Mir-290–295 deficiency in mice results in partially
penetrant embryonic lethality and germ cell defects
Lea A. Medeirosa,b,1, Lucas M. Dennisa,b,1, Mark E. Gilla,b,c,1, Hristo Houbaviyd, Styliani Markoulakia, Dongdong Fua,
Amy C. Whitee,2, Oktay Kiraka, Phillip A. Sharpb,e, David C. Pagea,b,c, and Rudolf Jaenischa,b,3
aWhitehead Institute for Biomedical Research, Cambridge, MA 02139;bDepartment of Biology, Massachusetts Institute of Technology, Cambridge,
MA 02139;cHoward Hughes Medical Institute anddDepartment of Cell Biology, University of Medicine and Dentistry of New Jersey, Stratford,
NJ 08084; andeDavid H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139
Contributed by Rudolf Jaenisch, July 21, 2011 (sent for review January 24, 2011)
Mir-290 through mir-295 (mir-290–295) is a mammalian-specific
microRNA (miRNA) cluster that, in mice, is expressed specifically
in early embryos and embryonic germ cells. Here, we show that
mir-290–295 plays important roles in embryonic development as
indicated by the partially penetrant lethality of mutant embryos.
In addition, we show that in surviving mir-290–295-deficient em-
bryos, female but not male fertility is compromised. This impair-
ment in fertility arises from a defect in migrating primordial germ
cells and occurs equally in male and female mutant animals. Male
mir-290–295−/−mice, due to the extended proliferative lifespan of
their germ cells, are able to recover from this initial germ cell loss
and are fertile. Female mir-290–295−/−mice are unable to recover
and are sterile, due to premature ovarian failure.
required for normal embryogenesis (1–3) and embryonic germ
in early embryos and embryonic germ cells have been established,
the role of individual miRNAs in the development of these cell
types remains unclear. Six miRNA families comprise the majority
of miRNA species cloned from mouse embryonic stem (ES) cells,
with miRNAs from the mir-290 cluster, mir-290 through mir-295
(mir-290–295), being the most abundant (5). Members of this
cluster are the first embryonic miRNAs up-regulated in the zygote
(6). It has previously been shown that the mir-290 cluster miRNAs
are processed from a single primary transcript (7) and possess
highly similar pre-miRNA sequences (8).
Several studies have addressed the role of mir-290–295 in
embryonic stem (ES) cells where this cluster is a direct target of
the Oct4, Sox2, and Nanog regulatory network (9). The mir-290
cluster is correlated with developmental potency. Mir-290–295
expression decreases as ES cells differentiate (8). Furthermore,
certain members of the miR-290 family were found to increase
the efficiency of reprogramming by Oct4, Sox2, and Klf4 ∼10 fold
(10). In addition, members of the miR-290 family promote the
G1–S transition and thereby the rapid proliferation characteristic
of ES cells (11). The mir-290 cluster was also implicated in in-
direct control of de novo DNA methylation in ES cells (12, 13).
Taken together, these data imply important roles for mir-290–295
in ES cells and by extension, early mouse development. In this
study, we examined the in vivo consequences of targeted disrup-
tion of mir-290–295 in the developing mouse.
n themouse, miRNA-mediated posttranscriptional regulation is
mir-290–295 Is Specifically Expressed in the Early Embryo and Embry-
onic Germ Cells. We have shown that mir-290–295 is expressed in
ES cells and not in adult somatic tissues (8). To address the
timing of mir-290 cluster expression, we performed RT-PCR for
the mir-290–295 primary transcript (pri-mir-290–295) throughout
early embryonic development on pools of embryos. We observed
onset of expression of the primary transcript at the 4–8 cell stage
(Fig. 1A), consistent with the finding that expression of these
miRNAs is up-regulated postzygotically (6). Expression of the
mir-290–295 primary transcript decreased after embryonic day
To more closely examine the expression of the mir-290 cluster
miRNAs after gastrulation, we performed RT-PCR for the pri-
mary transcript on a panel of embryonic tissues from E14.5 em-
bryos. Expression of mir-290–295 was detectable in the embryonic
testis but not in any other tissue examined (Fig. 1B). Because mir-
290–295 has been shown to be expressed in primordial germ cells
(4) we analyzed mir-290 cluster expression in germ cells during
embryonic development. Expression of the mir-290 cluster was
observed in gonads of both sexes at E12.5. The primary mir-290–
295 transcript was down-regulated in female gonads between
E13.5 and E14.5, whereas in male gonads expression persisted
through E14.5 and became undetectable by E15.5 (Fig. 1C).
