig DRC, VUB

(in-house)

IHC 1:5000

/

Collagen-IV Rabbit Millipore

(AB756)

IHC 1:200 HIER, 10mM citrate pH6

FLT1 Rabbit Santa Cruz

(sc-9029)

IHC 1:50

WB 1:200

/

CD31 Rat (IgG2a) BD

(550274)

IHC 1:500 PIER, Proteinase K, 6min

+ TSA amplification

ESM Table 3. Details of the two-way ANOVA of glucose clearance, beta cell proliferation, total beta cell volume and individual beta cell size. AUC IPGTT 13.5 dpc

(mmol/l x min) Ki67+/INS+ 14.5 dpc

(%) Beta cell volume 14.5 dpc

(µl) Beta cell size 14.5 dpc

(µm²) Group

dTg NP-Dox 1428 ± 89.9 [n=11] 0.44 ± 0.11 [n=6] 0.52 ± 0.034 [n=6] 160.0 ± 5.1 [n=3]

dTg NP+Dox 1916 ± 98.1 [n=18] 0.42 ± 0.30 [n=6] 0.65 ± 0.048 [n=6] 170.0 ± 7.6 [n=3]

dTg P-Dox 1833 ± 211.3 [n=6] 3.27 ± 0.31 [n=6] 0.94 ± 0.13 [n=7] 184 ± 5.43 [n=6]

dTg P+Dox 1701 ± 142.2 [n=10] 2.91 ± 0.35 [n=5] 0.95 ± 0.09 [n=7] 189.5 ± 9.0 [n=6]

Two-way ANOVA

Interaction F (1, 41) = 5.46, * F(1,19) = 0.51 F(1,22) = 0.55 F(1,14) = 0.09

Dox-administration F (1, 41) = 0.51 F(1,19) = 0.64 F(1,22) = 0.82 F(1,14) = 0.84

Pregnancy F (1, 41) = 1.81 F(1,19) = 125.3,*** F(1,22) = 21.08, *** F(1,14) = 7.05, *

Data are presented as mean ± SEM. *: p<0.05 and ***: p<0.001.

ESM Fig 1.

ESM Fig 1. Mouse genotyping and full hGH coding region amplified from genomic tail DNA. Double transgenic status of two RIPrtTA–tetO-sFLT1 mice was confirmed via

amplification of the RIPrtTA target sequence (~430bp) (lanes 1-2) and tetO-sFLT1 target

sequence (~450bp) (lanes 3-4). PCR for hGH (~1468bp) was performed for both

samples (lanes 5-6) and for two positive controls (lanes 7-8), one negative control (lane

9) and a no primer control (lane 10). No hGH sequence could be amplified from genomic

tail DNA of RIPrtTA–tetO-sFLT1 mice.

ESM Fig 2.

ESM Fig 2. Transgene expression and additional validation of the DOX-induced changes in intra-islet vascularization during pregnancy. (a) Beta cell-specific

expression of sFLT1 in dTg P-Dox and (b) dTg P+Dox (FLT1 [red] in insulin+ cells

[green]) (c) results in a significant 20-fold increase of insulin+ cells expressing sFLT1 at

G14.5 in dTg P+Dox mice as compared to dTg P-Dox mice. Data are shown as mean ±

SEM (n=3-4) (4.2 ± 0.5 % in dTg P-Dox vs. 80.1 ± 2.2 % in dTg P+DOX). White

squares: dTg P-Dox, black squares: dTg P+Dox. ***: p<0.001, Student’s t-test. (d) Transgenic production of a ~115-kDa soluble isoform of human FLT1 in islet protein

extracts. (e) Intra-islet endothelial cell staining (CD31 [red]) in dTg P+Dox and (f) dTg P

-Dox and (g) visualization of functional vasculature by i.v. injected biotinylated tomato

lectin (red) in dTg P-Dox and (h) in dTg P+Dox. (i) In addition, intra-islet hypoxia was

assessed by pimonidazole staining (brown) in dTg P–Dox and (j) dTg P+Dox, (k) with

liver tissue as internal positive control in dTg P-Dox and (l) dTg P+Dox.

