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First published online 29 August 2007
doi: 10.1242/dev.007906

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SOX3 activity during pharyngeal segmentation is required for craniofacial morphogenesis

Division of Developmental Genetics, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

View larger version (41K): [in this window] [in a new window]   Fig. 1. Craniofacial defects in Sox3 null mice. (A) Wild-type mouse. (B) Sox3 null mouse with teeth overgrowth and vibrissae paralysis on the left side. (C-H) Skeletal preparation of wild-type (C) and Sox3 null (D) newborns. Higher magnification of the middle ear in a control (E) and mutant (F), where the tympanic ring and distal part of the styloid bone are absent and the stapes and distal part of the malleus are malformed. Magnification of the hyoid bone in wild-type (G) and mutant (H) newborn with abnormal orientation of the lesser horn (asterisk) on the left side. (I) Statistical analysis of the craniofacial skeletal defects in Sox3 null mice (n=14). I, incus; M, malleus; Sp, stapes; St, styloid process; Tr, tympanic ring.

View larger version (98K): [in this window] [in a new window]   Fig. 2. Ncc migration defect in Sox3 null mouse embryos. (A-F) CRABPI immunofluorescence and 3D reconstruction. (A,B) Sox3 null embryo at 10 ss: only confocal sections through the region containing migrating ncc were used for 3D reconstruction (to avoid foregut diverticulum background). (A) Right side with PA2 migrating ncc. (B) Left side with ncc accumulating in front of PA2 (compare distance covered by ncc between both sides, arrows). (C) Wild-type and (D) Sox3 null embryo at 9.5 dpc, with ncc accumulating in front of PA2 (arrow). Staining is present in the distal part of the arch. (E) Wild-type and (F) Sox3 null embryo at 10.5 dpc with hypoplastic PA2. The bulge of ncc observed at 9.5 dpc is strongly reduced. (G,H) Dlx2 in situ hybridisation on 10.5 dpc wild-type (G) and Sox3 null embryos (H): reduction of Dlx2 staining in proximal PA2 (arrow). (I,J) PAAs staining by ink injection in wild-type (I) and Sox3 null (J) 9.5 dpc embryos, where PAA2 is missing. (K) Apoptosis quantification. An average of 42.7±1.95 CRABP/TUNEL-positive cells were present on the left side of affected embryos, whereas we counted 22.45±2 on the right side (P<0.0001, n=7). (L-O) TUNEL assay and CRABPI immunofluorescence. Coronal section of 9.5 dpc Sox3 null embryo affected on the left side only. DAPI (L). TUNEL assay (M). CRABPI (N). TUNEL/CRABPI merge picture (O). More apoptotic ncc are present on the left side of the embryo. Scale bar: 75 µm in L for L-O. a1-3, pharyngeal arch arteries 1-3; as, aortic sac; da, dorsal aorta.

View larger version (88K): [in this window] [in a new window]   Fig. 3. Marker analysis in the mouse hindbrain at 9.5 dpc. (A,B) Hoxa2 in situ hybridisation on flat mounted hindbrain in wild-type (A) and mutant (B) embryos. (B) Arrow points to the abnormal proximal pa2 on the left side. (C,D) EphA4 in situ hybridisation in wild-type (C) and mutant (D) embryos. (E,F) Hoxb1 in situ hybridisation in wild-type (E) and mutant (F) embryos. No significant difference was observed in the mutants for these three markers.

