A Transgenic DND1GFP Fusion Allele Reports in Vivo Expression and RNA-binding Targets in Undifferentiated Mouse Germ Cells†

Overview

Journal Biol Reprod

Publisher Oxford University Press

Specialty Reproductive Medicine

Date 2021 Jan 4

PMID 33394034

Citations 6

Authors

Victor A Ruthig

Tetsuhiro Yokonishi

Matthew B Friedersdorf

Sofia Batchvarova

Josiah Hardy

Jason A Garness

Jack D Keene

Blanche Capel

Affiliations

Soon will be listed here.

Abstract

In vertebrates, the RNA-binding protein (RBP) dead end 1 (DND1) is essential for primordial germ cell (PGC) survival and maintenance of cell identity. In multiple species, Dnd1 loss or mutation leads to severe PGC loss soon after specification or, in some species, germ cell transformation to somatic lineages. Our investigations into the role of DND1 in PGC specification and differentiation have been limited by the absence of an available antibody. To address this problem, we used CRISPR/Cas9 gene editing to establish a transgenic mouse line carrying a DND1GFP fusion allele. We present imaging analysis of DND1GFP expression showing that DND1GFP expression is heterogeneous among male germ cells (MGCs) and female germ cells (FGCs). DND1GFP was detected in MGCs throughout fetal life but lost from FGCs at meiotic entry. In postnatal and adult testes, DND1GFP expression correlated with classic markers for the premeiotic spermatogonial population. Utilizing the GFP tag for RNA immunoprecipitation (RIP) analysis in MGCs validated this transgenic as a tool for identifying in vivo transcript targets of DND1. The DND1GFP mouse line is a novel tool for isolation and analysis of embryonic and fetal germ cells, and the spermatogonial population of the postnatal and adult testis.

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References

Hogarth C, Evanoff R, Mitchell D, Kent T, Small C, Amory J . Turning a spermatogenic wave into a tsunami: synchronizing murine spermatogenesis using WIN 18,446. Biol Reprod. 2013; 88(2):40. PMC: 3589231. DOI: 10.1095/biolreprod.112.105346. View

Yoshinaga K, Ogawa M, Hayashi S, Kunisada T, Fujimoto T, Nishikawa S . Role of c-kit in mouse spermatogenesis: identification of spermatogonia as a specific site of c-kit expression and function. Development. 1991; 113(2):689-99. DOI: 10.1242/dev.113.2.689. View

Suzuki A, Niimi Y, Shinmyozu K, Zhou Z, Kiso M, Saga Y . Dead end1 is an essential partner of NANOS2 for selective binding of target RNAs in male germ cell development. EMBO Rep. 2015; 17(1):37-46. PMC: 4718414. DOI: 10.15252/embr.201540828. View

Yamaji M, Tanaka T, Shigeta M, Chuma S, Saga Y, Saitou M . Functional reconstruction of NANOS3 expression in the germ cell lineage by a novel transgenic reporter reveals distinct subcellular localizations of NANOS3. Reproduction. 2009; 139(2):381-93. DOI: 10.1530/REP-09-0373. View

Pui H, Saga Y . Gonocytes-to-spermatogonia transition initiates prior to birth in murine testes and it requires FGF signaling. Mech Dev. 2017; 144(Pt B):125-139. DOI: 10.1016/j.mod.2017.03.002. View

Nicholson C, Friedersdorf M, Bisogno L, Keene J . DO-RIP-seq to quantify RNA binding sites transcriptome-wide. Methods. 2016; 118-119:16-23. PMC: 5411286. DOI: 10.1016/j.ymeth.2016.11.004. View

Lin Y, Barske L, DeFalco T, Capel B . Numb regulates somatic cell lineage commitment during early gonadogenesis in mice. Development. 2017; 144(9):1607-1618. PMC: 5450849. DOI: 10.1242/dev.149203. View

Wang Z, Jiao X, Carr-Schmid A, Kiledjian M . The hDcp2 protein is a mammalian mRNA decapping enzyme. Proc Natl Acad Sci U S A. 2002; 99(20):12663-8. PMC: 130517. DOI: 10.1073/pnas.192445599. View

Law N, Oatley M, Oatley J . Developmental kinetics and transcriptome dynamics of stem cell specification in the spermatogenic lineage. Nat Commun. 2019; 10(1):2787. PMC: 6594958. DOI: 10.1038/s41467-019-10596-0. View

10.

Stevens L . Testicular teratomas in fetal mice. J Natl Cancer Inst. 1962; 28:247-67. View

11.

Youngren K, Coveney D, Peng X, Bhattacharya C, Schmidt L, Nickerson M . The Ter mutation in the dead end gene causes germ cell loss and testicular germ cell tumours. Nature. 2005; 435(7040):360-4. PMC: 1421521. DOI: 10.1038/nature03595. View

12.

van Dijk E, Cougot N, Meyer S, Babajko S, Wahle E, Seraphin B . Human Dcp2: a catalytically active mRNA decapping enzyme located in specific cytoplasmic structures. EMBO J. 2002; 21(24):6915-24. PMC: 139098. DOI: 10.1093/emboj/cdf678. View

13.

Cho Y, Iguchi N, Yang J, Handel M, Hecht N . Meiotic messenger RNA and noncoding RNA targets of the RNA-binding protein Translin (TSN) in mouse testis. Biol Reprod. 2005; 73(4):840-7. DOI: 10.1095/biolreprod.105.042788. View

14.

Toyooka Y, Tsunekawa N, Takahashi Y, Matsui Y, Satoh M, Noce T . Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development. Mech Dev. 2000; 93(1-2):139-49. DOI: 10.1016/s0925-4773(00)00283-5. View

15.

Kedde M, Strasser M, Boldajipour B, Vrielink J, Slanchev K, le Sage C . RNA-binding protein Dnd1 inhibits microRNA access to target mRNA. Cell. 2007; 131(7):1273-86. DOI: 10.1016/j.cell.2007.11.034. View

16.

Suzuki A, Igarashi K, Aisaki K, Kanno J, Saga Y . NANOS2 interacts with the CCR4-NOT deadenylation complex and leads to suppression of specific RNAs. Proc Natl Acad Sci U S A. 2010; 107(8):3594-9. PMC: 2840499. DOI: 10.1073/pnas.0908664107. View

17.

Duszczyk M, Wischnewski H, Kazeeva T, Arora R, Loughlin F, von Schroetter C . The solution structure of Dead End bound to AU-rich RNA reveals an unusual mode of tandem RRM-RNA recognition required for mRNA regulation. Nat Commun. 2022; 13(1):5892. PMC: 9537309. DOI: 10.1038/s41467-022-33552-x. View

18.

Bowles J, Feng C, Spiller C, Davidson T, Jackson A, Koopman P . FGF9 suppresses meiosis and promotes male germ cell fate in mice. Dev Cell. 2010; 19(3):440-9. DOI: 10.1016/j.devcel.2010.08.010. View

19.

Yamaji M, Jishage M, Meyer C, Suryawanshi H, Der E, Yamaji M . DND1 maintains germline stem cells via recruitment of the CCR4-NOT complex to target mRNAs. Nature. 2017; 543(7646):568-572. PMC: 5488729. DOI: 10.1038/nature21690. View

20.

Zhou Z, Shirakawa T, Ohbo K, Sada A, Wu Q, Hasegawa K . RNA Binding Protein Nanos2 Organizes Post-transcriptional Buffering System to Retain Primitive State of Mouse Spermatogonial Stem Cells. Dev Cell. 2015; 34(1):96-107. DOI: 10.1016/j.devcel.2015.05.014. View