ESM Fig 3.

sTg NP+ Dox

sTg P+ Dox

0

1

2

3

4

Ki6

7+/IN

S+

14.5

dpc

(%) **

3.5 7.5 10.5 14.5

0

10

20

30

40

dpc

Bod

y w

eigh

t (g)

dTg NP- Dox

dTg NP+ Dox

dTg P- Dox

dTg P+ Dox

0

125

150

200

250

Mea

n cr

oss-

sect

iona

l are

a pe

r bet

a ce

ll 14

.5 d

pc (µ

m²) *

15 30 60 1200

5

10

15

20

25

30

Time (min)

Blo

od g

luco

se G

0 (m

mol

/l)

15 30 60 1200

5

10

15

20

25

30

Time (min)

Blo

od g

luco

se 0

dpc

(mm

ol/l)

dTg P - Dox

dTg P + Dox

0

1×103

2×103

3×103

AU

C (m

mol

/l x

min

)

a b

dTg NP- Dox

dTg NP+ Dox

0

1×103

2×103

3×103

AU

C (m

mol

/l x

min

)

c d

e

ESM Fig 3. Baseline IPGTT, pregnancy-associated evolution of body weight and beta cell size in double transgenic mice and beta cell proliferation in single transgenic controls. (a) Glucose clearance was similar in both nonpregnant and (b) future pregnant double transgenic mice at baseline. Inset shows the AUC for each

IPGTT with line indication of mean ± SEM (n=11-13). (c) Evolution of body weight from

start until 14.5 dpc. Data are shown as mean ± SEM (n=10-17). (d) Beta cell

proliferation in Dox-administered single transgenic (sTg) mice shows similar replication

rates in the nonpregnant and pregnant state at 14.5 dpc (0.98 ± 0.18% Ki67+ insulin+

cells in sTg NP+Dox vs. 2.70 � 0.26% in sTg P+Dox). Data are shown as mean ± SEM

(n=3-4). (e) Mean cross-sectional area per beta cell (µm²), as a measure for beta cell

size, indicates a pregnancy-associated cell hypertrophy at G14.5. No significant

changes are seen upon conditional islet hypovascularization while no significant

interaction effect was observed between Dox-administration and (non)pregnancy. Data

are shown as mean ± SEM (n=3-6). Two-way ANOVA with Tukey post hoc comparison.

Post hoc multiple comparison did not reveal significant differences between groups,

therefore the asterix indicates statistical significance for the main effect of pregnancy on

beta cell size irrespective of Dox-administration (see also ESM Fig. 4). Circles, dTg NP;

squares, dTg P; downward triangles: sTg NP; upward triangles: sTg P; white symbols, −

Dox; black symbols, + Dox. *p<0.05; **p<0.01.

ESM Fig 4.

Nonpregnant Pregnant0

1´103

2´103

3´103

AUC

IPG

TT 1

3.5

dpc

(mm

ol/l

x m

in)

Nonpregnant Pregnant0

1

2

3

4

Ki67

+/IN

S+ 1

4.5

dpc

(%)

Nonpregnant Pregnant

0.5

1.0

1.5

Beta

cel

l vol

ume

14.5

dpc

(µl)

0Nonpregnant Pregnant

0

50

100

150

200

Mea

n cr

oss-

sect

iona

l are

ape

r bet

a ce

ll 14

.5 d

pc (µ

m²)

a b

c d

ESM Fig 4. Summary data from two-way ANOVA of glucose clearance, beta cell proliferation, total beta cell volume and individual beta cell size. (a) Two-way

ANOVA revealed a significant interaction between Dox-administration and pregnancy,

indicating a differential effect of Dox in dTg NP vs. dTg P mice, p<0.05. (b) Two-way

ANOVA revealed a significant main effect of pregnancy on beta cell proliferation,

p<0.001; but no significant effect of Dox; nor significant interaction between the two

factors. (c) Two-way ANOVA revealed a significant main effect of pregnancy on total

beta cell volume, p=0.001; but no significant effect of Dox; nor significant interaction

between the two factors. (d) Two-way ANOVA revealed a significant main effect of

pregnancy, p<0.05; but no significant effect of Dox; nor significant interaction between

the two factors. Solid line: dTg -Dox, dashed line: dTg +Dox.