Volume 15, Issue 1, Pages 49-61 (July 2008) Regulation of Activin/Nodal Signaling by Rap2-Directed Receptor Trafficking  Sun-Cheol Choi, Gun-Hwa Kim, Seung Joon Lee, Eunjoo Park, Chang-Yeol Yeo, Jin-Kwan Han  Developmental Cell  Volume 15, Issue 1, Pages 49-61 (July 2008) DOI: 10.1016/j.devcel.2008.05.004 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Rap2 Is Essential for Activin/Nodal Signaling (A) Uninjected control embryo. (B) Ventral injection of Rap2 DNA (200 pg). An arrowhead indicates ectopic dorsal axis. (C) Dorsal injection of Rap2 DNA. (D) Uninjected E., uninjected control embryo; Rap2-injected E., dorsomarginally Rap2 DNA (200 pg)-injected embryo. Actin serves as a loading control. (E) Animal cap explants injected with Xnr1 (100 pg) RNA alone or with the indicated combinations of Rap2 MO (40 ng), Co MO (40 ng), and flag-Rap2 RNA (100 pg) were subjected to RT-PCR analysis. ODC is a loading control. −RT, a control RT-PCR in the absence of reverse transcriptase; WE, whole embryo; AC, uninjected animal caps. (F and G) DE-lux (40 pg) or ARE-lux (40 pg) reporter construct was injected into the animal pole region of four-cell stage embryos with or without the combinations of Xnr1 (100 pg), Activinβ1 (5 pg), Rap2 MO (40 ng), and Co MO (40 ng) as indicated. Error bars represent the standard deviation (SD). (H) Animal caps expressing Activinβ1 RNA (5 pg) alone or with the combinations of Rap2 MO (40 ng), Co MO (40 ng), and flag-Rap2 RNA (100 pg) as indicated were subjected to western blotting using anti-phospho-Smad2 antibody. (I–L) Animal cap tissues injected with Myc-tagged Smad2 (400 pg) alone or with the combinations of Xnr1 RNA (100 pg), Rap2 MO (40 ng), and Co MO (40 ng) as indicated were subjected to immunostaining using primary anti-Myc and secondary FITC-conjugated antibodies. Arrowheads indicate the nuclear Smad2. Developmental Cell 2008 15, 49-61DOI: (10.1016/j.devcel.2008.05.004) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 In Vivo Requirement of Rap2 for Activin/Nodal-Dependent Gene Expression (A–D) Phenotypes of Rap2-depleted embryos. Four-cell stage embryos were injected radially in the marginal regions with the indicated combinations of Rap2 MO (40 ng), Co MO (40 ng), and Rap2 DNA (200 pg) or not injected as a control (A) and cultured until sibling embryos reached stage 32. The percentages of phenotypes of embryos shown in (B–D): (B) 91%, n = 42; (C) 65%, n = 60; (D) 58%, n = 52. (E–T) Four-cell stage embryos were injected in the dorsal or ventral marginal zone with the combinations of Rap2 MO (40 ng), Co MO (40 ng), and Rap2 DNA (200 pg) as indicated, cultured to stage 10.5 (E–P) or stage 30 (Q–T) and then subjected to whole-mount in situ hybridization against Chordin, Xbra, Wnt8, or MyoD genes. The percentages of affected embryos in (H–P) and (R–T): (H) 96%, n = 30; (I) 100%, n = 35; (J) 92%, n = 35; (K) 70%, n = 33; (L) 56%, n = 30; (M) 60%, n = 35; (N) 85%, n = 35; (O) 90%, n = 35; (P) 82%, n = 30; (R) 98%, n = 30 (S) 52%, n = 41 (T) 92%, n = 35. Developmental Cell 2008 15, 49-61DOI: (10.1016/j.devcel.2008.05.004) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Rap2 Is Required for Sorting of Activin/Nodal Receptors into a Recycling Pathway Rather Than a Degradative One (A) Four-cell stage embryos were injected in the animal pole with RNAs encoding Glu-Nodal (2 ng), ALK4(KR)-HA (2 ng), ActRIIB(KR)-Myc (2 ng), and Cripto-Flag (2 ng) with or without Rap2 MO (40 ng) or Co MO (40 ng). Kinase-dead ALK4(KR) and ActRIIB(KR) receptors were used to minimize receptor downregulation. Animal halves dissected at stage 9.5 were treated with DTSSP to covalently stabilize protein complexes at the plasma membrane and then lysates were immunoprecipitated for either ALK4(KR)-HA or ActRIIB(KR)-Myc with anti-HA and anti-Myc