Itoh K, Ossipova O, Sokol SY.

R01 HD092990 NICHD NIH HHS , R01 GM116657 NIGMS NIH HHS , R24 OD021485 NIH HHS

             phosphorylation
             notochord development
             SMAD binding

	Graphical Abstract
	Figure 1. Screening gastrula secretome for Pnhd-associated proteins (A) Experimental scheme. Embryos were injected four times animally with FLAG-Pnhd RNA (500 pg) and dissociated at stage 10. After 3 h, conditioned media (CM) from dissociated FLAG-Pnhd-expressing and control embryos were combined and immunoprecipitated with anti-FLAG beads. (B) Coomassie blue staining of FLAG-Pnhd-containing and control protein pulldowns. Two bands of 36–38 kDa correspond to FLAG-Pnhd (arrow). (C) Immunoblot analysis of immunoprecipitates, CM, and cell lysates with anti-FLAG antibody. Heavy and light antibody chains are visible in addition to the specific Pnhd bands (arrowhead). Anti-Erk1 antibody serves as loading control for the lysates. (D) Numbers of identified peptides for top candidate secreted Pnhd-interacting proteins that were identified by mass spectrometry. (E) CM were combined from embryos expressing HA-Pnhd and FLAG-Admp as described in (A) and precipitated with anti-FLAG antibody. Supernatant (S) or cell pellet (P) fractions from dissociated cell lysates are also shown. Anti-HA antibody recognizes HA-Pnhd as 37- to 39-kDa bands, whereas anti-FLAG antibody detects the unprocessed form of FLAG-Admp (45 kDa) and mature FLAG-Admp (17 kDa, arrows). Anti-Erk1 antibody validates the separation of cytoplasmic and secreted proteins. (F) Admp binds endogenous Pnhd. CM from dissociated normal embryos (stage 10) or sibling embryos expressing FLAG-Pnhd were immunoprecipitated with anti-FLAG or anti-Pnhd (5F9) antibodies. After gel separation of protein precipitates, immunoblotting was carried out with the indicated antibodies.
	Figure 2. Pnhd inhibits Admp ventralizing activity (A) Experimental scheme. Marginal zone of four-cell embryos was injected with admp or flag-bmp4 RNA (50 pg) and flag-pnhd RNA (150 pg) as indicated. (B–G) Typical embryo phenotypes at stage 20 are shown. Arrows (B, D) point to neural tubes, asterisk in (C) marks a ventralized embryo. Scale bar: 400 μm in (G). (H) Quantification of the experiments shown in (B–G). Ventralization activity in stage 20 embryos has been scored as indicated at the bottom of the panel. Numbers of scored embryos are shown above each bar. Data are representative of three independent experiments. (I and J) Pnhd inhibits Smad1 phosphorylation in response to Admp but not BMP4. Embryos were injected as shown in (A) and harvested at stage 11 for immunoblotting with anti-phospho-Smad1 (pSmad1) and anti-Smad1 antibodies. Normalized ratios of pSmad1 to Smad1 levels are shown. Independent biological replicas are included for each group. Erk1, and β-catenin (βCat) levels control loading.
	Figure 4. Admp activity is enhanced by Pnhd depletion (A) Experimental scheme. Marginal zone of four-cell embryos was injected with admp RNA (50 pg) or PnhdMO2sp (40 ng) as indicated. (B–F) Pnhd MO2sp enhanced ventralization caused by Admp. Representative embryos are shown. (F) Quantification of the data shown in (B–E). Phenotypic scoring was done when uninjected embryos reached stage 28 using dorsoanterior index (DAI). Bar in E, 200 µm. (G and H) Immunoblotting of lysates of stage 11 embryos with anti-pSmad1, anti-Smad1, and anti-Erk1 antibodies. (G), control MO; (H), PnhdMO2sp. Normalized ratio of band intensities of pSmad1 relative to total Smad1 is indicated. Erk1 serves as a loading control. Independent biological replicas are included for each group (G, H). Data represent two to three independent experiments. (I) The effect of Admp on marginal zone markers is enhanced in Pnhd morphants. RNA was extracted from injected embryos at stage 11.5. RT-qPCR analysis of ventral (szl and bambi) or dorsal (chrd1 and dkk1) markers is shown. These data are representative of three experiments. Means ± SD are shown. Significance was determined by the Student's t test, ∗∗p < 0.01, ∗∗∗p < 0.001.
