Tabler JM , Bolger TG , Wallingford J .

F32 DE023272 NIDCR NIH HHS, 081880/Z/06/Z Wellcome Trust , BB/E013872/1 Biotechnology and Biological Sciences Research Council , BB/I021922/1 Biotechnology and Biological Sciences Research Council , F32DE023272 NIDCR NIH HHS, GM074104 NIGMS NIH HHS , R01 HL117164 NHLBI NIH HHS , R01 GM074104 NIGMS NIH HHS , Howard Hughes Medical Institute , HL117164 NHLBI NIH HHS , Wellcome Trust

	Fig. 1. Hedgehog perturbation affects the size of the oral opening. (A) Schematic illustrating primary mouth development. Frontal view of Xenopus tadpole indicates sectional plane for schematics. Stage 12.5, primary mouth induction occurs anterior to the prechordal plate (PP), notochord (Nc) and neural plate (NP). Stage 19 foregut endoderm (Fg) abuts mouth ectoderm (Ec, pink), separated by fibronectin-rich basement membrane (BM, green), between forebrain (Fb) and cement gland (CG). BM dissolves and mesenchymal clearance thins stomodeum (green dashed line indicates BM). Stage 37, buccopharyngeal membrane (BPM) formation. Stage 40, BPM perforation. (B–D) Frontal view of stage 45 tadpoles incubated from 2-cell stage with 10 μM (B), 5 μM (C), or 2 μM SANT1 (D) (B, n=13/13, C, n=16/16, D, n=15/15). Primary mouth is indicated by red arrowhead. (E) Control tadpole, 0.07% DMSO (n=26/26). (F–H) Tadpoles incubated with 2 μM (F), 20 μM (G) or 100 μM purmorphamine (H). Increase in mouth size was observed with increasing concentrations of purmorphamine (F, n=70/70, G, n=43/43, H, n=154/154). (I) Quantification of mouth size for 10 μM, 5 μM, or 2 μM SANT1, 0.07% DMSO, 2 μM, 20 μM or 100 μM purmorphamine, where mouth perimeter is normalized to width of the head. ⁎⁎⁎⁎P<0.001. Scheme indicating primary mouth size (green) in relationship to Hh activity (red bar). (J) Stage 45 control tadpole. (Jʹ) Facial anatomy schematic. (K–N) Tadpoles incubated with 250 μM cyclopamine from 2-cell stage (K), between stages 12.5–19 (L), 19–37 (M), or from 37 (N). (O–R) Tadpoles treated with 100 μM purmorphamine from the 2-cell stage (O), between stages 12.5–19 (P), 19–37 (Q), or from stage 37 (R).
	Fig. 2. Hedgehog signaling is necessary and sufficient for basement membrane dissolution. (A–C, E–Gʹ) Sagittal sections through primary mouth stained for β-catenin in magenta, fibronectin (FN) and nuclei in green. (Fg) foregut, (BM) basement membrane, (Fb) forebrain, (Cg) cement gland. (A–C) Stage 24. (A) Embryo treated with 10 μM SANT1 from the 2-cell stage. (Aʹ) Magnified view of endoderm–ectoderm interface and basement membrane. FN is observed (white arrowhead) (n=7). (B) Control. (Bʹ) FN immunofluorescence indicates the presence of BM between foregut and ectoderm (white arrowhead, n=7). (C) Embryo treated with 250 μM purmorphamine. (Cʹ) No FN immuofluorescence is observed between foregut and ectoderm (open white arrowhead, n=6/7). (D–Dʹ) Schematic indicating anatomy of sections represented in B–Bʹ. Fibronectin-rich basement membrane separates foregut and ectoderm. (E–Gʹ) Stage 26. (E) Embryo treated with 10 μM SANT1. (Eʹ) shows persistent FN immunofluorescence (white arrowhead, n=8). (F) Stage 26 control. (Fʹ) Almost no BM FN is observed in controls (open arrowhead, n=12). (G) Stage 26 embryo treated with 100 μM purmorphamine. (Gʹ) No FN immuofluorescence is observed (open white arrowhead, n=10). (H–Hʹ) Schematic indicating anatomy of WT sections represented in F–Fʹ. Fibronectin-rich basement membrane is absent or broken, foregut and ectoderm cells mix (red arrow). (J–L) Sagittal sections of stage 39 tadpoles stained for β-catenin (magenta) and DAPI. (J) Tadpole treated with 10 μM SANT1 (n=5). (K) Control tadpole. BPM is observed as a single layer epithelium (n=6). (L) Tadpole treated with 100 μM purmorphamine. No BPM is present (n=4). Scale bars indicate 50 μm. (M) Schematic indicating anatomy of WT sections represented in (K). Buccopharyngeal membrane (BPM) separates foregut from external environment and indicates site of future mouth.
