Schuff M et al. (2007), FoxN3 is required for craniofacial and eye deve...

XB-ART-34864

Dev Dyn 2007 Jan 01;2361:226-39. doi: 10.1002/dvdy.21007.

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FoxN3 is required for craniofacial and eye development of Xenopus laevis.

Schuff M , Rössner A , Wacker SA , Donow C , Gessert S , Knöchel W .

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A functional knockdown of FoxN3, a member of subclass N of fork head/winged helix transcription factors in Xenopus laevis, leads to an abnormal formation of the jaw cartilage, absence or malformation of distinct cranial nerves, and reduced size of the eye. While the eye phenotype is due to an increased rate of apoptosis, the cellular basis of the jaw phenotype is more complex. The upper and lower jaw cartilages are derivatives of a subset of cranial neural crest cells, which migrate into the first pharyngeal arch. Histological analysis of FoxN3-depleted embryos reveals severe deformation and false positioning of infrarostral, Meckel's, and palatoquadrate cartilages, structural elements derived from the first pharyngeal arch, and of the ceratohyale, which derives from the second pharyngeal arch. The derivatives of the third and fourth pharyngeal arches are less affected. FoxN3 is not required for early neural crest migration. Defects in jaw formation rather arise by failure of differentiation than by positional effects of crest migration. By GST-pulldown analysis, we have identified two different members of histone deacetylase complexes (HDAC), xSin3 and xRPD3, as putative interaction partners of FoxN3, suggesting that FoxN3 regulates craniofacial and eye development by recruiting HDAC.

