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First published online 24 October 2007
doi: 10.1242/dev.010645

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Nuclear accumulation of Smad complexes occurs only after the midblastula transition in Xenopus

Yasushi Saka^1,2,*, Anja I. Hagemann^1,*, Olaf Piepenburg^1, and James C. Smith^1,

¹ Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.
² Interdisciplinary Research Institute and Institut de Biologie de Lille, 1 rue du professeur Calmette, BP447, 59021 Lille Cedex, France.

View larger version (87K): [in this window] [in a new window]   Fig. 1. Self-assembly of VENUS fragments is prevented by specific mutations. RNA encoding the indicated combinations of wild-type or mutated VENUS fragments (500 pg of each) was injected into Xenopus embryos at the one-cell stage in the presence of RNA encoding monomeric red fluorescent protein (mRFP1; 500 pg) as a lineage marker. Embryos were cultured to midblastula stage 9 and examined using a fluorescence dissecting microscope to visualise mRFP1 (top of each panel) or VENUS (bottom of each panel). (A) Strong fluorescence is observed following injection of mRNA encoding VN154 and VC155. (B) Mutation of VN154 to create VNm9 abolishes self-assembly and fluorescence. (C) Injection of mRFP1 alone. (D) Strong fluorescence is observed following injection of mRNA encoding VN144 and VC145. (E) VENUS fluorescence does not occur in embryos injected with RNA encoding VNm3 and VCm5 and only background level of fluorescence is observed. (F) An uninjected embryo illustrating background fluorescence.

View larger version (36K): [in this window] [in a new window]   Fig. 2. Tagged forms of Smad2 and Smad4 retain their biological activities. (A) Illustration of the principle of bimolecular fluorescence. In unstimulated cells, in which Smad2 is not phosphorylated, Smad2 and Smad4 do not interact, and the N-terminal and C-terminal portions of VENUS are not able to complement. Phosphorylation of Smad2 results in a conformational change and allows interaction with Smad4 and fluorescence complementation. (B) Tagged Smad constructs retain their biological activity. Xenopus embryos received no injections (embs, caps), or were injected at the one cell stage with RNA encoding tagged forms of Smad2 or Smad4 proteins (100 pg for VC155-Smad2/VNm9-Smad4 BiFC tags and 80 pg for HA tags), alone or in combination, or with 5 pg RNA encoding Activin. Animal caps were dissected at the midblastula stage and cultured to the equivalent of early gastrula stage 10.5. Note that when expressed individually, tagged forms of Smads do not induce expression of Xbra and gsc, but co-expression of Smad2 and Smad4 (100 pg RNA of each) activates both genes at levels similar to those induced by Activin.

View larger version (66K): [in this window] [in a new window]   Fig. 3. Smad BiFC constructs respond to Activin signalling. (A) Diagram comparing wild-type VC155-Smad2 with the control protein VC155-Smad2C. (B) The response of these Smad BiFC constructs to Activin. Embryos were injected at the one cell stage with RNA encoding the indicated BiFC reporter constructs, together with RNA encoding mRFP1 as a lineage marker. Animal pole regions were dissected at the midblastula stage and labelled cells were identified by mRFP1 fluorescence (not shown), disaggregated, and plated on a fibronectin-coated substrate in the presence or absence of Activin. They were cultured to the equivalent of the early gastrula stage. In the absence of Activin (left-hand column of images) cytoplasmic fluorescence is detectable that derives from Smad2 homodimers (a), Smad2-Smad4 heterodimers (e) and Smad4 homodimers (i). Some autofluorescent yolk platelets are detectable in cells expressing VC155-Smad2C rather than VC155-Smad2 (c,g). Note that levels of fluorescence in these samples is exaggerated because the cells are round; Activin treatment (right-hand column of images) causes cells to flatten (Smith et al., 1990). We note that some flattening also occurs in cells co-injected with RNA encoding Smad2 and Smad4 fusion proteins (see Be), consistent with the ability of these constructs to activate expression of Xbra and gsc (see Fig. 2B). In the presence of Activin there is slight accumulation of Smad2 homodimers in the nucleus of responding cells (b) and dramatic accumulation of Smad2-Smad4 heterodimers (f). Smad4 homodimers are excluded from the nucleus (j). Little fluorescence is detectable in cells expressing VC155-Smad2C rather than VC155-Smad2 (d,h). (C) Loss of nuclear BiFC fluorescence by late gastrula stage 13. Animal pole blastomeres were isolated from embryos that had been injected with RNA encoding VC155-Smad2 and VNm9-Smad4 together with RNA encoding ECFP, or from embryos expressing just ECFP. Dissociated blastomeres were left untreated or treated with 16 U/ml Activin for 10 minutes and then cultured on a fibronectin-coated substrate and observed at the equivalent of stage 10.5 or stage 13. Strong nuclear BiFC fluorescence in response to Activin is detectable at stage 10.5, but by stage 13 this has decreased to the levels observed in untreated cells. Fluorescence levels in cells not expressing Smad-BiFC constructs are shown for comparison.

