Nodal Signals to Smads through Cripto-Dependent and Cripto-Independent Mechanisms  Chang-Yeol Yeo, Malcolm Whitman  Molecular Cell  Volume 7, Issue 5, Pages 949-957 (May 2001) DOI: 10.1016/S1097-2765(01)00249-0

Figure 1 Cripto Enhances Nodal-Induced Activation of Smad2 and Downstream Transcription Synthetic mRNAs (amount/embryo) encoding Cripto (500 pg), Nodal (100 pg), and Activin βB (20 pg) were injected into Xenopus embryos. α-amanitin (α-Am) was injected to block transcription. Phosphorylation of Smad2 and activation of downstream transcription were measured from stage 10 ectodermal explants. (A) Smad2 activation. Activated Smad2 was detected by Western blot analysis using anti-phospho-Smad2 antibody (α-PSmad2) (Faure et al., 2000). Cytoskeletal actin was used as a loading control (α-Actin). Levels of total Smad2 were also compared (α-Smad2) among different conditions. The two bands, recognized by anti-phospho-Smad2 antibody and anti-Smad2 antibody, were identified as Smad2 and Smad2Δexon3, a splicing variant of Smad2 (Faure et al., 2000). (B) Downstream transcription. Expression of a pan meso-endodermal gene Mix.2 and a dorsal mesodermal gene goosecoid (Gsc) were measured by RT-PCR analysis. EF1α was used as the loading control. Stage 10 embryos were used for RT-PCR positive and negative controls (+RT and -RT) Molecular Cell 2001 7, 949-957DOI: (10.1016/S1097-2765(01)00249-0)

Figure 2 Cripto Interacts Specifically with the ALK4 Receptor Interaction between Cripto and type I receptors was determined by coimmunoprecipitation and Western blot analysis. Synthetic mRNAs (500 pg of each RNA/embryo) encoding Cripto/Flag, Nodal, BMP4, BMP7, ALK4/HA, ALK2/HA, and ALK3/HA were injected. Extracts from stage 9.5 embryos were immunoprecipitated for Flag-tagged Cripto (α-Flag IP). Coimmunoprecipitation of HA-tagged type I receptors were detected by anti-HA Western blot analysis (α-HA) Molecular Cell 2001 7, 949-957DOI: (10.1016/S1097-2765(01)00249-0)

Figure 3 Cripto Has Two Distinct Functional Domains (A) A schematic drawing of conserved regions of Cripto. The amino-terminal signal sequence (open box), the EGF-like motif and the CFC motif (gray boxes), and the carboxy-terminal hydrophobic tail (filled box) are shown. Relative positions of amino acids (bars) that were substituted or deleted in Cripto mutants are also indicated. (B) Cripto mutants and Smad2 activation. Synthetic mRNAs (500 pg of each RNA/embryo) encoding Nodal, wild-type Cripto/Flag and Cripto/Flag mutants were injected. Stage 10 ectodermal explants were used for anti-phospho-Smad2 Western blot analysis (α-PSmad2). (C) Interaction between Cripto mutants and ALK4. RNAs (500 pg of each RNA/embryo) encoding ALK4/HA, wild-type Cripto/Flag and Cripto/Flag mutants were injected. Embryo extracts were immunoprecipitated for Flag-tagged Cripto (α-Flag IP), and coimmunoprecipitation of ALK4 was determined by anti-HA Western blot analysis (α-HA) Molecular Cell 2001 7, 949-957DOI: (10.1016/S1097-2765(01)00249-0)

