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doi: 10.1242/10.1242/dev.00401

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XMAN1, an inner nuclear membrane protein, antagonizes BMP signaling by interacting with Smad1 in Xenopus embryos

Department of Biological Sciences, Graduate School of Science, University of Tokyo, and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan

View larger version (38K): [in a new window]   Fig. 1. Isolation of a novel neuralizing factor. (A) Animal caps expressing ß-globin (a,c) and A124C10 (b,d) at the equivalent of stage 25. Whole-mount in situ analysis for nrp1 expression was carried out in c,d. Arrowheads, cement glands. (B) RT-PCR analysis for neural marker expression in ß-globin or A124C10 expressing animal caps at early gastrula (stage 10.5) and tailbud (stage 25) stages. RT(+) and RT(-) indicate whole-embryo RNA transcribed with or without reverse transcriptase, respectively. EF-1 was used as a loading control. Doses of injected mRNA (pg/embryo): ß-globin, 500; A124C10, 500.

View larger version (57K): [in a new window]   Fig. 2. Structural features of XMAN1. (A) Schematic structural comparison of human (Accession Number, AF112299) and Xenopus MAN1 (Accession Number, AB100267). LEM domains (blue), putative transmembrane domains (TM, yellow), and RNA recognition motif (RRM, red) are indicated. RNP2 and RNP1, conserved regions among RRMs, are shown as black boxes. A black bar represents the coding region of A124C10. The total numbers of amino acid residues of each protein and sequence identities of each domain between the two proteins are shown. (B) The sequence of the LEM domain of Xenopus MAN1 in comparison with those of human MAN1, LAP2 and emerin. Asterisks and colons indicate identical and homologous amino acid residues, respectively, conserved among at least three molecules. (C) Sequence comparison between human and Xenopus MAN1 from the first transmembrane domain. Asterisks and colons are indicated as above. RRM domains are boxed. (D) Alignment of putative RRMs of human and Xenopus MAN1. Pfam00076 represents an RRM in a database for multiple sequence alignments (Bateman et al., 2002). A core sequence for RRM is reported previously (Birney et al., 1993). U, uncharged; Z, uncharged, serine or threonine; x, any amino acid residues.

View larger version (66K): [in a new window]   Fig. 3. Temporospatial expression of XMAN1. (A) Analysis of temporal expression pattern of XMAN1 by RT-PCR with RNA from various developmental stages. FGFR, a loading control. (B) Whole-mount in situ analysis of Xenopus embryos at early gastrula (a-c), neurula (d-f) and tadpole (g,h) stages. c and e show cross-sections of b and d, respectively. f represents a close view of the dorsal region of e. h shows a dorsal view of the head region of g. Arrows, dorsal blastopore groove. nc, neural crest; nt, notochord; fp, floor plate; el, epithelial layer; sl, sensory layer; ey, eye; ov, otic vesicle; ba, branchial arches.

View larger version (46K): [in a new window]   Fig. 4. Neuralizing activity of XMAN1 resides in the C-terminal region. (A) Schematic representation of XMAN1 deletion mutants. LEM domains, transmembrane domains and RRMs are depicted as in Fig. 2A. Circles indicate Myc tags. (B) Western blot for analyzing the expression level of the XMAN1 deletion mutants. Extracts prepared from stage 11 gastrula embryos expressing XMAN1 deletion mutants were immunoblotted with anti-Myc antibody (upper panel) or anti-ß-tubulin antibody (lower panel; loading control). (C) Mapping of neuralizing activity of XMAN1. RT-PCR analysis for nrp1 was performed with animal caps at the equivalent of the tailbud stage 25. Doses of injected mRNA: 500 pg per embryo. (D) Confocal microscopic analysis of the subcellular localization of Myc-tagged XMAN1 (a-c), Flag-tagged XMAN1-CT (d-f) and Myc-tagged human emerin (g-i) expressed in COS-7 cells (green). Nuclei (red) were visualized by co-transfecting with the DsRed2-Nuc vector. Scale bars: 10 µm.

View larger version (44K): [in a new window]   Fig. 5. XMAN1 dorsalizes the ventral mesoderm. (A) Embryos were injected with ß-globin (a,c,e) or XMAN1-CT (b,d,f) mRNA into the ventral marginal zone at the four-cell stage and observed at stage 20 (a,b) or stage 35 (c-f). Somites were stained with 12/101 antibody (e,f). Arrowheads, secondary axes. (B) RT-PCR analysis of RNA from ß-globin or XMAN1-CT mRNA (500 pg per embryo)-injected ventral marginal zones for expression of pan-mesodermal (Xbra), dorsal (gsc, chordin and -actin) and ventral (Xhox3, Msx1 and Xvent1) mesodermal markers.