Because the embryonic gonad is composed of both somatic and
germ cell populations, we examined mir-290–295 expression in
E14.5 Wv/Wvgonads to test whether mir-290–295 expression in
the E14.5 testis was dependent upon the presence of germ cells.
Wvhomozygotes harbor a mutation in the c-kit gene that impairs
primordial germ cell (PGC) migration to the gonad, resulting in
loss of germ cells before E14.5 (14). The primary mir-290–295
transcript was undetectable in embryonic Wv/Wvtestes,suggesting
that mir-290–295 expression in embryonic gonads is restricted to
the germ cells (Fig. 1D).
Generation of mir-290–295 Mutant Mice. To determine the function
of mir-290–295 in vivo, we generated mice deficient for the 2-kb
locus containing these miRNAs by targeted disruption in ES cells
(Fig. 2A). Two independent mir-290–295+/−ES cell lines, where
correct targeting had been validated by Southern blot and PCR,
were injected into B6DF2 host blastocysts to produce chimeras.
Transmission of the targeted allele through the male germline
was confirmed by Southern blotting and PCR analysis (Fig. 2B).
To verify that deletion of the mir-290–295 locus eliminated ex-
pression of the miR-290 family of miRNAs, mir-290–295 homo-
zygous knockout ES cells lines were produced. Northern blot
(Fig.2C) andreal-time RT-PCR (datanot shown)using probes to
detect mature miR-290 family miRNAs failed to detect any of
these miRNAs in mutant (mir-290–295−/−) ES cells, confirming
that the targeted deletion resulted in a null allele. These findings
suggest that mir-290–295 is dispensable for maintaining the plu-
ripotent state in embryonic stem cells.
Author contributions: L.A.M., L.M.D., M.E.G., H.H., S.M., P.A.S., D.C.P., and R.J. designed
research; L.A.M., L.M.D., M.E.G., H.H., S.M., D.F., A.C.W., and O.K. performed research;
L.A.M., L.M.D., M.E.G., H.H., S.M., and O.K. contributed new reagents/analytic tools; L.A.M.,
L.M.D., M.E.G., and S.M. analyzed data; and L.A.M., L.M.D., M.E.G., and R.J. wrote the
The authors declare no conflict of interest.
1L.A.M., L.M.D., and M.E.G. contributed equally to this work.
2Present address: Alnylam Pharmaceuticals, Cambridge, MA 02142.
3To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
www.pnas.org/cgi/doi/10.1073/pnas.1111241108 PNAS Early Edition
| 1 of 6
mir-290–295 Deficiency Results in Partially Penetrant Embryonic
Lethality. mir-290–295+/−mice were fertile and indistinguish-
able from wild-type littermates. When heterozygous animals were
intercrossed, we observed a significantly lower fraction of mir-
290–295−/−offspring than the 25% predicted by Mendelian seg-
regation (Table 1). Only 7% (32 of 452, P < 0.001, χ2) of 4-wk-old
postnatal progeny from mir-290–295+/−intercrosses were mir-
290–295−/−, suggesting that about three-quarters of the homozy-
gous knockout animals were lost during development. At E18.5,
just before birth, the percentage of mir-290–295−/−embryos ob-
served (7%, 3 out of 46, P < 0.01, χ2) was identical to that seen at
postnatal stages, indicating that perinatal lethality was not re-
sponsible for the loss of mir-290–295−/−embryos (Table 1).
To determine when during gestation mir-290–295−/−embryos
were lost, embryos from heterozygous intercrosses were isolated
at blastocyst (E3.5) and mid-late gestation (E8.5–E18.5) stages
of development. Mutant (mir-290–295−/−) blastocysts appeared
morphologically indistinguishable from their wild-type and het-
erozygous counterparts and were observed at the predicted
Mendelian ratio of 25% (32 out of 117 total blastocysts). Further
analysis of embryos at mid-late gestation suggested that mir-290–
295−/−embryos were lost over a period between E11.5 and E18.5.
