Xenopus furry contributes to release of microRNA gene silencing

Toshiyasu Goto; Akimasa Fukui; Hiroshi Shibuya; Ray Keller; Makoto Asashima

doi:10.1073/pnas.1008954107

. 2010 Oct 25;107(45):19344–19349. doi: 10.1073/pnas.1008954107

Xenopus furry contributes to release of microRNA gene silencing

Toshiyasu Goto ^a,^b,^c, Akimasa Fukui ^a,^d, Hiroshi Shibuya ^c, Ray Keller ^b, Makoto Asashima ^a,^e,¹

PMCID: PMC2984173 PMID: 20974966

Abstract

A transcriptional corepressor, Xenopus furry (Xfurry), is expressed in the chordamesodermal region and induces secondary dorsal axes when overexpressed on the ventral side of the embryo. The N-terminal furry domain functions as a repressor, and the C-terminal leucine zipper (LZ) motifs /coiled-coil structure, found only in vertebrate homologs, contributes to the nuclear localization. The engrailed repressor (enR)+LZ repressor construct, which has properties similar to Xfurry, induced several chordamesodermal genes. In contrast, an antisense morpholino oligonucleotide, Xfurry-MO, and the activating construct, herpes simplex virus protein (VP16)+LZ, had effects opposite those of Xfurry overexpression. Because blocking protein synthesis with cycloheximide superinduced several Xfurry transcriptional targets, and because expression of enR+LZ induced such genes under cycloheximide treatment, we analyzed the role of an Xfurry transcriptional target, microRNA miR-15. Cycloheximide reduced the expression of primary miR-15 (pri-miR-15), whereas miR-15 reduced the expression of genes superinduced by cycloheximide treatment. These results show that Xfurry regulates chordamesodermal genes by contributing to repression of pretranscriptional gene silencing by miR-15.

Keywords: axis formation, transcriptional corepressor, chordamesodermal genes, cycloheximide

In Drosophila, furry is an important gene for maintaining the integrity of cellular extensions during wing morphogenesis (1). Mutations in furry result in a strong multiple hair-cell phenotype characterized by clusters of epidermal hairs and branched hairs. Furry functions via interactions with a serine/threonine kinase, tricornered, through the furry domain (FD) (1, 2). Tricornered encodes the Drosophila nuclear DBF2-related (NDR) kinase, and mutations of this gene result in dramatic multiple wing hair, branched bristle, and lateral arista phenotypes (3). Tricornered/furry signaling also plays a key role in the development of sensory dendrites in Drosophila neurons (4). In fission yeast, Mor2/Furry and Orb6/Tricornered are essential for morphogenesis through regulation of the actin cytoskeleton (5), whereas in budding yeast transcriptional activator of OCH1 (Tao3)/Furry affects cell wall biosynthesis kinase (Cbk1)/tricornered kinase activity in polarized growth (6). Recently, it was reported that Furry and the human homolog of Drosophila tricornered, NDR1, attach to spindle microtubules and play a role in mitotic chromosome alignment in mammalian cultured cells (7). Thus, furry and its homologs function generally in morphogenesis and cell division through interactions with tricornered and its homologs.

Furry protein localizes to the cell membrane in fly wings (2) and to the cytoplasm and spindle microtubules in yeast and mammalian cultured cells (5, 7). However, Furry also is found in the nuclei of salivary gland and fat body cells in the fly, suggesting an additional function in gene regulation (2). Human furry also has shown transcriptional regulatory properties in acute myeloid leukemia patients (8). Together, these observations suggest that furry functions in the nucleus, possibly in transcriptional regulation, and also highlight that much is unknown about the function of furry, especially in vertebrate embryogenesis.

Axis formation is an important event in early vertebrate embryogenesis that involves complex regulation of gene expression. In Xenopus, signaling molecules such as β-catenin and siamois in the early canonical wingless (Wnt) pathway (9–12) induce complete secondary axes with anterior structures when expressed on the ventral side, whereas activin/nodal-like signaling molecules such as derriere, Xenopus nordal related-1 gene (Xnr1), Xnr5, and Xnr6 (13–15) induce secondary axes without anterior structures. MicroRNAs also play important roles in axis formation and organizer induction, especially in the context of nodal signaling. MicroRNAs miR-15 and miR-16 inhibit nodal signaling at the translational level during axis formation (16), and depletion of miR-427 inhibits organizer formation (17). Thus, small RNAs are involved in novel aspects of major signaling pathways during embryogenesis. Small RNAs function at both the translational and transcriptional levels in vertebrates. The transcriptional gene silencing operates by the antisense strand of the siRNA targeting chromatin-remodeling complexes to the specific promoter regions (18, 19). In mammalian cultured cells, miR-320 contains the promoter-region sequence of the cell-cycle gene polymerase (RNA) III (DNA directed) polypeptide D (POLR3D) in the antisense orientation and plays a cis-regulatory role in transcriptional silencing of POLR3D expression (20). Moreover, transcriptional activation can be induced through RNA duplexes targeting several of the promoter regions in mammalian cells (21, 22). Taken together, this evidence suggests that microRNA-mediated transcriptional gene silencing is an important process in vertebrate embryogenesis.

The current study demonstrated that Xenopus furry (Xfurry) functions as a transcriptional corepressor during embryogenesis. Direct reduction of microRNA expression and indirect induction of dorsal genes by Xfurry implicate microRNAs in the regulation of gene silencing during embryogenesis.

Results

Expression of Xfurry.

