The molecular controls that govern the differentiation of embryonic stem (ES) cells remain poorly understood. DGCR8 is an RNA-binding protein that assists the RNase III enzyme Drosha in the processing of microRNAs (miRNAs), a subclass of small RNAs. Here we study the role of miRNAs in ES cell differentiation by generating a Dgcr8 knockout model. Analysis of mouse knockout ES cells shows that DGCR8 is essential for biogenesis of miRNAs. On the induction of differentiation, DGCR8-deficient ES cells do not fully downregulate pluripotency markers and retain the ability to produce ES cell colonies; however, they do express some markers of differentiation. This phenotype differs from that reported for Dicer1 knockout cells, suggesting that Dicer has miRNA-independent roles in ES cell function. Our findings indicate that miRNAs function in the silencing of ES cell self-renewal that normally occurs with the induction of differentiation.
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Gene Expression Omnibus
We thank C. Beard and the Anita Sil Laboratory for technical advice; J. Dausman for technical help; J. Reiter and N. Shomron for reagents and M. Ramalho-Santos, J. Reiter and members of the Blelloch Laboratory for critically reading the manuscript. The primers for Oct4, Rex1, Gapdh, T (brachyury) and Krt18 were gifts from J. Reiter (University of California San Francisco) This work was supported by grants to R.B. from the University of California San Francisco Urology Department, the Sandler Foundation, the Lance Armstrong Foundation and the National Institutes of Health (K08 NS48118). Y.W. is supported by the California Institute of Regenerative Medicine postdoctoral fellowship program. C.M. is supported by the National Science Foundation graduate research fellowship program.
RNA blot and microarray analysis of miRNAs.
Analysis of apoptosis in wild-type, DGCR8 knockout and DGCR8 rescued ES cells.
Quantitative real-time PCR analysis of Nanog, Afp, Vasa, Eomes and Sox1 after EB differentiation at 0, 8 and 16 d.
Semiquantitative RT-PCR analysis of wild-type and rescued ES cells.
Colony differentiation assay.
Ratio of miRNA expression for δ/flox and δ/δ relative to wild-type.
Ratio of abnormal implants from DGCR8 wt/δ × wt/δ crosses at different time points in embryonic development.
Sequences of probes used in RNA blot analysis and primers used in quantitative real-time PCR.