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Fig. 2.
Fzd7-depletion disrupts foregut organogenesis. (A) Targeted injection of fzd7-MOs (50 ng) and red fluorescent tracer (RLDx) into the D1 cells of 32-cell stage Xenopus embryos. Bight field (BF) and fluorescent view of representative bisected stage 20 embryo showing that the injection targeted the foregut (fg) and avoided the axial mesoderm. (B-D) Confocal immunostaining confirmed that the fzd7-MOs resulted in a loss of Fzd7 protein from the foregut cell membrane (B,C), which was rescued by injection of Fzd7 mRNA lacking MO-target sequence (D). (E–M) In situ hybridization showed that fzd7-MO embryos failed to maintain hhex at stage 20 and did not express the liver (nr1h5) and pancreas/duodenum (pdx1) markers at stage 35, which could be partially rescued by injection of fzd7 mRNA. (N, O) Ventral view of stage 45 embryos showing foregut edema and defective intestinal coiling with shortened gut in fzd7-MO injected embryos. (P,Q) H&E-stained section of a control (P) and a fzd7-MO-injected embryo (Q) that lacks foregut organs including liver (lv) and stomach (st). (R, S) In situ hybridization of a2m in isolated stage 42 gut tubes showed loss of foregut organs such as liver (lv), stomach (st) and pancreas (p) and shortened intestine (In). The number of embryos with the illustrated phenotype is indicated in each panel.
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Fig. 1. Differential Wnt signaling patterns the Xenopus endoderm. Wnt signaling has different thresholds in the endoderm. Embryos were injected with a dose range of mRNA encoding Sfrp5; 500 pg (B,G), 800 pg (C,H), 2 ng (D,I) and 3 ng (E,J). In situ hybridization for hhex (A–E) and vent1 and vent2, a mixture of both probes referred to as vent1/2 (F–J) in stage 19 bisected embryos showed that low doses of Sfrp5 expanded the hhex expression domain (yellow dashed line) (B–D) at the expense of hindgut markers vent1/2 (G–I). The highest dose of Sfrp5 resulted in a loss of hhex (E). The number of embryos with the illustrated phenotype is indicated in each panel.
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Fig. 3. Fzd7-depletion causes defects in foregut cell morphology. (A,B) Bight field view of the foregut surface at stage 20 showed that Fzd7 morphants exhibit larger loosely adherent cells (B), compared to controls (A). (C) Confocal immunostaining of the foregut (anterior left, dorsal up) with β1-integrin (C,D), C-cadherin (E,F), β-catenin (G,H) and phalloidin (F-actin) (I,J) showed decreased cell adhesion molecules and reduced cytoskeleton in the enlarged foregut cells of Fzd7 morphants. All of the images were taken using same setting for control and fzd7-MO embryos. (K,L) Quantitation of cell size and orientation; foregut cell length, width and orientation in control (K) and fzd7- MO injected embryos (L) were measured from β-catenin immunostaining (green) using Image-J. Nuclei shown in blue, fzd7-MO/RLDx in red. All images were oriented with dorsal up. The yellow line marks the long axis of foregut cells, and quantification shows the frequency of orientations of the long axis of cells in Fzd7 morphants (L,L') and control (K,K') n1⁄4 the total number of foregut cells from 5 uninjected (K') and 5 fzd7-MO embryos (L' ). (M) The relative length, width, and length/width radio in control and Fzd7-depleted foregut cells.**po0.01 relative to controls in Student's t-test. (N) Western blot analysis shows no significant changes of total E-cadherin and C-cadherin level in the foregut explants.
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Fig. 4. Fzd7-depletion causes reduced cell proliferation. (A) Confocal immunos- taining of phospho-Histone h3 (PH3; green), nuclei (blue) and fzd7-MO/RLDx (red) at stage 12 (A,B) and stage 19 (C,D) show that Fzd7-depleted embryos exhibit reduced foregut (outlined in dashed yellow line) proliferation. (E) Mean number of PH3 positive cells in the foregut 7S.D. *po0.05 relative to sibling controls in Student's t-test (n1⁄44 embryos/ condition).
