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Figure 1. Pnhd is a secreted protein that promotes posterior development. (A) Alignment of CK domains from Pnhd and other secreted proteins. Spacing is indicated by numbers of non-conserved amino acids between conserved cysteine residues. X.l., Xenopus laevis; S.p., Stegastes partitus; H.s., Homo sapiens. (B) Secretion of Pnhd by Xenopus gastrula cells. Four-cell embryos were injected with 0.5 ng of Flag-Pnhd RNA for each blastomere, cultured to the onset of gastrulation and dissociated to individual cells. Pnhd levels were compared in the media conditioned for 3 h and the corresponding cell lysates. (C) Pnhd is secreted by transfected HEK293Tcells. Deletion of the putative signal peptide in PnhdSP prevents secretion. (D) Head defects in embryos injected dorsally with 2 ng of pnhd RNA at the four-cell stage. Frequencies of embryos with head defects and their total number are indicated. The results are representative of more than five independent experiments. A, anterior; cg, cement gland; CM, conditioned medium; IB, immunoblot; IP, immunoprecipitation; P, posterior.
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Figure 2. Pnhd transcript localization at different developmental stages. WISH was carried out with albino embryos using pnhd antisense and sense RNA probes. (A) Vegetal view of a stage 10 embryo (dorsal is up). Arrowhead points to vegetal endoderm. (B) Cross-section of a stage 10 embryo. (C) Control embryo, stage 10 (sense probe). (D) Vegetal view of a stage 11.5 embryo (dorsal is up). (E) Vegetal view of a stage 12 embryo (dorsal side is up). Arrows in A,D,E indicate mesodermal expression. (F) Dorsal view of a stage 14 embryo (anterior is up). Arrowhead marks the neuroectoderm. (G) Cross-section of the embryo shown in F. (H) Anterior view of a stage 19 embryo (dorsal is up). (I) Posterior view of a stage 19 embryo (dorsal is up). Arrow indicates staining in the tailbud. (J) Side view of a stage 25 embryo. (K) Head of a stage 25 embryo (anterior is left). Arrowhead points to the signal in the superficial ectoderm cells that are dorsal to the cement gland. (L) Cross-section corresponding to the midbrain level of embryo in J. (M) Dorsal view of a stage 25 embryo. (N) Stage 25, control sense probe. (O) Lateral view of a stage 25 embryo tailbud. Anterior is to the left in J,K,M-O. (P) Transverse section of a stage 25 embryo corresponding to J and O. Dashed lines mark the approximate level of corresponding sections (indicated by bold letters). Dorsoventral (D/V) and anteroposterior (A/P) embryonic axes are indicated. Abbreviations: bc, blastocoel; bv, brain ventricle; cg, cement gland; df, dorsal fin; dl, dorsal blastopore lip; en, endoderm; ev, eye vesicle; lm, lateral mesoderm; me, mesoderm; mhb, midbrain-hindbrain boundary; ne, neuroectoderm; nt, neural tube; ntb, neural tube border; no, notochord; ov, otic vesicle; s, somites; sm, somitic mesoderm; tb, tailbud. Scale bars: 50 µm (B,G,L); 25 µm (P).
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Figure 3. Pnhd induces mesoderm in ectodermal explants. (A-E) Early embryos were injected with 1-2 ng of Pnhd RNA. Ectoderm explants were dissected at late blastula stages and cultured until stage 12 to examine morphology (B,C) and gene expression by RT-PCR (D,E). (B,C) Pnhd RNA induced animal cap elongation by stage 12. Frequencies of elongated explants and their total number are indicated. The results represent more than five independent experiments. (D) Induction of selected mesodermal markers by Flag-Pnhd RNA (1 ng). (E) Flag-Pnhd RNA has the same ability to induce tbxt as untagged Pnhd RNA in RT-qPCR, but this activity is lost in Flag-PnhdSP lacking the signal peptide (2 ng of each RNA). (F) Pnhd protein was purified from the supernatants of transfected HEK293T cells. To assess its mesoderm-inducing activity, ectoderm explants were dissected from stage 10 embryos and cultured in 0.6×MMR solution containing 1.5 µg/ml or 6.5 µg/ml of Pnhd. RT-qPCR was carried out for tbxt at stage 11 or stage 14. Data are mean±s.d. Significance was determined by an unpaired two-tailed Student's t-test. **P<0.01, ***P<0.001.
