Whole-genome microRNA screening identifies let-7 and mir-18 as regulators of germ layer formation during early embryogenesis.
Tight control over the segregation of endoderm, mesoderm, and ectoderm is essential for normal embryonic development of all species, yet how neighboring embryonic blastomeres can contribute to different germ layers has never been fully explained. We postulated that microRNAs, which fine-tune many biological processes, might modulate the response of embryonic blastomeres to growth factors and other signals that govern germ layer fate. A systematic screen of a whole-genome microRNA library revealed that the let-7 and miR-18 families increase mesoderm at the expense of endoderm in mouse embryonic stem cells. Both families are expressed in ectoderm and mesoderm, but not endoderm, as these tissues become distinct during mouse and frog embryogenesis. Blocking let-7 function in vivo dramatically affected cell fate, diverting presumptive mesoderm and ectoderm into endoderm. siRNA knockdown of computationally predicted targets followed by mutational analyses revealed that let-7 and miR-18 down-regulate Acvr1b and Smad2, respectively, to attenuate Nodal responsiveness and bias blastomeres to ectoderm and mesoderm fates. These findings suggest a crucial role for the let-7 and miR-18 families in germ layer specification and reveal a remarkable conservation of function from amphibians to mammals.
PubMed ID: 23152446
PMC ID: PMC3521625
Article link: Genes Dev.
Grant support: P30 CA030199-30 NCI NIH HHS , P30 CA030199-30 NCI NIH HHS , P30 CA030199-30 NCI NIH HHS , P30 CA030199-30 NCI NIH HHS , P30 CA030199-30 NCI NIH HHS , P30 CA030199-30 NCI NIH HHS , R01 HL113601 NHLBI NIH HHS , R01 HL113601 NHLBI NIH HHS , R01 HL113601 NHLBI NIH HHS , R01 HL113601 NHLBI NIH HHS , R01 HL113601 NHLBI NIH HHS , R01 HL113601 NHLBI NIH HHS , R33 HL087375 NHLBI NIH HHS , R33 HL087375 NHLBI NIH HHS , R33 HL087375 NHLBI NIH HHS , R33 HL087375 NHLBI NIH HHS , R33 HL087375 NHLBI NIH HHS , R33 HL087375 NHLBI NIH HHS , R33 HL088266 NHLBI NIH HHS , R33 HL088266 NHLBI NIH HHS , R33 HL088266 NHLBI NIH HHS , R33 HL088266 NHLBI NIH HHS , R33 HL088266 NHLBI NIH HHS , R33 HL088266 NHLBI NIH HHS , P30 CA030199 NCI NIH HHS , P30 CA030199 NCI NIH HHS , P30 CA030199 NCI NIH HHS , P30 CA030199 NCI NIH HHS , P30 CA030199 NCI NIH HHS , P30 CA030199 NCI NIH HHS , R01 GM062848 NIGMS NIH HHS
Genes referenced: acvr1b myod1 nodal nodal1 nodal2 nodal3.1 smad2 t wnt8a
Morpholinos referenced: acvr1b MO2
Article Images: [+] show captions
|Figure 4. Endogenous localization of let-7a and miR-18a in early vertebrate embryos. (A) In situ hybridization showing endogenous let-7a and miR-18a in E7 mouse embryos viewed in whole-mount (A,D) or transverse histological section (B,E). (C,F) High-magnification view distinguishing epiblast mesoderm and endoderm layers. Note the abundant expression of let-7a (A) and miR-18a (D) in ectoderm and mesoderm but not in endoderm. (G) Endogenous Xlet-7a in cleavage stage Xenopus embryos showing expression in the animal hemisphere (presumptive ectoderm and mesoderm). (S) Endogenous Xlet-7a expression in gastrula stage (stage 10.5) Xenopus embryos. View of the mesoderm in the equatorial region marked by Xbra (S) and involuting endoderm marked by Xsox17α (T) reveals overlap with Xlet-7a prominently in mesoderm (U). (V) Transverse sections show expression domains of Xbra (V), XleftyA (W), and Xsox17α (X) relative to Xlet-7a (Y), revealing that the miR is present in both ectoderm and mesoderm but not in endoderm and is nonoverlapping with XleftyA but coincident with Xacvr1b (Z) mRNAs.|
|Figure 5. Endogenous Xlet-7 discriminates mesoderm and endoderm by repressing Xacvr1b. (A) Diagram of the TP loss-of-function strategy to block interaction of the miR with the mRE in vivo. (B) Either Xlet-7 TP or control morpholinos were injected equatorially into two blastomeres on one side of four-cell stage embryos. (C,D) Unilaterally injected embryos (as in B) cultured to gastrula stage (stage 10.5), bisected transversely, and probed for Xacrv1b (C) and XleftyA (D) expression. Injection of Xlet-7 TP morpholino up-regulated and expanded the domains of Xacrv1b and XleftyA expression in the marginal zone mesoderm and underlying deep endoderm. (E) Xlet-7 TP injection up-regulated Xnr-1, Xnr-2, Xnr-3, and Xnr-4 as well as XleftyA by qRTCR on dissected lateral marginal zone explants relative to the control morpholino, which has no effect on development or gene expression. (F) Unilateral Xlet-7 TP morpholino injection repressed a marker of mesoderm, Xbra (F), while concomitantly expanding the domain of endodermal marker genes Xsox17α (G) and Xfoxa2 (H) into the mesoderm domain. (I,J) Unilateral control morpholino injection had no effect on both mesodermal (I) and endodermal (J) markers. (K,L) qRTCR analyses on dissected lateral marginal zone explants show that the Xlet-7 TP morpholino up-regulated expression of endodermal genes (XSox17α, Xfoxa2, and Xmixer) (K) and repressed mesodermal genes (Xbra, XmyoD, and Xwnt8) (L) as compared with the control morpholino.|
|Figure 6. Blocking endogenous let-7 converts ectoderm to endoderm in embryos. (A) Embryos at the 16-cell stage were injected in one animal blastomere (presumptive ectoderm) together with Xlet-7 TP or control morpholino and LacZ mRNA dextran as a lineage label. Whereas the progeny of control morpholino-injected blastomeres contributed exclusively to epidermis (A), Xlet-7 TP morpholino-injected blastomeres contributed to endoderm, examined at the tailbud stage (stage 35) in lateral view (A,D), transverse bisection (B,F), dorsal view (C,G), and ventral view (D,H). (I) The same as in A, but examined at the tadpole stage (stage 45) and injected with Alexa Fluor546 dextran as a lineage label. Note the pronounced diversion of ectoderm to gut cell fate conversion, viewed ventrally.|