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Comparison of Gdf3 and Nodal signaling activities. (A) Schematic depiction of mouse Gdf3 and modified Gdf3 proteins; the sites of prodomain cleavage is indicated (arrows). (B) Gdf3 activity is Cripto and Foxh1 dependent. The indicated plasmids were co-transfected into 293T cells together with the A3-luc reporter. (Insets) Western blot detection of mature Nodal (∼12 kDa) and mature Gdf3 (∼17 kDa) proteins in conditioned media using α-Nodal antibody or α-Gdf3 antibody. (C) Gdf3 activity is facilitated by Cripto, but not Cryptic. 293T cells were co-transfected with expression vectors for Nodal or bf-Gdf3, Foxh1 and FLAG-epitope-tagged Cryptic, Cripto or Oep; control samples correspond to cells transfected with Nodal or bf-Gdf3 and Foxh1 without EGF-CFC proteins. (Inset) Western blot detection of EGF-CFC proteins in cell lysates usingα -FLAG antibody (FLAG-Cryptic, ∼22 and ∼20 kDa; FLAG-Cripto,∼ 21 and ∼19 kDa; FLAG-Oep, ∼21 kDa). (D) Lefty1 inhibits Gdf3 activity. Cells were co-transfected with expression constructs for Nodal or bf-Gdf3, Cripto and Foxh1, in the presence or absence of Lefty1; controls correspond to these cells without Nodal or bf-Gdf3 transfection. (Inset) Western blot detection of mature Lefty1 protein (∼30 kDa) in conditioned media. In all panels, assays were performed in triplicate; error bars represent s.d.
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Interactions of Gdf3 with activin receptors, Cripto and Lefty1. (A) Formation of a Gdf3 receptor complex. 293T cells were transfected with the indicated expression constructs, followed by crosslinking with the membrane-impermeable reagent DTSSP, immunoprecipitation of Myc-tagged ActRIIB from cell lysates and reversal of crosslinking; kinase-inactive activin receptor mutants were used in order to decrease receptor internalization. Western blots of immunoprecipitated and input proteins are shown, detected using the following antibodies: α-Gdf3 (bf-Gdf3 proprotein, ∼49 kDa; mature bf-Gdf3, ∼17 kDa); α-FLAG (f-Cripto, ∼21 kDa); α-HA (ActRIB-HA, ∼54 kDa); α-Myc (ActRIIB-6xMyc, ∼75 kDa). (B) Cripto interacts with Gdf3. Expression constructs were co-transfected into 293T cells, followed by crosslinking with DTSSP, immunoprecipitation of FLAG-tagged Cripto (f-Cripto) from cell lysates and reversal of crosslinking. Western blots are shown using the following antibodies: α-V5 (bv-Gdf3 proprotein, ∼50 kDa) and α-FLAG (f-Cripto, ∼21 kDa). (C) Gdf3 interacts with Lefty1 in conditioned media in the absence of crosslinking. The mature form of Lefty1 is preferentially immunoprecipitated; the expected positions of the Lefty1 proprotein (arrow) and a mature Lefty1 variant generated by use of an alternative prodomain cleavage site (asterisk) are indicated. Proteins were detected by western blotting using the following antibodies: α-FLAG (bf-Gdf3 proprotein, ∼49 kDa; mature bf-Gdf3,∼ 17 kDa) and α-Lefty1 (Lefty1 proprotein, ∼40 kDa; mature Lefty1, ∼30 kDa).
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Gdf3 induces secondary axis and mesendoderm formation in Xenopus embryos. (A-D) Secondary axis formation following ventral marginal zone injection. Morphology of control uninjected embryo (A) and embryos injected with the indicated doses of Nodal (B), Gdf3 (C) or bf-Gdf3 (D) mRNAs; arrows indicate secondary axis formation. (E-H) Gdf3 induces morphogenetic elongation in animal cap explants. Unlike control uninjected caps (E), animal caps injected with the indicated doses of Nodal (F), Gdf3 (G) or bf-Gdf3 (H) mRNAs undergo morphogenetic elongation, as scored at stage 20. (I) Mesendoderm formation in animal cap assays. High doses of Gdf3 (500-1000 pg) result in weak induction of Xbra and Mixer, while low doses of Nodal (50 pg) and bf-Gdf3 (50-100 pg) result in strong induction of markers for mesoderm (Xbra, Gsc, chordin and Xwnt8) and endoderm (Mixer), but not the dorsal marker Xnr3. Ornithine decarboxylase (Odc) is used as an internal control. -RT, no reverse transcriptase added.
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Gdf3 expression in pre-implantation and early post-implantation mouse embryos. (A,B) Expression of Gdf3 is found at low levels in the inner cells of compacted morula at the 16-cell stage (A), and is later expressed at higher levels in the inner cell mass (icm) at the blastocyst stage (B), with no expression in the trophoblast or primitive endoderm (pe). (C-F) After implantation, expression is restricted to the embryonic epiblast (epi) at 5.5 dpc (C,D) and 6.0 dpc (E,F), and is not found in the visceral endoderm (ve) or extra-embryonic ectoderm; expression disappears prior to primitive streak formation. Scale bars: 50μ m.
