Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
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.
???displayArticle.pubmedLink???
23152446 ???displayArticle.pmcLink???PMC3521625 ???displayArticle.link???Genes Dev ???displayArticle.grants???[+]
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.
Agius,
Endodermal Nodal-related signals and mesoderm induction in Xenopus.
2000, Pubmed,
Xenbase
Agius,
Endodermal Nodal-related signals and mesoderm induction in Xenopus.
2000,
Pubmed
,
Xenbase Armes,
The ALK-2 and ALK-4 activin receptors transduce distinct mesoderm-inducing signals during early Xenopus development but do not co-operate to establish thresholds.
1997,
Pubmed
,
Xenbase Bartel,
MicroRNAs: target recognition and regulatory functions.
2009,
Pubmed Ben-Ami,
A regulatory interplay between miR-27a and Runx1 during megakaryopoiesis.
2009,
Pubmed Branford,
Regulation of gut and heart left-right asymmetry by context-dependent interactions between xenopus lefty and BMP4 signaling.
2000,
Pubmed
,
Xenbase Branford,
Lefty-dependent inhibition of Nodal- and Wnt-responsive organizer gene expression is essential for normal gastrulation.
2002,
Pubmed
,
Xenbase Bushway,
High-throughput screening for modulators of stem cell differentiation.
2006,
Pubmed Castellano,
The estrogen receptor-alpha-induced microRNA signature regulates itself and its transcriptional response.
2009,
Pubmed Chang,
Neural induction requires continued suppression of both Smad1 and Smad2 signals during gastrulation.
2007,
Pubmed
,
Xenbase Chang,
Widespread microRNA repression by Myc contributes to tumorigenesis.
2008,
Pubmed Chen,
Developmental analysis of activin-like kinase receptor-4 (ALK4) expression in Xenopus laevis.
2005,
Pubmed
,
Xenbase Choi,
Target protectors reveal dampening and balancing of Nodal agonist and antagonist by miR-430.
2007,
Pubmed Colas,
Mix.1/2-dependent control of FGF availability during gastrulation is essential for pronephros development in Xenopus.
2008,
Pubmed
,
Xenbase Dale,
Fate map for the 32-cell stage of Xenopus laevis.
1987,
Pubmed
,
Xenbase Djiane,
Role of frizzled 7 in the regulation of convergent extension movements during gastrulation in Xenopus laevis.
2000,
Pubmed
,
Xenbase Dosch,
Bmp-4 acts as a morphogen in dorsoventral mesoderm patterning in Xenopus.
1997,
Pubmed
,
Xenbase Dougan,
The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm.
2003,
Pubmed Dunn,
Combinatorial activities of Smad2 and Smad3 regulate mesoderm formation and patterning in the mouse embryo.
2004,
Pubmed Dupont,
Germ-layer specification and control of cell growth by Ectodermin, a Smad4 ubiquitin ligase.
2005,
Pubmed
,
Xenbase Dyson,
The interpretation of position in a morphogen gradient as revealed by occupancy of activin receptors.
1998,
Pubmed
,
Xenbase Ema,
Deletion of the selection cassette, but not cis-acting elements, in targeted Flk1-lacZ allele reveals Flk1 expression in multipotent mesodermal progenitors.
2006,
Pubmed Filipowicz,
Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight?
2008,
Pubmed Gadue,
Wnt and TGF-beta signaling are required for the induction of an in vitro model of primitive streak formation using embryonic stem cells.
2006,
Pubmed Green,
Graded changes in dose of a Xenopus activin A homologue elicit stepwise transitions in embryonic cell fate.
1990,
Pubmed
,
Xenbase Green,
Morphogen gradients, positional information, and Xenopus: interplay of theory and experiment.
2002,
Pubmed
,
Xenbase Green,
Responses of embryonic Xenopus cells to activin and FGF are separated by multiple dose thresholds and correspond to distinct axes of the mesoderm.
1992,
Pubmed
,
Xenbase Gurdon,
Single cells can sense their position in a morphogen gradient.
1999,
Pubmed
,
Xenbase Gurdon,
Activin signalling and response to a morphogen gradient.
1994,
Pubmed
,
Xenbase Hudson,
Xsox17alpha and -beta mediate endoderm formation in Xenopus.
1997,
Pubmed
,
Xenbase Iratni,
Inhibition of excess nodal signaling during mouse gastrulation by the transcriptional corepressor DRAP1.
2002,
Pubmed Jones,
Nodal-related signals induce axial mesoderm and dorsalize mesoderm during gastrulation.
1995,
Pubmed
,
Xenbase Kikuchi,
Notch signaling can regulate endoderm formation in zebrafish.
