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Referee comments: Referee 1

Posted by PLOS_ONE_Group on 07 Mar 2008 at 18:49 GMT

Referee 1's review:

Hayashi and colleagues report a requirement of Dicer in mouse PGC and spermatogonia proliferation.

The authors report the phenotype of Dicer KO in mouse PGCs and spermatogonia. They reported that, in TNAP-Cre mediated Dicer cKO embryos, the numbers of PGCs and spermatogonia were reduced, and these reductions were caused by defect in proliferation of these cells. In mouse female germline, requirement of Dicer, which produces miRNAs, in the production of normal egg have been reported from this group (Tang et al., Genes Dev. 2007) and another (Murchison et al., Genes Dev 2007) using Zp3-Cre mediated Dicer cKO mouse. In male germline, however, the requirement of Dicer or miRNAs has not been reported. This is the first demonstration that Dicer or miRNA is required for development of male germ cells.

The authors also describe the detailed expression analysis of 214 miRNAs during PGC development by qPCR. They found that miR-17 and ES cell-specific 290-5 clusters were highly expressed in PGCs. Considereing the previous studies of the miR-17 cluster, it is possible that this miR-17 clusters are implicated in proliferation of PGCs and spermatogonia as suggested by the authors.

piRNAs, which are another classes of small RNAs and bound to Piwi protein, have a role in repression of retrotransposons during spermatogenesis. The authors show that Dicer is not involved in the repression of retrotransposons. This result show that production of mouse piRNAs is independent of Dicer as has been observed in worms and zebrafish, and that two distinct small RNA pathways are exist have different roles during mouse spermatogenesis.

This is interesting study, and suitable for publication in PLOS one. The authors need to discuss the following points.

1. It is possible that expression level of Gapdh mRNA, which was used as control mRNA in this study, was upregulated or downregulated in Dicer cKO cells. In fact, this group (Tang et al., Genes Dev. 2007) reported that many genes were upregulated in Dicer KO oocytes. The authors need to show that the level of Gapdh is unchanged in Dicer KO PGCs.

2. It is attractive model that miR-19 targets the tumor suppressor gene PTEN and promotes the proliferation of PGCs. Is the level of PTEN mRNA upregulated in Dicer cKO PGCs?

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N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.