ART I C L E S
Canonical and atypical E2Fs regulate the mammalianendocycleHui-Zi Chen1,2,10, Madhu M. Ouseph1,3,10, Jing Li1,11, Thierry Pécot1,4,5, Veda Chokshi1, Lindsey Kent1, Sooin Bae1,Morgan Byrne1, Camille Duran1, Grant Comstock1, Prashant Trikha1, Markus Mair1, Shantibhusan Senapati1,Chelsea K. Martin1, Sagar Gandhi1, Nicholas Wilson1, Bin Liu1,5, Yi-Wen Huang6, John C. Thompson1,Sundaresan Raman4, Shantanu Singh4, Marcelo Leone4, Raghu Machiraju4, Kun Huang5, Xiaokui Mo7,Soledad Fernandez7, Ilona Kalaszczynska8, Debra J. Wolgemuth9, Piotr Sicinski8, Tim Huang1, Victor Jin5
and Gustavo Leone1,2,12
The endocycle is a variant cell cycle consisting of successive DNA synthesis and gap phases that yield highly polyploid cells.Although essential for metazoan development, relatively little is known about its control or physiologic role in mammals. Usinglineage-specific cre mice we identified two opposing arms of the E2F program, one driven by canonical transcription activation(E2F1, E2F2 and E2F3) and the other by atypical repression (E2F7 and E2F8), that converge on the regulation of endocycles invivo. Ablation of canonical activators in the two endocycling tissues of mammals, trophoblast giant cells in the placenta andhepatocytes in the liver, augmented genome ploidy, whereas ablation of atypical repressors diminished ploidy. These twoantagonistic arms coordinate the expression of a unique G2/M transcriptional program that is critical for mitosis, karyokinesis andcytokinesis. These results provide in vivo evidence for a direct role of E2F family members in regulating non-traditional cell cyclesin mammals.
The archetypal cell division cycle culminates in the generation of geneti-1
cally identical diploid progenies. However, a number of cell-cycle varia-2
tions in nature exist, with the endocycle being a remarkable variant used3
by a wide range of organisms in diverse environmental contexts1. This4
modified cell cycle is characterized by alternatingDNA synthesis (S) and5
gap (G) phases in the absence of intervening mitosis (M), karyokinesis6
and cytokinesis. As a result of successive rounds of whole-genome7
duplication, cells may achieve genome polyploidy that exceeds 1000C8
(refs 2,3). The biological advantages of polyploidy have been proposed9
to include an augmented response to strenuous cellular metabolic10
demands; dampened sensitivity to apoptotic or checkpoint stimuli4;11
and buffering against deleteriousmutations thatmay cause cancer5,6.Q1 12
The molecular mechanisms that control mitotic and variant cell13
cycles among species are presumed to be similar. In the textbook view14
1Solid Tumor Biology Program, Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, The Ohio State University,Columbus, Ohio 43210, USA. 2Medical Scientist Training Program and Integrated Biomedical Graduate Program, College of Medicine, The Ohio State University,Columbus, Ohio 43210, USA. 3Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA. 4Computer Science and Engineering, TheOhio State University, Columbus, Ohio 43210, USA. 5Biomedical Informatics, The Ohio State University, Columbus, Ohio 43210, USA. 6Department of Obstetrics andGynecology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. 7Center for Biostatistics, Office of Health Sciences, The Ohio State University,Columbus, Ohio 43210, USA. 8Department of Cancer Biology, Dana-Farber Cancer Institute, and Department of Genetics, Harvard Medical School, Boston,Massachusetts 02215, USA. 9Department of Genetics and Development, Department of Obstetrics and Gynecology, The Institute of Human Nutrition, and The HerbertIrving Comprehensive Cancer Center, Columbia University Medical Center, New York, New York 10032, USA. 10These authors contributed equally to this work.11Present address: Roche Molecular Systems, 4300 Hacienda Drive, Pleasanton, California 94588, USA..12Correspondence should be addressed to G.L. (e-mail: [email protected])
Received 13 September 2011; accepted 3 September 2012; published online XX Month XXXX; DOI: 10.1038/ncb2595
of cell-cycle control, E2F transcriptional activators and repressors are 15
portrayed to orchestrate a gene expression program essential formitotic 16
cell-cycle progression7, with E2F activators and repressors oppositely 17
regulating a common set of target genes. However, careful analyses 18
of mouse knockout tissues demonstrate that E2F function in vivo 19
does not strictly adhere to this elegant dichotomous paradigm8–16. 20
This is perhaps not surprising given the large number of genes in the 21
mammalian E2F family, which consists of eight distinct genes encoding 22
at least nine structurally related protein products that can be divided 23
into activator or repressor subclasses17. Canonical E2F activators (E2F1, 24
E2F2, E2F3a and E2F3b) have a single DNA-binding domain (DBD), 25
heterodimerize with DP1/DP2 proteins and associate with co-activator 26
proteins to robustly induce RNA polymerase II-dependent gene 27
expression18. Canonical repressors (E2F4, E2F5 and E2F6) also have a 28
NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 1
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TGC: NanoString Liver: quantitative rtPCRa
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2–4C 8C 16C 32C 64C 128C 256C
Figure 1 Loss of E2f activators promotes TGC and hepatocyteendocycles. (a) NanoString analysis of TGC-specific E2f1–3 expression inlaser-capture-microdissected wild-type cells; n = 2 placentae analysedper time point. (b) Quantitative rtPCR analysis of E2f activatorsin pre- (E13.5) and postnatal (P0–12mo) wild-type livers, withn per time point as indicated. 3wk*, 1 day post-weaning (3wk).(c) Representative E9.5 H&E-stained placenta sections showing controland E2f1−/−;E2f2−/−;E2f3−/− (123tko) TGCs. The arrows point toselected TGCs. (d) Feulgen quantification of ploidy in E9.5 control and123tko TGCs; n = 3 per genetic group. One-way ANOVA, ∗P ≤ 0.05,∗∗P ≤ 0.01, ∗∗∗P ≤ 0.001. (e) Representative H&E-stained sections
showing control and Mx–cre;E2f1−/−;E2f2−/−;E2f3f / f (Mx-123tko) liversof 8-week-old mice ten days after pIpC injection. (f) Measurementsshowing enlargedMx-123tko livers (top) and hepatocytes (bottom) relativeto control tissues and hepatocytes, with n per genetic group as indicated.Two-tailed Student t -test, ∗∗P ≤0.01. (g) Flow cytometry of control andMx-123tko liver nuclei from 8-week-old mice, with n per genetic group asindicated. One-way ANOVA, ∗∗P ≤0.01, ∗∗∗P ≤0.001. c control, wild-typeTGCs; e control, pIpC-injected Mx-cre;E2f1−/−;E2f2−/−;E2f3+/+ livers;f,g control, wild-type livers. Data in a,b,d,f,g are average values, ± s.d.are included where n>2 samples were analysed. Scale bars, 12.5 µm (c)and 10 µm (e).
