Tabler JM et al. (2014), Hedgehog activity controls opening of the prima...

XB-ART-49476

Dev Biol 2014 Dec 01;3961:1-7. doi: 10.1016/j.ydbio.2014.09.029.

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Hedgehog activity controls opening of the primary mouth.

Tabler JM , Bolger TG , Wallingford J , Liu KJ .

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To feed or breathe, the oral opening must connect with the gut. The foregut and oral tissues converge at the primary mouth, forming the buccopharyngeal membrane (BPM), a bilayer epithelium. Failure to form the opening between gut and mouth has significant ramifications, and many craniofacial disorders have been associated with defects in this process. Oral perforation is characterized by dissolution of the BPM, but little is known about this process. In humans, failure to form a continuous mouth opening is associated with mutations in Hedgehog (Hh) pathway members; however, the role of Hh in primary mouth development is untested. Here, we show, using Xenopus, that Hh signaling is necessary and sufficient to initiate mouth formation, and that Hh activation is required in a dose-dependent fashion to determine the size of the mouth. This activity lies upstream of the previously demonstrated role for Wnt signal inhibition in oral perforation. We then turn to mouse mutants to establish that SHH and Gli3 are indeed necessary for mammalian mouth development. Our data suggest that Hh-mediated BPM persistence may underlie oral defects in human craniofacial syndromes.

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Species referenced: Xenopus laevis
Genes referenced: fn1 frzb frzb2 gli3 shh sox2
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	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.

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