View larger version (34K): [in a new window]   Fig. 6. XMAN1 antagonizes the BMP pathway. XMAN1 does not suppress the expression of BMP4 (A) or induce BMP antagonists (B). RT-PCR analyses for BMP4 (A), noggin and chordin (B) with animal caps at the equivalent of stage 10.5. (C) XMAN1 acts synergistically with Noggin in neural induction. Real-time RT-PCR analyses for nrp1 and Xotx2 with animal caps at the equivalent of stage 25. EF-1 was used as an internal control, and each bar was normalized to the level of EF-1 expression. Representative data from three independent experiments are shown. Doses of injected mRNA (pg/embryo): XMAN1, 200; noggin, 2. (D) XMAN1 blocks the BMP pathway. RT-PCR analysis for Xhox3, Msx1 and Xbra with animal caps at the equivalent of stage 10.5. (E) Suppression of BMP-dependent Xvent2-Luc activation by XMAN1 constructs. Alk3*, activated form of type I BMP receptor; GL3-Luc, control luciferase construct.

View larger version (73K): [in a new window]   Fig. 7. XMAN1 preferentially associates with BMP-responsive Smads. (A) XMAN1 associates with XSmad1 in vivo. Myc-tagged wild-type (MT-XMAN1), C-terminal (MT-CT) and N-terminal (MT-CT) constructs of XMAN1 were co-expressed with HA-tagged XSmad1 in Xenopus embryos as indicated. Extracts prepared from stage 10.5 embryos were immunoprecipitated with an anti-HA antibody. The precipitates were analyzed by western blotting with an anti-Myc antibody (upper panel). Middle and lower panels show the expression levels of XMAN1 and XSmad1 proteins, respectively. (B) XMAN1 associates with endogenous XSmad1. Extracts prepared from stage 10.5 embryos injected with or without MT-CT were immunoprecipitated with anti-Smad1 antibodies, and the precipitates were immunoblotted with anti-Myc antibody (upper panel). Middle and lower panels show the expression levels of MT-CT and endogenous XSmad1 proteins, respectively. (C) XMAN1 interacts preferentially with BMP-responsive Smads in vitro by GST pull-down assays. [35S]Met-labeled in vitro translated Smad and GFP products were incubated with purified GST or GST-XMAN1-CT and subjected to SDS-PAGE. % binding, percentage of the intensity of each band in the lane of GST-CT against the corresponding total input.

View larger version (62K): [in a new window]   Fig. 8. Roles of XMAN1 during Xenopus development. (A) XMAN1 antisense morpholino oligo (XMAN1-MO) inhibits the translation from synthetic XMAN1 mRNA. Extracts prepared from stage 10.5 and stage 25 embryos injected with Myc-tagged XMAN1 mRNA possessing the XMAN1-MO annealing sequence (5'UTR-XMAN1-MT) together with XMAN1-MO or XMAN1-4mmMO (doses in ng per embryo are in parentheses) were subjected to immunoblotting with anti-Myc antibody (upper panel) and with anti-ß-tubulin antibody (lower panel). (B) XMAN1-MO suppresses neural induction by XMAN1. Real-time RT-PCR analysis for nrp1 with animal caps at the equivalent of stage 25. Doses of injected mRNA, 500 pg per embryos. Doses of XMAN1-MO are indicated in parentheses (ng per embryo). (C) Morphological appearance of injected embryos. XMAN1-4mmMO (b,f), XMAN1-MO (c,g) and XMAN1-MO plus MT-XMAN1 mRNA (d,h) were injected together with ß-gal mRNA as a tracer (b-d,f-h) into two right animal blastomeres at the four-cell stage. XMAN1-MO injection caused a reduced eye on the injected side (c,g), which is rescued by co-injection of MT-XMAN1 mRNA (d,h). Injected doses (per blastomere): MOs, 25 ng; MT-XMAN1 mRNA, 67.5 pg; ß-gal mRNA, 15 pg. a-d, dorsal view (anterior is upwards); e-h, lateral view of a-d, respectively, showing the right side of the embryos. (D) Whole-mount in situ analysis for Dlx3 (epidermis, stage 13), Rx2A (eye, stage 20), Xemx1 (telencephalon, stage 20) and nrp1 (pan-neural, stage 20) expression in uninjected, XMAN1-4mmMO- and XMAN1-MO-injected embryos as indicated. a-c, dorsal view (anterior is upwards); d-l, anterior view (dorsal is upwards). ß-Gal mRNA was co-injected in b,c,e,f.