View larger version (62K): [in this window] [in a new window]   Fig. 4. Cranial nerves and epibranchial placode formation in mice. (A-C) Anti-neurofilament immunochemistry at 10.5 dpc. Wild-type embryo (A). (B) Sox3 null embryo with a fusion of the IX and X cranial nerves. (C) Sox3 null embryo, in which nerves from the distal VII and IX ganglia do not reach PA2 and PA3, respectively (inset corresponding to the boxed region). (D,E) Phox2a in situ hybridisation at 9.5 dpc, showing the epibranchial placodes. (D) Wild type. (E) Sox3 null embryo with a reduced geniculate placode and a fusion of the petrosal and nodose placodes. (F,G) Ngn2 in situ hybridisation at 10.5 dpc. (F) Wild-type embryo (magnification of petrosal/nodose placodes, inset). (G) Sox3 null embryo, in which petrosal and nodose placodes are connected (magnification, inset). (H) Statistical analysis of the placodal and cranial nerves defects in Sox3 null embryos.

View larger version (90K): [in this window] [in a new window]   Fig. 5. SOX3 and SOX2 expression in the pharyngeal region in wild-type and mutant mouse embryos. (A,E,I) Whole-mount immunofluorescence and 3D reconstruction of embryo sagittal halves. (B-D,F-H,J-L) Immunofluorescence on sections. (A,E) Anti-SOX3 immunofluorescence. (A) Expression at 8 ss in the neural plate and pharyngeal region. (B-D) Transverse section at 8-10 ss at PP1 level (section plan shown in A). (B) DAPI. (C) SOX3 is detected in the proximal pharyngeal ectoderm and underlying lateral endoderm (note non-specific staining in foregut diverticulum); inset magnified PP1 region boxed. (D) SOX2 is present more widely than SOX3 in the pharyngeal endoderm. (E) At 9.5 dpc SOX3 expression is restricted to the posterior pouch margins. (F-H) Coronal section in the proximal pharyngeal region at 9.5 dpc. (F) DAPI. (G) SOX3 is detected in both epithelia in the posterior pouch margin. This regionalisation is acquired as the arches develop: in the more caudal PA3, SOX3 expression is not as restricted as in PA2. (H) SOX2 is present in the pharyngeal endoderm and weakly in the ectoderm. (I) Anti-GFP immunofluorescence on a Sox3 null embryo at 9.5 dpc. GFP (from the Sox3 locus) is present across the reduced proximal region of PA2 (arrow). (J-L) Coronal section in the proximal pharyngeal region at 9.5 dpc in a Sox3 null embryo. (J) DAPI staining (inset: magnified abnormal PA2, boxed). (K) GFP is present all around the hypomorphic proximal region of PA2. (L) SOX2 expression also highlights the abnormal morphology of PA2 (expression is stronger in the ectoderm than in H as the section is more distal). Scale bars: 50 µm in B, for B-D; in F, for F-H and J-L.

View larger version (117K): [in this window] [in a new window]   Fig. 6. Marker analysis in mouse pharyngeal pouches at 9.5 dpc. (A,B) Pax1 in situ hybridisation in wild type (A) and Sox3 null (B) embryos. Note the abnormal appearance of proximal PA3 (arrow). (C,D) Coronal sections through the proximal pharyngeal region of wild-type (C) and Sox3 null (D) embryos hybridised with Pax1. The domain of expression is expanded throughout PA2 endoderm (arrow). (E,F) Bmp7 in situ hybridisation in wild-type (E) and Sox3 null (F) embryos. (G,H) Coronal sections of wild-type (G) and Sox3 null (H) embryos hybridised with Bmp7. (I,J) Fgf8 in situ hybridisation in wild type (I) in the anterior pouch margin (arrow) and Sox3 null embryo (J), where it is present across the reduced PA2 (bracket). (K,L) Coronal sections of wild-type (K) and Sox3 null (L) embryos hybridised with Fgf8. (M,N) Fgf3 in situ hybridisation in wild type (M) in the posterior pouch margin (arrow) and Sox3 null embryo (N), where it is present across the reduced PA2 (bracket). (O,P) Bmp4 in situ hybridisation in wild-type (O) and Sox3 null (P) embryos with uninterrupted expression between first and second pharyngeal clefts, arrow. All these markers of the pouch endoderm and/or ectoderm show an expansion of their expression across proximal PA2 in the mutant embryos. Scale bar: 100 µm in C for all sections.

