Supplementary Information - Nature Research · Figure S4. Impaired oogenesis in ATRS/S mice. a,...

Supplementary Information

A mouse model of the ATR-Seckel Syndrome reveals that replicative stress

during embryogenesis limits mammalian lifespan.

Matilde Murga, Samuel Bunting, Maria F. Montaña, Rebeca Soria, Francisca Mulero, Marta

Cañamero, Youngsoo Lee, Peter J. McKinnon, Andre Nussenzweig and Oscar Fernandez-

Capetillo.

Nature Genetics: doi:10.1038/ng.420

Figure S1. Cranial anomalies in Seckel mice. a, Side and top views of CT scans

of 3 month old ATR+/+ and ATRS/S littermates. The skull surface of the mutant

animals shows less marked osseous relieves, with the indentation of the sutures

between the cranial bones being significantly reduced. In addition, the mutant

animals presented dental malocclusion which was due to a smaller curvature-

radius of the upper incisors (red arrowhead, 1), together with a rounding of the

crown of the lower incisors (red arrowhead, 2). The previous phenotypes were

100% penetrant. A deficient closure of the fontanelles was also frequently

detected (6/9 mice analysed). Scale bar indicates 2 cm. b, Head circumference

measurements in 3 month old ATR+/+ and ATRS/S mice. Note: A perimeter is

drawn around the head circumference of the animals for this analyses (see

dashed red lines in a). Due to the differences between the mice and humans, we

used the region of the skull that surrounds the brain, which is equivalent to the

occipitofrontal measurement done in humans. On average, Seckel animals

present a decrease on the head circumference of -8.81 SD, which is bigger to

that of height (-4.88 SD) or weight (-3.4 SD).


Figure S2. Neurological defects on ATRS/S mice. a, Picture of the brains from a

pair of one month old wt and Seckel animals. Scale bar indicates 0.5 cm. b, The

figure illustrates the generalized loss of cellularity (Niss1 staining, blue) and

acute depletion of astrocytes (GFAP staining, green) in the corpus callosum of 6

week old ATRS/S mice.


Figure S3. Analysis of the organ sizes of ATRS/S mice. a, Comparison of the

sizes of the different organs extracted from 3 month old ATR+/+ and ATRS/S

animals. The decrease in size of organs from the mutant animals is also

expressed in terms of the number of standard deviations below the wild type

average (last column; <>SD). b, ATR western blot in several organs from 1

month-old ATR+/+ and ATRS/S mice. ATR levels in newborns (p5) are also

shown for lung and testes.


Figure S4. Impaired oogenesis in ATRS/S mice. a, Immunohistochemistry of

ATR+/+ and ATRS/S ovaries with an oocyte recognizing antibody (DDX4). The

arrowhead points to the only maturing oocyte recognized in this section. Scale

bar (black) indicates 0.5 mm. b, Quantification of the number of DDX4 positive

cells found in a region of in ovaries from newborn and 1 month old animals. c,

Primary follicles of the newborn ATRS/S ovaries. Red arrows indicate the

presence of vacuoles. Scale bar (black) indicates 15 μm.


Figure S5. Functional complementation of ATR with the humanized murine

allele. a, PCR genotyping; and b, RT-PCR of ATR with primers at E8 and E10 in

littermate MEF lines. c, ATR western blot in several organs from ATR+/+ and

ATRHS/HS mice. d, A closer picture of the heads from a couple of 3 month-old

ATR+/+ and ATRHS/HS littermates. e, Weight of ATRHS/HS and control mice at 3

months of age (n=10).


Figure S6. Analysis of the frequency and functional capacity of Seckel HSCs.

a, Representative FACS plots from ATR+/+, ATRS/S and 18 month old ATR+/+

live, lineage- BM showing: (left) Lineage- Sca1+ c-Kit+ (LSK) population; (right)

LT-HSC, ST-HSC and MPP populations. b, Schema illustrating the

differentiation process of HSCs. c, Frequencies of LSK, LT-HSC and MPP

populations as a % of total BM. d, Timecourse showing the reconstitution of the

granulocyte population of lethally irradiated wt recipients after competitive

transplantation of control or Seckel CD45.2 BM with CD45.1 wt BM. e,

Contribution of ATR+/+ and ATRS/S CD45.2 BM to the reconstitution of B cell

(B220+), T cell (CD3+) and granulocyte (GR1+) compartments of lethally

irradiated wt host, 12 weeks after competitive transplantation.


Figure S7. Dampening of the IGF1/GH axis on Seckel organs. Expression level

differences between livers and brains of 3 month old ATR+/+ and ATRS/S were

analyzed by microarray analysis (see methods). The data for the genes of the

IGF‐1/GH previously associated to progeria 1, 2 is presented in the table.


Figure S8. RS on Seckel embryos. γH2AX IHC on sagital sections of 13.5 dpc

littermate embryos of the indicated genotypes. The digitalized slides at high

resolution are available upon request for closer inspection and can be viewed

with a free viewer (MIRAX, Zeiss).


Figure S9. Aggravation of the Seckel phenotypes in a p53-deficient

background. Summary of the effects of p53 elimination on the dwarfism,

craniofacial abnormalities, progeroid phenotypes and overall lifespan of ATRS/S

animals.


Figure S10. Increase of RS on Seckel embryos by elimination of p53. γH2AX

IHC on sagital sections of 13.5 dpc littermate embryos of the indicated

genotypes. The digitalized slides at high resolution are available upon request

for closer inspection and can be viewed with a free viewer (MIRAX, Zeiss).


Supplementary Information - Nature Research · Figure S4. Impaired oogenesis in ATRS/S mice. a,...

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Transcript of Supplementary Information - Nature Research · Figure S4. Impaired oogenesis in ATRS/S mice. a,...