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Figure 4. RNA sequencing defines Pnhd target genes. (A) Heatmap of gene expression in the Pnhd-expressing and control uninjected animal pole cells that were cultured until stages 11 and 12. The duplicate samples are highly similar. (B) Volcano plot shows top genes upregulated by Pnhd. (C) Differentially expressed genes that are induced by Pnhd RNA (1.5 ng) in animal caps. The list was derived from the top 100 genes induced by Pnhd in four independent RNA-seq experiments. (D) qRT-PCR validation of Pnhd targets in animal caps. Data are mean±s.d. Significance was determined by an unpaired two-tailed Student's t-test. **P<0.01, ***P<0.001, ****P<0.0001.
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Figure 5. Pnhd is required for mesoderm formation. (A-D) WISH validates changes in cdx4 expression in embryos with manipulated Pnhd levels in one half of each embryo. Red-gal was used as a β-galactosidase substrate (red) for lineage tracing of the injected area. Compare gene expression (dark staining) between the injected (red) and uninjected sides. (E) Quantification of changes in cdx4 RNA in Pnhd-depleted or overexpressing embryos. (F) RT-qPCR confirmation of the downregulation of cdx4, hoxd1, msgn1 and tbxt in stage 10.5 marginal zone explants depleted of pnhd. Data are mean±s.d. Significance was determined by an unpaired two-tailed Student's t-test. *P<0.05, **P<0.01.
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Figure 6. Pnhd response requires FGF but not Wnt signaling. (A-C) Embryos were injected in the animal pole region at the two-cell stage with 1-2 ng of Pnhd RNA, FGFR1-Fc, FGFR4-Fc or dnFGFR1 RNA (2 ng each), 1 ng of Wnt8 or 300 pg of Dkk1 RNA, as indicated. Ectoderm explants were dissected at stages 9-10 and cultured until stages 11-11.5 for gene expression analysis by RT-qPCR. (A) The induction of tbxt and cdx4 by Pnhd is blocked by the FGF inhibitor SU5402 (100 µm). Stimulation with bFGF was used as a positive control. (B) Gene target activation by Pnhd was prevented by DN-FGFR1 and secreted forms of FGFR1-Fc and FGFR4-Fc. (C) The Wnt inhibitor Dkk1 did not affect Pnhd signaling but effectively blocked Wnt8 responses. Data are mean±s.d. Significance was determined by an unpaired two-tailed Student's t-test. *P>0.05, **P>0.01, ***P>0.001, ****>0.0001.
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Figure 7. Pnhd inhibits Akt, but activates Erk1. (A) Schematic of the experiments shown in B-D. Embryos were injected with RNAs encoding Flag-Pnhd (1.5 ng), p110CAAX (0.5 ng), or treated with FGF protein, as indicated. Ectoderm explants were dissected at stages 8, 9.5 or 10, and cultured until the desired stage for immunoblotting with indicated antibodies. (B) Comparison of Pnhd and FGF effects on blastula ectoderm. Pnhd inhibits Akt phosphorylation in ectoderm explants isolated at stage 8 and analyzed at stage 10. FGF has no effect on Akt, but activates Erk. (C) Pnhd inhibits Akt but induces Erk phosphorylation in ectoderm isolated at stage 9.5 and cultured until stage 10.25. This result has been obtained in at least ten experiments. The separator line serves to indicate that several irrelevant gel lanes have been omitted. (D) Pnhd-dependent stimulation of Erk is not affected by the Akt activator p110CAAX. (E) Erk1 phosphorylation in Pnhd-expressing embryos at stage 11. Embryos were injected with RNAs encoding Flag-Pnhd or Flag-PnhdSP (1.5 ng each). (F) Downregulation of Erk1 phosphorylation in lysates of stage 11 embryos injected with Pnhd MOsp. Embryos were injected with RNAs encoding dnFGFR1 (1.5 ng each) or 40 ng of Pnhd MOsp, as indicated. There are no detectable changes in β-catenin or phospho-Smad1. (G) Pnhd promotes Smad2 phosphorylation by Activin. Ectoderm explants were dissected from the injected embryos at stage 8 and cultured for 1 h with or without Activin. Immunoblot analysis with anti-pSmad2 antibodies is shown. Pnhd is detected by anti-Flag antibodies (arrowhead). Erk1 is a control for loading. WE, whole embryo controls in C,D,F.
