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MicroRNA-mediated posttranscriptional regulation is required for maintaining undifferentiated properties of blastoderm and primordial germ cells in chickens.

ABSTRACT: MicroRNAs (miRNAs) play a critical role in determining the differentiation fate of pluripotent stem cells and germ cells in mammals. However, the mechanism(s) of miRNA-mediated posttranscriptional regulation with regard to lineage specification and differentiation in chick development require further investigation. Therefore, we conducted miRNA expression profiling to explore specific miRNA signatures in undifferentiated blastoderm and primordial germ cells (PGCs). We identified seven miRNAs that are highly expressed in blastoderm and 10 that are highly expressed in PGCs. In this study, miR-302a and miR-456 for blastoderm and miR-181a* for PGCs were analyzed further for their target transcripts and regulatory pathways. Both miR-302a and miR-456 bound directly to the sex-determining region Y box 11 transcript and could act as posttranscriptional coregulators to maintain the undifferentiated state of the chicken blastoderm through the suppression of somatic gene expression and differentiation. Moreover, miR-181a* showed a bifunctional role in PGCs by binding to two different transcripts. miR-181a* inhibited the somatic differentiation of PGCs by silencing homeobox A1 expression. Additionally, miR-181a* prevented PGCs from entering meiosis through the repression of the nuclear receptor subfamily 6, group A, member 1 transcript. Collectively, our data demonstrate that in chickens miRNAs intrinsically regulate the differentiation fate of blastoderms and PGCs and that the specific timing of germ cell meiosis is controlled through miRNA expression.

SUBMITTER: Lee SI

PROVIDER: S-EPMC3127938 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

Project description:Eyal-Giladi and Kochav stage X (EGK.X) blastoderm mostly consists undifferentiated progenitor (pluripotent) cells that have the potency to differentiate into all cell types. Analyzing the transcriptional programs of blastoderm is critical to understand the molecular mechanisms that underlie the developmental fate of early embryos, and practical application of blastoderm-derived pluripotent cells, including ESC. Primordial germ cells (PGC) are also undifferentiated progenitor cells that later differentiate into male and female germ cells. Analyzing the transcriptional programs of PGC is critical to understand the molecular mechanisms that underlie the developmental fate of early germ cells, and practical application of PGC in generating transgenic chickens. Therefore, we performed whole-transcriptome sequencing (WTS) in the chicken EGK.X blastoderm and PGCs together with the chicken embryonic fibroblasts (CEF, as a reference sample). CEF are differentiated and an abundant somatic cells. The generated WTS data of CEF, EGK.X blastoderm and PGC are rich resources to utilize for various focused research. As a first focused research, we utilized the generated WTS data for analyzing the expression profiling of DNA repair and apoptosis pathway genes in EGK.X blastoderm and PGC compared with CEF. DNA is the fundamental unit of inheritance and is susceptible to damage by various exogenous and endogenous sources. When the DNA is damaged, the cell sense the nature of damage through multiple surveillance mechanisms and select an appropriate DNA repair pathway to repair the damage and avoid passing the damage to a progeny of cells. When the cell fails to repair DNA damage, cell cycle progression is halted and apoptosis is initiated. Despite several genes are involved in five major DNA repair pathways and two major apoptosis pathways, a comprehensive understanding on the expression of those genes are not well-understood in chicken tissues. Through WTS analysis in CEF, stage X blastoderm and PGC, we identified that most of the DNA repair pathway genes were expressed more high in blastoderm and high in PGC than CEF. At the other hand, most of the apoptosis pathway genes were expressed low in blastoderm and PGC than CEF.

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MicroRNA-mediated posttranscriptional regulation is required for maintaining undifferentiated properties of blastoderm and primordial germ cells in chickens.

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