Rankin SA , Zorn AM .

R01 DK070858 NIDDK NIH HHS

Xla Wt + fn1 MO + sdc4 MO (Fig S6 D)
Xla Wt + fn1 MO + sdc4 MO (Fig S6 I)
Xla Wt + fn1 MO + sdc4 MO (Fig S6 K)
Xla Wt + fzd7 MO (Fig 2 J)
Xla Wt + fzd7 MO (Fig S6 C)
Xla Wt + fzd7 MO + sdc4 MO (Fig 2 K)
Xla Wt + fzd7 MO + sdc4 MO (Fig 2 M)
Xla Wt + fzd7 MO + sdc4 MO (Fig 3 E J)
Xla Wt + fzd7 MO + sdc4 MO (Fig 3 F J)
Xla Wt + fzd7 MO + sdc4 MO (Fig 3 H J)
Xla Wt + fzd7 MO + sdc4 MO (Fig S2 A column 5)
Xla Wt + fzd7 MO + sdc4 MO (Fig S2 A column 5)
Xla Wt + fzd7 MO + sdc4 MO (Fig S2 A column 5)
Xla Wt + fzd7 MO + sdc4 MO (Fig S2 A column 6)
Xla Wt + fzd7 MO + sdc4 MO (Fig S2 A column 6)
Xla Wt + fzd7 MO + sdc4 MO (Fig S2 A column 6)
Xla Wt + fzd7 MO + sdc4 MO (Fig S2 A column 6)
Xla Wt + PD173074 (Fig S5 A3)
Xla Wt + sdc4 MO (Fig 1. F.)
Xla Wt + sdc4 MO (Fig 1. I)
Xla Wt + sdc4 MO (Fig 1. L)
Xla Wt + sdc4 MO (Fig 1. O)
Xla Wt + sdc4 MO (Fig 2 B)
Xla Wt + sdc4 MO (Fig 2 F)
Xla Wt + sdc4 MO (Fig 2 G)
Xla Wt + sdc4 MO (Fig.2.M)
Xla Wt + sdc4 MO (Fig 3 B J)
Xla Wt + sdc4 MO (Fig 3 C J M)
Xla Wt + sdc4 MO (Fig 3 I)
Xla Wt + sdc4 MO (Fig 4 B)
Xla Wt + sdc4 MO (Fig 7 B)
Xla Wt + sdc4 MO (Fig 7 J)
Xla Wt + sdc4 MO (Fig 7 N)
Xla Wt + sdc4 MO (Fig S2 A column 3)
Xla Wt + sdc4 MO (Fig S2 A column 4)
Xla Wt + sdc4 MO (Fig S2 A column 4)
Xla Wt + sdc4 MO (Fig S2 A column 4)
Xla Wt + sdc4 MO (Fig S6 B)
Xla Wt + sdc4 MO (Fig S6 G)
Xla Wt + sdc4 MO (Fig S6 L)
Xla Wt + sdc4 MO + szl MO (Fig S6 E)
Xla Wt + sdc4 MO + szl MO (Fig S6 J)
Xla Wt + sdc4 MO + szl MO (Fig S6 O)
Xla Wt + su5402 (Fig S5 A2)

	Fig. 1. Sdc4 is required for foregut organogenesis (A) Schematic of a NF19 stage Xenopus embryo in mid-sagittal section with the foregut (fg) endoderm shown in dark grey and mid/hindgut endoderm in light grey. (B) In situ hybridization showing sdc4 expression at stage NF19. (C) Confocal image showing Alexa lineage tracer (red) in a bisected stage NF19 embryo confirming the targeted microinjection of the foregut endoderm. (D) Western blot analysis of NF19 dissected foreguts confirming Sdc4 depletion by Sdc4-MO injection. (E–G) Lateral view of stage 45 embryos showing foregut hypoplasia in Sdc4-depleted embryos that is rescued by sdc4 mRNA injection. (H–J) In situ hybridization of a2m in isolated stage 45 gut tube showing hypoplastic liver (lv), stomach (st) and pancreas (p). (K–P) In situ hybridization with liver (nr1h5) and pancreas/duodenum (pdx1) markers at stage NF35 showing defects in organ specification, which are rescued by sdc4 RNA injection. In all panels, numbers indicate the embryos exhibiting the phenotype.