We isolated a Xenopus homolog of furry (Xfurry) encoding a conserved, N-terminal FD (Fig. S1) with high identity to Drosophila furry and other furry homologs (Fig. S1). The nonconserved region of Xfurry has low identity with invertebrate furry but high identity with vertebrate furry. Xfurry has two C-terminal leucine zipper (LZ) motifs and a coiled-coil structure that are conserved only in vertebrates, suggesting that the function of Xfurry differs from its invertebrate homologs. Transcripts of Xfurry were present maternally at the same levels until the late blastula stage (stage 9), after which they decreased through gastrulation and then increased gradually again through the neurula and tailbud stages (Fig. 1A). Whole-mount in situ hybridization and RT-PCR revealed that Xfurry is expressed weakly in the dorsal mesodermal region (Fig. 1 B and C) at the early gastrula stage. The expression then increases gradually in the chordamesoderm during gastrulation (Fig. 1D) and is restricted to the notochord at late stages (Fig. 1 E and F). These expression patterns implicate Xfurry in axis formation (23–26).

Xfurry Induces Chordamesodermal Genes.

Overexpression of Xfurry mRNA in ventral blastomeres induced a secondary axis (Fig. 1H, arrowhead), whereas dorsal overexpression had no effect on axis formation (Fig. S2). Chordamesodermal marker genes were induced in animal caps dissected from embryos injected with Xfurry mRNA (Fig. 1I). In contrast, dorsal injection of Xfurry morpholino (Xfurry-MO) (Fig. S3) had effects opposite those of Xfurry mRNA overexpression, including interference with head formation, shortened dorsal axes (Fig. 1K), and reduced expression of chordamesodermal marker genes (Fig. 1L). The phenotype of Xfurry-MO–injected embryos was rescued by coinjection with Xfurry mRNA (Fig. S4C). These results indicate that Xfurry plays an important role in axis formation during Xenopus development by inducing expression of chordamesodermal genes.

Xfurry Functions as a Transcriptional Corepressor.

To investigate the functional domains of Xfurry, we overexpressed various deletion constructs of Xfurry. A conjugate of the FD and the LZ/coiled-coil domain (FD+LZ) had an effect on secondary axis induction very similar to that of Xfurry mRNA (Fig. S5 and Table S1). These results suggest that the FD and LZ domains are essential and sufficient to induce a secondary axis.

Furry protein is expressed in a cell type-specific manner in Drosophila, notably in the cell membrane and cytoplasm of wing cells and in the nuclei of salivary gland and fat body cells (2). To investigate the function of Xfurry in Xenopus mesodermal cells during axis formation, we observed fluorescence expression of overexpressed full-length Xfurry conjugated with GFP at the C terminus (Xfurry+GFP cDNA) at gastrula stages. The fluorescent protein was detected throughout the nucleus and cytoplasm of mesodermal cells (Fig. 2A, Top Row and Fig. S5). Overexpressed FD+LZ+GFP, which has a function similar to that of Xfurry (Table S1), is localized in nuclei and cytoplasm (Fig. 2A, Second Row and Fig. S5). In contrast, LZ+GFP is localized mainly in nuclei and FD+GFP is localized in the cytoplasm (Fig. 2A, Third and Bottom Rows). We also confirmed the localization of GFP constructs by Western blotting of nuclear and cytoplasm extracts from the injected embryos (Fig. S6). The blot results suggest that Xfurry localizes to nuclei via the LZ structure and that the localization of Xfurry depends on the localization of partner proteins interacting via its FD and LZ domains. This evidence, together with the evidence that human furry has transcriptional properties in acute myeloid leukemia patients (8), supports the hypothesis that Xfurry enters the nucleus and functionally resembles a transcription factor. However, the LZ domain alone had no effect on axis formation (Table S1), indicating that another domain, the FD, is necessary for axis formation.

Fig. 2. — Function of *Xfurry* as a transcriptional corepressor. (A) Fluorescent signals of Xfurry constructs in stage-10 mesodermal cells (Fig. S5). Red signals indicate cell nuclei stained with DAPI. (*Top Row*) Xfurry+GFP. (*Second Row*) FD+LZ+GFP construct. (*Third Row*) LZ+GFP. (*Bottom Row*) FD+GFP. Arrowheads show colocalization of GFP and DAPI signals. Arrows indicate noncolocalized signals. (B) Control embryo (stage 27). (C) Dorsal injection of *FD+SiaHD* interfered with head development. (D) Expression of *FD+SiaHD* reduced transcripts of *goosecoid* (Gsc) and *Cerberus* (Cer). (E–G) Phenotypes of injected stage-29 embryos. (E) Control embryo. (F) Ventral injection of *enR+LZ* (400 pg/blastomere) induced secondary axes in all embryos (n = 59). (G) Dorsal injection of *VP16+LZ* (400 pg/blastomere) shortened the dorsal axis in all embryos (n = 60). (H) Dorsal expression of *enR+LZ* induced chordamesodermal and head organizer genes, and expression of *VP16+LZ* reduced those genes (stage 10).

On the other hand, the FD fused to the Siamois homeodomain (FD+SiaHD) showed effects opposite to Siamois and the same as a fusion of the engrailed repressor domain and the homeodomain of Siamois (enR+SiaHD) (27). Dorsal expression of FD+SiaHD ventralized the injected embryos (Fig. 2C) and repressed expression of the organizer/head organizer genes goosecoid and cerberus, which are downstream of Siamois (27, 28) (Fig. 2D). The FD therefore seems to function as a transcriptional repressor, and these results suggest that Xfurry forms a complex with transcription factors having both DNA-binding and furry-interaction domains, including ones interacting with the LZ structure, and that this complex functions as a transcriptional repressor via the FD. Expression of the FD alone weakly induced secondary axes (Table S1), suggesting that it might interact weakly with other transcription factors in such a complex. The LZ motif and coiled-coil structure are found only in vertebrate furry homologs, and a transcriptional regulation function has not been reported for invertebrate furry.

Additionally, several reports in Drosophila indicate that furry and its homologs interact and function with tricornered and its homologs (2–7). To determine whether Xenopus homologs of tricornered, the NDR1 and NDR2 kinases, affect axis formation, we overexpressed these genes in ventral blastomeres at the four-cell stage. Neither NDR1 nor NDR2 affected axis formation in Xenopus (Fig. S7), suggesting that the function of Xfurry in axis formation is distinct from that shown in reports on other systems and does not involve Tricornered as a downstream component.

