April 1, 2009;
The miR-430/427/302 family controls mesendodermal fate specification via species-specific target selection.
The role of microRNAs in embryonic cell fate specification is largely unknown. In vertebrates, the miR-430/427/302 family shows a unique expression signature and is exclusively expressed during early embryogenesis. Here, we comparatively address the embryonic function of miR-302 in human embryonic stem cells (hESCs) and its ortholog miR-427 in Xenopus laevis. Interestingly, we found that this miRNA family displays species-specific target selection among ligands of the Nodal
pathway, with a striking conservation of the inhibitors, Lefties, but differential targeting of the activators, Nodals. The Nodal
pathway plays a crucial role in germ layer
specification. Accordingly, by gain and loss of function experiments in hESCs, we show that miR-302 promotes the mesendodermal lineage at the expense of neuroectoderm
formation. Similarly, depletion of miR-427 in Xenopus embryos hinders the organizer
formation and leads to severe dorsal mesodermal patterning defects. These findings suggest a crucial role for the miR-430/427/302 family in vertebrate embryogenesis by controlling germ layer
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Figure 1. miR-430/427/302 microRNA Family Expression(A) Sequences of human and mouse miR-302a, Xenopus laevis miR-427, and zebrafish miR-430a, representative miRNAs that belong to the miR-430/427/302 family. The seed sequence is indicated in red.(B and C) Northern blot analysis of miRNA expression during differentiation of the RUES1 and RUES2 hESC lines in (B) monolayer or (C) embryoid bodies. The probes used are indicated on the right of each panel. The small nuclear RNA U2 was used as loading control. CM, MEF-conditioned medium; 2D, differentiation in two-dimensional culture.(D) Temporal expression pattern of miR-427 during early X. laevis development. The 5.8S ribosomal RNA is used as loading control.(E) Spatial expression of miR-427 in a X. laevis embryo analyzed by whole-mount in situ hybridization. The arrow indicates the dorsal lip; arrowheads indicate the neural plate border.
Figure 4. miR-427 Loss of Function Affects Mesodermal Patterning and Organizer Induction in Vivo(A) Ventralization of mesodermal explants by miR-427 depletion. Xenopus eight-cell-stage embryos were injected into both dorsal (DMZ) or ventral (VMZ) marginal blastomeres with 10 ng α-miR-427-Mo. Uninjected and Mo-injected DMZ and VMZ explants were dissected at stage 10 and assayed for expression of the indicated markers by RT-PCR analysis.(B) Whole-mount in situ hybridization for the organizer markers, chordin and goosecoid (purple staining). Embryos injected dorsally with LacZ RNA together with either α-miR-427-Mo or lefty mRNA or left untreated were stained at stage 10. Light-blue staining represents expression of the lineage tracer LacZ in the injected side of the embryo.(C) Decreased Nodal activity in miR-427-depleted embryos. Different doses of Xnr1 RNA, as indicated, were injected alone or together with α-miR-427-Mo (10 ng) into the animal pole of two-cell-stage embryos. Animal caps were examined at the gastrula stage (stage 11) for the expression of Xnr target genes by RT-PCR. Chd, chordin; gsc, goosecoid; ODC, ornithine decarboxylase; xbra, brachyury.
Semiconserved regulation of mesendoderm differentiation by microRNAs.