Neilson KM , Pignoni F , Yan B , Moody SA .

R01 EY1316709 NEI NIH HHS , R03 HD055321 NICHD NIH HHS , R01 EY013167-09 NEI NIH HHS , R03 HD055321-01A1 NICHD NIH HHS , R01 EY013167 NEI NIH HHS

             face development

           OTOFACIOCERVICAL SYNDROME 1; OTFCS
           BRANCHIOOTIC SYNDROME 3; BOS3

	Figure 2. Expression of Eya genes. A,B: Eya1 is diffusely expressed throughout the ectoderm (ecto) of the animal (an) cap of the blastula (A) and the gastrula (B; side views). veg, vegetal pole; bl, blastopore lip; endo, endoderm. C–E: Eya3 is detected in the animal blastomeres of the 32-cell embryo (C), the animal cap of the blastula (D), and enhanced in the dorsal ectoderm of the late gastrula (E; side views). F: At neural plate stages, Eya3 is expressed throughout the neural plate (np) and preplacodal ectoderm (PPE), but not the cement gland (cg; anterolateral view). G–O: Eya2 expression. G: Eya2 is diffusely expressed throughout the neural plate and PPE (Anterior view). H: Upon neural tube closure, Eya2 is expressed in the adeno-hypophyseal (AH), olfactory (olf), trigeminal (Vp), and dorsolateral (dlp) placodes. I: It also is detected in the caudal neural tube (nt) and somites (so) (H, anterior view; I, dorsal view). J–L: At tail bud (J) and larval (K,L) stages, Eya2 is strongly expressed in the olfactory (olf), otic (oto), and various other placodes (pl) and their cranial ganglion derivatives (crg). Note that the lens placode (L) does not express Eya2. There is additional expression in the nephric mesoderm (ne) and hypaxial muscle precursors (hyp; J, K, side views; L, anterior view). M–O: In transverse sections at the levels of the diencephalon (M), otocyst (N), and caudal hindbrain (O), Eya2 expression is notable in the cranial ganglia (profundal, Pg; maxillo-mandibular branch of the trigeminal, Vg; VIIIg, IXg), otocyst, and somitic and nephric mesoderm.
	Figure 3. Expression of Groucho-related genes (Grg). A: stage VI oocyte with Grg4 transcripts enhanced on animal (an) side (side view). B: An eight-cell embryo with maternal ESG1 transcripts enhanced in the animal blastomeres (side view). C: In the blastula, animal cap ectoderm expresses Grg5 (side view). D: In the gastrula, the entire ectoderm expresses Grg4, whereas the preinvoluted mesoderm (meso) and endoderm do not (side view). E–G: At neural plate stages, Grg4 (E), ESG1 (F), and Grg5 (G) are expressed in the neural plate and preplacodal ectoderm (PPE); Grg5 is also expressed in the cement gland (anterior views). H: ESG1 is expressed throughout the neural tube, but most strongly in the anterior part (ant. nt). It is also detected in the neural crest (nc), dorsal epidermis (epi), and somites (dorsal view). I: At tail bud stages, ESG1 expression is strong in the entire neural tube, migrating neural crest, somites, otocyst, and tail bud (tb; side view). J: At tail bud stages, Grg4 is weakly detected in the cement gland (ventral view). K: At larval stages, Grg4 is expressed throughout the brain (b), retina (r), placode derivatives (crg, L, olf, oto), and the branchial arches (BA; side view). L: ESG1 is expressed in the same tissues, and also is detected in the heart (h), ventral gut (vg), nephric mesoderm and the initial outgrowth of the lateral line (LL; side view). M–R: In transverse sections at the levels of the anterior trunk (M), forebrain (N–P) and hindbrain (Q,R), expression of the various Grg-related genes is noted in the nephric mesoderm, forebrain (fb), hindbrain (hb), retina, branchial arches, cement gland, various placodes (olf, oto, IXg, epibranchial [ebp]) and heart.
	Figure 4. Expression of a CG17265-related gene, Xt-ccd85c. A: Diffuse expression of Xt-ccd85c throughout the anterior neural plate and PPE (anterior view). B: At neural tube closure, there is diffuse expression throughout the neural tube, retina, dorsal epidermis, otocyst, and branchial arches (side view). C: At late tail bud stages, the pineal (p), retina, midbrain, and hindbrain are stained. There also is weak staining in the lens, otocyst, and branchial arches (side view). D: Dorsal view at larval stage showing expression extending into the forebrain. E–G: Transverse sections at forebrain (E), hindbrain (F), and caudal hindbrain (G) demonstrate restricted neural expression.
	Figure 5. Expression of SOBP-related genes. A: zfp198 mRNA is concentrated in the animal blastomeres at the eight-cell stage (side view). B: zfp198 is expressed throughout the ectoderm of the gastrula (side view). C: At early neural plate, LOC414497 is expressed throughout the neural plate, preplacodal ectoderm (PPE), and dorsal epidermis (anterolateral view). D: At neural tube closure, neural crest and lens placode (Lp) expression of zfp198 becomes apparent (anterolateral view). E–G: At late tail bud, zfp198 (E) and LOC414497 (F) are expressed throughout the brain, retina, placode derivatives (olf, L, crg, oto) and branchial arches, whereas Xt-Sobp (G) is expressed in patches in the brain and in several placode derivatives (olf, crg, oto; side view). H: At larval stages, Xt-Sobp expression extends into the forebrain (side view). I–K: Transverse sections at forebrain (I), hindbrain (J), and spinal (K) levels demonstrate expression of zfg198 and LOC414497 in neural (fb, r, hb, sc), branchial arch, otocyst and somites. L: Transverse section at hindbrain demonstrates Xt-Sobp more restricted expression in lateral hindbrain, otocyst, and cranial ganglia.