To investigate further the function of Xfurry as a transcriptional corepressor, we made cDNA constructs in which a repressor domain (enR+LZ) or an activator domain (VP16+LZ), was fused to the C terminus of Xfurry (Fig. S5). Ventral expression of enR+LZ induced secondary axes similar to those induced by overexpression of Xfurry mRNA (Fig. 2F). In contrast, dorsal expression of VP16+LZ had opposite effects, interfering with head formation and shortening the dorsal axis (Fig. 2G), similar to the phenotype generated by Xfurry depletion (Fig. 1K). Expression of several zygotic marker genes (23–28), including chordamesodermal genes, was increased by expression of enR+LZ and reduced by expression of VP16+LZ (Fig. 2H). Expression of enR+LZ rescued the phenotype of Xfurry-MO–injected embryos, and overexpression of Xfurry rescued the phenotype of VP16+LZ-injected embryos (Fig. S4 D–F). We thus proposed that Xfurry is a transcriptional corepressor that up-regulates chordamesodermal gene expression on the dorsal side of the embryo by downregulating an as yet unidentified general repressor of these chordamesodermal genes. We used constructs of enR+LZ and VP16+LZ to examine this hypothesis in the following experiments.

Overexpression of the early, dorsal-inducing genes β-catenin (12), vegetal T-box transcription factor (VegT) (29), and Xnr5 (14) increased the levels of Xfurry transcript (Fig. 3A). Expression of Xfurry also was increased in goosecoid-injected embryos and in activin-treated animal caps (Fig. S8 A and B). These results suggest that Xfurry induction is a general response to dorsalization of the embryo. To identify potential repressors that are direct target genes of Xfurry, we performed a microarray assay using RNA extracted from pre-midblastula transition (MBT) cycloheximide-treated animal caps expressing enR+LZ or VP16+LZ (SI Materials and Methods). Unexpectedly, enR+LZ still induced early chordamesodermal marker genes in treated animal caps (Fig. 3B), suggesting that Xfurry functions as a transcriptional corepressor without zygotic protein synthesis.

Direct Target Gene of Xfurry.

Cycloheximide treatment superinduces the transcription of several genes (30, 31) including some Xfurry-induced genes (Fig. 3C, Left). The other protein synthesis inhibitor, anisomycin, also induced such cycloheximide-induced genes (Fig. 3C, Right). These findings indicate that Xfurry could be involved in the superinduction of genes via protein synthesis inhibitors. It has been suggested that the superinduction could occur by inhibition of protein synthesis of transcriptional repressors, enzymes such as ribonucleases, and components of gene silencing. Here, the transcriptional corepressor enR+LZ induced early chordamesodermal marker genes under cycloheximide treatment (Fig. 3B); thus Xfurry would increase the transactivational effect of superinduction without zygotic protein synthesis. This suggests that Xfurry does not function by down-regulating proteins such as other transcriptional repressors and enzymes such as ribonucleases.

In further experiments, we found that blocking translation of specific cycloheximide-inducible genes with antisense morpholino oligonucleotides (MOs) also increased their transcripts. For example, injection of pintallavis-MO and goosecoid-MO increased transcripts of pintallavis and goosecoid, respectively (Fig. 3 D and E). In the case of goosecoid, it is possible that the negative feedback loop, in which goosecoid protein works as a repressor of itself (32), is related to this transactivation. However, our data revealed that injection of pintallavis-MO and goosecoid-MO increased expression of a transcriptional corepressor, Xfurry (Fig. 3F). These results indicate that reduced levels of specific proteins are required for the increase of Xfurry. Moreover, cycloheximide and anisomycin treatment also induced Xfurry expression (Fig. 3C). Taken together, these findings suggest that Xfurry is necessary for repressing nontranslational (i.e., transcriptional) gene silencing to activate the transcription of early chordamesodermal genes.

We next isolated small (20- to 120-base) RNAs and injected them into dorsal blastomeres at the four-cell stage. Small RNAs from the fraction with 100–120 bases or less than 60 bases did not severely affect axis formation (Fig. 4 B and D), but small RNAs from the fraction with 60–100 bases, including premicroRNAs (around 70 bases), severely interfered with axis formation (Fig. 4C). Small RNAs, including premicroRNAs, strongly reduced early chordamesodermal gene expression (Fig. 4E). Because small RNAs from the fraction with less than 60 bases did not severely affect axis formation, we considered that appropriate processing would be necessary for function of matured small RNAs.

In yeast and mammalian cultured cells, microRNA forms an RNA-induced, transcriptional gene-silencing (RITS) complex that inhibits transcription via DNA methylation at the promoter region of its gene (16–22). However, the role of microRNA/RITS complexes in embryogenesis is still unexplained. To investigate whether small RNAs, including premicroRNAs, have RITS function in Xenopus embryogenesis, we injected small RNAs into four animal blastomeres of 32-cell embryos in a solution containing cycloheximide to prevent any translational inhibition by small RNAs. Because most of these embryos treated with cycloheximide died before the 32-cell stage, we began this experiment after the 32-cell stage. Small RNAs strongly reduced expression of superinduced genes in the injected animal cap cells and also slightly reduced expression of Xfurry (Fig. 4F). This suggests that small RNAs, including premicroRNAs, have RITS properties for the transcription of several genes. In Xenopus, mir-15 inhibits nodal signaling at the translational level (16), and the phenotype of Xfurry-induced secondary axes lacking heads was similar to that produced when nodal signaling is activated (Fig. 1H) (33). Therefore we investigated whether Xfurry controls the expression of mir-15 and whether mir-15 functions via the RITS complex.