View larger version (128K): [in this window] [in a new window]   Fig. 4. Association of Smad2-Smad4 heterodimers with nuclear membranes in interphase cells and with chromatin in mitotic cells. (A) Cells derived from an embryo expressing VC155-Smad2 and VNm9-Smad4 cultured in the absence of Activin. There are weak strands of BiFC fluorescence in the cytoplasm and around the nucleus. Blue staining derives from co-expressed ECFP lineage tracer that tends to accumulate slightly in the nucleus. (B) Strands of BiFC nuclear fluorescence in untreated animal pole cells are colocalised with nuclear membranes. Left-hand panel shows staining of ECFP-tagged emerin, middle panel shows BiFC fluorescence, and right-hand panel shows the merged image. (C) Cells identical to those in A but treated with Activin. Note strong nuclear fluorescence. Arrows indicate spots referred to in text. (D) Three images of a mitotic Xenopus animal pole blastomere derived from an embryo injected at the one cell stage with RNA encoding ECFP-tagged histone H2B (specific fluorescence visible in left-hand panel) and RNA encoding VNm9-Smad4 and VC155-Smad2 (specific fluorescence visible in centre panel). Note that histone and BiFC fluorescence colocalise (right-hand panel). (E) Smad complexes are associated with chromatin during mitosis. The cell illustrated is derived from an embryo injected with BiFC constructs designed to reveal heteromeric interactions between molecules of Smad2exon3 and Smad4. Images were taken at intervals of 1.5 minutes. Note fluorescence associated with chromatin. Cells co-express an ECFP-GPI membrane marker. (F) Localisation of Smad-BiFC complexes to nuclear membranes and chromosomes is specific. Injection of RNA encoding the complementary VENUS fragments VC155 and VNm9 (200 pg of each, representing a 20 fold molar excess over concentrations used in Smad-BiFC experiments) reveals weak fluorescence in the cytoplasm and at the periphery of cells, but not in the nuclei or on chromosomes. Arrows indicate mitotic chromatin. Higher levels of fluorescence at the periphery of cells may be due to displacement of material by yolk platelets.

View larger version (46K): [in this window] [in a new window]   Fig. 5. BiFC Nuclear fluorescence increases as the concentration of Activin is increased; BiFC can reveal endogenous TGF-ß signalling during normal development of Xenopus. (A) Animal pole blastomeres derived from an embryo injected with RNA encoding VNm9-Smad4 and VC155-Smad2, together with RNA encoding ECFP, were exposed to the indicated concentrations of Activin [measured in units/ml (Cooke et al., 1987)] and examined by fluorescence microscopy 4 hours later. Note that as the concentration of Activin increases, nuclei become brighter. Cytoplasmic fluorescence in cells treated with 16 U/ml activin derives from yolk platelets. (B) Quantification of the data in A. The values represent the ratio of nuclear BiFC fluorescence to ECFP fluorescence in the nucleus averaged over 10-15 cells. The error bars represent standard deviations. (C) Endogenous TGFß signalling in the Xenopus embryo revealed by BiFC. Xenopus embryos were injected with 100 pg RNA encoding VNm9-Smad4 and VC155-Smad2 together with RNA encoding ECFP-tagged histone H2B. They were cultured to the early gastrula stage before being bisected, as shown in the diagram, into animal and vegetal halves and observed under the fluorescence microscope. Note low levels of fluorescence largely confined to nuclear membrane in the animal hemisphere of the embryo, and higher levels in the nuclei of cells in the vegetal region.

View larger version (74K): [in this window] [in a new window]   Fig. 6. Entry of Smad2-Smad4 complexes into the nucleus does not occur until the midblastula transition, irrespective of the stage of treatment with Activin. (A) Animal pole cells derived from embryos injected with RNA encoding VNm9-Smad4 and VC155-Smad2 and ECFP lineage marker were treated with Activin at early blastula stage 7 (left-hand column) or late blastula stage 9 (right-hand column), and were cultured for the indicated times. Note that nuclear translocation of Smad complexes in cells treated with Activin at stage 7 occurs only at 75 minutes after treatment, corresponding to stage 8.5 (c), but that the delay in cells treated with Activin at stage 9 is less than 10 minutes (f). (d,h) Control cells that were not treated with Activin. (B) Gene activation in response to Activin also only occurs after stage 9. Animal pole regions were dissected from Xenopus embryos at stage 7, 8 or 9 and cultured in the presence or absence of Activin for either 30 minutes or until control embryos reached stage 9 before being assayed for expression of chordin and goosecoid. Following treatment at stage 7, gene activation does not occur within 30 minutes of culture, but is clearly detectable by stage 9. The same is true of animal caps treated at stage 8, but treatment of animal pole regions at stage 9 results in gene activation within 30 minutes. (C) Lack of nuclear BiFC in embryos at stage 7 is not a consequence of the slow maturation of BiFC constructs or delayed complex formation. Compared with uninjected cells (a), weak cytoplasmic fluorescence is clearly visible in cells expressing VC155-Smad2 and VNm9-Smad4 as early as stage 7 (b). (D) Phosphorylation of endogenous Smad2 occurs within 30 minutes of treatment with Activin even at stage 7. The western blot was performed with protein extracts of animal pole regions at the indicated stages. (E) VC155-Smad2 is expressed and can be phosphorylated in response to Activin as early as stage 7. Positions of VC155-Smad2 and of endogenous Smad2 are indicated. Protein extracts are derived from animal pole regions excised from embryos previously injected with RNA encoding VC155-Smad2 and VNm9-Smad4.

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