View larger version (67K): [in this window] [in a new window]   Fig. 7. Morphology of proximal PA2 in Sox3 null mouse embryos. (A,B,D,E,G-I) Confocal sections of phalloidin-stained 9.5 dpc wild-type (A) and Sox3 null (D) embryos. PP1 and PP2 appear connected in the mutant. (B,E) Higher magnification and deeper sections of a wild-type (B) and Sox3 null (E) embryos with a `stem' connecting distal PA2 (arrow). (C,F) Confocal sections of phalloidin and anti-CRABPI double-stained 9.5 dpc wild-type embryo (C) and Sox3 null mutant (F), where ncc migrate through the `stem'. (G-I) High magnification of PP1 posterior margin in wild type (G) and of the `stem' in mutant embryos (H,I). Superficial section (H), where margin-like cells form a `stem' but show a disruption of apical actin accumulation. Deeper section (I), where two margins flank what is left of PA2. Actin polarisation is also disrupted. (J-M) Anti-N-cadherin immunofluorescence on 9.5 dpc embryos. (J) 3D reconstruction of a wild-type embryo sagittal half, inside view. Expression is seen in the neural tube and pharyngeal endoderm. (K) High magnification of a confocal section (region boxed in H) with apical accumulation of N-cadherin in the pouch margin. (L) 3D reconstruction of a mutant embryo sagittal half, inside view. (M) High magnification of confocal section (region boxed in J) with a disruption of apical N-cadherin accumulation. (N) Anti-GFP immunohistochemistry of a 17 ss Sox3 null embryo (transverse section, PA2 level). The close apposition between ectoderm and endoderm on one side (arrow) interrupts PA2. (O,P) Spry-1 in situ hybridisation on 8/9 ss wild-type (O) and 7/8 ss Sox2+/-; Sox3Y/- (P) embryos sectioned at pharyngeal level, posterior to PA1; the gene is expressed in pharyngeal epithelia and mesenchyme. No significant difference is observed between wild type and double mutant. Scale bar: 100 µm in A for A,D; 50 µm for B,E and O,P; 25 µm for C,F; 10 µm for G,H,I,K,M. ov, otic vesicle.

View larger version (51K): [in this window] [in a new window]   Fig. 8. Ncc migration defects in Sox3; Fgfr1 double mutants. (A) Migration defects were analysed at 9.5 dpc by CrabpI in situ hybridisation. Mouse embryos were littermates obtained from Sox3+/- females mated with Fgfr1n7/+ males. Introduction of the hypomorphic Fgfr1 allele increases the penetrance of the ncc defects on the Sox3 mutant background. (Note: the penetrance of the Sox3 null phenotype is reduced in this cross, perhaps due to the introduction of the different background on which the Fgfr1 mutation is maintained, but the sample size is also small.) (B-E) CrabpI in situ hybridisation at 9.5 dpc on (B) wild-type, (C) Sox3 null, (D) Sox3+/-; Fgfr1n7/+ and (E) Sox3 null; Fgfr1n7/+ embryos showing increasing severity of PA2 defects in double mutants. Insets: PA2 domain magnification.

View larger version (25K): [in this window] [in a new window]   Fig. 9. Model illustrating the origin of the Sox3 null PA2 phenotype. The position of future PP1 is determined by a focalised endodermal outpocketing at 8.0-8.5 dpc (A). Studies in urochordates (Manni et al., 2002) have shown that ectodermal and endodermal cell interdigitations then result in the opening of the pouch, surrounded by a newly formed epithelium, the pouch margin. At this stage SOX3 is restricted to the posterior pouch margin and ncc are migrating in PA2. In the absence of Sox3 (B), the endoderm contacts the ectoderm over a much wider region, resulting in the formation of an enlarged pouch when the perforation occurs, and also an enlarged pouch margin. As a consequence, ncc migration is impaired.

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