Even though mutant embryos were observed at the predicted
Mendelian ratio at E8.5, E9.5, E10.5, E11.5, and E13.5, ∼50–
60% of these embryos (Table 2) displayed abnormalities not
observed in their wild-type or heterozygous littermates. Two
abnormal phenotypes were observed. Before E10.5, about 16%
of the mutant embryos were partially or completely localized
outside the yolk sac (Fig. 3). Such abnormal embryos were not
observed at later stages presumably because they had died and
The second abnormal phenotype, comprising about 40% of
the mutant embryos, showed general developmental delays as
early as E8.5. These mutants had fewer somites than their wild-
type or heterozygous littermates and showed delays in chorio-
allantoic attachment, axial turning, and neural tube closure.
Adult mir-290–295−/−Females Are Sterile, Whereas Adult mir-290–
295−/−Males Are Fertile. Surviving mir-290–295−/−animals were
healthy and phenotypically normal, although homozygous knock-
out females were infertile. Ovaries from adult (5–12 wk post-
partum) mir-290–295−/−animals were small, having a volume less
than 20% that of wild-type littermates (Fig. 4A). Ovaries from
homozygous knockouts older than 10 wk contained no observable
e t y
- +- + - +- + - +- + - +
E12.5 XYE13.5 XX
RT - +
RT-PCR of primary mir-290–295 transcript and Actin control in early embryos.
(B) RT-PCR of mir-290–295 and Rps15 control in a tissue panel from E14.5
embryos. (C) RT-PCR of mir-290–295 and Tbp control in E12.5–E15.5 em-
bryonic gonads from males and females. (D) RT-PCR of mir-290–295 and
Rps15 control in E14.5 testis isolated from wild-type and homozygous c-kit
mir-290–295 is expressed in the early embryo and in germ cells. (A)
7 kb10 kb
for generation of the mir-290–295 allele. (B) Southern blot and PCR confir-
mation of correct targeting of the mir-290–295 locus. (C) Northern blot
validation of mir-290–295 targeting.
Targeted disruption of the mir-290–295 locus. (A) Targeting strategy
mir-290–295 deficiency results in partially penetrant
Postnatal (4 wk)
29: 56: 32 (27)
2: 4: 2 (25)
7: 21: 13 (32)
18: 30: 21 (30)
14: 36: 12 (19)
10: 19: 6 (17)
16: 26: 10 (19)
8: 35: 3 (7)*
168: 252: 32 (7)†
Embryos from heterozygous matings were dissected at the specified time-
points and genotyped. Reabsorbed embryos refer to embryos that were too
disintegrated to separate from the maternal tissue and were therefore not
*P < 0.01, χ2test.
†P < 0.001, χ2test.
mir-290–295-deficient embryos display defects at
Total number of
Number of abnormal or
mir-290–295−/−embryos analyzed in this table are progeny from both mir-
290–295+/−intercrosses and mir-290–295+/−× mir-290–295−/−crosses. “Ab-
normal” refers to embryos that were either observed outside the yolk sac
or were developmentally delayed compared with wild-type littermates.
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| www.pnas.org/cgi/doi/10.1073/pnas.1111241108Medeiros et al.
follicular structures (data not shown). In the ovaries of 5- to 8-wk-
old mir-290–295−/−animals, small numbers of follicles were oc-
casionally observed (Fig. 4 C and E).
Whereas testes from adult mir-290–295−/−males were relatively
periodic acid-Schiff (PAS)–stained sections of mir-290–295−/−
males showed empty seminiferous tubules (black rectangle in Fig.
4H) alongside tubules filled with germ cells at various stages of
spermatogenesis (Fig. 4 H and J). Mir-290–295−/−males regularly
fathered litters with both wild-type and mir-290–295+/−females
and can therefore be considered fertile.
Both Male and Female mir-290–295−/−Early Postnatal Gonads Show
Reduced Germ Cell Numbers. Examination of ovaries and testes
from postnatal day 5 (P5) animals revealed a reduced number of
gonocytes in both males and females. At P5, mir-290–295−/−
ovaries contained less than 20% as many oocytes as wild-type
ovaries, as determined by histological analysis and immunos-
taining for mouse vasa homolog (MVH), a germ cell marker (15)
(Fig. S1 A–D). In contrast to wild-type ovaries (Fig. S1 A and C),
mir-290–295−/−ovaries displayed very few primordial follicles
(Fig. S1 B and D). Consistent with these data, P10 mir-290–295−/−
ovaries showed severe depletion of the primordial follicle pool
(Fig. S1 F and H compared with Fig. S1 E and G). Because the
primordial follicle pool is a finite population (16), once all of the
follicles have been recruited from the pool for maturation and/or
death, the female will no longer be fertile.
