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. 2011 Aug 23;108(34):14163-8.
doi: 10.1073/pnas.1111241108. Epub 2011 Aug 15.

Mir-290-295 deficiency in mice results in partially penetrant embryonic lethality and germ cell defects

Lea A Medeiros  1 , Lucas M Dennis, Mark E Gill, Hristo Houbaviy, Styliani Markoulaki, Dongdong Fu, Amy C White, Oktay Kirak, Phillip A Sharp, David C Page, Rudolf Jaenisch
Affiliations

Mir-290-295 deficiency in mice results in partially penetrant embryonic lethality and germ cell defects

Lea A Medeiros et al. Proc Natl Acad Sci U S A. .

Abstract

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 embryos, female but not male fertility is compromised. This impairment 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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
mir-290–295 is expressed in the early embryo and in germ cells. (A) 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 embryonic 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 (Wv) mutants.
Fig. 2.
Fig. 2.
Targeted disruption of the mir-290–295 locus. (A) Targeting strategy for generation of the mir-290–295 allele. (B) Southern blot and PCR confirmation of correct targeting of the mir-290–295 locus. (C) Northern blot validation of mir-290–295 targeting.
Fig. 3.
Fig. 3.
Some mir-290–295−/− embryos were observed outside the yolk sac. (A) Wild-type E9.5 embryo. (B) mir-290–295−/− E9.5 embryo located outside of the yolk sac. (Scale bars, 500 μM.)
Fig. 4.
Fig. 4.
Adult male mir-290–295−/− testes show reduced germ cells compared 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, 100 μM.)
Fig. 5.
Fig. 5.
Both male and female mir-290–295−/− embryonic gonads show reduced 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.
Fig. 6.
Fig. 6.
Primordial germ cells (PGCs) are mislocalized in mir-290–295−/− embryos. (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 phosphatase 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.
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