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Suppression of head formation by Xmsx-1 through the inhibition of intracellular nodal signaling

¹ Department of Developmental Biology, National Institute for Basic Biology, 38 Nishigonaka, Myodaiji, Okazaki, 444-8585, Japan
² Department of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies, Okazaki, 444-8585, Japan

View larger version (43K): [in a new window]   Fig. 1. Ectopic expression of Xmsx-1 in the anterior endoderm suppresses head structure formation. Xenopus embryos were either uninjected (A and C, lanes 1, 4, 5) or injected with 500 pg of Xmsx-1 (B and C, lane 2) or 200 pg of BMP-4 (C, lane 3) mRNA in the submarginal zone of the two dorsal blastomeres at the 4-cell stage. (A,B) The embryos were cultured until stage 40 to observe the phenotypes. (C) RT-PCR analysis for the early anterior endomesodermal markers. The dorso-vegetal quarters of the embryos were dissected at stage 10.25 (lanes 1-3) and then subjected to RT-PCR analysis for the indicated markers. Lanes 4 and 5, whole embryo control with (lane 4) or without (lane 5) the RT step.

View larger version (50K): [in a new window]   Fig. 2. Ventral overexpression of HI-Xmsx-1 induces an ectopic head. (A) Schematic drawing of the different forms of Xmsx-1. (Top) Wild-type Xmsx-1 protein. Activating forms of Xmsx-1, HI-Xmsx-1 (middle), and TI-Xmsx-1 (bottom), were constructed by fusing the 81 amino acids of the VP16 activation domain (black box, VP16 AD) with the full-length and C-terminal 137 amino acid region of Xmsx-1 protein, respectively. The amino acid numbers of several junction sites are shown above each drawing. The homeodomain is indicated by the gray box (HD). (B-D) Phenotypes of mRNA-injected embryos. (E,F) Staining with the notochord- and otic vesicle-specific antibody, MZ15. Four-cell stage Xenopus embryos were ventrally injected with 500 pg of TI-Xmsx-1 (B,E) or HI-Xmsx-1 (C,F) mRNA alone or coinjected with 500 pg of HI-Xmsx-1 and 1 ng of wild-type Xmsx-1 mRNA (D). HI-Xmsx-1-injected embryos formed a secondary axis with an ectopic head containing eye(s), cement gland and short notochord in the ventral side. The ectopic head region is magnified in the right side of C. The ectopic head phenotype was rescued by wild-type Xmsx-1. cg, cement gland; ey, eye; 2°, secondary axis.

View larger version (47K): [in a new window]   Fig. 3. HI-Xmsx-1 induces head organizer marker genes in the ventral endomesoderm. Two ventral blastomeres of 4-cell-stage embryos were coinjected marginally with 100 pg of HI- or TI-Xmsx-1 mRNA and 50 pg of ß-galactosidase mRNA as a lineage tracer, then processed for RT-PCR (A) and whole-mount in situ hybridization (B) at stage 10.25. (A) The ventro-vegetal quarter of the embryos injected with the indicated mRNA (top, lanes 1, 2, 3) was dissected at stage 10.25 and RNA was extracted immediately, for early stage, or after being cultured until sibling embryos reached stage 18, for late stage. RNA extracted from each explant was analyzed by RT-PCR. Lane 4 shows the expression of each marker in whole embryos and lane 5 shows the control reactions with no RT step. (B) The injected embryos were stained by red Gal for lineage tracing and then analyzed by whole-mount in situ hybridization for Xotx-2, Xhex and cerberus gene expression. Anterior organizer genes were induced in the ventral endomesoderm. The dorsal blastopore lip is indicated by black arrowheads. Arrows indicate the RNA-injected ventral marginal zone, in which the nuclei of cells are stained red. AS, antisense probe; S, sense probe.

View larger version (50K): [in a new window]   Fig. 4. Xmsx-1 does not suppress nodal-related gene expression. Embryos were either uninjected (lanes 1, 4, 5) or injected with 500 pg of Xmsx-1 (lane 2) or 200 pg of BMP-4 (lane 3) mRNA into the equatorial region of the two dorsal blastomeres at the 4-cell stage. Dorsal marginal zone explants (DMZ; lanes 1, 2, 3) were cultured until sibling embryos reached stage 10.5, then nodal-related gene expression was analyzed by RT-PCR.