2004,
Pubmed
,
Xenbase King,
Putting RNAs in the right place at the right time: RNA localization in the frog oocyte.
2005,
Pubmed
,
Xenbase Latinkić,
The Xenopus Brachyury promoter is activated by FGF and low concentrations of activin and suppressed by high concentrations of activin and by paired-type homeodomain proteins.
1997,
Pubmed
,
Xenbase Lee,
Micropatterning of human embryonic stem cells dissects the mesoderm and endoderm lineages.
2009,
Pubmed Li,
MiR-17-92 cluster regulates cell proliferation and collagen synthesis by targeting TGFB pathway in mouse palatal mesenchymal cells.
2012,
Pubmed Luxardi,
Distinct Xenopus Nodal ligands sequentially induce mesendoderm and control gastrulation movements in parallel to the Wnt/PCP pathway.
2010,
Pubmed
,
Xenbase Marjoram,
Rapid differential transport of Nodal and Lefty on sulfated proteoglycan-rich extracellular matrix regulates left-right asymmetry in Xenopus.
2011,
Pubmed
,
Xenbase Mayr,
Disrupting the pairing between let-7 and Hmga2 enhances oncogenic transformation.
2007,
Pubmed Müller,
Differential diffusivity of Nodal and Lefty underlies a reaction-diffusion patterning system.
2012,
Pubmed Murry,
Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development.
2008,
Pubmed Newman,
Lin-28 interaction with the Let-7 precursor loop mediates regulated microRNA processing.
2008,
Pubmed Nieuwkoop,
The "organization centre". 3. Segregation and pattern formation in morphogenetic fields.
1967,
Pubmed Peng,
Xenopus laevis: Practical uses in cell and molecular biology. Solutions and protocols.
1991,
Pubmed
,
Xenbase Perea-Gomez,
Nodal antagonists in the anterior visceral endoderm prevent the formation of multiple primitive streaks.
2002,
Pubmed Peyriéras,
Conversion of zebrafish blastomeres to an endodermal fate by TGF-beta-related signaling.
1998,
Pubmed Piccolo,
The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals.
1999,
Pubmed
,
Xenbase Re'em-Kalma,
Competition between noggin and bone morphogenetic protein 4 activities may regulate dorsalization during Xenopus development.
1995,
Pubmed
,
Xenbase Reissmann,
The orphan receptor ALK7 and the Activin receptor ALK4 mediate signaling by Nodal proteins during vertebrate development.
2001,
Pubmed
,
Xenbase Rosa,
The miR-430/427/302 family controls mesendodermal fate specification via species-specific target selection.
2009,
Pubmed
,
Xenbase Roush,
The let-7 family of microRNAs.
2008,
Pubmed Ruiz i Altaba,
Sequential expression of HNF-3 beta and HNF-3 alpha by embryonic organizing centers: the dorsal lip/node, notochord and floor plate.
1993,
Pubmed
,
Xenbase Rybak,
A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stem-cell commitment.
2008,
Pubmed Schier,
The one-eyed pinhead gene functions in mesoderm and endoderm formation in zebrafish and interacts with no tail.
1997,
Pubmed Schneider,
Spatially distinct head and heart inducers within the Xenopus organizer region.
,
Pubmed
,
Xenbase Shen,
Nodal signaling: developmental roles and regulation.
2007,
Pubmed Smith,
Identification of a potent Xenopus mesoderm-inducing factor as a homologue of activin A.
1990,
Pubmed
,
Xenbase Stathopoulos,
Dorsal gradient networks in the Drosophila embryo.
2002,
Pubmed Sweetman,
Specific requirements of MRFs for the expression of muscle specific microRNAs, miR-1, miR-206 and miR-133.
2008,
Pubmed Tabata,
Morphogens, their identification and regulation.
2004,
Pubmed Tada,
Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture.
2005,
Pubmed Tadros,
The maternal-to-zygotic transition: a play in two acts.
2009,
Pubmed Takahashi,
Ascorbic acid enhances differentiation of embryonic stem cells into cardiac myocytes.
2003,
Pubmed Varlet,
nodal expression in the primitive endoderm is required for specification of the anterior axis during mouse gastrulation.
1997,
Pubmed Viswanathan,
Selective blockade of microRNA processing by Lin28.
2008,
Pubmed Vize,
Assays for gene function in developing Xenopus embryos.
1991,
Pubmed
,
Xenbase Wang,
Regulation of the let-7a-3 promoter by NF-κB.
2012,
Pubmed Wilkinson,
Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts.
1993,
Pubmed Willems,
Patterning of mouse embryonic stem cell-derived pan-mesoderm by Activin A/Nodal and Bmp4 signaling requires Fibroblast Growth Factor activity.
2008,
Pubmed
