An increase in the PGC number

An increase in the PGC number caused by dnd RNA injection does not exceed 3-fold, even if a high level of Dnd expression occurs in many cells. It is likely that an additional factor essential for PGC specification exists in medaka, which shows expression and PGC-specifying activity similar to dnd but dnd-independent asymmetric segregation. Alternatively, PGC-competent SAG supplier are limited in the medaka embryo for the ultimate fate decision. It is also possible that only a limited number of PGCs are permissive for embryonic development and survival until observation for PGCs. This possibility is small, because dnd RNA-injected embryos are not different from controls in survival rate and cell death as well as survival rate of their blastomeres in single-cell culture. A similar situation has also been recorded for the Drosophila PGC specifier oskar in vivo (Ephrussi and Lehmann, 1992) and the human PGC specifier Sox17 in vitro (Irie et al., 2015). During early development of diverse animals, miRNAs play an essential role in maternal-zygotic transcription and PGC development (Giraldez et al., 2006; Schier, 2007). Through inhibiting the accessibility of miRNAs including MiR-430 to target mRNAs, Dnd protects maternal RNAs from clearance in cultured human germ cells and PGCs of zebrafish (Kedde et al., 2007) and medaka (Tani et al., 2009). In this study, by reporter assays we reveal that Dnd counteracts with miRNA-430 to regulate maternal RNA turnover and translation for medaka PGC formation. This suggests that Dnd protects germ plasm RNAs from miRNA-mediated degradation to ensure PGC specification. Our identification of Dnd as a critical PGC specifier in medaka provides insights into cell fate decisions during germline-soma separation in medaka as a lower vertebrate model. Altering Dnd activity could be a strategy for analyzing and manipulating germ cell formation. Our finding that dnd RNA asymmetrically segregates via particle partition reveals the presence of a previously unidentified mechanism that controls asymmetric segregation of macromolecules in early development.
Experimental Procedures
Author Contributions
Acknowledgments We thank Dr. A.F. Schier for pGFPmiR430, Jiaorong Deng for fish breeding, Veronica Wong and Choy Mei Foong for laboratory management. This work was supported by grants to Y.H. from the National Research Foundation of Singapore (NRF-CRP7-2010-03), to J.S. from the Ministry of Education of Singapore (R-154-000-678-112), and to M.L. from the National Natural Science Foundation of China (31372520) and the Shanghai Universities First-Class Disciplines Project of Fisheries.
Introduction Neurological diseases have mainly been studied using animal models and immortalized neural cell lines due to the difficulties associated with examining the CNS of patients. Recent advances in human induced pluripotent stem cell (hiPSC) technologies have enabled neurological diseases to be modeled by culturing patient-specific neural cells in dishes (Imaizumi and Okano, 2014; Marchetto and Gage, 2012). The first hiPSCs were generated from cultured dermal fibroblasts by inducing reprogramming factors (Takahashi et al., 2007). hiPSCs derived from fibroblasts have been recognized as the standard iPSCs for several years. Therefore, most previously reported patient-specific hiPSC lines were generated from skin fibroblasts (Brennand et al., 2011; Imaizumi et al., 2012). Skin biopsies of patients are required to generate dermal fibroblast lines, and this can cause bleeding, infection, and scarring. Therefore, patient-specific hiPSCs should ideally be generated using less invasive procedures, but the resulting cells must have a similar pluripotency as dermal fibroblast-derived hiPSCs. Yamanaka and colleagues first reported that iPSCs can be generated from various types of somatic cells, including hepatocytes (Aoi et al., 2008). Since then, several groups have generated hiPSCs from peripheral blood nuclear cells (PBMC) (Loh et al., 2010; Mack et al., 2011; Seki et al., 2010), which can be easily obtained from patients using minimally invasive methods. Among these reports, Fukuda and colleagues showed that a small number of CD3-positive T cells can be efficiently reprogrammed into iPSCs using Sendai virus (SeV) vectors (Seki et al., 2010). CD3-positive T cells can be cultured in vitro using plates coated with an anti-CD3 monoclonal antibody (mAb) and in the presence of recombinant interleukin-2 (rIL-2). These cells can be stored in frozen vials and thawed several months later. Thus, CD3-positive T cells can be obtained non-invasively, are easily stored and efficiently reprogrammed, and might therefore be an ideal source of patient-specific iPSCs.