Expression of enR+LZ reduced transcripts of primary miR-15 (pri-miR-15) in cycloheximide-treated animal caps (Fig. 4G), whereas overexpression of mir-15 reduced expression of goosecoid, Xenopus not gene (Xnot), and pintallavis and slightly reduced the expression of chordin with the cycloheximide treatment (Fig. 4H). Expression of Xfurry was not altered by mir-15 expression (Fig. 4H). Moreover, expression of pri-mir-15 was reduced by both cycloheximide and anisomycin treatments (Fig. 4I and Fig. S8C). Taken together, these results suggest that Xfurry functions to reduce expression of mir-15, which in turn, down-regulates both translational and transcriptional gene silencing for some early chordamesodermal genes and thus induces expression of these genes.

Discussion

The slight increase in Xfurry expression induced by enR+LZ without cycloheximide treatment in this study could be attributed to the complexity of related signal transduction events, including nodal/activin signaling (Figs. 2H and 3A). However, injection of Xfurry-MO or expression of VP16+LZ or small RNAs causes an increase in Xfurry transcripts (Figs. 1L, 2H, and 4E). The protein synthesis inhibitors also induced Xfurry transcripts (Fig. 3C), although Xfurry transcripts were not translated to protein. Therefore, inhibiting protein synthesis activates the corepressor function of Xfurry and other genes having a DNA-binding domain, and interaction with the FD would contribute to this activation. As stated, the requirements for increasing and activating Xfurry/Xfurry occur when the protein levels of Xfurry target genes are low. Expression of enR+LZ and VP16+LZ (Fig. 3B) and overexpression of mir-15 (Fig. 4H) under the cycloheximide treatment did not alter Xfurry expression. These facts indicate that direct control of Xfurry expression is independent of Xfurry itself or of mir-15. Because small RNAs, including premicroRNAs, directly reduced expression of Xfurry (Fig. 4F), the release from transcriptional gene silencing by other microRNAs would increase Xfurry transcription under low-protein conditions such as that induced by the cycloheximide and anisomycin treatments.

Under low-protein conditions, both pretranscriptional and translational silencing by microRNAs should be inhibited for recovery to the normal protein levels. To decrease expression of microRNAs, some transcriptional repressors and related genes must be increased or activated, and one of those genes is Xfurry. We hypothesize that this requirement for repressor increase/activation would occur under the low-protein condition of one gene and might lead not only to the transcriptional activation of the low-protein gene but also to the silencing of other functionally related genes, indirectly increasing the transcription of the low-protein gene. In this study, injection of pintallavis-MO increased the expression of pintallavis, Xfurry (Fig. 3 D and F), goosecoid, and Xnot and decreased the expression of pri-mir-15, but the expression of chordin was not increased (Fig. 4J). Taken together, these observations indicate that the transcription of several genes might be regulated simultaneously by a single microRNA.

Normal development requires the precise spatial and temporal regulation of protein expression from various genes. When the amount of a specific protein is reduced and an increase is required, release of both translational and pretranscriptional gene silencing would occur more quickly than by the classical system involving an intermediate step of protein synthesis. Such a mechanism for the release of gene silencing would be suitable for the immediate and diverse pattern of gene regulation that characterizes embryogenesis. Translational gene silencing requires preexisting transcripts, such as maternal mRNAs, but transcriptional gene silencing does not require those transcripts. Our results show that cycloheximide treatment from the pre-MBT stage greatly reduced expression of mir-15 (Fig. 4I and Fig. S8C) and, eventually, the up-regulation of a suite of chordamesodermal genes. This finding indicates that correct mir-15 levels are necessary for correct levels of dorsal chordamesodermal proteins. We therefore propose that microRNA-mediated transcriptional gene silencing is necessary for regulation of zygotic protein levels. Xfurry does not function directly to induce chordamesodermal genes but instead regulates their expression to maintain their protein levels in a regulated band via a mechanism to repress microRNAs (Fig. 4K).

Because of the expression patterns of Xfurry, we posited that it functions in chordamesodermal gene regulation. However, weak expressions of Xfurry were observed in ventral, ectodermal, and endodermal regions (Fig. 1B). We therefore examined the expressions of several other marker genes by RT-PCR. Without protein synthesis inhibitors, expression of enR+LZ or VP16+LZ increased or decreased ectodermal (keratin), ventral (bone morphogenetic protein 4, ventricular size 1, and ventricular size 2), mesodermal [Xenopus brachyury (Xbra)], and endodermal (cerberus and Siamois) genes, respectively, whereas expression of myogenic differentiation (MyoD) was not affected (Fig. 2H and Fig. S8D). Although the cycloheximide and anisomycin treatments reduced expression of keratin in the animal caps, most of these genes were increased by such inhibition of protein synthesis (Fig. S8 E and F). We also found that enR+LZ induced those marker genes (except for MyoD) in the cycloheximide-treated animal caps (Fig. S8G). Expression of Siamois was not observed in animal caps (Fig. S8 E–G). Taken together, these findings suggest that the gene regulation by Xfurry is not specific for chordamesodermal genes. However, the regulations of keratin and MyoD expression were different from those of other marker genes, and induction was observed only with expression of enR+LZ (Fig. S8 D and G) or treatment with protein synthesis inhibitors (Fig. S8 E and F), suggesting that gene regulation in the chordamesodermal region by Xfurry is not conserved completely in other regions. The Xfurry transcripts gradually accumulated in the chordamesodermal region (Fig. 1), and the dorsal injection of Xfurry did not induce ventralization (Fig. S2). Based on this study, we propose that Xfurry functions mainly in chordamesodermal gene regulation and that other transcriptional factors operate in the gene regulation mechanisms of other tissues.

The release of microRNA-mediated transcriptional gene silencing could up-regulate transcripts of the targeted genes. However, small RNAs, including microRNA, also are involved in mRNA stabilization (34, 35); mir-16 contains a UAAAUAUU sequence that is complementary to the adenine- and undine-rich elements and is required for mRNA degradation (34). Moreover, inactivation of miR-427 leads to stabilization of the mRNAs and might be involved in the turnover of maternal mRNAs (36). Our data revealed that Xfurry transcripts are maternally expressed and decrease temporally after the MBT (Fig. 1A). The Xfurry-mediated decrease in microRNAs therefore might prevent message degradation for several genes and at least temporarily control the levels of mRNAs.