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Figure 8. Pnhd cooperates with endogenous-inducing signals to promote mesoderm formation during gastrulation. (A) Schematic of the experiments presented in B,C. Mesoderm-inducing signals are indicated by orange arrows, and the cells receiving them are in light brown. Each animal blastomere of two-cell embryos received 1 ng of Pnhd RNA. Pnhd-expressing or control ectoderm explants were isolated at stage 8 or stage 10, as indicated. When the control embryos reached stage 12.5, the explants were lysed for immunoblotting with antibodies specific for pErk1 and Erk (B) and for RT-qPCR analysis of cdx4 and tbxt transcripts (C). (B) Erk is synergistically activated by Pnhd and endogenous signals in ectoderm explants isolated at stage 10. (C) Cooperative activation of mesodermal gene targets by Pnhd and endogenous signals in ectoderm dissected at stage 10. (D,E) Pnhd is required for mesoderm formation in response to endogenous-inducing signals. (D) Schematic of the experiment shown in E. Model for Pnhd function (right). Pnhd is activated in the marginal zone by Nodal and FGF signaling and functions within the newly induced mesodermal layer. D, dorsal mesoderm; V, ventral mesoderm. The green color indicates presumptive endoderm in the vegetal pole, the gray color indicates presumptive ectoderm at the animal pole, and the future mesoderm is represented by brown shading. (E) Pnhd is required for mesoderm formation in animal-vegetal conjugates. Pnhd MOsp-injected or control animal pole explants were combined with vegetal explants at stage 8. After culture until stage 11, levels of cdx4 and hoxd1 transcripts were determined in the conjugates by RT-qPCR. Data are mean±s.d. Significance was determined by an unpaired two-tailed Student's t-test. **P<0.01.
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Figure S1. Alignment of Pnhd homologs. Multiple protein sequences that are similar to Pnhd from different species have been aligned using ClustalOmega from EBI. Percent identity to Xenopus laevis Pnhd.L protein is shown for Pnhd homologs from Danio rerio, Pelecanus crispus, Apis mellifera, and Daphnia magna. All three cystine knot domains marked by characteristic Cys residues appear conserved in different Pnhd proteins (asterisks).
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Figure S2. Pnhd secretion in cultured cells. A, HEK293T cells were transfected with Pnhd-Flag-pCS2 DNA or control pCS2 DNA and cultured for 48 hrs. Proteins from cell lysates or corresponding conditioned media were separated by SDS- PAGE and immunoblotted with anti-Flag antibodies. b-catenin is a control for protein loading.
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Figure S3. Validation of pnhd knockdown. A, Translation-blocking MO inhibits pnhd-HA RNA translation in vivo. Embryos were coinjected with pnhd-HA-RNA and HA-vangl2 RNA (negative control) in the presence of a control (Co) or pnhd MOatg (10 ng each) as indicated. Immunoblotting with anti-HA antibodies shows specific inhibitory effect of pnhd MOatg on Pnhd protein levels. B, pnhd MOsp (40- 60 ng) interferes with endogenous Pnhd RNA splicing in morphants. C-F, Morphological phenotypes of embryos depleted of Pnhd (C-E). F, pnhd RNA (10 pg) rescues the morphant phenotype (F). G. Quantification of data presented in (C-F)
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Figure S4. Regulation of wnt8a transcription by Pnhd. A-D, Whole mount in situ hybridization validates changes in wnt8a expression in embryos with manipulated pnhd. Injection experiments were performed as described in Fig. 5B- F. Wnt8 transcripts are visible as dark blue staining. RedGal is a lineage tracer. Representative embryos are shown. E, quantification of the results shown in A-D. F, RT-qPCR confirms downregulation of wnt8a.L, tbxt.s, msgn1.L and hoxd1.s in stage 10.5 marginal zone explants depleted of Pnhd. By contrast, the ventral marker admp2 is not affected by Pnhd depletion.
