We used two complementary methods MO based

We used two complementary methods, MO-based knockdown and cell transplantation, to generate zebrafish containing a spectrum of PGC numbers. Our findings revealed that a threshold number of PGCs is required for the stability of ovarian fate. Furthermore, no compensatory proliferation of germ Deforolimus in the PGC-depleted morphants was observed, thus maintaining the morpholino-induced phenotype during development. In organogenesis, the number of tissue-specific stem cells might regulate the final size of the organs (Stanger et al., 2007). Our results suggest that the ability of PGCs to proliferate is constrained by their number, which consequently changes the size and identity of the gonad. The development of PGC-depleted gonads appeared to be inadequate and protracted, which seems to delay differentiation further. When PGC number increased, compensatory growth occurred, indicating the presence of potential feedback molecules, possibly growth factors derived from somatic cells. Interestingly, we also observed 10%–20% of zebrafish with a PGC number below the threshold developed as females, suggesting that these individuals might possess a distinct genetic makeup, leading to different response to the size of germ cell pool. We do not know whether there is any connection between the initial PGC count and the response to the depletion. It is possible that the “additional males” appearing in the MO-depleted batch are genetic females with a lower number of initial PGCs (within the normal female range) prior to injection that forced them to develop a testis. Germ cell sex determination and meiotic initiation are tightly coupled events during the early stage of sexual differentiation (Lesch and Page, 2012). Our histological analysis and gene expression profiles (zp2, org, sycp3, dazl) confirmed that zebrafish gonads are in a meiotic ovarian stage at 14 dpf. Concurrently, we also detected putative testis markers expressed in WT gonads, indicating the plastic nature of early immature gonads expressing “lineage priming” genes of both sexes as shown in mammals (Jameson et al., 2012). Comparative transcriptome analysis showed that under unbiased selection, WT individuals display similar expression patterns, indicating that zebrafish undergoing either mode of proliferation (and with either sexual fate) will enter meiosis. On the other hand, PGC-depleted gonads were not able to express ovarian or meiotic markers at that stage; thus, the juvenile ovary stage might be accordingly delayed. In mammals, the mutual antagonism between CYP26B1/FGF9 and retinoic acid (RA) regulates the meiotic entry of germ cells (Kocer et al., 2009). Our data suggest that zebrafish might use alternative molecules or mechanisms for executing meiosis since we did not detect dimorphic expression of cyp26a1 either at the beginning of sexual differentiation (14 dpf) or during meiotic progression of ovary (22 dpf). It should be noted that immune response genes were predominantly upregulated in PGC-depleted morphants at 14 dpf, and this may correlate with PGC depletion inducing a regenerative response similar to that of injured tissues (Godwin and Brockes, 2006). Moreover, we observed that immune response genes were upregulated in both WT and the PGC-depleted morphants at 22 dpf, suggesting that these genes may serve key regulatory roles during gonadal development, as in rainbow trout (Yano et al., 2012). The comparison of transcriptomes among WT zebrafish identified seven DE genes at 14 dpf and 945 DE genes at 22 dpf, manifesting the increasing divergence of promale versus profemale pathways during development. At 22 dpf, the expression of multiple signaling pathways revealed a complex regulatory network in gonadal differentiation. Several developmental pathways have been shown to be involved in germline development and the juvenile ovary to testis transformation in zebrafish, including the canonical Wnt, Tp53/Fancl, nuclear factor-κB, and Piwi/piRNA pathways (Houwing et al., 2007; Pradhan et al., 2012; Rodríguez-Marí et al., 2010; Sreenivasan et al., 2014). Our data demonstrate that genes/pathways supporting ovarian follicle development are coupled with genes associated with meiotic progression and mRNA translation, further promoting the female fate.