Mir-290–295−/−testes also showed fewer germ cells than con-
trols at P5 (Fig S1 I–L). Chains of 3–4 gonocytes were observed in
P5 homozygous knockout males, suggesting that the male mir-
290–295−/−germ cells were still proliferating. Furthermore, at
P10, tubules in the homozygous knockout male contained more
suggesting that the male mir-290–295−/−germ cells continued to
proliferate (Fig. S1 M–P). These data are not surprising, consid-
ering that the expression data in Fig. 1C indicated that the mir-
290 cluster is not expressed after E14.5 in males.
The above data, combined with the observation of empty
seminiferous tubules in the adult male homozygous knockout
(Fig. 4 H and J), suggest that the original germ cell defect is ini-
tially sex neutral and that males are able to regain fertility by
clonal expansion of surviving germ cells.
Both Male and Female mir-290–295−/−Embryonic Gonads Show Germ
Cell Depletion. We sought to determine the timepoint during de-
velopment when a difference in germ cell number or localization
became apparent between homozygous knockouts and their wild-
type siblings. Because mir-290–295 expression becomes un-
detectable after E13.5 in females and E14.5 in males (Fig. 1C), we
examined E13.5 gonads by immunostaining for the germ cell
marker MVH. Inspection of mir-290–295−/−ovaries and testes at
E13.5 revealed a dramatic reduction in the number of MVH+
cells relative to control embryonic gonads (Fig. 5 A–E). To ensure
that the loss of MVH expression was indicative of decreased germ
cell number, and not a gene-specific effect of mir-290–295 de-
ficiency, we examined expression of another germ cell marker,
germ cell nuclear antigen (GCNA) (17) in E13.5 homozygous
knockout gonads and found a similar reduction in the number of
GCNA+cells (data not shown).
We then examined E11.5 embryos to determine whether re-
duced numbers of mir-290–295−/−PGCs were colonizing the
(A) Wild-type E9.5 embryo. (B) mir-290–295−/−E9.5 embryo located outside
of the yolk sac. (Scale bars, 500 μM.)
Some mir-290–295−/−embryos were observed outside the yolk sac.
with wild-type testes, whereas adult female mir-290–295−/−ovaries are
atrophied. (A) Ovaries and reproductive tracts from 8-wk-old wild-type and
mir-290–295−/−females. (Scale bar, 1 mm.) (B–E) Hematoxylin and eosin-
stained ovary sections from adult wild-type (B and D) and mir-290–295−/−(C
and E) animals. (Scale bars, 100 μM.) (F) Testes from 8-wk-old wild-type and
mir-290–295−/−males. Note that the smaller testes size of the knockout male
is at least partially due to the smaller body weight of the knockout animal.
(Scale bar, 1 mm.) (G–J) Periodic acid-Schiff (PAS)–stained testes sections
from adult wild-type (G and I) and mir-290–295−/−(H and J) males. Black
rectangle in H highlights area with empty seminiferous tubules. (Scale bars,
Adult male mir-290–295−/−testes show reduced germ cells compared
Total Germ Cell Number
wild-type mir-290-295 -/-
Germ Cell Density
(% of wild-type)
duced germ cell numbers as early as E11.5. (A–D) MVH immunostaining of
sections of E13.5 ovaries (A and B) and testes (C and D) from wild-type (A and
C) and mir-290–295−/−(B and D) embryos. (Scale bars, 50 μM.) (E) Germ cell
density (germ cells/area of gonadal section for ovaries and germ cells/testis
cord area for testes) as determined by MVH staining. Each bar represents
a single embryo with black bars indicating male embryos and white bars,
female embryos. (F–I) MVH immunostaining of E11.5 embryo sections from
wild-type (F and H) and mir-290–295−/−(G and I) embryos. (Scale bars, 50
μM.) White arrows point to germ cells in G and I. The additional green cells in
I are blood cells. (J) Total germ cell numbers in E11.5 gonads as determined
by serial sectioning and MVH staining. Each bar represents a single embryo
with black bars indicating male embryos and white bars, female embryos.