This study could not reveal how cells recognize specific low-protein-level conditions. Defining the mechanisms of activation and isolation of Xfurry-related genes might be fruitful areas of investigation.

Materials and Methods

Cloning and Constructs of Xfurry.

The Xfurry cDNA fragments (GenBank AB294244) were obtained by RT-PCR, including 5′- and 3′-RACE, with RNAs extracted from stage-10 embryos using degenerate primers. The Xfurry constructs were synthesized by PCR and were subcloned into a modified pCS2⁺ vector. The primers used are described in SI Materials and Methods.

Antisense MOs and microRNA.

We obtained the antisense MOs Xfurry-MO (5′-cgggcaggtaagaggctcacacact-3′), control-MO (5′-tgctgttgttttgatccatgttgtg-3′), pintallavis-MO (37), and goosecoid-MO (38) from Gene Tools, LLC. We used the mature mir-15 and control mutant mir-15 (Invitrogen) previously described (16). Small RNAs were extracted from stage-10 embryos using a mirVanaTM miRNA Isolation Kit (Ambion) and were separated on 15% acrylamide gels. Small RNAs reextracted from gels were purified by Microcon-Y30 columns (Millipore). The developmental stages are according to Nieuwkoop and Faber (39). The microinjection and microsurgery methods used are described in SI Materials and Methods.

Whole-Mount in Situ Hybridization.

Whole-mount in situ hybridization was performed as previously described (40).

Microarray Analysis.

Total RNAs were extracted from animal caps of enR+LZ-injected and VP16+LZ-injected embryos (plus cycloheximide treatment) that were dissected at stage 8 and cultured until stage 10. The microarray assay was performed as previously described (41).

Supplementary Material

Supporting Information

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Acknowledgments

We thank Paul Skoglund and Takuya Nakayama for helpful suggestions on the manuscript. This work was supported in part by grants from the Japan Science and Technology Agency, the Ministry of Health, Labour and Welfare, and the Ministry of Education, Culture, Sports, Science and Technology (to M.A.) and by Grant R37 HD025594 from the National Institutes of Health (to R.K.).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1008954107/-/DCSupplemental.

