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Fig. 1. Facial development in Xenopus laevis from stages 32–41 (40–76 hpf). A, C, E, G, I) Frontal views of tadpole faces. The arrow points to the stomodeum (outlined in yellow dots) or embryonic mouth (outlined in red dots). B, D, F, H, J) Transverse sections through the facial prominences. E-cadherin labels epithelium (red), and all tissue is counterstained with phalloidin (which labels F-actin, green). Abbreviations: BA1; 1st branchial arch, cg; cement gland, mx; maxillary prominence, mnp; medial nasal prominence, ma; mandibular prominence, np; nasal placode or pit.
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Fig. 2. A–D) frontal views of tadpole faces showing whole mount in situ hybridizations of RALDH2 (A,C) and RARγ (B,D) mRNA expression. The cement gland (cg) is pseudo-colored brown. The arrow points to the stomodeum. E,F) Transverse sections of RARγ mRNA expression and neural crest marker AP-2 in the orofacial region. G,H) DiI (red) fate mapping and phalloidin counterstaining labeling all F-actin (green). G) A frontal view of a tadpole fixed immediately post labeling with DiI in the RARγ expression domain at stage 28 (corresponding to panel D). The arrow points to the stomodeum. H) DiI labeling in transverse section approximately 40 h post labeling. DiI is observed in the primary palate region dorsal to the oral cavity. Abbreviations: cg; cement gland.
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Fig. 3. A) Schematic showing experimental plan for B–E. B,C) Frontal view of tadpoles at 4 dpf showing the embryonic mouth outlined in red dots. D,E) Transverse sections of the orofacial region. E-cadherin labels the epithelium (red), counterstained with phalloidin (green). F) Schematic showing experimental plan for face transplants from morphant to un-injected sibling. G,H) Frontal views of tadpoles showing the embryonic mouth outlined in red dots 2 days after transplant procedure. I) Schematic showing experimental plan for face transplants from un-injected to morphant sibling. J,K) Frontal views of tadpoles showing the embryonic mouth outlined in red dots 2 days after transplant procedure. Abbreviations: cg; cement gland.
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Fig. 4. A) Schematic showing experimental plan for B–I. B,C) Frontal view of tadpoles at 4 dpf showing the shape of the embryonic mouth (dotted in red). D,E) Transverse sections of the orofacial region. E-cadherin labels the epithelium (green), counterstained with propidium iodide (red). F–G) In situ hybridizations for the neural crest marker AP-2 (blue). F,H) Frontal views. G,I) Lateral views. The arrow points to the stomodeum or oral cavity. Abbreviations: cg; cement. PI; propidium iodide, e-cad; e-cadherin, mhb; midbrain–hindbrain boundary.
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Fig. 5. A–F) Frontal views of whole mount in situ hybridizations for lhx8 and msx2, (mRNA in blue). The arrow points to the stomodeum. G) Schematic showing the experimental plan H. H) RT-PCR using lhx8, msx2 and actin specific primers for control and RAR inhibitor treated tadpoles. Abbreviations: cg; cement gland.
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Fig. 6. A) Schematic showing experimental plan for B–E. B,C) Frontal view of tadpoles at 4 dpf showing the embryonic mouth outlined in red dots. D,E) Transverse sections of the orofacial region. E-cadherin labels the epithelium (red), counterstained with phalloidin (green). F) Schematic showing experimental plan to determine synergy of RAR inhibition and Lhx8/Msx2 loss of function. G–I) Frontal views of tadpoles showing the embryonic mouth outlined in red dots, 2 days after RAR treatment. Abbreviations: cg; cement gland.
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Fig. 7. A–D) Transverse sections through the dorsal facial prominences showing phospho-histone H3 immunohistochemistry (pH3; green) counterstained with propidium iodide (red). The brain and nasal pits are outlined in white dots. pH3 positive cells were counted in the region below the white dots. E) Schematic showing experimental plan for F–I. F–I) Frontal views of tadpoles at 4 dpf showing the embryonic mouth outlined in red dots. Abbreviations: cg = cement gland.
