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Zebrafish dead end possesses ATPase activity that is required for primordial germ cell development.

ABSTRACT: Zebrafish dead end (dnd) mRNA is specifically expressed in primordial germ cells (PGCs) and is required for PGC migration and survival. Previous studies have shown that zebrafish Dnd functions by protecting the 3'UTRs of nanos1 and TDRD7 from miR-430b-mediated RNA deadenylation. In this work, we demonstrate that zebrafish Dnd protein possesses Mg(2+)-dependent ATPase activity that is required for PGC formation. Michaelis-Menten analysis revealed that the ATPase has a k(cat) of 0.632 +/- 0.036/min under optimal conditions, and mapping studies using Dnd truncates showed that ATPase resides in the last 91 aa of the Dnd C terminus. Internal deletion and point mutagenesis analysis of this region were used to identify key amino acids required for ATPase activity. Rescue experiments conducted by injecting mRNAs encoding the Dnd ATPase mutants into embryos in which the endogenous dnd expression was inhibited demonstrated that the ATPase activity is required for normal zebrafish PGC survival. Real-time PCR analysis showed that the expression of PGC markers nanos1 and TDRD7 but not vasa were down-regulated when dnd mutant proteins lacking ATPase were expressed in the rescued embryos, indicating that the Dnd ATPase is involved in protecting nanos1 and TDRD7 transcripts.

SUBMITTER: Liu W

PROVIDER: S-EPMC3230528 | biostudies-other | 2010 Aug

REPOSITORIES: biostudies-other

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Zebrafish dead end possesses ATPase activity that is required for primordial germ cell development.

Liu Weiyi W Collodi Paul P

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20100406 8

Zebrafish dead end (dnd) mRNA is specifically expressed in primordial germ cells (PGCs) and is required for PGC migration and survival. Previous studies have shown that zebrafish Dnd functions by protecting the 3'UTRs of nanos1 and TDRD7 from miR-430b-mediated RNA deadenylation. In this work, we demonstrate that zebrafish Dnd protein possesses Mg(2+)-dependent ATPase activity that is required for PGC formation. Michaelis-Menten analysis revealed that the ATPase has a k(cat) of 0.632 +/- 0.036/mi ...[more]

PMID: 20371629

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Project description:BackgroundMicroRNAs (miRNAs) are critical regulators of transcriptional and post-transcriptional gene silencing, which are involved in multiple developmental processes in many organisms. Apart from miRNAs, mouse germ cells express another type of small RNA, piwi-interacting RNAs (piRNAs). Although it has been clear that piRNAs play a role in repression of retrotransposons during spermatogenesis, the function of miRNA in mouse germ cells has been unclear.Methodology/principal findingsIn this study, we first revealed the expression pattern of miRNAs by using a real-time PCR-based 220-plex miRNA expression profiling method. During development of germ cells, miR-17-92 cluster, which is thought to promote cell cycling, and the ES cell-specific cluster encoding miR-290 to -295 (miR-290-295 cluster) were highly expressed in primordial germ cells (PGCs) and spermatogonia. A set of miRNAs was developmentally regulated. We next analysed function of miRNA biogenesis in germ cell development by using conditional Dicer-knockout mice in which Dicer gene was deleted specifically in the germ cells. Dicer-deleted PGCs and spermatogonia exhibited poor proliferation. Retrotransposon activity was unexpectedly suppressed in Dicer-deleted PGCs, but not affected in the spermatogonia. In Dicer-deleted testis, spermatogenesis was retarded at an early stage when proliferation and/or early differentiation. Additionally, we analysed spermatogenesis in conditional Argonaute2-deficient mice. In contrast to Dicer-deficient testis, spermatogenesis in Argonaute2-deficient testis was indistinguishable from that in wild type.Conclusion/significanceThese results illustrate that miRNAs are important for the proliferation of PGCs and spermatogonia, but dispensable for the repression of retrotransposons in developing germ cells. Consistently, miRNAs promoting cell cycling are highly expressed in PGCs and spermatogonia. Furthermore, based on normal spermatogenesis in Argonaute2-deficient testis, the critical function of Dicer in spermatogenesis is independent of Argonaute2.

Dataset Information

Zebrafish dead end possesses ATPase activity that is required for primordial germ cell development.

Publications

Zebrafish dead end possesses ATPase activity that is required for primordial germ cell development.

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