	Fig. 2. Sdc4 is required for early foregut progenitor gene expression and proliferation (A–L) In situ hybridization with foregut progenitor marker hhex on embryos injected with indicated dose of control-MO, Sdc4-MO and/or Fzd7-MO at stage NF11 (A-D) and stage NF19 (E–L). (M) Loss of Sdc4 and Fzd7 results in reduced anterior endoderm proliferation at NF11. The mitotic index (pH3+ cells/total foregut cells) was quantified from confocal phosphor-histone H3 immunostaining of bisected NF11 embryos injected with the indicated MO and mRNAs. The average mitotic index ±S.D. *p<0.05 and**p<0.01 relative to age matched controls in Student’s t-test, (n=4 embryos/condition with 100 cells/embryo).
	Fig. 3. Sdc4 interacts with Fzd7 to regulate cell adhesion and morphology of foregut progenitors (A–H) Confocal immunostaining of cytoskeletal β–catenin in the foregut of NF19 bisected embryos injected with the indicated MO and/or mRNAs. (I) Western blot analyses of stage NF19 dissected foregut tissue shows no obvious changes of total β-catenin, E-cadherin and Tubulin levels, whereas Cdh3 and Itgb1 levels are reduced by 71% and 48%, respectively (quantified by Image-J). (J) Quantitation of foregut cell size in control MO, Sdc4-MO and Fzd7-MO injected embryos were measured from β–catenin immunostaining using Image-J. *p<0.05 in pairwise student T-tests. K) Quantification of foregut cell orientation (direction of the long axis of the cell relative to the Dorsal (D)–Ventral (V) axis) in control and Sdc4-depleted embryos (4 embryos of each condition). (L–N) Confocal immunostaining of the anterior endoderm at stage NF11 showing Cdh3 (C-cadherin) in green and Sdc4-MO/RLDx in red.
	Fig. 4. Sdc4 mediates Fn1 matrix deposition in the foregut (A–C) Confocal Fn1 immunostaining in the foregut of control-MO, Sdc4-MO and Fzd7-MO injected embryos at stage NF19. In controls peri-cellular Fn1 is observed in the foregut around the closing blastocoel and two Fn1 matrix layers: one between the foregut endoderm (en) and cardiac mesoderm, (m) and the other layer between foregut mesoderm (m) and ectoderm (ec). The Fn1 layer between the endoderm and mesoderm is largely absent (arrows) in Sdc4-depleted embryos. (D) Western blot analysis of dissected NF19 foreguts tissue shows that total Fn1 protein levels are unchanged in Sdc4-depleted embryos as compared to controls.
	Fig. 5. Sdc4 is required for both Wnt/JNK and BMP/Smad signaling in the foregut (A) Luciferase reporter assays in control-MO, Sdc4-MO, and Fz7-MO injected embryos. Firefly luciferase reporter plasmids (BRE for BMP activity, ATF2 for Wnt/JNK activity, and TOP:flash for canonical Wnt/β−catenin activity) were co-injected with pRL-TK:Renilla luciferase into D1 presumptive foregut cells of 32-cell stage embryos and assayed at stage NF19. Luciferase activity was normalized to renilla activity and the mean relative activity of triplicate samples is shown ±S.D; injections were repeated a minimum of three times and a representative result is shown. *p<0.05 and **p<0.01 in pairwise student t-tests compared to control-MO reporter levels. (B–D) Quantification of confocal immunostaining of pSmad1/5/8, pJNK and nuclear β−catenin. Mean pixel intensity of normalized to nuclear sytox green measured using Image-J. Arbitrary units (A.U.) with control-MO samples set to 1.0±S.D. *p<0.05 in repeated measure ANOVA (n=5 embryos/condition with 50–150 cells/embryo).
	Fig. 6. BMP signaling regulates foregut identity while Wnt/JNK signaling is necessary for both foregut identity and morphology (A–E) Confocal immunostaining of phosphorylated JNK (pJNK) or (F–J) phosphorylated Smad1/5/8 (pSmad1) in the foregut of bisected embryos at stage NF19. Quantification showed in Supplemental Fig. S7 (K–O) Confocal immunostaining of cortical β-catenin as cell membrane marker to show foregut cell morphology at NF19. (P–T) In situ hybridization with hhex in bisected NF19 embryos. The number of embryos with representative phenotype on the left versus total embryos examined on the right.
	Fig. 7. The Sdc4 PDZ-binding domain is dispensable for Fn1 matrix assembly and BMP signaling, but required cell morphology (A–D) Confocal Fn1 immunostaining in the foregut region of bisected embryos at stage NF19. (E–H) Confocal immunostaining of pSmad1/5/8 at NF19. Quantification showed in Supplemental Fig. S7 (I–L) Confocal immunostaining of cytoskeletal β-catenin showing foregut cell morphology at NF19. (M–P) In situ hybridization with hhex. Numbers in the figure show the number of embryos with representative phenotype on the left versus total embryos examined on the right.