Both male and female mir-290–295−/−embryonic gonads show re-
Medeiros et al.PNAS Early Edition
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genital ridges. At E11.5, most male and female mir-290–295−/−
genital ridges exhibited less than 5% as many germ cells as found
in wild-type littermate controls (Fig. 5 F–J), as determined by
mir-290–295−/−Animals Show Many Mislocalized Primordial Germ
Cells. On the basis of the reduced number of germ cells coloniz-
ing the genital ridges in E11.5 mutants and the fact that mir-290–
295 is expressed in migrating primordial germ cells (4) we ex-
plored the possibility that mutant embryos exhibited defective
germ cell migration. PGCs in the developing mouse embryo mi-
grate from their origin in the proximal epiblast, through the de-
veloping hindgut, to the genital ridges (18, 19). We used two
different germ cell markers, alkaline phosphatase (AP) (20) and
Oct4, to locate germ cells in the midgestation embryo. Alkaline
phosphatase expression was detected by whole-mount staining,
whereas an Oct4-GFP transgene was used to monitor Oct4 ex-
pression (21). In wild-type animals at E9.5, PGCs were located
almost exclusively in the hindgut and dorsal mesentery (Fig. 6 A,
C, and D). Although there was great variability among PGC
numbers, both mir-290–295−/−males and females had about one-
fourth as many germ cells in the hindgut and mesentery as wild-
type animals of the same developmental stage (Fig. 6 B–D).
Surprisingly, mutant males and females exhibited AP+Oct4+
cells on the ventral surface of the embryo near the hindlimb buds
and the base of the tail (Fig. 6B). On average, mutant embryos
had 40 of these ectopic PGCs, whereas their wild-type counter-
parts had 10 or fewer (Fig. 6D). These ectopic PGCs were ob-
served as late as E11.5 (data not shown). A difference between
germ cell localization in mutants and control littermates could be
observed as early as E8.5. In Oct4-GFP wild-type embryos at the
7-somite stage, Oct4+cells had already moved into the hindgut
(Fig. S2A). In contrast, Oct4+cells in the 7-somite stage Oct4-
GFP mir-290–295−/−embryos were clustered together at the base
of the allantois (Fig. S2 B and C). Taken together, these data
suggest that loss of germ cells in mir-290–295−/−animals is at least
partially due to improper germ cell migration.
mir-290–295−/−Germ Cells Do Not Undergo Premature Cell Cycle
Arrest or Apoptosis. Because the mir-290 cluster has been shown
to regulate the G1–S transition (11), and protect against apo-
ptosis in ES cells (22), we explored the possibility that mir-290–
295−/−germ cells might have undergone apoptosis or premature
cell cycle arrest. We studied two timepoints during germ cell
development: E9.5, when germ cells are migrating toward the
developing gonad and E12.5, after germ cells have arrived in the
gonad butbefore they have undergonemitotic arrest(in males)or
meiotic arrest (in females). E9.5 embryos were serially sectioned
and stained for either Ki-67 or cleaved caspase-3. In all sections,
SSEA-1 was used to identify the migrating PGCs (23). At least
90% of both wild-type and mir-290–295−/−migrating PGCs were
Ki-67+(Fig. S3 A–G). Ki-67 protein is expressed in all pro-
liferating cells during the late G1, S, G2, and M phases of the cell
cycle. Only cells in the G0phase ofthe cell cycle do not express Ki-
67 (24, 25). Therefore, the data from E9.5 animals indicate that
mir-290–295−/−PGCs are actively cycling. Furthermore, neither
(Fig. S3 H–O), indicating that mir-290–295−/−PGCs are not un-
dergoing apoptosis. This is consistent with previous reports that
germ cell apoptosis is mediated by the caspase-3 pathway (26).