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Fig. 5. Fzd7 depletion results loss of both Wnt/β-catenin and Wnt/JNK activity in the foregut. (A) Fzd7-depletion resulted in a reduction of β-catenin/Tcf and JNK/AP1 activity in the foregut. TOP:flash or AP1:Luciferase reporter plasmids were injected into either the D1 foregut endoderm cells or the D4 hindgut endoderm cells at the 32-cell stage, with or without fzd7-MO as indicated. The TOP:Flash reporter is an indicator of β-catenin/Tcf activity, while the AP1:luciferase reporter is an indicator of JNK-mediated c-Jun/ c-Fos (AP1) activity. At stage 20 luciferase activity was measured, in triplicate. The average relative luciferase activity, normalized to co-injected pRTK:Renila, from three biological replicates per condition is shown 7S.D. *po0.05 and ** po0.01 relative to control foregut in Student's t-test. (B) Western blot showed decreased phospho-JNK1/2 (p-JNK) and a loss of dephosphorylated active β-catenin and total cytosolic β-catenin in the foregut explants at stage 19. (C): Confocal immunostaining showed reduced nuclear β-catenin levels in Fzd7 morphant foregut tissue (D) relative to controls (C), at stage 20. (E) Mean pixel intensity of nuclear β-catenin staining measured using Image- J 7S.D (foregut cells were scored from 5 embryos/ condition).
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Fig. 6. Fzd7 signals via both β-catenin and JNK coordinate foregut cell proliferation, gene expression and cell morphology. (A) Inhibition of either Wnt/β-catenin or JNK pathways reduced foregut cell proliferation. Embryos were either injected with RNA encoding Dkk1 (500 pg) to block the Wnt/β-catenin pathway or treated with the cell soluble JNK inhibitor SP600125 (JNKi; 100 μM). Mean number of PH3 positive cells in the foregut +/−S.D. *po0.05 and ** po0.01compared to controls (n1⁄44 embryos/ condition). (B) Activation of either β-catenin or JNK signaling rescued cell proliferation in Fzd7 morphants. Embryos were injected with fzd7-MOs (50 ng) with or without RNA encoding a constitutively active JNK (c.a. JNK; 200 pg) or a hormone inducible GR:Lef-βCTA (β-cat; 200 pg) activated by 1 μM dexamethasone at stage 11. Mean number of PH3 positive foregut cells at stage 12 7S.D. *po0.05 relative to control and **po0.05 relative to fzd7-MO alone in Student's t-test (n1⁄44 embryos/ condition). (C) Fzd7/ JNK signaling regulates cell shape. Confocal immunostaining of β-catenin at stage 20 showed that c.a.JNK injection (F) rescued the cell-size defects in Fzd7 morphants (D), whereas activation of the GR:Lef-βCTA (β-cat) did not (E). The JNK inhibitor (JNKi) caused a reduction of cytoskeletal β-catenin and increased cell size (H), phenocopying fzd7- MO (D), whereas Dkk1 (1.5 ng) had no effects on cell morphology (G). (I) Both Fzd7/β-catenin and Fzd7/JNK regulate gene expression. In situ hybridization to stage 20 embryos showed that co-injection of either GR:Lef-βCTA (β-cat) (K) or c.a.JNK (L) restored hhex expression in Fzd7 morphants (J), whereas the JNK inhibitor (N) or high levels of Dkk1 (1.5 ng) (M) suppressed hhex. The number of embryos with the illustrated phenotype is indicated in each panel.
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Fig. 7. Multiple thresholds of Wnt/Fzd7/β-catenin activity pattern the endoderm. (A) Fzd7/β-catenin signaling levels were modulated in a dose response with different combination of fzd7-MOs and/or treatment with BIO, a GSK3 inhibitor that stabilizes β-catenin. In situ hybridization of the foregut marker hhex and the mid/hindgut markers vent1/2 in stage 20 embryos showed that increasing dose of BIO and therefore increasing β-catenin activity decreased hhex while expanding vent1/2 (A, B) relative to untreated controls (C, D). A partial knockdown of Fzd7 (25 ng of fzd7-MOs) resulted in a modest increase in hhex and reduction of vent1/2 (E, F), whereas a complete Fzd7 knockdown by 50 ng of the fzd7-MO caused a loss of hhex, which was not accompanied by an expansion of vent1/2 (G, H). A low dose of BIO (5 uM in I,J) rescued hhex expression in fzd7-depleted embryos (I, J), whereas a higher BIO dose (10 uM) resulted in the foregut adopting a hindgut fate and expressing ectopic vent1/2 (K, L). The number of embryos with the illustrated phenotype is indicated in each panel.
