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FIGURE 1. Expression of XSMOC-1 during embryogenesis. A, RT-PCR analysis of XSMOC-1 expression at different stages of development. XSMOC-1 was not detectable until after stage 12. Histone H4 is shown as positive control, and ï°ˆRT indicates RT-PCR without reverse transcriptase as negative control. B, whole mount hybridization in situ analysis of XSMOC-1 (anterior to the left). B, ventral view of a stage 12.5 embryo showing anterior staining. C, ventro-lateral view of a stage 15 embryo showing anterior and lateral staining. Shown are lateral (D) and dorsal (E) view of a stage 17 embryo showing staining lateral to the neural plate. F, lateral views showing XSMOC-1 expression throughout the developing pronephros at stage 20 (F), stage 22 (G), and stage 24 (H). At stage 22 (G), additional expression was observed dorsal to the cement gland (arrowhead), and from stage 25 (H) onward, XSMOC-1 was expressed in the ventral region of the developing eye. I and J, dorsal views of H, following prolonged color development, showed expression in the mesencephalon, rhomben- cephalon (white arrows), and migrating neural crest (black arrows). K, transverse sections through the embryos shown in I and J in the region of the forebrain (K), hindbrain (L), and anterior trunk (M). Staining was prominent in the ventral aspect of the developing eye (K) and in the lateral regions of the hindbrain (L). Within the trunk (M), staining was observed in the pronephros and in subepithelial migrating neural crest cells. E, eye; FB, forebrain; HB, hindbrain; N, notochord; NC, neural crest; NT, neural tube; S, somite.
"This research was originally published THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.284(28) pp.18994-19005, July 10, 2009. Copyright: The American Society for Biochemistry and Molecular Biology."
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FIGURE 2. Xenopus embryos over-expressing XSMOC-1 exhibit a dorsalized phenotype. A, dorsal views of stage 17 embryos injected bilaterally at the two-cell stage with 300 pg of GFP (A and C) or XSMOC-1 (B and D) mRNAs. XSMOC-1-injected embryos have exaggerated anterior and diminished posterior structures (B) with laterally expanded expression of the neural plate marker Sox2 (D). The arrows indicate the position of the neural tube. E and F, transverse sections taken through the anterior regions of over-stained embryos C and D (white bars) show Sox2 expression throughout the dorsal tissues in XSMOC-1-injected embryos (F). The phenotypes shown for XSMOC-1 over-expression are typical for this stage and were observed in 95% of the embryos in three separate experiments (n = 96).
"This research was originally published THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.284(28) pp.18994-19005, July 10, 2009. Copyright: The American Society for Biochemistry and Molecular Biology."
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FIGURE 3. Dorsalization is pronounced in tadpoles overexpressing XSMOC-1. A, stage 26 Xenopus embryos injected bilaterally at the two-cell stage with 300 pg of GFP (A, C, and E) or XSMOC-1 mRNAs (B, D, and F). XSMOC-1-injected embryos were dorsalized, with exaggerated dorsal/anterior structures, particularly cement glands. The XSMOC-1 overexpression phenotypes shown in B were typical for this stage and were observed in 95% of the embryos in five independent experiments (n=218). C and D, histological 3-mm plastic sections (modified Von Gieson stain) showing hypertrophic cement gland cells in XSMOC-1-overexpressing embryos (D). E and F, 7-mm paraffin sections (Feulgen, light green, orange G method) showing enlargement of the neural tube and disorganized somites in XSMOC-1 over-expressing embryos (F).
"This research was originally published THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.284(28) pp.18994-19005, July 10, 2009. Copyright: The American Society for Biochemistry and Molecular Biology."