Similar results were obtained with E12.5 gonocytes. Male and
female wild-type and mir-290–295−/−E12.5 gonads were serially
antigen (PCNA) or SSEA-1 and cleaved caspase-3. PCNA, like
Ki-67, is a marker of actively cycling cells (27). (Because double
Ki-67 and MVH immunostaining proved difficult, PCNA was
least 85% of the gonocytes were PCNA+, and therefore ac-
tively cycling (Fig. S4). In addition, neither wild-type nor mir-290–
(Fig. S5). Taken together these data show that decreased numbers
of germ cells in the mir-290–295−/−animal cannot be explained by
apoptosis or failure of the mutant germ cells to proliferate.
In this study, we investigated the biological function of the mir-290
cluster by targeted deletion in the mouse. Although miRNAs of
the mir-290 cluster are the first miRNAs up-regulated in the de-
velopingembryo, this cluster wasnot required forpreimplantation
development or ES cell pluripotency. Instead, we found that mir-
290–295 deficiency had a significant effect between implantation
and midgestation and during germ cell development. Approxi-
mately three-quarters of mir-290–295-deficient embryos were
lost during embryonic development. The surviving quarter of ho-
mozygous knockouts showed a germ cell loss. Adult male mu-
tants recover from this loss, whereas female mutants do not and
mir-290–295 Deficiency Confers an Incompletely Penetrant Embryonic
Lethality. The earliest abnormality in mir-290–295−/−animals was
observed at E8.5. Specifically, about 16% of mir-290–295-de-
ficient embryos at E8.5 (and E9.5 and E10.5) were found either
partially or completely outside of the yolk sac. The phenomenon
of postimplantation embryos located either partially or com-
pletely outside the yolk sac has been observed for mutants of
several genes involved in patterning the embryo during gastru-
lation. These mutants include the Type II Activin receptor
knockout (28), Hnf3β knockout (29), Otx2 knockout (30), Lpp3
knockout (31), Axin knockout (32), and Nodal hypomorph (33).
The exact mechanism by which these embryos end up outside the
yolk sac remains unclear (29, 34).
One intriguing possibility is that the mir-290–295 cluster might
play a role in embryonic patterning by regulating lefty1. Both lefty1
wild-type (20 sp)
mir-290-295 -/- (20 sp)
wild-type mir-290-295 -/-
Number of AP+ cells in
hindgut and mesentery
wild-type mir-290-295 -/-
Number of AP+ cells near
tail and hindlimbs
bryos. (A and B) Images of E9.5 Oct4-GFP wild-type (A) and Oct4-GFP mir-
290–295−/−(B) embryos. (Scale bars, 1 mm.) The numbers in parentheses
refer to the number of somite pairs in each embryo. Arrows point to PGCs
located near the tail. (C) Number of PGCs, as determined by alkaline phos-
phatase staining, in the hindgut and mesentery of E9.5 embryos. Black bars
indicate the average number of PGCs for each genotype. (D) Number of
PGCs, as determined by alkaline phosphatase staining, near the hindlimb
and base of tail, in E9.5 embryos. Black bars indicate the average number of
PGCs for each genotype.
Primordial germ cells (PGCs) are mislocalized in mir-290–295−/−em-
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and lefty2 have been shown to be targets of the miR-290 family
miRNAs (9, 12). In zebrafish, miR-430, which has the same
AAGUGC seed sequence as many of the miRNAs in the mir-290
cluster, balances the expression of the Nodal agonist squint and
the TGF-β Nodal antagonist lefty (35). In human ES cells, deple-
tion of miR-302, which targets lefty1 and lefty2, leads to a strong
decrease in the expression of mesodermal and endodermal
markers (36). The mir-290–295 mutant mouse would provide a
unique opportunity to test the hypothesis that AAGUGC seed
miRNAs are involved in balancing lefty-Nodal signaling.
mir-290–295 Deficiency Results in Germ Cell Loss. Approximately
25% of mir-290–295−/−animals survived to adulthood. Although
adult female homozygous knockouts were sterile and male ho-
mozygous knockouts were fertile, the germ cell loss leading to
sterility in the female was first observed in both sexes at E11.5.
This suggests that fewer germ cells were colonizing the gonads in
both mir-290–295−/−females and males. Observations of migrat-
ing PGCs revealed a reduction in the number of correctly local-
female mutants, the decreased number of PGCs properly local-
ized to the hindgut area correlated with the increased number of
ectopic germ cells observed on the ventral posterior surface of the
embryo. These data suggest that the germ cell loss observed in the
mutant is due to mislocalization of a subpopulation of primordial
germ cells, which are subsequently unable to colonize the gonad
and therefore cannot contribute to the germ cell pool.