References

1.Cong J, et al. The furry gene of Drosophila is important for maintaining the integrity of cellular extensions during morphogenesis. Development. 2001;128:2793–2802. doi: 10.1242/dev.128.14.2793. [DOI] [PubMed] [Google Scholar]
2.He Y, Fang X, Emoto K, Jan YN, Adler PN. The tricornered Ser/Thr protein kinase is regulated by phosphorylation and interacts with furry during Drosophila wing hair development. Mol Biol Cell. 2005;16:689–700. doi: 10.1091/mbc.E04-09-0828. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Geng W, He B, Wang M, Adler PN. The tricornered gene, which is required for the integrity of epidermal cell extensions, encodes the Drosophila nuclear DBF2-related kinase. Genetics. 2000;156:1817–1828. doi: 10.1093/genetics/156.4.1817. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Emoto K, et al. Control of dendritic branching and tiling by the Tricornered-kinase/Furry signaling pathway in Drosophila sensory neurons. Cell. 2004;119:245–256. doi: 10.1016/j.cell.2004.09.036. [DOI] [PubMed] [Google Scholar]
5.Hirata D, et al. Fission yeast Mor2/Cps12, a protein similar to Drosophila Furry, is essential for cell morphogenesis and its mutation induces Wee1-dependent G(2) delay. EMBO J. 2002;21:4863–4874. doi: 10.1093/emboj/cdf495. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Nelson B, et al. RAM: A conserved signaling network that regulates Ace2p transcriptional activity and polarized morphogenesis. Mol Biol Cell. 2003;14:3782–3803. doi: 10.1091/mbc.E03-01-0018. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Chiba S, Ikeda M, Katsunuma K, Ohashi K, Mizuno K. MST2- and Furry-mediated activation of NDR1 kinase is critical for precise alignment of mitotic chromosomes. Curr Biol. 2009;19:1–7. doi: 10.1016/j.cub.2009.02.054. [DOI] [PubMed] [Google Scholar]
8.Hayette S, et al. AF4p12, a human homologue to the furry gene of Drosophila, as a novel MLL fusion partner. Cancer Res. 2005;65:6521–6525. doi: 10.1158/0008-5472.CAN-05-1325. [DOI] [PubMed] [Google Scholar]
9.Heasman J, et al. Overexpression of cadherins and underexpression of beta-catenin inhibit dorsal mesoderm induction in early Xenopus embryos. Cell. 1994;79:791–803. doi: 10.1016/0092-8674(94)90069-8. [DOI] [PubMed] [Google Scholar]
10.Lemaire P, Garrett N, Gurdon JB. Expression cloning of Siamois, a Xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis. Cell. 1995;81:85–94. doi: 10.1016/0092-8674(95)90373-9. [DOI] [PubMed] [Google Scholar]
11.Wylie C, et al. Maternal beta-catenin establishes a ‘dorsal signal’ in early Xenopus embryos. Development. 1996;122:2987–2996. doi: 10.1242/dev.122.10.2987. [DOI] [PubMed] [Google Scholar]
12.Schohl A, Fagotto F. A role for maternal beta-catenin in early mesoderm induction in Xenopus. EMBO J. 2003;22:3303–3313. doi: 10.1093/emboj/cdg328. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Lustig KD, et al. A Xenopus nodal-related gene that acts in synergy with noggin to induce complete secondary axis and notochord formation. Development. 1996;122:3275–3282. doi: 10.1242/dev.122.10.3275. [DOI] [PubMed] [Google Scholar]
14.Takahashi S, et al. Two novel nodal-related genes initiate early inductive events in Xenopus Nieuwkoop center. Development. 2000;127:5319–5329. doi: 10.1242/dev.127.24.5319. [DOI] [PubMed] [Google Scholar]
15.Onuma Y, Takahashi S, Yokota C, Asashima M. Multiple nodal-related genes act coordinately in Xenopus embryogenesis. Dev Biol. 2002;241:94–105. doi: 10.1006/dbio.2001.0493. [DOI] [PubMed] [Google Scholar]
16.Martello G, et al. MicroRNA control of Nodal signalling. Nature. 2007;449:183–188. doi: 10.1038/nature06100. [DOI] [PubMed] [Google Scholar]
17.Rosa A, Spagnoli FM, Brivanlou AH. The miR-430/427/302 family controls mesendodermal fate specification via species-specific target selection. Dev Cell. 2009;16:517–527. doi: 10.1016/j.devcel.2009.02.007. [DOI] [PubMed] [Google Scholar]
18.Morris KV, Chan SW, Jacobsen SE, Looney DJ. Small interfering RNA-induced transcriptional gene silencing in human cells. Science. 2004;305:1289–1292. doi: 10.1126/science.1101372. [DOI] [PubMed] [Google Scholar]
19.Hawkins PG, Santoso S, Adams C, Anest V, Morris KV. Promoter targeted small RNAs induce long-term transcriptional gene silencing in human cells. Nucleic Acids Res. 2009;37:2984–2995. doi: 10.1093/nar/gkp127. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Kim DH, Saetrom P, Snøve O, Jr, Rossi JJ. MicroRNA-directed transcriptional gene silencing in mammalian cells. Proc Natl Acad Sci USA. 2008;105:16230–16235. doi: 10.1073/pnas.0808830105. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Morris KV. RNA-directed transcriptional gene silencing and activation in human cells. Oligonucleotides. 2009;19:299–306. doi: 10.1089/oli.2009.0212. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Suzuki K, Kelleher AD. Transcriptional regulation by promoter targeted RNAs. Curr Top Med Chem. 2009;9:1079–1087. doi: 10.2174/156802609789630875. [DOI] [PubMed] [Google Scholar]
23.Cho KW, Blumberg B, Steinbeisser H, De Robertis EM. Molecular nature of Spemann's organizer: The role of the Xenopus homeobox gene goosecoid. Cell. 1991;67:1111–1120. doi: 10.1016/0092-8674(91)90288-a. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Ruiz i Altaba A, Cox C, Jessell TM, Klar A. Ectopic neural expression of a floor plate marker in frog embryos injected with the midline transcription factor Pintallavis. Proc Natl Acad Sci USA. 1993;90:8268–8272. doi: 10.1073/pnas.90.17.8268. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.von Dassow G, Schmidt JE, Kimelman D. Induction of the Xenopus organizer: Expression and regulation of Xnot, a novel FGF and activin-regulated homeo box gene. Genes Dev. 1993;7:355–366. doi: 10.1101/gad.7.3.355. [DOI] [PubMed] [Google Scholar]
26.Sasai Y, et al. Xenopus chordin: A novel dorsalizing factor activated by organizer-specific homeobox genes. Cell. 1994;79:779–790. doi: 10.1016/0092-8674(94)90068-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Fan MJ, Sokol SY. A role for Siamois in Spemann organizer formation. Development. 1997;124:2581–2589. doi: 10.1242/dev.124.13.2581. [DOI] [PubMed] [Google Scholar]
28.Kessler DS. Siamois is required for formation of Spemann's organizer. Proc Natl Acad Sci USA. 1997;94:13017–13022. doi: 10.1073/pnas.94.24.13017. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Zhang J, et al. The role of maternal VegT in establishing the primary germ layers in Xenopus embryos. Cell. 1998;94:515–524. doi: 10.1016/s0092-8674(00)81592-5. [DOI] [PubMed] [Google Scholar]
30.Dawid IB, Otani H, Curtiss P, Taira M. Regulatory interactions during embryogenesis in Xenopus laevis. C R Acad Sci III. 1993;316:945–958. [PubMed] [Google Scholar]
31.Tadano T, Otani H, Taira M, Dawid IB. Differential induction of regulatory genes during mesoderm formation in Xenopus laevis embryos. Dev Genet. 1993;14:204–211. doi: 10.1002/dvg.1020140307. [DOI] [PubMed] [Google Scholar]
32.Danilov V, Blum M, Schweickert A, Campione M, Steinbeisser H. Negative autoregulation of the organizer-specific homeobox gene goosecoid. J Biol Chem. 1998;273:627–635. doi: 10.1074/jbc.273.1.627. [DOI] [PubMed] [Google Scholar]
33.Piccolo S, et al. The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals. Nature. 1999;397:707–710. doi: 10.1038/17820. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Jing Q, et al. Involvement of microRNA in AU-rich element-mediated mRNA instability. Cell. 2005;120:623–634. doi: 10.1016/j.cell.2004.12.038. [DOI] [PubMed] [Google Scholar]
35.Valencia-Sanchez MA, Liu J, Hannon GJ, Parker R. Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev. 2006;20:515–524. doi: 10.1101/gad.1399806. [DOI] [PubMed] [Google Scholar]
36.Lund E, Liu M, Hartley RS, Sheets MD, Dahlberg JE. Deadenylation of maternal mRNAs mediated by miR-427 in Xenopus laevis embryos. RNA. 2009;15:2351–2363. doi: 10.1261/rna.1882009. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Martynova N, et al. Patterning the forebrain: FoxA4a/Pintallavis and Xvent2 determine the posterior limit of Xanf1 expression in the neural plate. Development. 2004;131:2329–2338. doi: 10.1242/dev.01133. [DOI] [PubMed] [Google Scholar]
38.Sander V, Reversade B, De Robertis EM. The opposing homeobox genes Goosecoid and Vent1/2 self-regulate Xenopus patterning. EMBO J. 2007;26:2955–2965. doi: 10.1038/sj.emboj.7601705. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Nieuwkoop PD, Faber J. Normal Table of Xenopus laevis (Daudin) Amsterdam: North Holland; 1967. [Google Scholar]
40.Harland RM. In situ hybridization: An improved whole-mount method for Xenopus embryos. Methods Cell Biol. 1991;36:685–695. doi: 10.1016/s0091-679x(08)60307-6. [DOI] [PubMed] [Google Scholar]
41.Fukui A, Goto T, Kitamoto J, Homma M, Asashima M. SDF-1 alpha regulates mesendodermal cell migration during frog gastrulation. Biochem Biophys Res Commun. 2007;354:472–477. doi: 10.1016/j.bbrc.2007.01.007. [DOI] [PubMed] [Google Scholar]