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Fig. 8. A model of how Wnt/Fzd7/Sfrp5 regulate β-catenin and JNK signaling to coordinate endoderm fate, morphogenesis. (A) Schematic of a neurula embryo (anterior left) showing expression of the fzd7 (green), wnt11, wnt5 and wnt8 in the ventral mesoderm and endoderm (blue) and the Wnt-antagonist sfrp5 in the surface of the foregut endoderm (red). The spatial expression pattern of receptors, ligands and antagonists is postulated to establish differential Wnt activity in the endoderm. (B) Different thresholds of β-catenin/TCF activity (red line) pattern endoderm with high activity promoting hindgut progenitor fate (hg), where as a low but essential level of β-catenin/ TCF is required to maintain foregut (fg) fate. (C) Differential Fzd7/JNK activity might regulate cell shape, adhesion and morphogenesis in the foregut (green). Fzd7/JNK signaling in the deep endoderm promotes cell adhesion and the oriented cell shape required for gut elongation. Sfrp5 in the surface layer reduces JNK activity to a low but essential level to establish an epithelium. If JNK activity is too low (as in Fzd7 morphants) cells become enlarged, loosely adherent with a random orientation.
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Figure S1. Expression pattern of fzd7, sfrp5, and wnt11: (A) Schematic of an early somite stage Xenopus embryo showing foregut (fg) and hindgut (hg) progenitors, adapted from (Li et al., 2008). (B) In situ hybridization to stage 180 bisected embryos shows expression of sfrp5 (B), fzd7 (C) and wnt11 (D).
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Figure S2. Wnt signaling has different thresholds in the endoderm. (A-H) Embryos were treated with the following doses of XAV-939 to promote β-catenin degradation: DMSO vehicle (A,E), 10 μM (B,F), 30 μM (C,G), 80 μM (D,H). In situ hybridization to stage 19 bisected embryos showed that low doses of XAV-939 expanded the hhex expression domain (yellow dashed line) (B) at the expense of hindgut markers vent1 and vent2 (a mixture of both probes referred to as vent1/2) (F). The highest dose of XAV-939 resulted in a loss of hhex and vent1/2 (C,D,G, H). The number of embryos with the illustrated phenotype is indicated in each panel.
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Figure S3. Loss of Fzd7 in endoderm does not cause defects in Brachet's cleft formation or gastrulation (A,D,G) Bright field and fluorescent images (B, E, H) of bisected gastrula show that the formation of Brachet's cleft between the mesendoderm and the ectoderm (red arrows) of the dorsal lip (A, B) was inhibited by injection of fzd7-MOs into B1 cells at the 32-cell stage mesodermal cells (D, E), as previously published (Winklbauer, R., Medina, A., Swain, R. K. and Steinbeisser, H. (2001) 'Frizzled-7 signalling controls tissue separation during Xenopus gastrulation', Nature 413(6858): 856-60). However, injection of the fzd7-MOs into the D1 cells had little is any impact on tissue-separation (G,H). (C, F) At stage 32 B1-injected embryos exhibited typical bent axis and spina bifida, consistent with the known role of Fzd7 in convergent extension of the axial mesoderm. In contrast D1-injected embryos did not have axial defects but rather exhibited foregut edema.
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Figure S5. Loss of Fzd7 does not cause ectopic apoptosis: (A, C, E) TUNEL and (B, D, F) active-caspase-3 immunostaining (green) showed that fzd7-MO embryos did not exhibit elevated apoptosis at stage 19, whereas injection of a +control MO that is known to induce apoptosis exhibits robust caspase-3 activity (F). The number of embryos with the illustrated phenotype is indicated in each panel.
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Figure S7. Impact of non-canonical Wnt pathway inhibitors on foregut morphology and gene expression: (A) Confocal immunostaining of β-catenin to show foregut cell size and shape and in situ hybridization for hhex at stage 19 in the following embryos: (A, B) DMSO control, (C, D) fzd7-MO (50 ng), (E, F) Cdc42 inhibitor (50 ) (G, H) CamKinase inhibitor (20 ), (I, J) Rac1 inhibitor (100 ) and (K, L) Ca2+ dependant PKC inhibitor (40 ). The number of embryos with the illustrated phenotype is indicated in each panel.
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