Given that members of the mir-290–295 family have been
(22), in ES cells, we investigated whether increased apoptosis or
a block in proliferation could explain the germ cell loss in the
mutant animals. No cleaved caspase-3+PGCs were observed in
wild-type or mir-290–295−/−germ cells at E9.5 or E12.5. In E9.5
and E12.5 animals, at least 85% of migrating PGCs in both wild-
type and mir-290–295−/−animals were actively cycling. Whereas
the above results rule out the possibility of premature cell cycle
that slower proliferation kinetics of the mutant germ cells con-
tributes to the decrease in germ cells caused by mislocalization.
Nevertheless, whereas a defective G1–S transition might be
able to explain part of the germ cell loss realized at E11.5 and
E13.5, it would not explain the primary observation that some
mir-290–295−/−germ cells are mislocalized during migration.
Ectopic PGCs localized in or near the tail have been previously
reported in mice deficient for the proapoptotic protein Bax (26,
37, 38). Although the majority of ectopic PGCs in Bax−/−mice
were found within the abdominal midline dorsal body wall, some
ectopic PGCs were also observed on the tail. Unlike Bax−/−em-
bryos, mir-290–295−/−embryos do not display ectopic germ cells
in or near the abdominal midline body wall, suggesting that mis-
localization of mir-290–295−/−germ cells is not simply a conse-
quence of faulty apoptotic pathways.
Ectopic “tail” PGCs are hypothesized to arise from PGCs,
which fail to become incorporated into the hindgut during the
initial stages of germ cell migration (37). Our observations are
consistent with this model. At E8.5, although wild-type PGCs
have already entered the developing hindgut, the majority of
mutant PGCs have not yet started to migrate and instead are
stuck near the base of the allantois (Fig. S2). Whether the failure
of germ cells to disperse by E8.5 is due to cell autonomous defects
within the germ cells or defects in the surrounding soma still
remains unclear. Recently, miR-430, which has the same seed
sequence as many members of the mir-290–295 cluster, was im-
plicated in PGC migration in the zebrafish (39). Interestingly, loss
of miR-430 led to defective migration due to misexpression of
chemokines in the surrounding soma. The mir-290–295 locus is
expressed in migrating PGCs (4). Furthermore, the mir-290–295
primary transcript is expressed in the early (E6.5) embryo and its
expression decreases by E10.5 (Fig. 1A). Currently it is not known
whether or not miR-290 miRNAs are present in the tissues
through which PGCs must migrate. Therefore, we cannot rule out
the possibility that a subtle defect in the surrounding soma might
be the cause of the ectopic germ cells.
Little is known about the genes involved (both in the germ cells
themselves and the soma) in the early stages (E7.5–E8.5) of germ
cell migration. Although, on the basis of misexpression studies,
fragilis was thought to play a role in movement of the primordial
germ cells from the epiblast into the endoderm (40), a fragilis
knockout showed no defects in germ cell localization (41). To the
best ofour knowledge,themir-290–295deletionrepresentsthefirst
germ cell mutant where PGCs mislocalize to the ventral surface of
the embryo near the developing hindlimbs. Thus, it will be of in-
295 cluster is involved in the early stages of germ cell migration.
Our data cannot exclude the possibility that the migration de-
fect observed is an indirect effect of improper germ cell specifi-
cation. We do not know whether the same number of germ cells
are allocated in mir-290–295−/−animals and wild-type animals.
Further experiments beyond the scope of this work are required
to address these questions.
unique to the mir-290–295−/−. Male germ cells undergo mitotic
arrest around E13.5 but then resume mitosis a few days after birth
(43). In contrast, female germ cells enter meiosis around E13.5,
thereby establishing the total oocyte pool for adult life (16). The
extra proliferative time in the males allows for additional clonal
expansion of the few surviving mir-290–295−/−gonocytes, which
results in enough germ cells for the mir-290–295−/−males to
mir-290–295 Deficiency Confers Incompletely Penetrant Phenotypes.
The phenotypes conferred by mir-290–295 deficiency are charac-
terized by variable expressivity and incomplete penetrance. The
contribute to the incomplete penetrance of the embryonic lethality.