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Morphology and histology of Gdf3 mutant embryos. (A) Two Gdf3-/- embryos at 8.5 dpc, showing a phenotypically wild-type embryo (left) and a severe mutant that consists of an empty visceral yolk sac (right). (B) A Gdf3-/- mutant at 8.5 dpc displays an abnormal embryonic region and prominent constriction at the embryonic/extra-embryonic boundary (arrowheads); an allantois-like structure (al?) is present. (C) Two Gdf3-/- embryos at 8.0 dpc, with a phenotypically wild-type embryo (left) and an abnormal embryo (right) with reduced anterior structures (arrow) and an ectopic projection (arrowhead). (D-G) Hematoxylin-Eosin staining of paraffin wax-embedded sections through decidua containing Gdf3-/- embryos at 7.5 dpc (D,E) and 8.0 dpc (F,G). In comparison with the phenotypically wild-type embryos (D,F), the severely affected mutant in E shows an embryonic/extra-embryonic boundary constriction (arrowheads) and an abnormal embryonic region containing mesoderm (arrows). The less-affected embryo in G lacks an allantois (arrowhead) and has visceral yolk sac-like tissue in place of an amnion (arrows). Scale bars: 200 μm. Abbreviations: al, allantois; am, amnion; ch, chorion; fb, forebrain; mes, mesoderm; ne, neuroectoderm; vys, visceral yolk sac.
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Phenotypic defects and abnormal Nodal expression in Gdf3 mutants. (A,B) Foxa2 is normally expressed in the anterior primitive streak (ps) (A), but is often absent or greatly reduced in Gdf3 homozygotes (B, arrow). (C,D) Lhx1 is expressed in the primitive streak, nascent mesoderm and anterior visceral endoderm (ave) (C), but is frequently absent (D, arrow) or greatly reduced (D, arrowhead) in Gdf3 mutants. (E,F) Expression of Otx2 in the anterior neuroectoderm (ane) is unaffected in Gdf3 homozygotes. (G,H) Expression of Cerl marks the AVE and definitive endoderm (de), but the definitive endoderm is often absent in Gdf3 homozygotes (H, arrow). (I,J) Hesx1 expression in the AVE is sometimes reduced or absent in Gdf3-/- embryos (J, arrow). (K-M) Hex expression in the AVE is sometimes completely absent (L) or is misplaced distally (M, arrow). (N,O) Asymmetric expression of Nodal in the left lateral plate mesoderm (lpm) is observed in Gdf3-/- embryos with normal morphology at 8.0 dpc (N); a few abnormal mutants lack Nodal expression in the lateral plate, but retain expression around the node (nd) (O, arrow). (P,Q) Expression of T (brachyury) marks the tailbud (tb) and notochord (not) at 8.0 dpc (P), but is absent or greatly reduced in abnormal Gdf3-/- embryos (Q, arrow). (R,S) In rare Gdf3 homozygotes, expression of T reveals a partial axis duplication (R, arrows), corresponding to formation of twinned notochords (S, arrows) and multiple somites (arrowheads). (T-D′) Expression of Nodal is found in the proximal-posterior epiblast (ep) at 6.0 dpc (T,Z), but is downregulated in some Gdf3 mutants (U) while upregulated in others (V,A′). At 6.75 dpc, Nodal is expressed in the posterior epiblast and in the visceral endoderm (ve) overlying the primitive streak (W,B′), but some Gdf3-/- embryos show an expansion of the primitive streak (X,C′, double-headed arrow) that is accompanied by increased expression in the visceral endoderm (C′, arrow). Abnormal Gdf3-/- embryos with the `conical' morphology (Y) show high-level Nodal expression circumferentially in the visceral endoderm surrounding a thick mesodermal layer (mes) (D′, arrow). (E′,F′) Expression of Cripto in the proximal-posterior epiblast at 6.75 dpc is unaffected in most Gdf3 homozygotes, but is upregulated in `conical' mutants (F′). (G′,H′) Bmp4 expression in the extra-embryonic ectoderm (exe) at 6.5 dpc is unaffected in Gdf3 mutants. (I′,J′) Expression of Gdf1 in the pre-gastrulation epiblast is unaffected in Gdf3 homozygotes. Scale bars: 100 μm for A-Y,E′-J′; 50 μm for Z-D′.
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Model for Gdf3 function. (A) In wild-type embryos, Nodal activity promotes AVE induction and movement as well as primitive streak formation, and maintains its expression through an autoregulatory feedback loop. Gdf3 is also required for AVE formation and movement, and may additionally be necessary for streak formation and maintenance of Nodal expression. (B) In wild-type embryos (top), expression of Nodal antagonists by the AVE provides essential signals for patterning of the anterior epiblast; the distribution of Nodal pathway activity (including Gdf3) in the epiblast is depicted in blue, while that of Nodal antagonists is shown in red. In the absence of Gdf3, the AVE may form but be unable to move (middle), thereby resulting in embryos with downregulated Nodal expression that is restricted to the proximal epiblast. Alternatively, if the AVE fails to form (bottom), the absence of Nodal antagonists will result in upregulation of Nodal expression throughout the epiblast, potentially leading to expansion of the primitive streak and axis duplication.
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