antibodies. Association of these proteins was examined by western blotting analysis. (B and C) Animal cap cells injected with the indicated combinations of ALK4(KR)-HA (2 ng), DN Rab11 (2 ng), Rap2 MO (40 ng), and Co MO (40 ng) were dissociated and cell surfaces were biotinylated. Up to this stage, cells were kept at 4°C to prevent receptor internalization. After biotinylation, cells were washed with MesNa buffer to remove biotin from noninternalized receptors (lane 2) or cultured at 23°C for the times shown (hr) in the absence (B) or presence (C) of the proteasomal inhibitor MG132 (MG, 10 μM) or the lysosomal inhibitors NH4Cl (NC, 50 mM) or chloroquine (CQ, 100 mM). Biotinylated proteins were precipitated with streptavidin and analyzed by western blotting using anti-HA antibody. Lane 1 in (B) and (C) indicates total biotinylated ALK4(KR)-HA at the cell surface. (Lanes 3–12 in [B] and [C]) After the indicated times of 23°C incubation, MesNa wash was carried out to remove biotin from noninternalized and recycled ALK4 receptors at the plasma membrane. (D) Animal cap cells injected with the combinations of ALK4(KR)-HA (2 ng), WT Rap2 (200 pg), DN Rab11 (2 ng), and β-catenin MO (10 ng) were dissociated, cell surfaces biotinylated, and cultured at 23°C for the indicated times (hr) in the absence (lanes 3–9) or presence (lane 10) of MG132. Developmental Cell 2008 15, 49-61DOI: (10.1016/j.devcel.2008.05.004) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Rap2 Antagonizes Smad7 (A) Animal cap cells injected with ALK4(KR)-HA (2 ng) in combination with Rap2 (200 pg), Smad7 (500 pg) RNA, and Smad7 MO (40 ng) as indicated were subjected to recycling assay. (B) Embryos injected dorsomarginally with Rap2 DNA (200 pg), Smad7 (500 pg), or DN Rab11 (2 ng) RNA alone or their combinations were harvested at the early gastrula stages for western blotting analysis using anti-phospho-Smad2 antibody. (C) Animal cap cells from uninjected and Smad7 MO (40 ng) or Rap2 RNA (200 pg)-injected embryos were dissociated and then incubated for 30 min in the presence of Activin protein (100 ng/ml, lanes 5–7) or not (lanes 1–4) prior to cell surface biotinylation. Biotinylated receptors were induced to be internalized and recycled to the cell surface by culturing at 23°C for the indicated times (hr). (D) Quantification of the signals shown in (C). The biotinylated ALK4 is normalized to ALK4 in total extract. The data are expressed as the mean ± the standard deviation of three independent experiments. (E) Animal cap tissues injected with the indicated combination of Smad7-Myc (500 pg), Smurf2-HA (600 pg), and Rap2 (200 pg) were dissected at the mid-blastula stage and cultured in the presence of Activin protein (100 ng/ml) until stage 10.5. Cell lysates were subjected to western blotting analysis as indicated. Developmental Cell 2008 15, 49-61DOI: (10.1016/j.devcel.2008.05.004) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Rap2 and Smad7 Compete for Binding to the Receptor (A) HEK293T cells were transfected with the combinations of Rap2-GFP, ALK4(KR)-HA, ALK4(TD)-HA, and ActRIIB-Myc as indicated. Cell lysates were immunoprecipitated for ALK4(KR)-HA or Rap2-GFP, followed by western blotting analysis to examine their coprecipitations. (B) Animal cap tissues excised at the mid-blastula stages from uninjected and Smad7 MO (40 ng)-injected embryos were incubated in the presence or absence of Activin protein (100 ng/ml) until the sibling embryos reached stage 10.5. (C) HEK293T cells were transfected with the indicated combinations of Rap2-GFP (3 μg), Smad7-Myc (3 μg), and ALK4(KR)-HA (3 μg). Cell lysates were immunoprecipitated with anti-GFP, anti-Myc, or anti-HA antibodies as indicated and then subjected to immunoblotting analysis to observe their associations. (D) Dorsal (lanes 1–3) and ventral (lanes 4–6) maginal tissues excised at stage 8.5 were incubated for 1 hr in the presence or absence of Activin protein (100 ng/ml). (B and D) Cell lysates were subjected to immunoprecipitation with anti-Rap2 or anti-ALK4 antibodies, followed by western blotting analysis. Developmental Cell 2008 15, 49-61DOI: (10.1016/j.devcel.2008.05.004) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 Rap2 Is Required for the Dynamic Activation of Smad2 across the Dorsoventral Axis of the Embryo (A) Dorsal and ventral marginal zone tissues (DMZ and VMZ) were dissected at stage 9 from uninjected (lanes 1 and 2) and ventrally Rap2 RNA (200 pg) - or dorsally DN Rab11 RNA (2 ng)-injected (lanes 3 and 4) embryos. Dissociated cells were cell surface biotinylated and then cultured at 23°C for 1 hr to induce receptor internalization and recycling to the cell surface. After MesNa wash, intracellular biotinylated proteins were precipitated with streptavidin beads, followed by western blotting analysis using anti-ALK4 antibody. (B) As in (A), DMZ and VMZ from uninjected (lanes 1 and 2) and injected embryos (lanes 3–7) were subjected to recycling assays. DN Rab11-injected DMZs were also incubated at 23°C in the presence of MG132 (MG), chloroquine (CQ), or NH4Cl (NC) as shown. (C) Quantification of the signals in (B). The biotinylated ALK4 is normalized to ALK4 in total extract. The data are expressed as the mean ± the standard deviation of three independent experiments. (D) Four-cell stage embryos were injected in the dorsal and ventral marginal zones with Glu-Nodal (2 ng), ALK4(KR)-HA (2 ng), ActRIIB(KR)-Myc (2 ng), and Cripto-Flag (2 ng) in combination with Rap2 DNA (200 pg) as indicated, and then dorsal and ventral halves were excised at stage 9 and subjected to cell surface chemical crosslinking with DTSSP. Cell lysates were precipitated for ALK4(KR)-HA or ActRIIB(KR)-Myc proteins and subsequently, coimmunoprecipitation of nodal ligand and its receptors was examined by western blotting analysis. (E) Embryos were injected and subjected to the following processes as described in (D) except that the dorsal and ventral tissues were dissected at stage 10.5. (F and G) Four-cell stage embryos were injected in the dorsal (F) or ventral (G) marginal regions with DN Rab11 (2 ng), Co MO (40 ng), Rap2 MO (40 ng), or mixture of Rap2 MO and Rap2 DNA (200 pg) or not injected as a control. The embryos were then harvested at the indicated stages for western blotting analysis using an anti-phospho-Smad2 antibody. Actin serves as a loading control. Developmental Cell 2008 15, 49-61DOI: (10.1016/j.devcel.2008.05.004) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 7 Early Dorsal Expression of Rap2 in the Mid-Blastulae Depends on β-Catenin Signaling, but Not Activin/Nodal Signaling (A) Spatiotemporal expression of Rap2 and Smad7 along the dorsoventral axis of Xenopus embryo. Dorsal and ventral marginal zone tissues (DMZ and VMZ) dissected from uninjected embryos at stage 8.5 and 10 were cultured or not until stages 10.5 and 12, and then subjected to western blotting analysis using an anti-Rap2 or -Smad7 antibody. (B) Rap2 can be induced ectopically by coexpression of VegT and β-catenin. Animal cap explants injected with VegT (500 pg), β-catenin (500 pg), or both were dissected at the late blastula stages, cultured to stage 10 and subjected to RT-PCR. Co AC, uninjected control animal caps. (C and D) Ventral (VMZ) and dorsal marginal zone (DMZ) tissues were excised at stage 8.5 and 10 from uninjected or DN ALK4 (1 ng), FAST-EnR (800 pg), or β-catenin MO (30 ng)-injected embryos and subsequently subjected to western blotting analysis. Developmental Cell 2008 15, 49-61DOI: (10.1016/j.devcel.2008.05.004) Copyright © 2008 Elsevier Inc. Terms and Conditions