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FIGURE 4. XSMOC-1 induces neural markers in animal cap explants and acts non-cell-autonomously. A, RT-PCR analysis of animal caps obtained from embryos injected bilaterally with 300 pg of GFP (control) or XSMOC-1 at the two-cell stage. Animal caps were removed from stage 8 embryos and cultured until noninjected siblings reached stage 17. XSMOC-1 induced the neural markers N-CAM, NRP1, Otx2, and XAG1 and suppressed the expression of the epidermal marker, keratin. mRNA extracted from whole embryos (lane 3) was used as a positive control for the RT-PCRs, and reactions from which reverse transcriptase was omitted (ï°ˆRT; lane 4) were the negative con- trols. B and C, whole mount hybridization in situ of Otx2 in albino animal caps conjugated to wild-type caps. Wild-type embryos were injected bilaterally with 300 pg of GFP (B) or XSMOC1 (C). Animal caps were removed at stage 8 and conjugated to caps removed from stage 8 nonin- jected albino embryos. The conjugates were cultured until sibling embryos reached stage 17. Otx2 staining was not observed in the GFP control cap conjugates (B) but was present in the noninjected albino caps conjugated to XSMOC1-injected wild-type caps (C).
"This research was originally published THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.284(28) pp.18994-19005, July 10, 2009. Copyright: The American Society for Biochemistry and Molecular Biology."
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FIGURE 5. Unilateral injection of XSMOC-1 antisense morpholino (MO) produces mild ventralization and anophthalmia on the injected side. XSMOC-1 MO (6 ng) was injected into a single blastomere at the two-cell stage. At stage 17 (A and B), mild abnormalities were observed in the developing neural axis of XSMOC-1 MO-injected embryos (B). By stage 32 (C), MO-injected embryos were mildly ventralized (D and E) compared with controls (C). In addition, eyes were absent on the injected side (E); this was more apparent by stage 38 (G). Eye development appeared normal on the noninjected side (F). The XSMOC-1 MO phenotypes shown in D and E were typical for this stage and were observed in 90% of the embryos in five independent experiments (n=164). H, whole mount hybridization in situ analyses of Otx2 (H and I) and Tbx2 (J and K) in stage 32 control (G and J) and XSMOC-1 MO-injected (I and K) embryos. The injected sides are displayed on the right. The arrows indicate the location of the eye fields.
"This research was originally published THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.284(28) pp.18994-19005, July 10, 2009. Copyright: The American Society for Biochemistry and Molecular Biology."
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FIGURE 6. Complete loss of XSMOC-1 function leads to developmental arrest prior to neurulation. A-F, embryos injected bilaterally at the two-cell stage with 6 ng of 5-base mismatch control (A) or antisense (D-F) XSMOC-1 MO. Control MO-injected embryos developed normally (the position of the neural tube is indicated in C by an arrow), whereas antisense MO-injected embryos appeared normal up to the end of gastrulation (stage 12) but arrested prior to neurulation. The XSMOC-1 MO phenotypes shown in F were typical for this stage and were observed in 95% of the embryos in eight independent experiments (n = 326). G-I, RT-PCR analyses of markers expressed by control and antisense-XSMOC-1 MO-injected embryos at stage 10.5 (G),
12 (H), and 15 (I). Marker expression appeared normal up to stage 12 (G and H), but markers normally expressed after gastrulation were diminished (I). -RT, without reverse transcriptase.
"This research was originally published THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.284(28) pp.18994-19005, July 10, 2009. Copyright: The American Society for Biochemistry and Molecular Biology."
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FIGURE 7. Whole mount hybridization in situ of control (A, C, E, G, and I) and antisense XSMOC-1 MO-injected (B, D, F, H, and J) embryos showing expression of XNot (A and B) and XMyf5 (C and D) in stage 111.5 embryos, XSox2 (E and F) and XNot (G and H) in stage 12.5 embryos, and XSox2 (I and J) in stage 15 embryos. K and L, histological sections through I and J showing absence of archenteron (a) and any recognizable dorsal structures in antisense XSMOC-1 MO-injected embryos (modified Von Gieson stain).
"This research was originally published THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.284(28) pp.18994-19005, July 10, 2009. Copyright: The American Society for Biochemistry and Molecular Biology."