View larger version (30K): [in a new window]   Fig. 5. Xmsx-1 inhibits the Xnr-1 signaling for head formation. (A) Typical phenotypes of Xnr-1 and Xmsx-1 mRNA-injected embryos. Xnr-1 mRNA (100 pg) was injected, either alone (b) or coinjected with increasing amounts (c, 500 pg; d, 1 ng) of Xmsx-1 mRNA into the ventral marginal region of 4-cell stage embryos. Xmsx-1 suppressed the Xnr-1-induced complete secondary axis formation. (a) An uninjected control. (B) Gene expression analysis of head organizer markers in the ventral endomesoderm by RT-PCR. Xnr-1 and Xmsx-1 mRNAs were injected into the ventral marginal region of 4-cell-stage embryos and the ventro-vegetal quarters were dissected at stage 10.25 (lanes 1, 2, 3 and 4). Lane 1, uninjected control; lane 2, injected with 100 pg of Xnr-1 mRNA; lane 3, coinjected with 100 pg of Xnr-1 and 500 pg of Xmsx-1 mRNAs; lane 4, injected with 500 pg of Xmsx-1 mRNA. Lanes 5 and 6, uninjected whole embryo control, with (lane 5) or without (lane 6) the RT step.

View larger version (28K): [in a new window]   Fig. 6. The activin responsive element (ARE) is activated by HI-Xmsx-1 and suppressed by Xmsx-1. The construct -226gsc/Luc, which contains the luciferase gene under the control of the 226 bp goosecoid promoter containing the ARE was injected either alone or coinjected with the indicated mRNAs into the DMZ or ventral marginal zone (VMZ) of 4-cell-stage embryos. The injected or uninjected marginal zones were dissected at stage 10.25 (A) or 10 (B) and subjected to luciferase assays. (A) HI-Xmsx-1 activated the ARE in the ventral marginal region. (B) Xmsx-1 suppressed ARE activation in the dorsal side. Xnr-1 antagonized the suppressing activity of Xmsx-1 for the transcription through the ARE. Actual luciferase counts are presented.

View larger version (22K): [in a new window]   Fig. 7. Xmsx-1 physically interacts with Smads. (A) Xmsx-1/Xsmads complexes were detected by immunoprecipitation. 293T cells were transiently transfected with Flag-tagged Xmsx-1 alone (lane 2) or the indicated HA-tagged Xsmads alone (lanes 3-6), or cotransfected with Flag-tagged Xmsx-1 and each HA-tagged Xsmad (lanes 7-10). Lysates were assayed either by immunoprecipitation with an anti-Flag antibody followed by western blotting with an anti-HA antibody (upper panel) or by western blotting the whole lysate with an anti-HA antibody (lower panel). Lane 1 is a nontransfected control. (B) Xmsx-1 additively participates in the Xsmad2/Xsmad4ß complex. 293T cells were transiently cotransfected with glutathione S-transferase (GST)-tagged Xsmad4ß and HA-tagged Xsmad2 together with increasing amounts of HA-tagged Xmsx-1 (lanes 3-6). Lysates were assayed either by GST pull-down analysis followed by western blotting with an anti-HA antibody (upper panel) or by western blotting the whole lysate with an anti-HA antibody (lower panel). As negative controls, GSTvector expressing GST protein alone was cotransfected either with HA-tagged Xsmad2 (lane 1) or with HA-tagged Xmsx-1 (lane 2). (C) Xmsx-1 excludes xFAST-1 from Xsmad2/4 complex in a dose-dependent manner. Cells were cotransfected with the indicated amount of GST-xFAST-1, HA-Xsmad2, Xsmad4ß and HA-Xmsx-1 constructs. Lysates were assayed as in B.

View larger version (59K): [in a new window]   Fig. 8. Immunostaining of activated Smad1 and Xmsx-1 protein. Gastrula embryos were subjected to immunohistochemistry using anti-phosphoSmad1 (A) and anti-MSX-1 (B) antibodies. Embryos were bisected along the dorsoventral axis. The dorsal blastopore lip is indicated with an arrowhead.

View larger version (44K): [in a new window]   Fig. 9. Phenotype of embryos ventrally overexpressing inhibitors of BMP signaling. Four-cell stage Xenopus embryos were ventrally microinjected with the following synthetic mRNAs; (A) 200 pg of tBR and 500 pg of truncated BMPRII mRNAs; (B) as A plus 100 pg of truncated ALK2 mRNA; (C) 300 pg of chordin mRNA alone; (D) 100 pg of chordin, 0.3 pg of noggin and 20 pg of follistatin mRNAs. Both triple mRNA injections efficiently led to an ectopic head with eyes (white arrow) and a cement gland, while injection of increasing amount of any single mRNA did not induce ectopic head structures. White arrowheads, secondary trunk.