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Supporting Information

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[r1] 1.Cong J, et al. The furry gene of Drosophila is important for maintaining the integrity of cellular extensions during morphogenesis. Development. 2001;128:2793–2802. doi: 10.1242/dev.128.14.2793. [DOI] [PubMed] [Google Scholar]

[r2] 2.He Y, Fang X, Emoto K, Jan YN, Adler PN. The tricornered Ser/Thr protein kinase is regulated by phosphorylation and interacts with furry during Drosophila wing hair development. Mol Biol Cell. 2005;16:689–700. doi: 10.1091/mbc.E04-09-0828. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r3] 3.Geng W, He B, Wang M, Adler PN. The tricornered gene, which is required for the integrity of epidermal cell extensions, encodes the Drosophila nuclear DBF2-related kinase. Genetics. 2000;156:1817–1828. doi: 10.1093/genetics/156.4.1817. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r4] 4.Emoto K, et al. Control of dendritic branching and tiling by the Tricornered-kinase/Furry signaling pathway in Drosophila sensory neurons. Cell. 2004;119:245–256. doi: 10.1016/j.cell.2004.09.036. [DOI] [PubMed] [Google Scholar]

[r5] 5.Hirata D, et al. Fission yeast Mor2/Cps12, a protein similar to Drosophila Furry, is essential for cell morphogenesis and its mutation induces Wee1-dependent G(2) delay. EMBO J. 2002;21:4863–4874. doi: 10.1093/emboj/cdf495. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r6] 6.Nelson B, et al. RAM: A conserved signaling network that regulates Ace2p transcriptional activity and polarized morphogenesis. Mol Biol Cell. 2003;14:3782–3803. doi: 10.1091/mbc.E03-01-0018. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r7] 7.Chiba S, Ikeda M, Katsunuma K, Ohashi K, Mizuno K. MST2- and Furry-mediated activation of NDR1 kinase is critical for precise alignment of mitotic chromosomes. Curr Biol. 2009;19:1–7. doi: 10.1016/j.cub.2009.02.054. [DOI] [PubMed] [Google Scholar]

[r8] 8.Hayette S, et al. AF4p12, a human homologue to the furry gene of Drosophila, as a novel MLL fusion partner. Cancer Res. 2005;65:6521–6525. doi: 10.1158/0008-5472.CAN-05-1325. [DOI] [PubMed] [Google Scholar]

[r9] 9.Heasman J, et al. Overexpression of cadherins and underexpression of beta-catenin inhibit dorsal mesoderm induction in early Xenopus embryos. Cell. 1994;79:791–803. doi: 10.1016/0092-8674(94)90069-8. [DOI] [PubMed] [Google Scholar]

[r10] 10.Lemaire P, Garrett N, Gurdon JB. Expression cloning of Siamois, a Xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis. Cell. 1995;81:85–94. doi: 10.1016/0092-8674(95)90373-9. [DOI] [PubMed] [Google Scholar]

[r11] 11.Wylie C, et al. Maternal beta-catenin establishes a ‘dorsal signal’ in early Xenopus embryos. Development. 1996;122:2987–2996. doi: 10.1242/dev.122.10.2987. [DOI] [PubMed] [Google Scholar]

[r12] 12.Schohl A, Fagotto F. A role for maternal beta-catenin in early mesoderm induction in Xenopus. EMBO J. 2003;22:3303–3313. doi: 10.1093/emboj/cdg328. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r13] 13.Lustig KD, et al. A Xenopus nodal-related gene that acts in synergy with noggin to induce complete secondary axis and notochord formation. Development. 1996;122:3275–3282. doi: 10.1242/dev.122.10.3275. [DOI] [PubMed] [Google Scholar]

[r14] 14.Takahashi S, et al. Two novel nodal-related genes initiate early inductive events in Xenopus Nieuwkoop center. Development. 2000;127:5319–5329. doi: 10.1242/dev.127.24.5319. [DOI] [PubMed] [Google Scholar]

[r15] 15.Onuma Y, Takahashi S, Yokota C, Asashima M. Multiple nodal-related genes act coordinately in Xenopus embryogenesis. Dev Biol. 2002;241:94–105. doi: 10.1006/dbio.2001.0493. [DOI] [PubMed] [Google Scholar]

[r16] 16.Martello G, et al. MicroRNA control of Nodal signalling. Nature. 2007;449:183–188. doi: 10.1038/nature06100. [DOI] [PubMed] [Google Scholar]

[r17] 17.Rosa A, Spagnoli FM, Brivanlou AH. The miR-430/427/302 family controls mesendodermal fate specification via species-specific target selection. Dev Cell. 2009;16:517–527. doi: 10.1016/j.devcel.2009.02.007. [DOI] [PubMed] [Google Scholar]

[r18] 18.Morris KV, Chan SW, Jacobsen SE, Looney DJ. Small interfering RNA-induced transcriptional gene silencing in human cells. Science. 2004;305:1289–1292. doi: 10.1126/science.1101372. [DOI] [PubMed] [Google Scholar]

