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Genes Dev
2007 Dec 15;2124:3272-82. doi: 10.1101/gad.1623907.
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Long-range action of Nodal requires interaction with GDF1.
Tanaka C
,
Sakuma R
,
Nakamura T
,
Hamada H
,
Saijoh Y
.
???displayArticle.abstract??? GDF1 (growth/differentiation factor 1), a Vg1-related member of the transforming growth factor-beta superfamily, is required for left-right patterning in the mouse, but the precise function of GDF1 has remained largely unknown. In contrast to previous observations, we now show that GDF1 itself is not an effective ligand but rather functions as a coligand for Nodal. GDF1 directly interacts with Nodal and thereby greatly increases its specific activity. Gdf1 expression in the node was found necessary and sufficient for initiation of asymmetric Nodal expression in the lateral plate of mouse embryos. Coexpression of GDF1 with Nodal in frog embryos increased the range of the Nodal signal. Introduction of Nodal alone into the lateral plate of Gdf1 knockout mouse embryos did not induce Lefty1 expression at the midline, whereas introduction of both Nodal and GDF1 did, showing that GDF1 is required for long-range Nodal signaling from the lateral plate to the midline. These results suggest that GDF1 regulates the activity and signaling range of Nodal through direct interaction.
Figure 2. GDF1 is not an active ligand but enhances Nodal activity. (A–C) The activity of the Nodal-responsive reporter (n2)7luc in the Xenopus animal cap assay was determined after injection of mRNAs for Nodal (10 pg), GDF1 (1000 pg), or the Nodal coreceptor Cripto (20 pg) (A); of mRNAs for Nodal (2 pg) or GDF1 (40 pg) (B); or of mRNAs for Nodal (2 pg), GDF1 (40 pg), Lefty1 (50 pg), or Lefty2 (50 pg) (C). All embryos in B and C were also injected with 100 pg of the mRNA for the Nodal coreceptor Cryptic. (D) Xenopus embryos were injected with mRNAs for Nodal (++, 50 pg; +, 10 pg), GDF1 (40 pg), or Cryptic (100 pg), as indicated, after which animal caps were subjected to immunoblot analysis with antibodies to phospho-Smad2 (p-Smad2) or to α-tubulin (loading control). (E,F) The animal cap assay was also performed with mRNAs for zDVR1, Squint (Sqt), Cyclops (Cyc), or Flag-tagged OEP (OEP), as indicated. Injected mRNA amounts are shown in picograms (in parentheses).
Figure 3. GDF1 Interacts with Nodal. (A) Conditioned medium prepared from Xenopus oocytes expressing Flag-Nodal or GDF1, as indicated, was subjected to immunoprecipitation (IP) with antibodies to Flag (α-Flag). The resulting precipitates as well as the conditioned medium were subjected to immunoblot analysis (IB) under reducing conditions with antibodies to GDF1 (α-GDF1). (B) Conditioned medium prepared from Xenopus oocytes expressing Flag-GDF1 or Nodal, as indicated, was subjected to immunoprecipitation with antibodies to Flag. The resulting precipitates as well as the conditioned medium were subjected to immunoblot analysis under reducing conditions with antibodies to Nodal. (C) Conditioned medium prepared from Xenopus oocytes expressing Flag-Nodal or GDF1, as indicated, was subjected to immunoprecipitation with antibodies to Flag, and the resulting precipitates were subjected to immunoblot analysis under nonreducing conditions with antibodies to GDF1. Arrow indicates a Flag-Nodal–GDF1 heterodimer. (D) Conditioned medium prepared from Xenopus oocytes expressing Flag-GDF1 or Nodal, as indicated, was subjected to immunoprecipitation with antibodies to Flag, and the resulting precipitates were subjected to immunoblot analysis under nonreducing conditions with antibodies to Nodal or to GDF1. Arrow indicates a Flag-GDF1–Nodal heterodimer. The lower-mobility band corresponds to a heterodimer of Nodal and of Flag-GDF1 that was not cleaved at the proteolytic cleavage site.
Figure 4. Interaction with GDF1 increases Nodal activity. (A) Conditioned medium prepared from Xenopus oocytes expressing Nodal, GDF1, or Activin, as indicated, was assayed for activity in a Xenopus animal cap assay with the Nodal-responsive reporter (n2)7luc. (B) Immunoblot analysis of the conditioned media (10 μL) used for the assay in A. The GDF1 protein coexpressed with Nodal in frog oocytes migrated slightly faster than did that expressed in the absence of Nodal. This was also true when GDF1 was expressed with or without Nodal in COS cells (Supplementary Fig. S6). (C) Conditioned medium prepared from Xenopus oocytes expressing Nodal, GDF1, or both proteins (GDF1 + Nodal) was assayed for activity as in A. For “GDF1/Nodal (mix),†conditioned medium for GDF1 and that for Nodal were prepared separately and mixed. (D) Frog embryos were injected with (n2)7luc and mRNAs for Nodal (2 pg) or GDF1 (40 pg) as indicated (mRNA). Animal caps prepared from the embryos were then cultured in conditioned medium prepared from Xenopus oocytes expressing Nodal or GDF1 as indicated (Medium), after which the activity of (n2)7luc was determined. (Un) Uninjected. All embryos in A, C, and D were also injected with 100 pg of the mRNA for Cryptic.
Figure 5. GDF1 increases the range of the Nodal signal in frog embryos. (A–C) Experimental strategy. The Nodal-responsive reporter (f1)6lacZ, mRNAs for Cryptic (125 pg) and the Activin type I receptor ALK4 (50 pg), and TRLDx were injected into a single blastomere of a 32- or 64-cell stage Xenopus embryo. (A) Nodal mRNA (250 pg), with or without Gdf1 RNA (225 pg), was injected together with FLDx into either an adjacent blastomere or a blastomere separated by one or two cells. Animal caps were prepared at stage 8.5, cultured for 3 h, and stained with X-gal. The fluorescence of TRLDx and FLDx was also monitored to reveal descendents of the adjacent (B) or separated (C) injected cells. (D–H) X-Gal-stained animal caps for adjacent (D,F) or separated (E,G) blastomeres injected with Nodal mRNA alone (D,E) or with Nodal mRNA plus Gdf1 mRNA (F,G) in the effector mix. (H) An X-gal-stained animal cap derived from an embryo injected with only the reporter mix is also shown.
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