single DBD, heterodimerize with DP1/DP2 proteins and associate with1
chromatin-modifying proteins to repress gene expression18. The most2
recently identified family members encode the atypical repressors E2F73
and E2F8, which have two tandem DBDs and repress gene expression4
independent of DP1/DP2 heterodimerization19.Q2 5
Whereas flies and plants use endocycles as a mechanism for cell6
growth in a variety of tissue types1,20, endocycles in mammals are7
mainly restricted to trophoblast giant cells (TGCs) in the placenta8
and hepatocytes in the liver1,5. Endocycles in these mammalian tissues9
are believed to be integral to organ physiology1,5; however, surprisingly10
little evidence exists to support this contention. Here we developed11
new genetic tools in mice and show by depletion of either canonicalQ3 12
E2F activators or atypical E2F repressors that these two separate E2F13
transcriptional programs converge to control the endocycle through14
the regulation of cellular events important for mitosis, karyokinesis and15
cytokinesis. Surprisingly, placentae and livers with severely restricted16
polyploidization retained sufficient physiological function to carry17
them through apparently normal development.18
RESULTS19
E2f1–3 ablation results in hyperploidy20
E2F-mediated transcription contributes to endocycle control in flies21
and plants1,5,19–21, but its role in mammals has not been thoroughly22
evaluated. Whole-genome duplication in mammalian endocycling 23
cells is temporally regulated during development with major ploidy 24
increases in TGCs during mid gestation and in hepatocytes following 25
weaning at around three weeks of age22,23. The levels of E2f1 and E2f2 26
messenger RNAs in laser-capture-microdissected TGCs fromwild-type 27
placentae, as measured by NanoString technology (Fig. 1a), were low 28
throughout placental development, whereas the level of E2f3mRNA 29
was high before embryonic day (E)8.5 and following E13.5. Expression 30
analysis in livers by quantitative real-time polymerase chain reaction 31
(rtPCR) showed relatively high levels of E2f1 and E2f2 during fetal 32
development and a precipitous decrease following birth (postnatal 33
day 0, P0), whereas expression of E2f3 (E2f3a and E2f3b) decreased 34
gradually following birth and was almost undetectable post-weaning 35
(Fig. 1b). Consistentwith the expression pattern of theirmRNAs, E2F3a 36
and E2F3b protein levels decreased in newborn pups post-weaning 37
(Supplementary Fig. S1a), and E2F1 and E2F2 proteins could not be Q4 38
detected beyond the intrauterine period (data not shown). Thus, it 39
seems that total E2F1–3 activator levels are minimized when TGCs and 40
hepatocytes are most actively endocycling. 41
We used targeted gene inactivation strategies in mice to rigorously 42
evaluate the role of E2F activators in endocycling TGCs and 43
hepatocytes. To avoid functional compensation24, we evaluated TGCs 44
in embryos deficient for the entire E2F activator subclass (E2f1–3). 45
2 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION
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Visual inspection of haematoxylin and eosin (H&E)-stained E9.51
E2f1−/−;E2f2−/−;E2f3−/− (123tko) placentae revealed the presence2
of TGCs with abnormally large nuclei (Fig. 1c). Feulgen staining3
and quantification of genome ploidy by standardized methods4
(Supplementary Fig. S1b) showed a further shift of the 123tko TGC5
population towards higher ploidy (Fig. 1d and Supplementary Fig. S1c).6
Analysis of 123tko TGCs at later stages of development was precluded7
owing to embryo lethality at E10.5 (refs 25,26).8
To evaluate the role of the three activator E2Fs in hepatocytes9
we used polyinosine-polycytidine (pIpC)-inducible Mx–cre mice2710
and conditional alleles of E2f3 (Mx–cre;E2f1−/−;E2f2−/−;E2f3f / f;Mx-11
123tko) to examine the consequences of their combined ablation.12
Injection of pIpC in six-week-oldMx-123tkomice resulted in increased13
liver weights (Fig. 1f) and proportionately larger hepatocytes (Fig. 1e,f)14
than age-matched controls. Loss of E2f1 alone or in combination with15
E2f2 and E2f3 resulted in a gene-dose-dependent increase in hepatocyte16
ploidy (Fig. 1g and Supplementary Fig. S1d). The expression of E2f17
repressors (E2f4-8) was not affected by loss of E2f1–3 (Supplementary18
Fig. S1e). Together, these findings suggest that mammalian E2F19
activators have a physiologic role in suppressing endocycles.20
E2f7/E2f8 ablation reduces TGC ploidy21
Next, we explored the role of E2F repressors in endocycle control.22
Expression analysis of the canonical repressors (E2f4-6) showed23
relatively high levels of E2f4 in TGCs of E6.5 placentae that24
decreased with time, whereas E2f5-6 expression was relatively25
constant throughout placental development (Fig. 2a, left panel);26
expression of E2f4-6 was relatively high in fetal livers and their27
expression decreased after birth (Supplementary Fig. S2a). Inspection28
of E2f4−/− or E2f5−/− embryos and mice revealed normal placentae29
and livers (Supplementary Fig. S2b,c); E2f6−/− placentae and livers30
were not evaluated.31
We then focused our attention on the atypical repressors, E2F732
and E2F8. These E2Fs are expressed in TGCs throughout placental33
development (Fig. 2a, right panel). Immunohistochemistry showed34
robust staining of E2F7 and E2F8 proteins in a portion of TGCs (Fig. 2b,35
arrows), consistent with their established cell-cycle-dependent expres-36
sion in late S and G2 phases28–31. Visual inspection of E2f7−/−;E2f8−/−37
placentae at E10.5, one day before their embryonic lethality16, revealed38
a surprising number of TGCs at various stages of mitosis (Fig. 2c),39
implying an interruption of normal endocycles. Indeed, the ploidy40
of E2f7−/−;E2f8−/− TGCs never exceeded 64C (Fig. 2d, black bars),41
whereas wild-type TGCs with genomes >1,000C were readily detected42
(Fig. 2d, white bars). The combined inactivation of E2f7 and E2f843
led to ectopic bromodeoxyuridine (BrdU) incorporation (Fig. 2e,g),44
increased levels of G2/M (cyclin A2) and M-phase-specific proteins45
(cyclin B1 and phosphorylated H3, P-H3; Fig. 2f,g) and the appearance46
of numerous TGCs in metaphase and anaphase (Fig. 2h). Confocal47
microscopy and three-dimensional (3D) reconstruction of serially48
imaged E2f7−/−;E2f8−/− placentae showed that approximately 40%49
of TGCs contained at least two nuclei (Fig. 2i), which was confirmed50
by transmission electron microscopy (Fig. 2j). Placentae lacking E2f7,51
E2f8 or one allele of each (E2f7+/−;E2f8+/−) exhibited TGCs with52
an intermediate reduction in ploidy (Fig. 2d and Supplementary53
Fig. S2d,e) and a proportionate increase in G2/M- andM-phase-related54
events (Fig. 2g,j), once again suggesting a gene dosage effect.55
As the expression of trophoblast cell lineage markers was altered 56
in placentae globally deleted for E2f7 and E2f8 (TGCs, labyrinth 57
trophoblasts and spongiotrophoblasts)32, we could not formally rule 58
out a general disruption of differentiation as a possible cause for the 59
endocycle defects observed in mutant TGCs. To specifically ablate 60
E2f7 and E2f8 in TGCs of placentae we crossed Plfcre/+ knock-in 61
mice (Fig. 2k and data not shown)32 with E2f7f /f;E2f8f / f mice to 62
yield Plf-78dko embryos. We confirmed the TGC-specific ablation of 63
conditional alleles by PCR genotyping of laser-capture-microdissected 64
samples (Fig. 2l). The same abnormalities were observed in Plf- 65
78dko TGCs as in E2f7−/−;E2f8−/− TGCs, including the reduced 66
ploidy, increased cyclin A2, cyclin B1 and P-H3 expression levels, 67
and ectopic karyokinesis (Fig. 2d,g,j; red bars). In summary, these 68
data demonstrate cell autonomous functions for E2F7 and E2F8 in 69
facilitating TGC endocycles by repressing keymolecular events required 70
for mitosis and karyokinesis. 71
E2f7/E2f8 ablation prevents hepatocyte endocycles 72
The expression of E2f7 and E2f8 in fetal livers was high but decreased to 73
almost undetectable levels by weaning age (Fig. 3b and Supplementary 74
Fig. S3a). To examine their roles during postnatal liver development 75
we crossed E2f7f / f and E2f8f /f mice with Albumin–cre mice (Alb–cre; 76
ref. 33), which express cre in hepatocytes of late-term embryos and 77
newborn pups. The conditional inactivation of E2f7 (Alb-7ko), E2f8 78
(Alb-7ko) or both (Alb-78dko) did not significantly affect hepatic 79
mass (Supplementary Fig. S3b). However, evaluation of H&E- and 80
4,6-diamidino-2-phenylindole (DAPI)-stained liver sections from 81
Alb-8ko and Alb-78dko mice revealed markedly smaller hepatocytes 82
with smaller nuclei than in control animals (Fig. 3a,c). This reduction 83
in cell size was compensated by an increase in the total number of 84
hepatocytes (Fig. 3d). Moreover, Alb-8ko and Alb-78dko livers had 85
a decreased proportion of binucleated hepatocytes (Fig. 3e), which 86
is an early event believed to precede the onset of polyploidization34. 87
Strikingly, flow cytometry showed that hepatocytes in Alb-8ko and 88
Alb-78dko livers remained diploid over the entire lifetime of the 89
mouse, whereas hepatocytes in Alb-7ko livers had only a modest 90
reduction in genome ploidy (Fig. 3f). The percentages of Ki67-, 91
cyclin-A2-, cyclin-B1- and P-H3-positive hepatocytes were significantly 92
elevated in 2-month-old Alb-8komice and even more so in similarly 93
aged Alb-78dko mice (Fig. 3g). Analysis of mice globally deleted for 94
E2f7 (E2f7−/−) or E2f8 (E2f8−/−) yielded identical phenotypes as 95
conditionally deleted Alb-7ko and Alb-8komice (Supplementary Fig. 96
S3c,d). Surprisingly, Alb-78dkomice survived to old age in apparent 97
good health and retained the capacity to regenerate their livers on 98
chemical (carbon tetrachloride) or physical (partial hepatectomy) 99
injury (Supplementary Fig. S3e–h and data not shown). From these 100
results, we conclude that E2F8 is the principal atypical E2F that 101
functions to promote hepatocyte endocycles inmice. 102
The onset of genome endoreduplication in hepatocytes coincides 103
with the weaning of pups, but DNA content continues to increase 104
through adulthood even though very little E2f8 is expressed at this 105
time (see Fig. 3b). These observations raised the possibility that E2F8 106
might be required only for the programmed onset and not the 107
continuation of endocycles. We thus used a tamoxifen-inducible 108
system (SA-creERT2) to express cre in hepatocytes following birth 109
and/or the weaning of pups35. As determined by quantitative rtPCR, 110
NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 3
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×20
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32C 64C 128C 256C 512C 1,024C2–4C 8C 16C
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Figure 2 E2f7 and E2f8 promote TGC endocycles. (a) NanoStringanalysis of TGC-specific E2f 4–6 (left panel) and E2f7/E2f8 (right panel)expression in laser-capture-microdissected wild-type cells; n=2 placentaeanalysed per time point. (b) Immunohistochemistry demonstrating E2F7(top left) and E2F8 (bottom left) expression in wild-type E10.5 TGCsbut not mutant controls (right panels). The arrows point to selectedTGCs. (c) Representative H&E-stained sections of E10.5 control andE2f7−/−;E2f8−/− (78dko) placentae. Inset, higher magnification ofthe outlined area, showing a 78dko TGC in metaphase. (d) Feulgenquantification of genome ploidy in E10.5 TGCs; n =3 placentae analysedper genetic group. (e–g) Immunostaining and quantification of S and Mphase proteins in E10.5 control and 78dko TGCs. The arrows point to TGCs.n = 3 placentae analysed per genetic group. (h) Co-immunofluorescencemicroscopy showing E10.5 78dko TGCs in anaphase (left, P-H3) andmetaphase (right, PL-1). DAPI stained total DNA. (i) Representativeconfocal images of nuclei in E10.5 control (top right) and 78dko TGCs
(top left) and 3D reconstruction of a binucleated 78dko TGC (bottom).Draq5 stained total DNA pseudocoloured in green. (j) Left, transmissionelectron micrograph (TEM) of a 78dko E10.5 TGC (left top, arrows indicatetwo nuclei; left bottom, enlarged view of the area outlined in the top imageshowing separation between nuclear envelopes). Right, quantification ofbinucleated E10.5 TGCs; n = 3 placentae analysed per genetic group.(k) X-gal staining of E10.5 control and Plfcre/+ placentae carrying thereporter allele Rosa26LoxP. (l) PCR genotyping of genomic DNA isolatedfrom laser-capture-microdissected E10.5 control and Plfcre/+ TGCs. e,f,icontrol, E2f7+/+;E2f8+/+; e,f,h–j 78dko, E2f7−/−
;E2f8−/−; k control,Plf+/+;Rosa26LoxP / + ; l control, Plf+/+;E2f7f / f;E2f8f / f; d,g,j Plf-78dko,Plfcre/+;E2f7f / f;E2f8f / f. PL-1, placental lactogen 1; N.E., nuclear envelope.Data in a,d,g,j are average values, ±s.d. are included where n>2 sampleswere analysed. One-way ANOVA, ∗P ≤0.05; ∗∗P ≤0.01; ∗∗∗P ≤0.001. Scalebars, 10 µm (b), 12.5 µm (e,f), 5 µm (h) and 10 µm (i). Uncropped imagesof blots are shown in Supplementary Fig. SXX.