[r19] 19.Hawkins PG, Santoso S, Adams C, Anest V, Morris KV. Promoter targeted small RNAs induce long-term transcriptional gene silencing in human cells. Nucleic Acids Res. 2009;37:2984–2995. doi: 10.1093/nar/gkp127. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r20] 20.Kim DH, Saetrom P, Snøve O, Jr, Rossi JJ. MicroRNA-directed transcriptional gene silencing in mammalian cells. Proc Natl Acad Sci USA. 2008;105:16230–16235. doi: 10.1073/pnas.0808830105. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r21] 21.Morris KV. RNA-directed transcriptional gene silencing and activation in human cells. Oligonucleotides. 2009;19:299–306. doi: 10.1089/oli.2009.0212. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r22] 22.Suzuki K, Kelleher AD. Transcriptional regulation by promoter targeted RNAs. Curr Top Med Chem. 2009;9:1079–1087. doi: 10.2174/156802609789630875. [DOI] [PubMed] [Google Scholar]

[r23] 23.Cho KW, Blumberg B, Steinbeisser H, De Robertis EM. Molecular nature of Spemann's organizer: The role of the Xenopus homeobox gene goosecoid. Cell. 1991;67:1111–1120. doi: 10.1016/0092-8674(91)90288-a. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r24] 24.Ruiz i Altaba A, Cox C, Jessell TM, Klar A. Ectopic neural expression of a floor plate marker in frog embryos injected with the midline transcription factor Pintallavis. Proc Natl Acad Sci USA. 1993;90:8268–8272. doi: 10.1073/pnas.90.17.8268. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r25] 25.von Dassow G, Schmidt JE, Kimelman D. Induction of the Xenopus organizer: Expression and regulation of Xnot, a novel FGF and activin-regulated homeo box gene. Genes Dev. 1993;7:355–366. doi: 10.1101/gad.7.3.355. [DOI] [PubMed] [Google Scholar]

[r26] 26.Sasai Y, et al. Xenopus chordin: A novel dorsalizing factor activated by organizer-specific homeobox genes. Cell. 1994;79:779–790. doi: 10.1016/0092-8674(94)90068-x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r27] 27.Fan MJ, Sokol SY. A role for Siamois in Spemann organizer formation. Development. 1997;124:2581–2589. doi: 10.1242/dev.124.13.2581. [DOI] [PubMed] [Google Scholar]

[r28] 28.Kessler DS. Siamois is required for formation of Spemann's organizer. Proc Natl Acad Sci USA. 1997;94:13017–13022. doi: 10.1073/pnas.94.24.13017. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r29] 29.Zhang J, et al. The role of maternal VegT in establishing the primary germ layers in Xenopus embryos. Cell. 1998;94:515–524. doi: 10.1016/s0092-8674(00)81592-5. [DOI] [PubMed] [Google Scholar]

[r30] 30.Dawid IB, Otani H, Curtiss P, Taira M. Regulatory interactions during embryogenesis in Xenopus laevis. C R Acad Sci III. 1993;316:945–958. [PubMed] [Google Scholar]

[r31] 31.Tadano T, Otani H, Taira M, Dawid IB. Differential induction of regulatory genes during mesoderm formation in Xenopus laevis embryos. Dev Genet. 1993;14:204–211. doi: 10.1002/dvg.1020140307. [DOI] [PubMed] [Google Scholar]

[r32] 32.Danilov V, Blum M, Schweickert A, Campione M, Steinbeisser H. Negative autoregulation of the organizer-specific homeobox gene goosecoid. J Biol Chem. 1998;273:627–635. doi: 10.1074/jbc.273.1.627. [DOI] [PubMed] [Google Scholar]

[r33] 33.Piccolo S, et al. The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals. Nature. 1999;397:707–710. doi: 10.1038/17820. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r34] 34.Jing Q, et al. Involvement of microRNA in AU-rich element-mediated mRNA instability. Cell. 2005;120:623–634. doi: 10.1016/j.cell.2004.12.038. [DOI] [PubMed] [Google Scholar]

[r35] 35.Valencia-Sanchez MA, Liu J, Hannon GJ, Parker R. Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev. 2006;20:515–524. doi: 10.1101/gad.1399806. [DOI] [PubMed] [Google Scholar]

[r36] 36.Lund E, Liu M, Hartley RS, Sheets MD, Dahlberg JE. Deadenylation of maternal mRNAs mediated by miR-427 in Xenopus laevis embryos. RNA. 2009;15:2351–2363. doi: 10.1261/rna.1882009. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r37] 37.Martynova N, et al. Patterning the forebrain: FoxA4a/Pintallavis and Xvent2 determine the posterior limit of Xanf1 expression in the neural plate. Development. 2004;131:2329–2338. doi: 10.1242/dev.01133. [DOI] [PubMed] [Google Scholar]

[r38] 38.Sander V, Reversade B, De Robertis EM. The opposing homeobox genes Goosecoid and Vent1/2 self-regulate Xenopus patterning. EMBO J. 2007;26:2955–2965. doi: 10.1038/sj.emboj.7601705. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r39] 39.Nieuwkoop PD, Faber J. Normal Table of Xenopus laevis (Daudin) Amsterdam: North Holland; 1967. [Google Scholar]

[r40] 40.Harland RM. In situ hybridization: An improved whole-mount method for Xenopus embryos. Methods Cell Biol. 1991;36:685–695. doi: 10.1016/s0091-679x(08)60307-6. [DOI] [PubMed] [Google Scholar]

[r41] 41.Fukui A, Goto T, Kitamoto J, Homma M, Asashima M. SDF-1 alpha regulates mesendodermal cell migration during frog gastrulation. Biochem Biophys Res Commun. 2007;354:472–477. doi: 10.1016/j.bbrc.2007.01.007. [DOI] [PubMed] [Google Scholar]

PERMALINK

Xenopus furry contributes to release of microRNA gene silencing

Toshiyasu Goto

Akimasa Fukui

Hiroshi Shibuya

Ray Keller

Makoto Asashima

Abstract