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FIGURE 8. XSMOC-1 inhibits BMP2 activity, but not by direct ligand binding. A, embryos were injected bilaterally at the two-cell stage with 360 pg of GFP (control), 60 pg of BMP2 plus 300 pg of GFP, or 60 pg of BMP2 plus 300 pg of XSMOC-1 mRNAs and incubated until stage 26. In three independent experiments, BMP2-injected embryos were ventralized (82% ≤ dorso-anterior index of 1, n = 84), whereas those co-injected with BMP2 and XSMOC-1 showed partial to complete rescue (70% ≥ dorso-anterior index of 3 or greater, n =98). B and C, RT-PCR analysis of animal cap explants removed from embryos at stage 8 and cultured until sibling embryos reached stage 17. B, RT-PCR for the ventral marker XVent-1 was induced in caps over-expressing BMP2 but not in control injected caps or caps co-expressing BMP2 and XSMOC-1. C, RT-PCR analysis of animal cap explants removed from embryos injected bilaterally at the two-cell stage with 400 pg of GFP (control), 100 pg of activin plus 300 pg of GFP, or 100 pg of activin plus 300 pg of XSMOC-1 mRNAs incubated until stage 17. Expression of the mesodermal marker Brachyury (Bra), induced in caps overexpressing activin, was not inhibited by co-expression of XSMOC-1. D, immunoblot analysis of mouse 3T3 fibroblast cell lysates. 3T3 fibroblasts were transfected with or without XSMOC-1 and exposed to BMP2 for 1 h. Phosphorylation of Smad1, -5, and -8 by BMP2 was blocked in cells transfected with XSMOC-1. E, RT-PCR analysis of animal cap explants removed from embryos injected bilaterally at the two-cell stage with 450 pg of GFP (control), 150 pg of caBMPR1B plus 300 pg of GFP, or 150 pg of caBMPR1B plus 300 pg of XSMOC-1 mRNAs were incubated until stage 17. The expression of the ventral marker XVent-1, induced by over-expression of constitutively active BMP receptor IB (caBMPR1B), was blocked by co-expression with XSMOC-1 but not by noggin. -RT, without reverse transcriptase.
"This research was originally published THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.284(28) pp.18994-19005, July 10, 2009. Copyright: The American Society for Biochemistry and Molecular Biology."
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FIGURE 9. Evidence that XSMOC-1 signals through the MAPK pathway. A, XSMOC-1 activity was blocked by co-expression of LM-Smad1. RT-PCR anal- ysis of animal caps from embryos injected bilaterally at the two-cell stage with 300 pg of XSMOC-1 plus 600 pg of GFP, 300 pg of XSMOC-1 plus 600 pg of LM-Smad1, 6 pg of noggin plus 900 pg of GFP, or 6 pg of noggin plus 600 pg of LM-Smad1 and 300 pg of GFP mRNAs. Induction of the neural markers N-CAM, NRP1, and Otx2 by overexpression of XSMOC-1 was blocked by co- expression of LM-Smad1; expression of the epidermal marker keratin was maintained. Neural marker induction (and suppression of keratin) by over-expression of noggin was not affected by co-expression of LM-Smad1. B, immunoblot analysis of animal cap extracts from embryos over-eexpressing XSMOC-1 revealed elevated levels of dp-ERK. Equivalent amounts of protein (10 mg) were loaded per lane. C, anterior views of control (left) and XSMOC-1 MO-injected stage 12.5 embryos immunostained for dp-ERK, Note the absence of dp-ERK in the XSMOC-1 MO-injected embryo. D, RT-PCR analysis of animal caps from XSMOC-1-injected embryos incubated in the presence or absence of the MEK inhibitor U0126 (50uM) until control embryos reached stage 17. Anterior neuroectodermal (Otx2 and XAG-1) and panneural (NCAM and NRP-1) markers induced by XSMOC-1 were markedly reduced in the pres- ence of U0126. -RT, without reverse transcriptase.
""This research was originally published THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.284(28) pp.18994-19005, July 10, 2009. Copyright: The American Society for Biochemistry and Molecular Biology."
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SMOC1 (SPARC related modular calcium binding 1) gene expression in Xenopus laevis embryos, NF stage 24, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
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