supplying SA-creERT2;E2f8f / f mice with a tamoxifen diet beginning1
at either 7 days (2 weeks before weaning, pre-weaning) or 28 days2
of age (1 week following weaning, post-weaning) resulted in the3
tissue-specific efficient deletion of E2f8 (data not shown). Ablation of4
E2f8 pre-weaning prevented efficient polyploidization of hepatocytes5
(Supplementary Fig. S4a–c), whereas ablation post-weaning failed 6
to impact hepatocyte size, binucleation and DNA content (data not 7
shown and Supplementary Fig. S4a,b,d). These results suggest that 8
E2F8 is largely dispensable for the execution of endocycles following 9
the weaning of pups. 10
4 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION
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12 m
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3 wk n = 42 mo n = 912 mo n = 6 3 wk n = 4
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200
100
02 mo 6 mo3 wk 12 mo
12 mo
n =
3n
= 3
n =
3
∗∗
∗∗∗∗∗∗ ∗∗∗
∗∗∗∗∗∗
∗∗∗
0
7
14
21
28
35
n =
3
n =
9n
= 2
n =
6n
= 7
n =
9
n =
5n
= 2
n =
6
n =
5
Alb-78dko
Perc
en
tag
e o
f
Ki6
7-p
ositiv
e h
ep
.
Perc
en
tag
e o
f
p-H
3-p
ositiv
e h
ep
.
Perc
en
tag
e o
f
CycA
2-p
ositiv
e h
ep
.
Perc
en
tag
e o
f
CycB
1-p
ositiv
e h
ep
.
∗∗∗
∗∗∗
∗∗
∗
n =
19
0
0.5
0
1.0
n =
8n
= 7
n =
16
n =
8n
= 7
n =
7n
= 1
0n
= 6
n =
5n
= 8
n =
10
Control
Alb-78dko
E-cad/DAPI3 wk 2 mo 12 mo
n =
4 n =
8
n =
7n
= 6
3
2
1
0
1.0
0
0.5
0.8
0.4
1.2
Control
100
50
0
25
75
Perc
en
tag
e o
f liv
er
nu
cle
i g
ate
d
Perc
en
tag
e o
f liv
er
nu
cle
i g
ate
d
Perc
en
tag
e o
f liv
er
nu
cle
i g
ate
d
3 wk n = 42 mo n = 612 mo n = 5
4C 8C 16C
2C 4C 8C 16C2C
Control Alb-7koAlb-8ko Alb-78dko
12 mo 12 mo 12 mo
4C 8C16C2C 4C 8C 16C2C
2.0
0
2.5
1.5
1.0
0.5
Nu
mb
er
of
liver
nu
cle
i (×
1,0
00
)
Nu
mb
er
of
liver
nu
cle
i (×
1,0
00
)
0
1.5
1.0
0.5
2.0
0
1.5
1.0
0.5
2.0
2.5
1.5
1.0
0.5
0
a e
g
b
f
c d
Alb-78dAlb-8ko
Alb-7koControl
n =
6
n =
3n
= 3
n =
3n
= 3
n =
7n
= 2
n =
6n
= 8
n =
4n
= 3
n =
3n
= 5
n =
5n
= 5
n =
2
3 wk n = 42 mo n = 612 mo n = 2
100
50
0
25
75
4C 8C 16C2C 4C 8C2C 4C 8C 16C2C 4C 8C 16C2C16C
100
50
0
25
75
100
50
0
25
75
Figure 3 E2f8 is sufficient to promote hepatocyte endocycles.(a) Representative H&E-stained sections of aged (12 mo) livers.(b) Quantitative rtPCR analysis of E2f7 and E2f8 in pre- (E13.5)and postnatal (P0–12 mo) wild-type livers, with n per time pointanalysed as indicated. 3 wk*, 1 day post-weaning (3 wk). (c) Leftgraph, assessment of nuclei volume by confocal imaging and 3Dreconstruction in 6-month-old control and Alb-78dko hepatocytes. n,number of nuclei evaluated per genotype. Right panels, representativeconfocal images of DAPI-stained liver sections. (d) Assessment ofhepatocyte size in young (3 wk), adult (2–6 mo) and aged (12 mo)livers by quantifying the number of hepatocytes per image field,with n per genetic group analysed as indicated. (e) Left graph,quantification of binucleated hepatocytes in young, adult and aged
livers, with n per genetic group analysed as indicated. Right panels,immunofluoresence micrographs with anti-E-cadherin marking theboundary of mono- or binucleated hepatocytes. (f) Top row, flow cytometryof liver nuclei in young, adult and aged livers. n, number of liversanalysed per genotype and age group. Bottom row, representativefluorescence-activated cell sorting profiles of liver nuclei from aged (12mo) mice. (g) Immunohistochemistry and quantification of hepatocyteproliferation in 2-month-old livers, with n per genetic group analysed asindicated. Control, E2f7f / f;E2f8f / f; Alb-7ko, Alb–cre;E2f7f / f; Alb-8ko,Alb–cre;E2f8f / f; Alb-78dko, Alb–cre;E2f7f / f;E2f8f / f. Data in b–g areaverage values, ± s.d. are included where n >2 samples were analysed.One-way ANOVA, ∗P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗P ≤ 0.001. Scale bars,12.5 µm (a), 10 µm (c), 10 µm (e) and 10 µm (g).
E2F7/E2F8 regulate endocycle-related genes1
Given the role of E2F7 and E2F8 in transcriptional repression, we2
anticipated changes in gene expression as the principal underlying3
cause for the observed endocycle defects in E2f7/E2f8-deficient tissues.4
Gene expression profiling (Affymetrix Mouse Genome 430 2.0) using5
RNApurified from livers of 3-week-old wild-type,Alb-7ko,Alb-8ko and6
Alb-78dkomice showed that there were significantly more upregulated7
than downregulated genes in mutant tissues, consistent with their8
roles as repressors (Fig. 4a). Expression changes were confirmed9
by NanoString technology (Supplementary Fig. S5a). Supervised10
clustering methods identified three main classes of differentially 11
expressed genes (Fig. 4a). Class I includes genes differentially expressed 12
in all three mutant genetic groups relative to wild-type controls. These 13
genes may represent targets regulated by both E2F7 and E2F8 because 14
loss of eithermember led to their derepression. Class II represents genes 15
differentially expressed in two of the three mutant genetic groups and 16
includes target genes that may be uniquely repressed by either E2F7 17
or E2F8. Finally, class III represents genes differentially expressed in 18
only one mutant genetic group, and includes target genes that may be 19
synergistically repressed by E2F7 and E2F8. 20
NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 5
ART I C L E S
NanoString: E10.5 TGCs
Liv
er a
nd
TG
Cs (6
6)
G2
/M(3
3)
Genes a
nta
go
nis
tically re
gula
ted
P < 0.05
Genes antagonistically regulated
IPA: molecular and cellular functions
Number of genes
Affymetrix: liver
Cla
ss I
Cla
ss II
Cla
ss III
Syn
erg
.
targ
ets
E2F
8
targ
ets
E2F
7
targ
ets
E2F
7 a
nd
E2F
8 ta
rgets
Affymetrix: weaning age (3 wk) liver
1 2 3 4
P < 0.05
Cell cycle
Small molec. biochem.
Lipid metabolism
Nucleic acid metab.
Cell–cell signal.