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Fig. 8. A–H) Alcian blue staining showing cartilage in 4–5 dpf tadpoles. Top row shows the ventral views and the second row shows the dorsal views. I–N) Transverse sections at 3–4 dpf through the primary palate region. Collagen II immunohistochemistry (green) counterstained with propidium iodide (red). Abbreviations, sr; suprarostral, qua; quadrate, ir; infrarostral, Mk; Meckel's, ch; ceratohyal, bh; basihyal, eth; ethmoid, trab; trabecular, pq; palatoquadrate, np; nasal pit, oc; oral cavity.
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Fig. 9. Model of the experimental results. A) Frontal view of a representative embryo at stage 30 (35 hpf) showing expression domains of RALDH2, RARγ, lhx8 and msx2 overlaid onto the prospective facial prominences. B) A simple model of how retinoic acid and lhx8/msx2 regulate growth and differentiation of the prominences that contribute to the upper lip and primary palate. C) Decreased retinoic acid signaling results in decreased Lhx8 and Msx2 function. This in turn results in decreased growth and differentiation of the prominences that contribute to the upper lip and primary palate contributing to the formation of a median cleft. Abbreviations: mx; maxillary prominence, mnp; medial nasal prominence, ma; mandibular prominence, op; olfactory placode or pit.
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Supplemental Fig. 1. I. Retinoic acid reverses the RALDH2 morpholino induced cleft. A) Frontal views of embryos at stages 412. II. Retinoic acid reverses the RAR inhibitor (BMS-453) induced median dorsal cleft. E) Frontal views of embryos at stages 412. III. Citral treatment. I,J) Frontal views of embryos at stages 412. The mouth is outlined in red dots in all figures.
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Supplemental Fig. 2. A) Schematic showing the experimental plan for B. B) Transverse sections through the facial prominences showing phospho-histone H3 immunohistochemistry (red) counterstained with phalloidin (green). The brain and nasal pits or eyes are outlined in blue dots. F) Transverse sections through the dorsal facial prominences showing cleaved caspase-3 immunohistochemistry (green) counterstained with propidium iodide (red). The brain and nasal pits or eyes are outlined in blue dots.
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Supplemental Fig. 3. Dorsal views of the tadpole head showing tubulin immunohistochemistry (green) counterstained with propidium iodide (red), highlighting the brain, axon tracts and cilia. The major aim of this experiment was to show that the brain and nasal pits form after RAR inhibitor treatment. The width of the neural tube seems comparable in treated and controls (white bars). However, the space between the eyes and neural tube is reduced.
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Supplemental Fig. 4. Morpholino injections targeting lhx8 and msx2. A) Frontal view of tadpoles at 4 dpf showing the shape of the embryonic mouth (dotted in red) after injections of each of lhx8 MO, msx2 MO or both lhx8 and msx2 MOs. E,F) Frontal view of 4 dpf tadpoles showing the shape of the embryonic mouth (dotted in red) after lhx8/msx2 double morpholino injection and face transplants at stage 24. Abbreviation: cg; cement. Arrows point to the mouth in all figures. G) lhx8 MO rescues showing frontal views of the face. G) Representative of one of the 13% of embryos co-injected with lhx8 MO (90 ng) and control GFP RNA (200 pg) with a slight median orofacial cleft phenotype (n = 30, 2 experiments). H) Representative of one of the 62% of embryos co-injected with lhx8 MO (90 ng) and lhx8 RNA (200 pg) with normal facial phenotype (n = 84, 2 experiments). I) Representative of one of the 67.6% of embryos co-injected with control MO (90 ng) and lhx8 RNA (200 pg) with a smaller face and abnormally shaped mouth (n = 37, 2 experiments). D) Representative of one of the 100% of embryos co-injected with control MO and GFP RNA with normal facial phenotype (n = 53, 2 experiments).
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Supplemental Fig. 5. A) Transverse sections through the dorsal facial prominences at stages 370, showing cleaved caspase-3 immunohistochemistry (green) counterstained with propidium iodide (red) after either RAR inhibitor treatment (A,B) or lhx8/msx2 morpholino injections (C,D).
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Supplemental Fig. 6. A,B) frontal view of tadpoles at 3 dpf showing the shape of the embryonic mouth (dotted in red) after hydroxyurea/aphidicolin (HU/A) treatment. Arrows point to the mouth in all figures. Abbreviation: cg; cement.
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