	Figure 5. Pnhd cooperates with BMP inhibitors to trigger explant elongation (A) Two-cell-stage embryos were injected with pnhd (0.5 ng), chordin (0.15 ng), or truncated BMP receptor (tBR, 0.45 ng) RNA as indicated. Ectoderm explants were dissected at stages 9.5–10 and cultured until stage 14 for morphological examination. (B–G) Representative group morphology is shown. Arrows in (D and F) point to elongating explants. These experiments were repeated 3–7 times. Scale bar: 500 μm in (G).
	Figure 6. Pnhd synergizes with BMP inhibitors to induce notochord markers (A) Experimental scheme. Two-cell embryos were injected into animal pole region with pnhd (0.5 ng) RNA, chordin (0.15 ng), or truncated BMP receptor (tBR, 0.45 ng) RNA as indicated in (B, C). Ectoderm explants were dissected at stages 9.5–10 and cultured until stage 14 to examine notochord markers admp, shh, and emi3 by RT-qPCR. (B) Pnhd synergizes with Chordin to induce admp.S, shh.L, and emi3, but not sox2, at stage 14. (C) Pnhd synergizes with tBR to induce admp.S and shh.L, but not sox2. (D) Chordin inhibits the induction of cdx4.L by pnhd. These data are representative of three experiments. Means ± SD are shown. Significance was determined by the Student's t test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
	Figure 7. Pnhd is required for notochord specification (A–L) PnhdMOsp or PnhdMO2sp (40 ng each) were injected four times into marginal zone as shown in Figure 2A. When control embryos reached stage 14, injected or uninjected embryos were fixed for whole-mount in situ hybridization with the notochord-specific probes chrd.1 (A–D) and not (E–H) or the somitic marker myod1 (I–L). Dorsal view is shown, anterior is up. Arrows in (A, E, I) demarcate the width of chrd.1 or not expression domains or the gap in myod1 expression. (D, H, L) Quantification of notochord width (marked by chrd.1, not, or the gap in myod1) relative to embryo width. Means ± SD are shown. Number of scored embryos per group is shown above each bar. The data are representative of three independent experiments. Scale bar: 300 μm in (K).
	Figure 8. A model of Pnhd effects on dorsal mesoderm, Admp, and Smad1 activity Major activators of Smad1 phosphorylation in the Xenopus gastrula are ventrolaterally expressed Bmp4/7 and dorsal Admp. Pnhd transcripts are distributed in the ventrolateral marginal zone, complementary to the dorsal expression of admp. Dorsal organizer signals, such as Chordin, reduce Smad1 phosphorylation triggered by Bmp4/7. A similar antagonistic effect of Pnhd on Admp is proposed to further modulate Smad1 activity gradients.
	Suppl. Fig. 1. Both exogenous and endogenous Pnhd are recognized by specific antibodies, related to Figure 1. A, Embryos were injected with flag-pnhd RNA (75 pg) and the lysates of the injected and control uninjected embryos at stage 11.5-12 were immunoblotted with 5F9 antibodies. There is no visible signal in control embryos, but a strong 35-37 kDa band is detected in pnhd-expressing embryos. B, Lysates were prepared from normal embryos at stage 12, immunoprecipitated with the Pnhd-specific antibodies 5F9, 8G12,11D5 or anti-Myc as indicated and immunoblotted with 5F9.
	https://www.dropbox.com/s/1kqy0c287omzh15/Screen%20Shot%202021-06-28%20at%202.00.40%20PM.jpg?dl=0
	https://www.dropbox.com/s/534ejnyqd260wzt/Screen%20Shot%202021-06-28%20at%202.01.05%20PM.jpg?dl=0
	Suppl. Fig. 4. Validation of Pnhd knockdown by immunoprecipitation, related to Figure 4. Four-cell embryos were injected in the marginal zone with PnhdMO2sp or control MO (40 ng each) as indicated. Sixty injected embryos were dissociated at stage 10 and cultured for 3 hrs. Conditioned media were immunoprecipitated with anti-Pnhd (5F9) or anti-HA antibodies and immunoblotted with 5F9. Pnhd-specific band has been eliminated in embryos injected with PnhdMO2sp.