1 2 3 1 2 3 4 1 2 3 4
Control 78dko 8ko 7ko
Control Alb-78dko Control Mx-123tko
10 20 30 40 50 600P < 0.00245
n = 2 n = 2 n = 2 n = 2
1 2 3 1 2 31 2 1 23 4
a b
c
d
Control Alb-78dko Alb-8ko Alb-7ko
P < 0.05–1.5 +1.5
–3.0 +3.0
–1.5 +1.5
Figure 4 Canonical activator and atypical repressor E2Fs regulate keytranscriptional networks coordinating endocycles. (a) Heat map ofapproximately 4,500 differentially expressed genes in weaning age(3 wk) livers. Class I: genes regulated by E2F7 and E2F8. Class II:genes regulated by either E2F7 or E2F8. Class III: genes synergisticallyregulated by E2F7/E2F8. n = 3–4 livers analysed per genetic group.Control, E2f7f / f;E2f8f / f; Alb-78dko, Alb–cre;E2f7f / f;E2f8f/ f; Alb-8ko,Alb–cre;E2f8f / f; Alb-7ko, Alb–cre;E2f7f / f. (b) Top, heat map of TGCgene expression in a custom NanoString mRNA code set. RNA wasisolated from laser-capture-microdissected E10.5 TGCs in frozen
placental tissues. Bottom, G2/M-related genes in NanoString codeset with deregulated expression in TGCs. n = 2 placentae analysed pergenetic group. Control, E2f7+/+
;E2f8+/+;78dko, E2f7−/−;E2f8−/−; 8ko,E2f7+/+;E2f8−/−; 7ko, E2f7−/−;E2f8+/+. (c) Heat map of downregulatedgenes in Mx-123tko livers with significantly deregulated expression inAlb-78dko livers. n =3–4 livers analysed per genetic group. Alb-78dko,Alb–cre;E2f7f /f;E2f8f/f; Mx-123tko, Mx–cre;E2f1−/−;E2f2−/−;E2f3f / f.(d) Top five molecular and cellular functions revealed through IngenuityPathway Analysis (IPA) of genes antagonistically regulated by E2F1–3and E2F7/E2F8.
Further analysis focused on genes upregulated in both Alb-8ko1
and Alb-78dko livers (Class II), as hepatocytes in these two genetic2
groups exhibited the most pronounced reduction in genome ploidy,3
as well as genes upregulated in all three mutant cohorts (in Class4
I), because these could also contribute to the observed defect in5
polyploidization. Closer inspection of these genes by Ingenuity Pathway6
Analysis revealed a striking bias for functions related to cytokinesis,7
G2/M progression and various stages of mitosis, such as chromosome 8
condensation, stabilization and segregation (Supplementary Fig. S5b,c). 9
By manual annotation we also noted an increase in gene products with 10
GTPase-activating (GAP) or GTP exchange factor (GEF) functions 11
(data not shown), which regulate the Rho family of small GTPases 12
essential for successful cytokinesis36. NanoString technology was 13
then used to interrogate the expression of a panel of genes with 14
6 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION
ART I C L E S
Rela
tive o
ccup
ancy
Con. F7 F8 Con. F7 F8 Con. F7 F8 Con. F7 F8
ChIP E2F targets in HepG2 cells
Rcho-1 TS
Wild-type liver
HepG2
Rela
tive o
ccup
ancy
ChIP E2F targets in vivo
24 h post
TV inject.:
Flag
Tub.
IB:
Cdk1 Ccna2 TubCcne2 Chek1
ChIP E2F targets in Rcho-1 TS cells
Flag
Tub.
IB:
IB:
Flag
Tub.
Transf: Con. F7 F8
Con. F7 F8
Con. F8
Transf:
24
18
12
6
0
Rela
tive o
ccup
ancy
Con. F7 F8
Con. F7 F8 Con. F7 F8
16
12
6
0
14
7
0
12
8
4
0
12
8
4
0
E2f1 Ccna2 TubCcne2 Chek1
24
18
12
6
0
24
18
12
6
0
20
15
10
5
0
45
30
15
0 0
10
20
30
40
Con. F7 F8 Con. F7 F8 Con. F7 F8
10
5
0
5
3
0
4
2
1
3
0
2
1
Ccne2 Cdk1E2f1
a b
c d
e f
Con. F8 Con. F8 Con. F8
IgG
Anti-Flag
IgG
Anti-Flag
IgG
Anti-Flag
Figure 5 Atypical repressors E2F7/E2F8 directly bind gene targetsinvolved in endocycle control. (a) Immunoblot of transfected HepG2cells showing exogenous expression of Flag-tagged E2F7 and E2F8proteins with tubulin as the control. The arrows indicate tagged protein.(b) Representative ChIP assay in transfected HepG2 cells with anti-Flagantibodies demonstrating enhanced occupancy of Flag-tagged E2F7 andE2F8 proteins on E2F-binding sites in the promoter of G1/S (E2f1and Ccne2 ) and G2/M (Ccna2 and Chek1) genes. The tubulin (Tub)gene, a non-E2F target, demonstrates specific recruitment of Flag-taggedE2F7 and E2F8 to target promoters containing consensus E2F-bindingsequences. n = 3 independent transfection–ChIP experiments performed.(c) Immunoblot of transfected Rcho-1 trophoblast stem (TS) cells showingexogenous expression of Flag-tagged E2F7 and E2F8 proteins with tubulinas the control. The arrows indicate tagged protein. (d) RepresentativeChIP assays in transfected Rcho-1 trophoblast stem cells of G1/S and
G2/M genes as in HepG2 cells. n = 3 independent transfection–ChIPexperiments performed. (e) Immunoblot of liver lysates from 6-week-oldmice demonstrating expression of tagged E2F8 protein. A total of 10 µgof plasmid DNA was delivered through tail vein (TV) injection. Lysateswere prepared from livers 24h after injection. Tubulin served as a loadingcontrol. n = 3 and n = 5 wild-type mice were injected with control(empty) plasmid and plasmid expressing Flag–E2F8, respectively. Oneout of 5 had detectable expression of Flag-tagged E2F8 as shown bywestern blotting. (f) ChIP assay in liver lysates with anti-Flag antibodiesdemonstrating enhanced occupancy of Flag-tagged E2F8 on E2F-bindingsites in the promoter of G1/S (E2f1 and Ccne2 ) and G2/M (Cdk1) genes.a–f Con., empty plasmid; F7, plasmid expressing Flag-tagged E2F7; F8,plasmid expressing Flag-tagged E2F8. Data in b,d,f from the representativeexperiment are average values from triplicate quantitative rtPCR reactions±s.d. Uncropped images of blots are shown in Supplementary Fig. SXX.
cell-cycle- and G2/M-related functions in TGCs from E10.5 wild-type,1
E2f7−/−, E2f8−/− and E2f7−/−;E2f8−/− placentae. As shown in Fig. 4b,2
66 of the 99 genes interrogated had predominantly increased expression3
in E2f7/E2f8-deficient TGCs, suggesting a commonmechanism for how4
E2F7 and E2F8may regulate endocycles in TGCs and hepatocytes.5
A significant portion of common upregulated genes in E2f7/E2f8-6
deficient TGCs and hepatocytes have E2F-binding elements on their7
promoters, raising the possibility that these may represent direct8
targets of E2F7/E2F8. To test this hypothesis, we transfected human9
HepG2 cells and rat Rcho-1 trophoblast stem cells with plasmids10
expressing Flag-tagged E2F7 or E2F8 and performed chromatin11
immunoprecipitation (ChIP) assays with anti-Flag antibodies. As12
shown in Fig. 5b,d, Flag-specific antibodies, but not control IgG,13
co-immunoprecipitated promoter sequences containing E2F-binding 14
elements, but not irrelevant sequences lacking E2F-binding sites (Tub). 15
The above results were corroborated by further ChIP assays in human 16
embryonic kidney (HEK) 293 cells transfected with either Flag-tagged 17
E2F7 or E2F7 (Supplementary Fig. S5d) and in wild-type liver tissues 18
transfected by hydrodynamic-based methods with plasmids expressing 19
Flag-tagged E2F8 (Fig. 5e,f). Together, these results suggest that E2F7 20
and E2F8 repress a core group of genes with prominent involvement in 21
the regulation of G2/M-related events. 22
E2F1 functionally opposes atypical E2F7/E2F8 23
Given that ablation of E2f1–3 and E2f7/E2f8 led to opposite effects 24
on hepatocyte ploidy, we explored the possibility of cross-regulation 25
NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 7
ART I C L E S
1ko Alb-178tkoAlb-78dkoControl
6 m
o
H &
E
Num
ber
of
hep
. p
er
×40
fie
ld
Perc
en
tag
e o
f h
ep
atic m
ass
Perc
enta
ge o
f liv
er
nucle
i g
ate
d
Control n = 4
Alb-178tko n = 3
1ko n = 5Alb-78dko n = 5
∗∗
∗∗ ∗ ∗∗∗
∗
∗∗∗∗∗
∗
∗∗∗
∗∗∗
Perc
en
tag
e o
f T
GC
s w
ith
estim
ate
d p
loid
y
Perc
enta
ge o
f p
ositiv
e
TG
Cs ∗∗∗
E1
0.5
H
& E
Plf-178tko
Perc
en
tag
e o
f p
ositiv
e
hep
ato
cyte
s
Perc
en
tag
e o
f
bin
ucela
ted
hep
.