	Fig. 3. Wnt inhibition acts downstream of Hh signaling during BPM dissolution. (A–F) Stage 38 tadpoles. (A) Control tadpole incubated with 0.7% DMSO continuously from 2-cell stage. (B) Tadpole incubated with 0.7% DMSO, then 15 μM BIO from stage 12.5 (n=34). (C) Tadpole incubated with 0.7% DMSO, then 15 μM BIO from stage 19 (n=34). (D) Tadpole incubated with 100 μM purmorphamine, then 0.07% DMSO from stage 12.5 (n=34). Primary mouth is enlarged. (E) Tadpole incubated with 100 μM purmorphamine, then 15 μM BIO from stage 12.5 (n=34). (F) Tadpole incubated with 100 μM purmorphamine, then 15 μM BIO from stage 19 (n=34). Scale bars indicate 100 μm. (G) Schematic indicating drug application scheme and outcome. Red arrowheads (B and E) indicate absence of primary mouth.
	Fig. 4. Sonic hedgehog and Gli3 are required for perforation of the mammalian BPM. (A) Schematic frontal view of E9.0 mouse embryo. Dashed line indicates section plane. (B) Schematic of section. Red box indicates region represented in panels (C) and (Dʹ). (Fb) forebrain, (Fg) pharyngeal foregut, (H) Heart. (C–D) Nuclei are stained with DAPI (cyan) (B) Sagittal section of E9.0 (20 somite) Shh+/+ embryo (n=3). (Bʹ) Schematic illustrating anatomy in (B). (C) Sagittal section through E9.0 Shh−/− embryo (n=3). (Cʹ) Schematic illustrating anatomy depicted in (C). (D–E) Sagittal sections through E9.0 (19 somite) Gli3+/+ and Gli3xt/xt embryos, respectively. Remnant buccopharyngeal membrane (BPM) is observed in Shh−/−and Gli3xt/xt embryos. Nuclei are stained with DAPI (cyan). Scale bars indicate 100 μm. B–C are littermates, as are D–E. (F) Proposed model of primary mouth formation in Xenopus. Frontal schematic indicates sectional plane of diagrams. Cyan bar indicates stages of primary mouth competence. Red bar indicates level of Hh activation while blue indicates Wnt inhibition, where white is none, and bright color is high. Wnt inhibitors Crescent and Frzb-1 are expressed in primary mouth ectoderm (blue). Primary mouth ectoderm is induced adjacent to Hh signal where Wnt inhibition is highest (cyan bracket). By stage 28, BM is indicated by white outlines, and mesenchymal cells are illustrated in black. Hh signal activation (red) is highest ventral to the forebrain (Fb). (Cg) and (Fg) indicate cement gland and foregut, respectively. Wnt inhibitors are no longer expressed, and stomodeum is refractive to Wnt activation. By stage 39, endoderm–ectoderm intercalation forms monolayer buccopharyngeal membrane (BPM). Hh signal activation is required through intercalation stages.
	Supplementary Fig. 1. related to Figure 1: Activation and inhibition of Hh signaling narrow the head. (A–C) Dorsal views of stage 45 tadpoles. (A) Control treated tadpole. (B) 250 μM cyclopamine treated from the 2-cell stage. Eyes (black) are close-set and head is narrow compared to controls. (C) Tadpole treated with 100 μM purmorphamine from the 2-cell stage. Eyes (black) are close-set and head is narrower than controls. (D) Graph showing total width of head at the level of the eye (blue) and the distance between eyes (yellow) in stage 45 tadpoles. According to both measurements cyclopamine and purmorphamine treatment significantly decreased the width of tadpole heads compared to control treated tadpoles (p<0.001, cyclopamine treated compared to control, and p<0.001, purmorphamine compared to control). Diagram indicates measurement planes. (E) Graph indicating head length from the anterior-most extent of the head to either the posterior (green) or anterior extent of the eye (red). According to both measurements cyclopamine and purmorphamine treatment significantly decreased head length compared to controls (p<0.001, cyclopamine treated compared to control, and p<0.001, purmorphamine compared to control). Diagram indicates measurement planes.
	Supplementary Fig. 2. related to Figure 1: Hh signaling exhibits dose dependent regulation of primary mouth size. (A–G) Frontal view of stage 45 tadpoles incubated from the 2-cell stage with 250 μM cyclopamine (A), 50 μM cyclopamine (B), or 5 μM cyclopamine (C) (A, n=35, B, n=35, C, n=150). (D) Control tadpole incubated with 0.7% DMSO from the 2-cell stage. (E–G) Tadpoles incubated with 2 μM purmorphamine (E), 20 μM purmorphamine (F) or 100 μM purmorphamine (G). Primary mouth is indicated by red arrowheads. Cyclopamine caused a loss of the primary mouth perforation. A dose dependent increase in mouth size was observed with increasing concentrations of purmorphamine (E, n=35, F, n=35, G, n=140). Red gradient indicates Hh activation, and green bar indicates primary mouth size relative to Hh activation.