However, given what is known about miRNAs, the partially pene-
trant embryonic lethality might also be explained by the function of
the mir-290 cluster itself. MiRNAs have been shown to confer ro-
bustness to developmental systems (44–48). It was recently shown
that random fluctuations in gene expression can result in an in-
completely penetrant phenotype when a certain level of gene ex-
pression is required to pass a threshold to cause an outcome (49).
Consistent with this,deletionsofvarious miRNAs havebeenshown
to confer partially penetrant phenotypes (50–53). Taken together,
these data lead us to speculate that the loss of mir-290–295 ex-
pression might cause fluctuations in gene expression patterns.
Those mutants with gene expression patterns that differ greatly
from wild-type would not survive, whereas mutants with gene ex-
to develop normally during this period. Presumably after this time
window, the role of mir-290–295 becomes less critical. Therefore,
any mutants surviving this time period also survive to adulthood.
Materials and Methods
RT-PCR. RNA samples were isolated by homogenizing tissue or cells in TRIzol
(Invitrogen) following the manufacturer’s suggested protocol. Five micro-
grams of total RNA was DNase I treated using the DNA-Free RNA kit (Zymo
Research). One microgram of DNase I-treated RNA was reverse transcribed
usinga FirstStrandSynthesiskit(Invitrogen). PCRwasperformedusing1/80of
the reverse transcription reaction. The following primer sequences were used
to determine pri-mir-290–295 expression: 5′-GAACCTCACGGGAAGTGACC-3′
(forward primer) and 5′-TGCCCACAGGAGAGACTCAA-3′ (reverse primer).
Medeiros et al.PNAS Early Edition
| 5 of 6
Northern Blot Analysis. RNA was extracted using TRIzol (Invitrogen) following
the manufacturer’s instructions. A total of 30 μg of total RNA was electro-
phoresed for 45 min at 35 W, and semidry transferred to Hybond-NX nylon
membrane (Amersham Biosciences) at 18 V for 1.5 h at 4 °C. RNA was cross-
linked and incubated with locked nucleic acid (LNA) probe as previously de-
scribed (54, 55). After washing, membranes were exposed to a phosphor-
imager screen for 1–3 d, depending on the probe. Probes were synthesized by
IDT: (i) miR-17, C+TAC+CTG+CAC+TGT+AAG+CAC+TTT+G; (ii) miR-295, A+
GAC+TCA+AAA+GTA+GTA+GCA+CTT+T; and (iii) tRNA glu, TGGAGGTTCCAC-
CGAGAT, where + indicates that the following nucleotide is an LNA.
Generation of mir-290–295−/−Mice. Mice deficient for mir-290–295 were
generated by targeted disruption of the endogenous mir-290–295 locus via
homologous recombination in ES cells. Upstream and downstream arms were
PCR amplified from RPCI-23–222D1 BAC DNA, resulting in a construct where
2.1 kb of the mir-290–295 locus (including the mature miRNA sequences) was
replaced by a 1.6-kb neomycin resistance selection cassette (Fig. 2A). This
targeting construct was electroporated into V6.5 ES cells that were then
subjectedtoselection with G418.After 10dofselection,G418resistant clones
were analyzed by Southern blotting with external probes. Clones exhibiting
correct targeting were injected into B6D2F2 recipient blastocysts for sub-
sequent chimera generation. For this study, mice were maintained on
a 129Sv/J × C57BL/6 mixed genetic background. The committee on animal
care at the Massachusetts Institute of Technology approved all experiments
ACKNOWLEDGMENTS. We thank Jessica Dausman and Ruth Flannery for
technical assistance with the generation of chimeras and animal husbandry;
George Enders for GCNA antisera; and members of the R.J., D.C.P., and P.A.C.
laboratories for critical discussions. This work was supported by National
Institutes of Health Grants 5-F32-HD051190 (to A.C.W.), RO1-GM34277 (to
P.A.S.), and 5R37CA084198 and 5R01-HD045022 (to R.J), National Cancer
Institute Grant PO1-CA42063 (to P.A.S.) and Core Grant P30-CA14051 (to
Koch Institute), and the Howard Hughes Medical Institute (D.C.P.).
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| www.pnas.org/cgi/doi/10.1073/pnas.1111241108 Medeiros et al.