	Figure 6. Expression of a CG1135-related gene. A: LOC100049093 is expressed diffusely through the neural plate, preplacodal ectoderm (PPE), and dorsal epidermis (epi; anterior view). B: At neural tube closure, it is slightly enhanced in the neural tube, PPE, and neural crest (anterior view). C: At larval stages, it is detected in the brain, retina, several placode derivatives (L, olf, oto, crg), branchial arches, and nephric mesoderm (side view). D–F: Transverse sections at forebrain (D), hindbrain (E), and spinal (F) levels demonstrate extensive neural (fb, r, hb, sc), placodal (L, oto, IXg), and branchial arch, and weak expression in the nephric mesoderm.
	Figure 7. Expression of a CG7878-related gene. A: Ddx43 is diffusely expressed throughout the neural plate, preplacodal ectoderm (PPE), and dorsal epidermis (anterior view). B: At neural tube closure, it is very weakly expressed in the neural tube, a few placodes (dlp, L) and migrating neural crest (side view). C: At larval stages, it is expressed throughout the brain, retina, several placode derivatives (olf, oto, L), branchial arches, somites, heart, and nephric mesoderm (side view). D–F: Transverse sections at midbrain (D), hindbrain (E), and spinal (F) levels demonstrate neural (mb, r, hb, sc), lens, branchial arch, heart, otocyst, somite, and nephric mesoderm expression.
	Figure 8. Expression of a CKIIβ-related gene. A: CKIIβ is expressed throughout the ectoderm of the gastrula (dorsal view). B: At neural plate stages, epidermal staining continues, but expression is enhanced in the neural plate and preplacodal ectoderm (PPE; anterolateral view). C: At neural tube closure, CKIIβ is expressed throughout the neural tube, in placodes (AH, olf, L) and neural crest (anterior view). D: At tail bud stages, brain, retina, placodal structures (olf, L, crg, oto), and branchial arches are stained (side view). E: At larval stages, the nephric mesoderm is additionally stained (side view). Transverse sections at hindbrain (F) and spinal (G) levels demonstrate neural (hb, sc), branchial arch, otocyst, cranial ganglion (IXg), somite, and nephric mesoderm expression.
	Figure 9. Expression of a CG10576-related gene. A: At late gastrula stages, the ectodermal expression of 2G4 is enhanced on the dorsal side (d. ecto; v. ecto, ventral ectoderm; side view). B: At neural plate stages, 2G4 is expressed diffusely throughout the neural plate, preplacodal ectoderm (PPE), and dorsal epidermis (anterolateral view). C: 2G4 is expressed throughout the neural tube, retina, PPE, cranial neural crest, and epidermis (anterior view). D: At tail bud stages, 2G4 is intensely expressed in the retina, diencephalon (di), hindbrain, placodes (olf, L, oto), branchial arches, and weakly in the nephric mesoderm (side view). E: At larval stages, the neural tube expression expands to the dorsal spinal cord, and nephric mesoderm staining is prominent (side view). F,G: Transverse sections at hindbrain (F) and spinal (G) levels demonstrate neural (hb, sc), branchial arch, otocyst, epidermal and nephric mesoderm expression.
	Fig. 10. Expression of CG5033-related genes. A: MGC68939 mRNA is detected in animal blastomeres of the 16-cell embryo (side view). B: It is expressed throughout the ectoderm of the gastrula, but not in the endoderm (side view). C: Bop-1 is expressed throughout the neural plate, preplacodal ectoderm (PPE), and dorsal epidermis; anterolateral view). D: MGC68939 is expressed in the anterior neural tube, cranial neural crest, PPE, and some placodes (e.g., olf; anterolateral view). E: At early tail bud, MGC68939 is expressed in placodes (L, oto), the migrating neural crest, somites and tail bud (side view). F: At late tail bud, Bop-1 is expressed in the brain, retina, placodes (L, oto), branchial arches, somites, nephric mesoderm, tail bud, and ventral gut (side view). G: At late tail bud stages, MGC68939 staining is very similar to that of Bop-1, ventral gut staining is not detected until larval stages (side view). H: Transverse sections at forebrain (H), hindbrain (I), rostral spinal (J), and more caudal spinal (K) levels demonstrate neural (fb, r, hb, sc), branchial arch, lens, otocyst, somite, nephric mesoderm, and ventral gut expression.