Control Plf-78dko1ko
20
40
0
60Control n = 3
1ko n = 3Plf-78dko n = 3
Plf-178tko n = 3
45
0
36
27
18
9
35
0
28
14
7
25
0
20
15
10
5
60
0
40
20
BrdU CycA2P-H3Karyok.
7
0
6
5
4
3
2
1
400
200
0
300
100
100
75
50
0
25
4C 8C 16C2C 32C
45
0
36
27
18
9
3.0
0
1.5
1.4
0
0.7
1.2
0
0.6
1.0
0
0.5
2-4C 8C 16C 32C 64C 128C 256C 512C 1,024C
Ki67 P-H3 CycB1CycA2
a
c
d
b
e
g h
f
Figure 6 Loss of E2f1 restores endocycles in E2f7/E2f8 deficient TGCsand hepatocytes. (a) Representative H&E-stained sections of E10.5 TGCs.The lower left insets show higher magnification views of the areas outlinedin the main panels. (b) Feulgen quantification of genome ploidy inE10.5 TGCs; n = 3 placentae per genetic group analysed. Note thesignificant increase in the proportions of 64C and 128C Plf-178tko TGCsrelative to Plf-78dko TGCs. (c) Quantification of E10.5 TGCs undergoingkaryokinesis and expressing S/M phase markers; n = 3 placentae pergenetic group analysed. Note the significant reduction in the proportionof Plf-178tko TGCs undergoing karyokinesis relative to Plf-78dko TGCs.(d) Representative H&E-stained liver sections from 6-month-old mice.(e) Measurements showing no significant change in the percentageof hepatic mass in 6-month-old mice and a significant decrease inthe number of hepatocytes per image field in Alb-178tko relative to
Alb-78dko livers, with n per genetic group analysed as indicated inf. (f) Flow cytometry of liver nuclei in 6-month-old mice, with n pergenetic group analysed as indicated in the legend. Note the significantincrease and decrease in the proportion of 4C and 2C nuclei, respectively,in Alb-178tko livers relative to Alb-78dko livers. (g) Quantification ofbinucleated hepatocytes in 2-month-old livers, with n per genetic groupanalysed as indicated in f. (h) Quantification of hepatocyte proliferationin 2-month-old livers, with n per genetic group analysed as indicatedin Fig. 6f. a–c Control, E2f7+/+;E2f8+/+; d–h Control, E2f7f / f;E2f8f / f;1ko, E2f1−/−; Plf-178tko, Plfcre/+;E2f1−/−;E2f7f /f;E2f8f / f; Alb-178tko,Alb–cre;E2f1−/−;E2f7f / f;E2f8f / f. Data in b,c,e–h reported as average± s.d.b,f one-way ANOVA, ∗P ≤0.05;∗∗P ≤0.01; ∗∗∗P ≤0.001. c,e,g,h two-tailedStudent t -test, ∗P ≤0.05; ∗∗P ≤0.01; ∗∗∗P ≤0.001. Scale bars, 12.5 µm(a) and 10 µm (d).
between these two E2F subclasses. Loss of E2f1–3 did not impact1
the expression of other E2Fs (E2f4-8; see Supplementary Fig. S1e);2
however, loss of E2f7/E2f8 resulted in a transient increase of E2f13
expression soon after birth in the liver (Supplementary Fig. S5e).4
Interestingly, loss of E2f7/E2f8 had no significant effect on the5
expression of E2f1–6 in TGCs (Supplementary Fig. S5f). We then6
profiled global gene expression in hepatocytes lacking the three E2F7
activators (Mx-123tko). Strikingly, this analysis showed that most genes8
upregulated in E2f7/E2f8-deficient hepatocytes were downregulated9
in E2f1–3-deficient hepatocytes (Fig. 4c), and many of these genes10
had annotated cell-cycle functions related to G2/M transitions or11
mitoses and had E2F-binding sites in their promoters (Fig. 4d and12
Supplementary Fig. S5g,h).13
On the basis of the above results we considered the hypothesis14
that canonical E2F activators may functionally antagonize atypical15
E2F repressors in the control of endocycles. To rigorously test this16
possibility we generated and analysed mice with TGCs or hepatocytes17
deficient for E2F7, E2F8 and E2F1 (E2f1−/−). The ablation of E2f7f / f 18
and E2f8f / f alleles in TGCs was achieved by Plfcre/+ (Plf-178tko) and 19
their ablation in hepatocytes was achieved by Alb–cre (Alb-178tko). 20
Placenta sections from E10.5 Plf-178tko embryos exhibited TGCs 21
with large nuclei having increased ploidy (Fig. 6a), with some TGCs 22
having 128C and 256C genomes (Fig. 6b), which was never observed 23
in Plf-78dko TGCs. This increase in ploidy was accompanied by 24
a significant decrease in the number of multinucleated TGCs and 25
a slight decrease in BrdU- and P-H3-positive cells, even though 26
the number of cyclin-A2-positive TGCs was not reduced (Fig. 6c). 27
Similar results were obtained when placentae globally deleted for 28
E2f1, E2f7 and E2f8 (178tko) were examined (Supplementary Fig. 29
S6a–c). Evaluation of Alb-178tko livers revealed larger hepatocytes 30
containing greater DNA content than in Alb-78dko livers, with 31
some reaching 8C ploidy (Fig. 6d–f). The Alb-178tko livers also 32
had many more binucleated hepatocytes (Fig. 6g), and importantly, 33
had fewer Ki67-, P-H3- and cyclin-B1-positive hepatocytes, even 34
8 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION
ART I C L E S
Perc
enta
ge o
f T
GC
s w
ith
estim
ate
d p
loid
yP
erc
en
tag
e o
f h
ep
. w
ith
estim
ate
d p
loid
y
Control n = 3
Plf-78dko n = 3Plf-A12dko n = 3
Plf-A1278qko n = 3
E10.5
H &
E
Plf-78dkoControl Plf-A12dko Plf-A1278qko
Liv
er
H &
ELiv
er
Feulg
en
Control n = 2
Alb-78dko n = 2Alb-A12dko n = 3
Alb-A1278qko n = 3
20
40
0
60
∗∗∗
∗∗∗
∗∗∗
∗∗∗ ∗∗∗
2–4C 8C 16C 32C 64C 128C 256C 512C 1,024C
Alb-A12dko Alb-A1278qko Alb-78dkoControl
100
0
75
50
25
2–4C 8C 16C 32C 64C
a b
c
d
Figure 7 Cyclin A ablation reinstates genome ploidy of E2f7/E2f8 -deficientTGCs and hepatocytes. (a) Images of H&E-stained E10.5 TGCs. Thecombined loss of Ccna1 and Ccna2 (Plf-A12dko) led to TGCs similarin appearance to control TGCs. The further loss of Ccna1 and Ccna2in E2f7/E2f8 -deficient TGCs (Plf-A1278qko) led to TGCs with anormal (wild-type-like) appearance, in contrast to Plf-78dko TGCsthat seemed small or were binucleated (yellow arrows). The lower leftinsets show higher magnifications of the areas outlined in the mainpanels. (b) Feulgen quantification of genome ploidy in E10.5 TGCs. Theintensities (that is, estimated genome content) of 120–140 TGCs werequantified per placenta sample, with n placenta samples analysed pergenetic group as indicated. A number of TGCs quadruply deficient forCcna1, Ccna2, E2f7 and E2f8 reach ploidy levels of 128C and 256C.Control, E2f7f / f;E2f8f / f; Plf-A12, Plfcre/+;Ccna1−/−;Ccna2f / f; Plf-78dko,Plfcre/+;E2f7f / f;E2f8f / f;Plf-A1278qko, Plfcre/+;Ccna1−/−;Ccna2f / f;E2f7f / f;E2f8f / f. (c) Images of H&E-stained 3-month-old liver sections, which
were stained with the Feulgen technique to facilitate quantification of DNAcontent, showing an increase in both cellular and nuclear size of hepatocyteslacking Ccna1 and Ccna2 (Alb-A12dko) when compared with controlhepatocytes. Hepatocytes quadruply deficient for Ccna1, Ccna2, E2f7 andE2f8 (Alb-A1278qko) had nuclei resembling control hepatocytes. (d) Feulgenquantification of genome ploidy in livers of 3-month-old mice. The intensities(that is, estimated genome content) of 100 hepatocyte nuclei were quantifiedper liver sample, with n liver samples analysed per genetic group as indicated.Ccna1/Ccna2 -deficient livers had an increased proportion of hepatocyteswith higher ploidy levels (16C, 32C and 64C) relative to control livers.Quadruple deficient livers had a significantly elevated number of hepatocyteswith 16C genomes relative to Alb-78dko livers. Control, E2f7f /f;E2f8f / f;Alb-A12,Alb–cre;Ccna1−/−;Ccna2f / f; Alb-78dko, Alb–cre;E2f7f /f;E2f8f / f;Alb-A1278qko, Alb–cre;Ccna1−/−;Ccna2f / f;E2f7f / f;E2f8f / f. Data in b,dare average values, ±s.d. are included where n >2 samples were analysed.One-way ANOVA, ∗P ≤0.05; ∗∗∗P ≤0.001. Scale bars, 12.5 µm.