	Suppl. Fig. 5. Enhancement of Admp ventralizing activity by PnhdMOsp, related to Figure 4. A, Experimental scheme. Marginal zone of four-cell embryos was injected with admp RNA (50 pg) or Pnhd MOsp (60 ng) as indicated. B-E, PnhdMOsp enhanced ventralization caused by Admp. Representative embryos are shown. F. Quantification of the data shown in B-E. Phenotypes were scored at stage 28 using dorsoanterior index (DAI). G, H, Immunoblotting of lysates of stage 11 embryos with anti-pSmad1, anti- Smad1 and anti-Erk1 antibodies. Normalized ratio of band intensities of pSmad1 relative to total Smad1 is indicated. Both PnhdMOsp (60 ng) and PnhdMOatg (10 ng) enhanced pSmad1 levels by Admp. Erk1 serves as a loading control. Data represent two to three independent experiments.
	Suppl. Fig. 6. Pnhd promotes elongation of ectodermal explants in the presence of Chordin, related to Figure 5. A-D. Two cell embryos were injected with 0.5 ng of pnhd and/or 0.15 ng of chordin RNA as indicated. Ectodermal explants were dissected at stage 9.5-10 as shown in Fig. 5A. The explants were cultured until stage 11. Scale bar in D is 500 μm. E. Embryos were injected as described in A-D. Ectoderm explants were dissected at stages 9.5-10 and cultured until stage 11 to examine admp and shh by RT-qPCR. Data are representative of three experiments. Means +/- standard errors are shown. Significance was determined by the Student’s t-test, p<0.05 (*), p<0.01 (**).
	Figure 1. Screening gastrula secretome for Pnhd-associated proteins(A) Experimental scheme. Embryos were injected four times animally with FLAG-Pnhd RNA (500 pg) and dissociated at stage 10. After 3 h, conditioned media (CM) from dissociated FLAG-Pnhd-expressing and control embryos were combined and immunoprecipitated with anti-FLAG beads.(B) Coomassie blue staining of FLAG-Pnhd-containing and control protein pulldowns. Two bands of 36–38 kDa correspond to FLAG-Pnhd (arrow).(C) Immunoblot analysis of immunoprecipitates, CM, and cell lysates with anti-FLAG antibody. Heavy and light antibody chains are visible in addition to the specific Pnhd bands (arrowhead). Anti-Erk1 antibody serves as loading control for the lysates.(D) Numbers of identified peptides for top candidate secreted Pnhd-interacting proteins that were identified by mass spectrometry.(E) CM were combined from embryos expressing HA-Pnhd and FLAG-Admp as described in (A) and precipitated with anti-FLAG antibody. Supernatant (S) or cell pellet (P) fractions from dissociated cell lysates are also shown. Anti-HA antibody recognizes HA-Pnhd as 37- to 39-kDa bands, whereas anti-FLAG antibody detects the unprocessed form of FLAG-Admp (45 kDa) and mature FLAG-Admp (17 kDa, arrows). Anti-Erk1 antibody validates the separation of cytoplasmic and secreted proteins.(F) Admp binds endogenous Pnhd. CM from dissociated normal embryos (stage 10) or sibling embryos expressing FLAG-Pnhd were immunoprecipitated with anti-FLAG or anti-Pnhd (5F9) antibodies. After gel separation of protein precipitates, immunoblotting was carried out with the indicated antibodies.
	Figure 2. Pnhd inhibits Admp ventralizing activity(A) Experimental scheme. Marginal zone of four-cell embryos was injected with admp or flag-bmp4 RNA (50 pg) and flag-pnhd RNA (150 pg) as indicated.(B–G) Typical embryo phenotypes at stage 20 are shown. Arrows (B, D) point to neural tubes, asterisk in (C) marks a ventralized embryo. Scale bar: 400 μm in (G).(H) Quantification of the experiments shown in (B–G). Ventralization activity in stage 20 embryos has been scored as indicated at the bottom of the panel. Numbers of scored embryos are shown above each bar. Data are representative of three independent experiments.(I and J) Pnhd inhibits Smad1 phosphorylation in response to Admp but not BMP4. Embryos were injected as shown in (A) and harvested at stage 11 for immunoblotting with anti-phospho-Smad1 (pSmad1) and anti-Smad1 antibodies. Normalized ratios of pSmad1 to Smad1 levels are shown. Independent biological replicas are included for each group. Erk1, and β-catenin (βCat) levels control loading.