	Fig. 8. A model suggesting how Sdc4 might coordinate Fn1-mediated BMP signaling and Fzd7-mediated Wnt/JNK signaling in the foregut The schematic on top shows a section through a Xenopus embryo with a peri-cellular Fn1 matrix (blue) present through out the foregut (fg) and a prominent Fn1 layer between the endoderm and the BMP-expressing cardiac mesoderm (red). The blowup shows a foregut progenitor cell with integrin (Itg) and Sdc4 complexes promoting Fn1 matrix assembly (blue). We postulate that the Fn1 matrix might enhance BMP-receptor interactions to stimulate Smad1/5/8 phosphorylation (pSmad1), which are required to maintain a positive BMP feedback loop and hhex expression. In this model Sdc4-Fn1 also promotes non-canonical Wnt signaling by forming a co-receptor complex with Fzd7 to recruits Dishevelld (Dvl) and activate JNK. Activation of JNK would then promote hhex expression, stimulate foregut cell proliferation (pH3) and stabilize Cdh on the foregut cell surface.
	sdc4 (syndecan 4) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 19, mid-sagittal section, anterior right, dorsal up.
	Supplemental Fig S1. Expression of hhex, sdc4 and fzd7in Xenopus embryo In situ hybridization showing the expression pattern of hhex, sdc4 and fzd7 at late gastrula stage, 0–1 somite stage and 4–6 somite stage.
	Supplemental Fig S2. Dorsal mesodermal injection (B1) of Sdc4-MOs or Sdc4/FZd7-MOs cause gastrulation defects. (A) Comparison of B1 (dorsal mesoderm) and D1 (anterior endoderm) injection of control MO (50 ng), Sdc4-MOs (50 ng) and Sdc4/Fzd7-MOs (25 ng each). In situ hybridization showing hhex expression at s2stage 11 and 19 after B1 or D1 injection of Sdc4/Fzd7-MOs (25 ng each).
	Supplemental Fig S3. Loss of Cdh3 is not sufficient to phenocopy Sdc4-depletion (A and B) Confocal immunostaining of Cdh3 confirms the efficacy of the Cdh3-MO. (C-D) Confocal immunostaining of cytoplasmic β-catenin showing that cortical β-catenin is reduced in Cdh3-depleted cells but cells are not enlarged. (E and F) Cdh3 depletion does not impact hhex expression in the foregut at NF19.
	Supplemental Fig S4. Inhibition of BMPR activity with LDN193189 does not reduce pJNK levels (A and B) Confocal immunostaining of pJNK and (C and D) hhex expression in embryo treated with a BMPR inhibitor LDN1931189 or DMSO vehicle control. (E and F) Confocal immunostaining of pSmad in embryo treated with a BMPR inhibitor LDN1931189 or DMSO vehicle control.
	Supplemental Fig S5. FGF signaling is not regulated by Sdc4 signaling, and not required for early foregut identity (A)In situ hybridization of nkx2.5 and hhex, and confocal immunostaining of pJNK on DMSO and two different FGFR inhibitor treated embryos. (B) Confocal immunostaining of pFGFR and pERK on con-MO and Sdc4-MOs injected embryos.
	Supplemental Fig S6. Sdc4 regulates a positive BMP feedback loop to maintain bmp ligand expression. In situ hybridization (A–E) hhex, (F–J) bmp2, (K–O) bmp4+7 or (P–T) wnt11 on bisected NF19 embryos injected with the indicated morpholinos. The number of embryos that exhibited the illustrated phenotype is indicated.
	Supplemental Fig S7. Quantification of pJNK and pSmad forFig. 6 and Fig. 7(A) Quantification of pJNK and pSmad pixel intensity for Fig. 6. (B) Quantification of pSmad pixel intensity for Fig. 7.
	Supplemental Fig S8. Recombinant BMP2 protein rescue hhex and bmp ligand expression in Sdc4-depleted embryosIn situ hybridization of for hhex(A–J) and bmp4+7 (P–T) at NF19 in embryos that were injected with the indicated morpholino at the 32-cell stage and then with or without recombinant human BMP2 protein injected into the presumptive foregut at stage NF12. BMP2 rescues Sdc4-MO, Fn1-MO and Szl-MO, but not the Fzd-MO.

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