though again, the percentage of cyclin-A2-positive cells was not1
reduced (Fig. 6h). Thus, it seems that loss of E2f1 suppressed some,2
but not all, mitotic defects in TGCs and hepatocytes caused by3
E2f7/E2f8 deficiency.4
Loss of cyclin A2 reinstates a G2/M block and polyploidy in5
E2f7/E2f8 -deficient cells6
As the inactivation of E2f7/E2f8 results in ectopic mitoses without7
inhibiting DNA replication, we predicted that re-establishing a8
mitotic block would restore polyploidy in E2f7/E2f8-deficient cells.9
Previous analysis of trophoblast stem cell cultures showed that p57KIP210
activity participates in driving TGC differentiation and endocycles11
by inhibiting Cdk1 function37. However, we failed to detect any12
measurable change in p57KIP2 mRNA or protein levels that might13
underlie the phenotype of E2f7/E2f8-deficient TGCs or hepatocytes14
(Supplementary Fig. S6d). Given that cyclin A activity is critical15
for G2/M progression by regulating the APC (also known as theQ5 16
cyclosome)38, we sought to evaluate the consequence of inactivating17
cyclin A1/A2 in E2f7/E2f8-deficient TGCs and hepatocytes. We thus18
intercrossed Ccna1−/−;Ccna2f / f mice39 with Plfcre/+;E2f7f /f;E2f8f / f and19
generated embryos with quadruply deficient TGCs (Plf-A1278qko).20
Remarkably, TGCs in E10.5 Plf-A1278qko embryos had larger nuclei21
with greater DNA content than TGCs in Plf-78dko embryos, with some22
reaching 1024C (Fig. 7a,b). We also intercrossed Ccna1−/−;Ccna2f / f 23
mice with Alb–cre;E2f7f / f;E2f8f / f mice to generate offspring with 24
quadruply deficient hepatocytes (Alb-A1278qko). As shown in Fig. 7d, 25
hepatocytes in 3-month-old Alb-A1278qko mice had similar ploidy 26
levels as in age-matched wild-type mice. Interestingly, the loss of 27
Ccna1/Ccna2 alone (Alb-A12dko) in hepatocytes resulted in hyperploidy 28
(Fig. 7c,d). In summary, inhibiting the transcriptional network that 29
signals G2/M progression (by E2f1 inactivation) or interfering with 30
the mitotic machinery (by Ccna1/Ccna2 inactivation) re-established 31
a mitotic block and reinstated higher levels of polyploidy in 32
E2f7/E2f8-deficient cells. 33
DISCUSSION 34
In multi-cellular organisms there exists diversity in the kinetics and 35
composition of cell cycles and, presumably, in the mechanisms that 36
regulate them. The endocycle is commonly used in flies and plants and, 37
hence, more is known about how they are controlled in these organisms 38
than in mammals40–45. The balanced action of the E2F activator (dE2f1) 39
and repressor (dE2f2) in Drosophila is critical for the tight regulation of 40
cyclin E expression and successful completion of S phase in mitotic and 41
endocycling cells21,42,43. Although E2Fs are also involved in controlling 42
endocycles in plants, their role seems to be different from in mammals. 43
For example, the atypical E2Fe repressor in Arabidopsis is required to 44
NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 9
ART I C L E S
prevent endocycles44, whereas we show that the atypical E2F7 and E2F81
family members in mice promote endocycles. A comparison of the2
regulation of how atypical E2Fs are expressed during development3
and identification of their direct targets might shed light on these4
differences. In summary, our present work exposes an intricate E2F5
network involving balanced and antagonistic activities of canonical E2F6
activators (E2F1–3) and atypical E2F repressors (E2F7-8) that controls7
mammalian endocycles. Perhaps the most pronounced manifestation8
of altering the balance in the E2F network is the ectopic mitosis and9
karyokinesis observed in E2f7/E2f8-deficient TGCs.10
The results presented here expose two distinct arms of the E2F11
network, one arm involving canonical activators (E2F1–3) and a12
second involving atypical repressors (E2F7/E2F8), that converge to13
antagonistically regulate a common gene expression program critical14
for mammalian endocycles in vivo. A similar antagonistic role for E2F15
activators and atypical repressors seems to be at play in the control of a16
distinct program that is critical for mitotic cell cycles during placental17
development32. As loss of E2f4 or E2f5 in mice12–14 did not seem toQ6 18
affect the polyploidization of TGCs and hepatocytes (Supplementary19
Fig. S2c and data not shown) we propose that the most ancient20
E2F repressor arm consisting of atypical E2f7 and E2f8 evolved to21
specifically regulate variant cell cycles inmetazoans in amanner distinct22
from the function of canonical E2F repressor proteins. One reason23
for this might be to keep E2F7/E2F8-mediated repression outside24
the influence of the cyclin–CDK axis. As E2F7/E2F8 DNA-binding25
activity is independent of DP proteins, atypical repressors would26
remain unresponsive to CDK-mediated phosphorylation of DP, which27
negatively regulates the DNA binding and functions of canonical28
repressors45–47, allowing repression at a time (S/G2) when CDK29
activity is high. However, the exact molecular mechanism of how30
canonical activators and atypical repressors coordinate gene expression31
in endocycling tissues remains obscure.32
It has been assumed that polyploidy in mammals, as in flies and33
plants, is integral to the physiologic functions of placentae and livers.34
Here we generated mouse models with significantly reduced TGC35
and hepatocyte ploidy to assess this hypothesis. Surprisingly, TGCs36
and hepatocytes having significantly restricted ploidy levels seemed to37
impart placentae and livers, respectively, with adequate organ function38
to carry animals through embryonic and adult development. Whether39
altered liver ploidy in mice impacts ageing-related processes such as40
tumour development remains to be evaluated. In summary, genetic,41
systems-based and biochemical approaches revealed a fundamental42
role for the canonical activator and atypical repressor arms of the E2F43
network in orchestrating mammalian endocycles in vivo. �44
METHODS45
Methods and any associated references are available in the online46
version of the paper.47
Note: Supplementary Information is available in the online version of the paper48
ACKNOWLEDGEMENTS49
We thank L. Rawahneh, J. Moffitt and N. Lovett for excellent technical assistance50
with histology, and P. Wenzel for generating and collecting 123tko placentae.51
We also thank these individuals from OSUCCC Shared Resources: T. Wise, J.52
Palatini, H. Alders, P. Yan, P. Fada and B. Rodriguez (Microarray and Nucleic Acid53
Shared Resources); B. McElwain (Analytic Cytometry); R. Burry, K. Wolken, and B.54
Kemmenoe (Microscopy and Imaging); and K. La Perle (Comparative Pathology
and Mouse Phenotyping). We are grateful for PL-1 antibodies provided by F. 55
Talamantes, Rcho-1 trophoblast stem cells from M. Soares and HepG2 cells from Q7 56
S. Jacobs. This work was funded by NIH grants to G.L. (R01CA85619, R01CA82259, 57
R01HD047470, P01CA097189) and P.S. (R01CA132740). G.L. is a recipient of 58
The Pew Charitable Trust Scholar Award and the Leukemia & Lymphoma Society 59
Scholar Award. H-Z.C., M.M., S.S., S.R. and T. P. are recipients of the Pelotonia 60
Fellowship Award. 61
AUTHOR CONTRIBUTIONS 62
H-Z.C., M.M.O., J.L. and G.L. designed the experiments. H-Z.C., M.M.O., J.L., 63
T.P., V.C., L.K. and S.B. performed experiments. H-Z.C. and M.M.O. co-wrote the 64
paper with G.L.; all other authors listed helped perform experiments. Specifically, 65
J.C.T., S.R., S.S., M.L., R.M. and K.H. helped with experiments relating to 66
confocal microscopy and 3D reconstruction. X.M. and S.F. helped analyse data and 67
performed statistics. I.K., D.J.W. and P.S. provided mice for the study and reviewed 68
drafts of the manuscript. T.H., B.L. and V.J. contributed to discussion relating to 69
Affymetrix/NanoString analyses. 70
COMPETING FINANCIAL INTERESTS 71
The authors declare no competing financial interests. Q8 72
Published online at www.nature.com/doifinder/10.1038/ncb2595 73
Reprints and permissions information is available online at www.nature.com/reprints 74
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NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 11
METHODS DOI: 10.1038/ncb2595
METHODS1
Mouse husbandry and genotyping. All mice were housed in barrier conditions2
with a 12-h light/dark cycle and had access to food/water ad libitum. Genotyping3
primers are provided in Supplementary Table S1.4
Feulgen analysis. Placenta/liver sections of 5 µm were treated with 1N hy-5
drochloric acid and stained with Schiff reagent. Cytoplasm was counterstained6
with aqueous light green (1%). Nuclear intensity was quantified using ImageJ7
(http://rsbweb.nih.gov/ij/; ref. 48). TGC ploidy was expressed relative to 2C–4C8
labrynthine trophoblast cells. For binucleated TGCs, ploidy levels of individual9
nuclei were summed to derive cumulative ploidy. Hepatocyte ploidy was expressed10
relative to 2C–4C hepatocytes deficient for E2f7/EfF8. Hepatocyte ploidy was11
expressed on a per nucleus basis.12
Flow cytometry analysis. Nuclei suspensions were obtained from frozen liver13
tissue as previously described49. The total DNA content of a minimum of 30,00014
nuclei per liver samplewas analysed using LSR II (BDBiosciences). Cell cycle profiles15
