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A family of inner nuclear membrane proteins is implicated in gene regulation by interacting with chromatin, nuclear lamina and intranuclear proteins; however, the physiological functions of these proteins are largely unknown. Using a Xenopus expression screening approach with an anteriorneuroectoderm cDNA library, we have identified an inner nuclear membrane protein, XMAN1, as a novel neuralizing factor that is encoded by the Xenopus ortholog of human MAN1. XMAN1 mRNA is expressed maternally, and appears to be restricted to the entire ectoderm at the early gastrula stage, then to the anteriorneuroectoderm at the neurula stage. XMAN1 induces anterior neural markers without mesoderm induction in ectodermal explants, and a partial secondary axis when expressed ventrally by dorsalizing the ventralmesoderm. Importantly, XMAN1 antagonizes bone morphogenetic protein (BMP) signaling downstream of its receptor Alk3, as judged by animal cap assays, in which XMAN1 blocks expression of downstream targets of BMP signaling (Xhox3 and Msx1), and by luciferase reporter assays, in which XMAN1 suppresses BMP-dependent activation of the Xvent2 promoter. Deletion mutant analyses reveal that the neuralizing and BMP-antagonizing activities of XMAN1 reside in the C-terminal region, and that the C-terminal region binds to Smad1, Smad5 and Smad8, which are intracellular mediators of the BMP pathway. Interference with endogenous XMAN1 functions with antisense morpholino oligos leads to the reduction of anteriorneuroectoderm. These results provide the first evidence that the nuclear envelope protein XMAN1 acts as a Smad-interacting protein to antagonize BMP signaling during Xenopus embryogenesis.
lemd3 (LEM domain containing 3 gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 14, dorsal view, anteriorleft.
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.
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-β-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 β-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; β-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). β-Gal mRNA was co-injected in b,c,e,f.
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-β-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.
Fig. 5. XMAN1 dorsalizes the ventralmesoderm. (A) Embryos were injected with β -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 β-globin or XMAN1-CT mRNA (500 pg per embryo)-injected ventral marginal zones for expression of pan-mesodermal (Xbra), dorsal (gsc, chordin and α-actin) and ventral (Xhox3, Msx1 and Xvent1) mesodermal markers.
lemd3 (LEM domain containing 3) gene expression in Xenopus laevis embryo via in situ hybridization, NF stage 27, lateral view, anteriorleft, dorsal up.