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igure S5. Regulation of mesodermal gene expression by Pnhd .
For pnhd depletion, each blastomere of four-cell embryos has been injected with 40 ng of pnhd MOsp. The uninjected controls (A, C, E) or pnhd morphants (B, D, F) have been cultured until the indicated stages, fixed and processed for wholemount in situ hybridization with anti-sense probes that are specific for myod (A, B), a-globin (C, D) and chordin (E, F). Number of embryos with the presented phenotype (top) and the total number of embryos per group (bottom) are shown.
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Figure S6. Pnhd transcripts are induced in ectoderm by FGF and Wnt signals. RT-qPCR data from animal caps treated with FGF or expressing Wnt3a.
Ectoderm explants were dissected at stage 9-10 and cultured until stage 11. A, B, The induction of pnhd and cdx4 by FGF. C, Pnhd transcription is activated by
Wnt3a and this effect is blocked by Dkk1. Means +/- standard errors are shown. Significance was determined by the two-tailed Student’s t-test, p<0.01 (**)
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Figure S7. Secreted dominant-interfering form of FGFR4 rescues head deficiency caused by Pnhd. Four-cell embryos have been injected with indicated RNAs, morphological phenotypes were scored at stage 39. A, Uninjected control embryos. B. Lack of head structures (*) in embryos injected with pnhd RNA (0.9 ng). C. Mild posterior defects in embryos injected with FGFR4-Fc RNA (20 pg). D. Coinjection of FGFR4-Fc RNA rescued the headless phenotype. Arrows and arrowheads in A and D point to eyes (e) and cement glands (CG), respectively. E. Quantification of the results. The data are representative of two different experiments.
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Figure S8. Akt inhibition by Pten does not influence Pnhd signaling.
Embryos were injected with RNAs encoding Flag-Pnhd (1.5 ng) or Pten (0.5 ng), as indicated. Ectoderm explants were dissected at stage 9.5, and cultured until stage 10.25 for immunoblotting with indicated antibodies. Pnhd inhibits Akt, but induces Erk phosphorylation in ectoderm at late blastula stages. When coinjected with Pnhd, Pten further inhibits Akt but does not alter Pnhd-dependent Erk activation.
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Figure S9. Pnhd activity is blocked by the Nodal/Activin inhibitor SB505124.
Animal caps were dissected at stage 9-10 and stimulated with Activin or obtained from embryos injected with pnhd RNA. RT-qPCR analysis of tbxt.S transcripts was carried out in animal caps incubated in the presence or absence of SB505124. A,
Effect of SB505124 on Activin- or Pnhd-dependent mesoderm induction. Frequencies of the shown morphological changes and the total number of explants
per group are indicated. Data are representative of three independent experiments. B, RT-qPCR analysis of tbxt.S transcripts in animal caps treated with Activin in the presence or absence of SB505124. C, Pnhd-dependent induction of tbxt.S in the presence or absence of SB505124. Means +/- standard errors are shown. Significance was determined by the two-tailed Student’s t-test, p<0.01 (**).
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Figure S10. Pnhd-dependent elongation of animal caps isolated at the onset of gastrulation. Animal cap explants were prepared at stage 8 (A, B) or stage 10 (C, D) from the control uninjected (A, C) or pnhd RNA (2 ng)-injected (B, D) embryos and were cultured until stage 12.5, at which point explant morphology was imaged. Frequencies of explant elongation and total numbers of explants per group are shown. Data are representative of three independent experiments.
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Figure S11. Pnhd is required for FGF-dependent mesoderm induction.
A, B, RT-qPCR was carried out for cdx4 and tbxt in ectoderm lysates from Pnhd- depleted or control embryos after FGF stimulation as indicated. Embryos were injected into each of four animal blastomeres with Pnhd MOsp (30 ng) at the 8-cell stage. Ectoderm explants were dissected at stage 8-8.5 and cultured until stage 11. Means +/- standard errors are shown.
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