	Figure 3. Pnhd regulates Admp-dependent dorsoventral markers(A–F) Embryos were injected as described in Figure 2A. RNA was extracted from injected embryos at stage 11.5. RT-qPCR analysis of ventral (vent1, vent2, szl, and bambi, A–D) or dorsal (chrd1 and dkk1, E, F) markers is shown. These data are representative of four experiments. Means ± SD are shown. Significance was determined by the Student's t test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, p > 0.05, non-significant (N.S.).
	Figure 4. Admp activity is enhanced by Pnhd depletion(A) Experimental scheme. Marginal zone of four-cell embryos was injected with admp RNA (50 pg) or PnhdMO2sp (40 ng) as indicated.(B–F) Pnhd MO2sp enhanced ventralization caused by Admp. Representative embryos are shown. (F) Quantification of the data shown in (B–E). Phenotypic scoring was done when uninjected embryos reached stage 28 using dorsoanterior index (DAI). Bar in E, 200 µm.(G and H) Immunoblotting of lysates of stage 11 embryos with anti-pSmad1, anti-Smad1, and anti-Erk1 antibodies. (G), control MO; (H), PnhdMO2sp. Normalized ratio of band intensities of pSmad1 relative to total Smad1 is indicated. Erk1 serves as a loading control. Independent biological replicas are included for each group (G, H). Data represent two to three independent experiments.(I) The effect of Admp on marginal zone markers is enhanced in Pnhd morphants. RNA was extracted from injected embryos at stage 11.5. RT-qPCR analysis of ventral (szl and bambi) or dorsal (chrd1 and dkk1) markers is shown. These data are representative of three experiments. Means ± SD are shown. Significance was determined by the Student's t test, ∗∗p < 0.01, ∗∗∗p < 0.001.
	Figure 5. Pnhd cooperates with BMP inhibitors to trigger explant elongation(A) Two-cell-stage embryos were injected with pnhd (0.5 ng), chordin (0.15 ng), or truncated BMP receptor (tBR, 0.45 ng) RNA as indicated. Ectoderm explants were dissected at stages 9.5–10 and cultured until stage 14 for morphological examination.(B–G) Representative group morphology is shown. Arrows in (D and F) point to elongating explants. These experiments were repeated 3–7 times. Scale bar: 500 μm in (G).
	Figure 6. Pnhd synergizes with BMP inhibitors to induce notochord markers(A) Experimental scheme. Two-cell embryos were injected into animal pole region with pnhd (0.5 ng) RNA, chordin (0.15 ng), or truncated BMP receptor (tBR, 0.45 ng) RNA as indicated in (B, C). Ectoderm explants were dissected at stages 9.5–10 and cultured until stage 14 to examine notochord markers admp, shh, and emi3 by RT-qPCR.(B) Pnhd synergizes with Chordin to induce admp.S, shh.L, and emi3, but not sox2, at stage 14.(C) Pnhd synergizes with tBR to induce admp.S and shh.L, but not sox2.(D) Chordin inhibits the induction of cdx4.L by pnhd. These data are representative of three experiments. Means ± SD are shown. Significance was determined by the Student's t test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
	Figure 7. Pnhd is required for notochord specification(A–L) PnhdMOsp or PnhdMO2sp (40 ng each) were injected four times into marginal zone as shown in Figure 2A. When control embryos reached stage 14, injected or uninjected embryos were fixed for whole-mount in situ hybridization with the notochord-specific probes chrd.1 (A–D) and not (E–H) or the somitic marker myod1 (I–L). Dorsal view is shown, anterior is up. Arrows in (A, E, I) demarcate the width of chrd.1 or not expression domains or the gap in myod1 expression. (D, H, L) Quantification of notochord width (marked by chrd.1, not, or the gap in myod1) relative to embryo width. Means ± SD are shown. Number of scored embryos per group is shown above each bar. The data are representative of three independent experiments. Scale bar: 300 μm in (K).
	Figure 8. A model of Pnhd effects on dorsal mesoderm, Admp, and Smad1 activityMajor activators of Smad1 phosphorylation in the Xenopus gastrula are ventrolaterally expressed Bmp4/7 and dorsal Admp. Pnhd transcripts are distributed in the ventrolateral marginal zone, complementary to the dorsal expression of admp. Dorsal organizer signals, such as Chordin, reduce Smad1 phosphorylation triggered by Bmp4/7. A similar antagonistic effect of Pnhd on Admp is proposed to further modulate Smad1 activity gradients.

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