	Fig. 11. Expression of Gadd45-related genes. A: Weak expression of Gadd45g in the animal cap ectoderm of the blastula (side view). B: Gadd45a is diffusely expressed in the ectoderm and noninvoluted mesoderm of the gastrula (side view). C,D: Gadd45a is expressed throughout the neural plate, PPE and cement gland (C, anterior view; D, dorsal view). E,F: At neural tube closure, Gadd45a expression in the anterior part of the embryo (E) is enhanced in the cement gland and pineal, and in the posterior part (F) in a patch of paraxial mesoderm (pm) and in mesoderm surrounding the cloaca (arrowhead). G: At neural tube stages, Gadd45g is expressed in the neural tube, including pineal, and multiple placodes (olf, L, Vp, dlp; anterior view). H: At neural plate, Gadd45g is expressed in stripes of primary neurons (pn) and the trigeminal placode (Vp; dorsal view). I: At larval stages, Gadd45a expression becomes weak and diffuse throughout the head and anterior trunk, and is lost from the cement gland (side view). J: At larval stages, Gadd45g expression is confined to the brain, retina, anterior spinal cord, and placode derivatives (olf, L, crg, oto, ebp; side view). K,L,M: Transverse sections at forebrain (L), hindbrain (M), rostral spinal (K) levels demonstrate neural (fb, r, hb, sc) and placodal (L, oto, crg, ebp) expression.
	tle4 (transducin-like enhancer of split 4 (E(sp1) homolog)) gene expression in Xenopus tropicalis embryos, NF stage 28, assayed by in situ hybridization, head and upper trunk region, lateral view, anterior left, dorsal up.
	bop1 (BOP1 ribosomal biogenesis factor) gene expression in Xenopus tropicalis embryos, NF stage 28, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	bop1 (BOP1 ribosomal biogenesis factor) gene expression in Xenopus tropicalis embryos, NF stage 15, as assayed by in situ hybridization, anterodorsal-lateral view, anterior left, dorsal up.
	ccdc85c (coiled-coil domain containing 85C) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 18, anterior view, dorsal up.
	ccdc85c (coiled-coil domain containing 85C ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, head and trunk region, anterior left, dorsal up.
	zmym2 (zinc finger, MYM-type 2) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 5 (16 cell), lateral view, animal pole up.
	zmym2 (zinc finger, MYM-type 2) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 20/21, anterior-lateral view, dorsal up.
	mcrs1 (microspherule protein 1) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, A: NF stage 17/18, dorsal view, anterior right. B: NF stage 20/21, anterior view dorsal up.
	mcrs1 (microspherule protein 1) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage , lateral view, anterior left, dorsal up.
	mcrs1 (microspherule protein 1) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 28, transverse section through head region view, dorsal up.
	ddx43 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 43) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 17/18, anterior view, dorsal up.
	ddx43 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 43) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 20/21, anterior- lateral view, dorsal up.
	ddx43 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 43) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	csnk2b (casein kinase 2, beta polypeptide) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 9/10, lateral view, anterior left, dorsal up.
	csnk2b (casein kinase 2, beta polypeptide) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 20/21, lateral view, anterior left, dorsal up.
	csnk2b (casein kinase 2, beta polypeptide) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
	pa2g4 (proliferation-associated 2G4, 38kDa) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 17/18, anterior-lateral view, dorsal up.
	pa2g4 (proliferation-associated 2G4, 38kDa) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage , lateral view, anterior left, dorsal up.
	eya2 (EYA transcriptional coactivator and phosphatase 2 ) gene expression in X. laevis embryo, assayed via in situ hybridization, NF stage 17, anterior view, dorsal up.
	eya2 (EYA transcriptional coactivator and phosphatase 2 ) gene expression in X. laevis embryo, assayed via in situ hybridization.
	eya2 (EYA transcriptional coactivator and phosphatase 2 ) gene expression in X. laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior left.
	eya2 (EYA transcriptional coactivator and phosphatase 2 ) gene expression in X. laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view of head region, anterior left, dorsal up. key: pl: cranial placodes, ne: pronephric mesenchyme, oto: otic vesicle, olf: olfactory placode. L: lens placode with no expression.
	eya2 (EYA transcriptional coactivator and phosphatase 2 ) gene expression in X. laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view of head and trunk region, anterior left, dorsal up.

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