Figure 4 Nodal Activates Smad through ALK4 Receptor and Binds to ALK4/ActR-IIB Receptor Complex in a Cripto-Dependent Manner (A) Smad activation by Nodal through ALK4. Synthetic mRNAs (500 pg of each RNA/embryo) encoding Noggin, Nodal, Cripto/Flag, ALK4, and ALK4 (L45-ALK3) were injected. Activation of Smad1 and Smad2 in stage 10 ectodermal explants was measured by Western blot analysis using anti-phospho-Smad1 antibody (α-PSmad1) and anti-phospho-Smad2 antibody (α-PSmad2) (Faure et al., 2000). Levels of total Smad1 (α-Smad1) and Smad2 (α-Smad2) were also examined. Total protein levels among samples were equalized using cytoskeletal actin as a loading control (not shown). (B) Nodal binding to the receptor complex. RNAs (2.5 ng of each RNA/embryo) encoding ActR-IIB (KR)/Myc, ALK4 (KR)/HA, Cripto/Flag, Cripto ΔEGF/Flag, and Glu/Nodal were injected. Animal halves of stage 9.5 embryos were treated with DTSSP, and extracts were immunoprecipitated for HA-tagged ALK4 (KR) (α-HA IP). Coimmunoprecipitation of Glu-tagged Nodal (α-Glu), Myc-tagged ActR-IIB (KR) (α-Myc), and Flag-tagged Cripto (α-Flag) were examined by Western blot analysis Molecular Cell 2001 7, 949-957DOI: (10.1016/S1097-2765(01)00249-0)

Figure 5 Nodal Antagonizes Smad1 Phosphorylation by BMP in a Cripto-Independent Manner Smad activation was measured from stage 10 (A, B, and C) or stage 9.5 (D) ectodermal explants by Western blot analysis (α-PSmad1 and α-PSmad2). Protein loading was equalized using cytoskeletal actin as a loading control; total Smad1 and Smad2 were equal in all lanes (not shown). (A) Cripto-independent inhibition of Smad1 phosphorylation by Nodal. Synthetic mRNAs (500 pg of each RNA/embryo) encoding Nodal, wild-type Cripto/Flag, and Cripto/Flag mutants were injected. (B) Smad2 signaling and transcription-independent inhibition of Smad1 phosphorylation by Nodal. RNAs (amount/embryo) encoding Nodal (500 pg) and ALK4 (KR) (1 ng) were injected. α-amanitin was injected to block transcription. (C) Dose-dependent inhibition of Smad1 phosphorylation by Nodal. Increasing amounts of RNAs (20 pg, 100 pg, or 500 pg/embryo) encoding Nodal (lanes 2–4) or cm-Nodal (lanes 5–7) were injected. (D) Antagonism between BMP and Nodal. RNAs encoding Nodal (500 pg) and BMP7 (1 ng) were injected Molecular Cell 2001 7, 949-957DOI: (10.1016/S1097-2765(01)00249-0)

Figure 6 Nodal Forms Heterodimers with BMP7 Synthetic mRNAs (500 pg of each RNA/embryo) encoding HA/cm-Nodal, Flag/cm-Nodal, and Flag/cm-BMP7 were injected. Embryo extracts were immunoprecipitated for HA-tagged cm-Nodal (α-HA IP). Immunoprecipitated proteins were subjected to SDS-PAGE under reducing conditions with β-mercaptoethanol (β-ME) (A) or under nonreducing conditions without β-ME (B). Coimmunoprecipitation of Flag-tagged cm-Nodal and cm-BMP7 was examined by anti-Flag Western blot analysis (α-Flag WB). Positions of molecular weight markers (in KDa) are indicated Molecular Cell 2001 7, 949-957DOI: (10.1016/S1097-2765(01)00249-0)

Figure 7 Model for Cripto-Dependent and Cripto-Independent Nodal Signaling Cripto, through its CFC domain, forms a ligand-independent coreceptor complex with ALK4. Nodal binds to receptor complexes consisting of Cripto/ALK4 and ActR-IIB. In this complex, ActR-IIB activates ALK4 and, in turn, ALK4 phosphorylates, and activates Smad2. BMP7 transduces signals to Smad1 through the BMP type II and type I receptors. Nodal and BMP7 antagonize mutual signaling by forming heterodimers intracellularly Molecular Cell 2001 7, 949-957DOI: (10.1016/S1097-2765(01)00249-0)