Akiyoshi, Gli1, downregulated in colorectal cancers, inhibits proliferation of colon cancer cells involving Wnt signalling activation. 2006, Pubmed

Akiyoshi, Gli1, downregulated in colorectal cancers, inhibits proliferation of colon cancer cells involving Wnt signalling activation. 2006, Pubmed
Blaess, Gli3 coordinates three-dimensional patterning and growth of the tectum and cerebellum by integrating Shh and Fgf8 signaling. 2008, Pubmed
Cain, GLI3 repressor controls nephron number via regulation of Wnt11 and Ret in ureteric tip cells. 2009, Pubmed
Chen, Small molecule modulation of Smoothened activity. 2002, Pubmed
Danker, V(+)-fibronectin expression and localization prior to gastrulation in Xenopus laevis embryos. 1993, Pubmed , Xenbase
Démurger, New insights into genotype-phenotype correlation for GLI3 mutations. 2015, Pubmed
Dessaud, Interpretation of the sonic hedgehog morphogen gradient by a temporal adaptation mechanism. 2007, Pubmed
Dickinson, Positioning the extreme anterior in Xenopus: cement gland, primary mouth and anterior pituitary. 2007, Pubmed , Xenbase
Dickinson, The Wnt antagonists Frzb-1 and Crescent locally regulate basement membrane dissolution in the developing primary mouth. 2009, Pubmed , Xenbase
Dickinson, Development of the primary mouth in Xenopus laevis. 2006, Pubmed , Xenbase
Ekker, Distinct expression and shared activities of members of the hedgehog gene family of Xenopus laevis. 1995, Pubmed , Xenbase
Gradl, The Wnt/Wg signal transducer beta-catenin controls fibronectin expression. 1999, Pubmed , Xenbase
Hardin, Short-range cell-cell signals control ectodermal patterning in the oral region of the sea urchin embryo. 1997, Pubmed
He, Suppressing Wnt signaling by the hedgehog pathway through sFRP-1. 2006, Pubmed , Xenbase
Hollemann, Manipulation of hedgehog signaling in Xenopus by means of embryo microinjection and application of chemical inhibitors. 2007, Pubmed , Xenbase
Hui, A mouse model of greig cephalopolysyndactyly syndrome: the extra-toesJ mutation contains an intragenic deletion of the Gli3 gene. 1993, Pubmed
Jacox, Facial transplants in Xenopus laevis embryos. 2014, Pubmed , Xenbase
Kennedy, Quantitative analysis of orofacial development and median clefts in Xenopus laevis. 2014, Pubmed , Xenbase
Lee, SHH-N upregulates Sfrp2 to mediate its competitive interaction with WNT1 and WNT4 in the somitic mesoderm. 2000, Pubmed
Legius, Holzgreve-Wagner-Rehder syndrome: Potter sequence associated with persistent buccopharyngeal membrane. A second observation. 1988, Pubmed
Lewis, Cholesterol modification of sonic hedgehog is required for long-range signaling activity and effective modulation of signaling by Ptc1. 2001, Pubmed , Xenbase
Lewis, Reagents for developmental regulation of Hedgehog signaling. 2014, Pubmed , Xenbase
McClay, Pattern formation during gastrulation in the sea urchin embryo. 1992, Pubmed
Meijer, GSK-3-selective inhibitors derived from Tyrian purple indirubins. 2003, Pubmed , Xenbase
Peyrot, A revised model of Xenopus dorsal midline development: differential and separable requirements for Notch and Shh signaling. 2011, Pubmed , Xenbase
Pillai, Persistent buccopharyngeal membrane with cleft palate. A case report. 1990, Pubmed
Poelmann, Cell degeneration and mitosis in the buccopharyngeal and branchial membranes in the mouse embryo. 1985, Pubmed
Sinha, Purmorphamine activates the Hedgehog pathway by targeting Smoothened. 2006, Pubmed
Soukup, Development and evolution of the vertebrate primary mouth. 2013, Pubmed
Stanton, Small-molecule modulators of the Sonic Hedgehog signaling pathway. 2010, Pubmed
Takahama, Morphological changes in the oral (buccopharyngeal) membrane in urodelan embryos: development of the mouth opening. 1988, Pubmed
Theiler, [On the anlage of the fore-gut in the house mouse and the formation of furrows on the surgace of the egg-cylinder]. 1969, Pubmed
Varnat, Hedgehog pathway activity is required for the lethality and intestinal phenotypes of mice with hyperactive Wnt signaling. 2010, Pubmed
Verma, Persistent buccopharyngeal membrane: report of a case and review of the literature. 2009, Pubmed
Wang, The Shh-independent activator function of the full-length Gli3 protein and its role in vertebrate limb digit patterning. 2007, Pubmed
Watanabe, The ultrastructure of oral (buccopharyngeal) membrane formation and rupture in the anuran embryo. 1984, Pubmed
Waterman, The ultrastructure of oral (buccopharyngeal) membrane formation and rupture in the chick embryo. 1980, Pubmed
Waterman, Formation and perforation of closing plates in the chick embryo. 1985, Pubmed
Waterman, Ultrastructure of oral (buccopharyngeal) membrane formation and rupture in the hamster embryo. 1977, Pubmed
Wheway, Aberrant Wnt signalling and cellular over-proliferation in a novel mouse model of Meckel-Gruber syndrome. 2013, Pubmed
Williams, Identification of a small molecule inhibitor of the hedgehog signaling pathway: effects on basal cell carcinoma-like lesions. 2003, Pubmed