were generated using FlowJo (Tree Star).16
Affymetrix analyses. Total RNA was extracted from a minimum of 3 livers per17
genetic group using TRIzol (Invitrogen). The RNA quality was verified using an18
Agilent 2100 Bioanalyzer, and samples were submitted to the OSUCCCMicroarray19
Shared Resource facility for biotinylated complementary RNA generation and20
MOE4302.0 hybridization. AGeneChip Scanner 3000 (Affymetrix)was used for data21
acquisition. Pre-processing of raw signal intensities was performed using the robust22
multi-chip average method50. BRB-Array Tools 3.8.1 was then used to identify23
differentially expressed genes51. For heat map images, the geometric mean of genes24
after robust multi-chip average normalization from control liver samples was set25
as a reference, with colours corresponding to the ratio between expression of genes26
in mutant samples and the reference. Functional annotation was performed using27
Ingenuity Pathway Analysis (Ingenuity Systems).28
NanoString analyses. From each tissue sample, total RNA (200 ng) was profiled29
on a custom code set containing 200 selected genes following the manufacturer’s30
instructions (Supplementary Table S3). For TGC gene expression analysis, we31
isolated TGCs from frozen placenta sections using laser capture microdissection32
(PALM MicroLaser system). Total RNA was then extracted using the Arcturus33
PicoPure RNA Isolation Kit (Applied Biosystems 12204-01). For validation of34
gene expression changes in the liver (Supplementary Fig. S4a), the same set of35
TRIzol-isolated RNA was used in both Affymetrix and NanoString assays. The36
NanoString data are first normalized to the geometric mean of positive control spike37
counts in each lane of the cartridge, followed by subtracting the minimum negative38
control spike counts in each lane to remove the background. Finally, expression39
of each gene in the code set is normalized with respect to the geometric mean of40
the housekeeping genes. Heat maps were generated in the same manner as for the41
Affymetrix gene expression data.42
Confocal microscopy and 3D reconstruction. Tissues sections of 70 µm were43
stained with 1mM Draq5 (Biostatus) at 4 ◦C overnight. Sections were examined44
by confocal laser scanning microscopy (Zeiss LSM 510) at 63× and 0.7× scanQ9 45
zoom. Optical sections with between-plane-plane resolution of 0.40 µm and axial46
resolution of 0.42 µm were acquired. Four to five non-overlapping regions per47
placenta were imaged with a field view of 207 µm×207 µmand a depth of 40±2 µm,48
resulting in a stack of 100 images for each region. This process was identically49
repeated for liver samples. For 3D reconstruction, the image stacks were processed50
using Insight Toolkit and ITK-SNAP (ref. 52). Each nucleus was segmented by active51
contour segmentation using region competition as the stopping criterion53. Nuclei52
of binucleated TGCs were constructed separately and assigned different colours.53
Only the volumes of individual hepatocyte nuclei were measured.54
Transmission electron microscopy. Placentae were fixed/processed following a55
standard protocol (http://cmif.osu.edu/419.cfm) and embedded in Eponate resin.56
Placental cross-sections of 70 nm were mounted on copper grids, double stained57
with uranyl acetate and lead citrate, and observed with a Technai G2 Spirit58
transmission electron microscope (FEI).59
Transient transfections. For HepG2, subconfluent cells were transfected with60
1:3 ratio of plasmid DNA/reagent (X-tremeGENE HP DNA Transfection Reagent61
(Roche)) in 10% FBS RPMI-1640. Cells were collected 48 h after transfection. In62
one experiment, three 100 cm plates of transfected cells were combined for lysis and63
sonication as one sample in the ChIP assay. For Rcho-1, transfection conditions64
were the same as described for HepG2 cells. Rcho-1 cells were maintained as65
previously described54. In one experiment, two 150 cm plates were combined as66
one sample used in the ChIP. A total of three independent experiments for each67
cell line was performed. For HEK293 cells, subconfluent cells in 10% FBS DMEM
were transfected using calcium phosphate with 25 µg of plasmid DNA expressing 68
wild-type Flag–E2F7/Flag–E2F8 or mutant Flag–E2F7/Flag–E2F8. 69
Systemic administration of plasmid DNA. Six-week-old wild-type male mice 70
were injected with either sterile PBS or sterile PBS containing 10 µg plasmid DNA 71
through the tail vein following a published protocol55. Mice were euthanized by CO2 72
asphyxiation 24 h after tail-vein injection. Whole-body perfusion with warm saline 73
was performedusing the intracardiacmethod before excision of the liver, subsequent 74
derivation of a single-celled suspension by passing the perfused tissue through a 75
40 µmmesh and fixation/processing for ChIP assays. 76
ChIP assays. For HepG2 and Rcho-1 trophoblast stem cell lines, transfected 77
cells were crosslinked in growth media containing 1% formaldehyde followed 78
by neutralization with 1.25M glycine. The cytoplasmic fraction was discarded 79
(cytoplasmic lysis buffer: 5mM PIPES pH 8.0, 85mM KCl, 0.5% NP40 and fresh 80
protease inhibitors). Nuclear lysates were collected by incubating the nuclei pellet in 81
lysis buffer containing 50mM Tris at pH 8.0, 10mM EDTA, 0.3% SDS and a fresh 82
cocktail of protease/phosphatase inhibitors. Sonicated chromatin (∼100–300 base 83
pairs (bp)) was incubated with 10 µg of anti-Flag mouse monoclonal antibodies 84
(Sigma F1804) or normal mouse IgG (Santa Cruz sc-2025) at 4 ◦C overnight. 85
Antibody–protein–DNA complexes were recovered by the addition of 50% protein 86
G agarose slurry (Millipore 16-266) pre-cleared with 10mgml−1 transfer RNA and 87
10mgml−1 BSA. De-crosslinking was performed at 65 ◦C overnight with periodic 88
agitation. Recovered DNA was purified using the Qiagen PCR extraction kit. 5% 89
input and 2 µl of eluted DNA was used for quantitative rtPCR with gene-specific Q10 90
primers and SYBR green (BioRad 170-8880). For HEK293 cells, the EZ-CHIP assay 91
kit (Millipore 17-371)was used following themanufacturer’s instructions. Sonicated 92
chromatin was incubated with 2 µg of anti-Flag antibodies (Sigma F1804) or 93
normal mouse IgG (Santa Cruz sc-2025) at 4 ◦C overnight. Antibody–protein–DNA 94
complexes were recovered by the addition of salmon sperm DNA/protein G agarose 95
slurry (Millipore 16-201). Immunoprecipitated DNA fractions were de-crosslinked 96
at 65 ◦C overnight and purified using QIAquick columns (QIAGEN). Quantitative 97
rtPCR was performed as above except 1% input was used. All quantitative rtPCRs 98
were performed in triplicate using the following conditions: 1 cycle at 95 ◦C 5min, 99
followed by 20–40 cycles of 95 ◦C 15 s, 60–64 ◦C 30 s and 72 ◦C 30 s. 100
Immunoblot assays. Whole-cell lysates were prepared from lysis of HepG2 and 101
Rcho-1 trophoblast stem cells in a standard radioimmunoprecipitation buffer 102
containing a freshly diluted cocktail of protease and phosphatase inhibitors. For 103
preparation of liver lysates, frozen tissue (∼100mg) was thawed on ice followed 104
by homogenization in radioimmunoprecipitation buffer and then centrifugation 105
at maximum speed for 15min in 4 ◦C. Total protein concentration was measured 106
using the Bio-Rad Protein Assay kit (BioRad 500-0006). Denatured samples were 107
separated in 8–10% SDS–polyacrylamide gels, transferred onto PDVF membranes, 108
blocked in 5%milk–PBS solution and incubated overnight in 4 ◦Cwith the following 109
primary antibodies: anti-E2F3 1:5,000 (Santa Cruz sc-878x), anti-Flag 1:1,000 110
(Sigma F1804), anti-tubulin 1:5,000 (Sigma T6199) and anti-HSP70 1:5,000 (BD 111
Transduction Laboratories 610608). HRP-conjugated species-specific secondary 112
antibodies were applied to membranes followed by detection with the SuperSignal 113
West Femto kit (Thermo Scientific 34095). 114
Immunohistochemistry and immunofluoresence microscopy. After antigen 115
retrieval using Target Retrieval Solution (DAKO S1699), 5 µm deparaffinized 116
sections of placenta or liver tissues were incubated with primary antibodies at 117
4 ◦C overnight against E2F7 1:100 (Abcam ab56022), E2F8 1:50 (polyclonal against 118
residues 576–595 of murine E2F8), BrdU 1:100 (DAKOMO-0744), Ki67 1:100 (BD 119
Pharmingen 550609), phospho-histone 3 (Ser10) 1:100 (Millipore 06-570), cyclin 120
A2 1:100 (Santa Cruz sc-596), cyclin B1 1:100 (Santa Cruz sc-752), E-cadherin 1:100 121
(Abcam ab53033), p57Kip2 1:100 (Santa Cruz sc-8298; refs 56,57) and placental 122
lactogen 1:200 (PL-1, F. Talamantes). TGCs in M phase (Fig. 2h) were identified 123
by detecting P-H3 or the lineage-specific PL-1 protein. For immunofluorescence 124
signal detection, fluorophore-conjugated secondary antibodies were applied to the 125
slides. For immunohistochemistry, the VECTASTAINABC systemwas used (Vector 126
Laboratories). 127
BrdU incorporation assays. Pregnant females (E10.5) received a single intraperi- 128
toneal injection of BrdU (Sigma B5002) in sterile PBS at 100 µg g−1 body weight 1 h 129
before being euthanized for collection of placentae. Deparaffinized tissue sections of 130
5 µm were used for BrdU detection after incubation in 2N HCl and neutralization 131
with 10mM sodium borate (pH 8.0). Primary antibody was then applied overnight. 132
pIpC injections. Six-week-old mice received 5 intraperitoneal injections of 250 µg 133
of pIpC (Sigma P1530) dissolved in sterile PBS every alternate day. Mice were 134
euthanized 24 h after the last injection, and their livers were collected for flow 135
cytometry and microarray analyses. 136
NATURE CELL BIOLOGY
DOI: 10.1038/ncb2595 METHODS
Carbon tetrachloride injection. Six-month-old mice received a single intraperi-1
toneal injection of 10% carbon tetrachloride (Sigma 319961) dissolved in corn oil.2
Mice were euthanized at 0, 24, 48, 60 and 168 h (when liver regeneration is complete3
in rodents58) post-injection. Mice were administered an intraperitoneal injection of4
BrdU solution (100 µg g−1 body weight) to label hepatocytes in S phase of the cell5
cycle 1 h before being euthanized.6
Tamoxifen induction of cre. Tamoxifen chow (Teklad Lab Animal Diets, Harlan7
Laboratories) was prepared by mixing 3 kg of irradiated standard chow with 1 g8
tamoxifen (Sigma T5648). For pre-weaning induction of cre, 7-day-old pups and9
theirmother were switched from regular to tamoxifen chow for 1 week. After 1 week,10
they were again fed regular chow until weaning and up to time of euthanization (age11
2 months). For post-weaning induction of cre, pups were weaned from their mother12
at age 21 days and maintained on tamoxifen chow starting from age 28 days up to13
age 2 months. Tamoxifen chow was stored in the dark at 4 ◦C and changed weekly.14
It was provided to mice at 40–80mg kg−1 body weight59.15
X-gal staining. OCT-embedded placentae were sectioned and fixed in 0.2%16
PBS–glutaraldehyde solution (1.25mMEGTA at pH 7.3 and 2mMMgCl2). Sections17
were rinsed with wash buffer (2mMMgCl2, 0.01% sodium deoxycholate and 0.02%18
IGEPAL CA-630 (Sigma I8896)) and stained in LacZ solution (4mM potassium19
ferricyanide, 4mM potassium ferrocyanide and 1mgml−1 X-gal in wash buffer)20
at 37 ◦C overnight with protection from light. Nuclear Fast red was used as a21
counterstain.22
E2F-binding-site analysis. Promoter sequences (−3000 bp ∼+2000 bp) of genes23
antagonistically regulated by E2F1–3 and E2F7/E2F8 were obtained from the24
UCSC genome browser (http://genome.ucsc.edu/). consensus E2F-binding sites25
were identified using TFSearch (http://www.cbrc.jp/research/db/TFSEARCH.html).Q11 26
Statistical analyses. Binucleated TGCs, as a percentage of a minimum of 143 total27
TGCs assessed in each genetic group, were quantified in confocal images using the28
LSM image browser (Zeiss). All other immunohistochemistry/immunofluorescence-29
microscopy-associated quantification of TGCs/hepatocytes was performed using30
Metamorph 6.1 in photomerged images (placenta) or 5–7 random images (liver)31
acquired at 40× from a minimum of three sections per sample. The results are32
reported as an average percentage± s.d. of positive cells in the number (n) ofQ12 33
control and mutant samples analysed. Flow cytometry profiles (for example, Fig. 3f)34
are representative of a minimum of three independent experiments per genotype35
and age group. Flow cytometry results are reported as an average percentage± s.d.
of nuclei with a certain DNA content out of a minimum of 30,000 nuclei gated 36
per sample in n control and mutant samples analysed. Pairwise comparisons 37
were evaluated by two-tailed Student’s t -tests, whereas multiple comparisons were 38
evaluated by one-way analysis of variance (ANOVA). When appropriate, P values 39
were adjusted by the Holm’s method within each ploidy category. 40
Accession numbers. The gene expression microarray data used in this study can 41
be found in GEO using accession numbers GSE32353, GSE32354 and GSE32355. 42
48. Hardie, D. C., Gregory, T. R. & Hebert, P. D. From pixels to picograms: a beginners’ 43
guide to genome quantification by Feulgen image analysis densitometry. J. 44
Histochem. Cytochem. 50, 735–749 (2002). 45
49. Mayhew, C. N. et al. Liver-specific pRB loss results in ectopic cell cycle entry and 46
aberrant ploidy. Cancer Res. 65, 4568–4577 (2005). 47Q1350. Irizarry, R. A. et al. Summaries of Affymetrix GeneChip probe level data. Nucl. Acids 48
Res. 31, e15 (2003). 49
51. Bolstad, B. M., Irizarry, R. A., Astrand, M. & Speed, T. P. A Comparison of 50
normalization methods for high density oligonucleotide array data based on bias 51
and variance. Bioinformatics 19, 185–193 (2003). 52
52. Yushkevich, P. A. et al. User-guided 3D active contour segmentation of anatomical 53
structures: significantly improved efficiency and reliability. NeuroImage 31, 54
1116–1128 (2006). 55
53. Zhu, S. C. & Yuille, A. Region competition: unifying snakes, region growing, 56
and bayes/mdl for multiband image segmentation. IEEE Trans. Pattern Anal. 18, 57
884–900 (1996). 58
54. Sahgal, N., Canham, L. N., Canham, B. & Soares, M. J. Rcho-1 trophoblast stem 59
cells: a model system for studying trophoblast cell differentiation.Methods Mol. Med. 60
121, 159–178 (2006). 61
55. Liu, F., Song, Y. K. & Liu, D. Hydrodynamic-based transfection in animal by systemic 62
administration of plasmid DNA. Gene Ther. 6, 1258–1266 (1999). 63
56. Ullah, Z., Kohn, M. J., Yagi, R., Vassilev, L. T. & DePamphilis, M. L. Differentiation 64
of trophoblast stem cells into giant cells is triggered by p57/Kip2 inhibition of CDK1 65
activity. Gen. Dev. 22, 3204–3236 (2008). 66
57. Nagahama, H. et al. Spatial and temporal expression patterns of the cyclin- 67
dependent kinase (CDK) inhibitors p27Kip1 and p57Kip2 during mouse 68
development. Anat. Embryol. 203, 77–87 (2001). 69
58. Schultze, B., Gerhard, H. & Maurer, W. Liver Regeneration after Experimental Injury 70
(Grune and Stratton, 1975). 71
59. Kiermayer, C., Conrad, M., Schneider, M., Schmidt, J. & Brielmeier, M. Optimization 72
of spatiotemporal gene inactivation in mouse heart by oral application of tamoxifen 73
citrate. Genesis 45, 11–16 (2007). 74
NATURE CELL BIOLOGY
Page 1
Query 1:Further information given in the corresponding
author address not included in affiliation 1. OK?
Page 2
Query 2:Should ‘independent’ be ‘independently’ here?
Query 3:‘novel’ changed to ‘new’ here, and deleted from
before ‘lineage-specific’ in the third sentence of thefirst paragraph, according to style. OK?Query 4:‘while’ changed to ‘and’ here. OK (or is, for example,
‘whereas’ more appropriate)?
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Query 5:Text amended to ‘APC (also known as the
cyclosome)’ here, according to style. Please check.
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Query 6:Text amended to ‘As loss of ... of TGCs and’ here.
OK?Query 7:Please provide affiliations for F. Talamantes, M.
Soares and S. Jacobs.Query 8:Please confirm statement: ‘The authors declare no
competing financial interests.’
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Query 9:So the text can be formatted appropriately, please
clarify what is meant by ‘between-plane-plane’.Query 10:Please amend the text here to avoid the sentence
starting with a number.
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Query 11:Text amended to ‘consensus E2F-binding sites’
here. Is this correct? And, also please ensurethat the hyphenation of ‘-binding’ is appropriatethroughout.
Query 12:Please check that the intended meaning of the text
‘The results are ... mutant samples analysed.’ has beenretained after editing.Query 13:Please provide page range/article id for ref. 50.
General Queries
Query 14:For the representation of gene symbols and geno-
types we follow the standard scientific conventionsand nomenclature found in databases such as HUGOfor humans, MGI for mice or Flybase for Drosophila.Accordingly, many changes may have been madethroughout the text and figures. Please check thatwe have interpreted each instance correctly.
Query 15:Please note that reference numbers are formatted
according to style in the text, so that any referencenumbers following a symbol or acronym are givenas ‘ref. XX’ on the line, whereas all other referencenumbers are given as superscripts.
Query 16:The ‘Control’ for figure 1d does not seem to be
defined in the caption. Please check.
Query 17:Please check sentence ‘Uncropped images...’ added
to the end of the captions of figures 2 and 5, andsupply Supplementary Fig. number
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caption of figure 2 has been retained after editing.Also, the scale bar in 2j is not defined. Please amendcaption.
Query 19:The insets in figure 3a are not mentioned in the
caption—please amend caption. (And please checktext added to captions of 6a,7a to define those insets).
Query 20:Acronyms ‘F7’, ‘F8’ and ‘TS’ removed from text,
according to style, and the caption of figure 5amended so a single definition is given, and the termsare used in full throughout the rest of the caption.Please check that the intended meaning has been
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Query 21:Can ‘∗P ≤ 0.05;’ be deleted from the end of the
caption of figure 7, as it does not seem to be usedin the figure.
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