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Species referenced: Xenopus laevis
Genes referenced: foxd3 foxn2 foxn3 hdac3 hoxa2 mxd1 nkx3-2 rax sin3a snai1 sox3 tfap2a uqcc6
GO keywords: neural crest cell migration [+]
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	Figure 3. Analysis of the FoxN3 small eye phenotype. Thirteen nanograms of FoxN3-MO were co-injected with 200 pg GFP RNA in one blastomere at 2-cell stage. Asterisks denote the injected side. A,B: Whole mount in situ hybridization of unilaterally FoxN3-MO-injected embryos showing xRx staining in the eye field. A: Frontal view of a stage-16 embryo. B: Dorsal view of a stage-30 embryo. The insert shows a magnification of the right and left eye. No difference in xRx expression between injected and uninjected side is noted. C,C': Comparison of the injected (FoxN3-MO) and uninjected side of an embryo at stage 35. C' shows the injected side. No difference in the size of the forming eyes is observed. D,D': Right and left view of an unilaterally injected embryo, stage 39. D' shows the injected side. The eye at the FoxN3-MO injected side is clearly reduced in comparison to the uninjected side. E, F: Sections of Alcian blue-stained eyes of the uninjected control side (E) and the FoxN3-MO-injected side (F) of an embryo at stage 48. F: The eye is smaller but shows a normal structure. The different layers of the retina are clearly visible indicating that the differentiation of the retina is not affected by FoxN3-MO injection. The black arrows denote the optic nerve, indicating identical planes of section in E and F.
	Figure 5. Analysis of cranial nerve structures in FoxN3-depleted embryos. Thirteen nanograms FoxN3-MO was injected into each blastomere of 2-cell stage embryos. Developing nerves were visualised by 3A10 monoclonal antibody staining in whole embryos at stage 48. A: Ventral view on a bilaterally injected embryo shows a complete absence of hypoglossal nerves at both sides of the body. B: Ventral view on wild-type embryo. C: Dorsal view on a FoxN3-depleted embryo demonstrates a diffuse arrangement of cranial nerves. The nervus nasociliaris, a branch of the trigeminal nerve, and the maxillary nerve are strongly reduced, while the facial nerve shows normal appearance. The nervus frontalis is completely absent. D: Dorsal view on wild-type embryo. fa, nervus facialis; fo, nervus frontalis; hg, nervus hypoglossus; max, nervus maxillare; md, nervus mandibulare; nc, nervus nasociliaris. Roman numbers indicate the cranial nerves II (optic), III (oculomotorius), and V (trigeminus).
	Figure 6. Effects of FoxN3 depletion on neural crest formation. Embryos were unilaterally injected with 13 ng FoxN3-MO and 500 pg GFP RNA. After sorting for right/left injections, embryos were submitted at different developmental stages to in situ whole mount hybridization. The asterisks indicate the side of injection. A: FoxD3, stage 23. B:AP2 alpha , stage 23. C:snail, stage 23. D:AP2 alpha , stage 29. E:snail, stage 29. No difference in the expression domains between injected and uninjected sides can be observed. F,G: Whole mount in situ hybridization of unilaterally injected embryos stained for HoxA2 (F) and Sox3 (G). Embryos were injected at their right sides. H-K: Whole mount in situ hybridization of bilaterally FoxN3-MO or Co-MO-injected embryos (13 ng pro blastomere). H:Xbap, Co-MO-injected, stage 38. I:Xbap, FoxN3-MO injected, stage 38. Xbap expression is visible close to the cement gland in the controls (see arrow). The FoxN3-MO-injected embryos show reduced expression of Xbap in the prospective jaw (see arrow). The ventral part of this expression domain is completely absent. J:Xbap, Co-MO-injected, stage 43, ventral view. K:Xbap, FoxN3-MO-injected, stage 43, ventral view. The Xbap expression in the controls is restricted at stage 43 within the head to Meckel's cartilage. FoxN3-MO-injected embryos show severe reduction of staining (see arrows).
	Xenopus snai1 / snail gene expression
	Figure 1. Inhibition of in vivo translation of a FoxN3/GFP fusion construct by FoxN3‐MO. The 5′‐coding sequence (300 bp) of FoxN3 including the translation start codon were fused to GFP (FoxN3/GFP). Alternatively, six silent mutations in the putative morpholino binding site were introduced (FoxN3Res/GFP). RNA of these constructs was injected into both blastomeres at the 2‐cell stage and embryos were analysed at stage 31. A: 5′‐coding sequence (25 bp) of FoxN3 is reverse complementary to the FoxN3 morpholino (FoxN3‐MO) sequence. Silent mutations in the FoxN3Res/GFP construct are indicated. B,D,F: Lateral views on embryos injected with 0.5 ng FoxN3/GFP RNA (B), co‐injected with 13 ng Co‐MO (D) or with 13 ng FoxN3‐MO (F) into each blastomere. C,E,G: Lateral views on embryos injected with 0.5 ng FoxN3Res/GFP RNA containing the silent mutations (C), co‐injected with 13 ng Co‐MO (E) or with 13 ng FoxN3‐MO (G) into each blastomere. Embryos are photographed under normal light (left) and under UV light (right). Fluorescence is visible in embryos with FoxN3Res/GFP RNA injection and FoxN3‐MO (G), but not with FoxN3/GFP RNA and FoxN3‐MO (F).
	Phenotype of FoxN3‐MO injected embryos during embryogenesis. Embryos were injected with morpholino (13 ng/blastomere) either in two (A–C) or in one blastomere (D) at 2‐cell stage. A: Comparison of FoxN3‐MO‐injected embryos (right side) and Co‐MO‐injected embryos (left side) at stage 41, lateral view. The FoxN3‐MO‐injected embryos reveal smaller eyes, while the trunk appears to be quite normal. B: Same analysis at stage 43. The control embryos (left side) grow normal, while FoxN3‐MO‐injected show severe defects. The endophthalmus of the eyes is clearly visible. The head becomes malformed and the whole embryos become oedemic. C: Magnification of the head of a stage‐45 embryo (dorsal view). Cranial structure of Co‐MO‐injected embryo (left side) is normal. The FoxN3‐MO‐injected embryo (right side) is smaller. The nose and mouth region is invaginated. D: Dorsal view of an unilaterally FoxN3‐MO‐injected embryo at stage 45 (bottom side is injected). The left eye is clearly reduced, as well as the anterior cartilage. E: Diagram of eye and cartilage phenotypes at stage 48 presented as dose dependency of FoxN3‐MO. Indicated amounts of FoxN3‐MO were injected into each blastomere at the 2‐cell stage. Embryos were scored for eye phenotype (red) and for cartilage phenotype (blue). The numbers at the bottom indicate injected embryos and the percentage of surviving embryos at stage 48.
	Figure 4. Craniofacial cartilage defects of FoxN3‐depleted embryos. Embryos were injected with 13 ng FoxN3‐MO in each blastomere at 2‐cell stage. A: Dorsal view on an Alcian blue–stained wild‐type embryo at stage 48. B: The corresponding schematic drawing of the craniofacial cartilage. C: Dorsal view on a FoxN3‐MO injected and Alcian blue–stained embryo at stage 48. D: The corresponding schematic drawing of the craniofacial cartilage. Frontal view (E, G) and sections (F, H) of Alcian blue–stained Co‐MO (E, F) and FoxN3‐MO‐injected embryos (G, H). FoxN3‐MO‐injected embryos reveal an invagination of the olfactory region, characterised by a large cleft. Many of the cranial cartilage elements are malformed or absent. The white lines in E and G denote plane of sections shown in F and H. ac, auditory cap; apc, anterior parachordal cartilage; ba, branchial arches; bb, basobranchiale; br, brain; ch, ceratohyale; fo, foramen olfactorium; ic, infrarostal cartilage; it, intestine; mc, Meckel's cartilage; mpp, muscular process of palatoquadrate; nc, notochord; pcqm, processus cornu quadratus medialis; pq, palatoquadrate; sbp, subocular bars of the palatoquadrate; sfen, subocular fenestra; sp, suprarostral plate; ta, tectum anterius; tr, trabecula; tenc, tentacular cartilage; vpp, ventrolateral process of palatoquadrate.
	Cranial structures in Xenopus. A: Dorsal view of an Alcian blue–stained wild‐type Xenopus embryo at NF stage 48. B: The corresponding schematic drawing of the craniofacial cartilages and other head structures and organs. Key: ac, auditory cap; apc, anterior parachordal cartilage; ba, branchial [pharyngeal] arches; bb, basobranchiale; br, brain; ch, ceratohyale; fo, foramen olfactorium; ic, infrarostal cartilage; it, intestine; mc, Meckel's cartilage; mpp, muscular process of palatoquadrate; nc, notochord; pcqm, processus cornu quadratus medialis; pq, palatoquadrate; sbp, subocular bars of the palatoquadrate; sfen, subocular fenestra; sp, suprarostral plate; ta, tectum anterius; tr, trabecula; tenc, tentacular cartilage; vpp, ventrolateral process of palatoquadrate.
	Cranial structures in Xenopus. E. Frontal view and F. section of Alcian blue–stained section through head of wild‐type Xenopus embryo at NF stage 48. Key: ba, branchial [pharyngeal] arches; bb, basobranchiale; ch, ceratohyale; mc, Meckel's cartilage; mpp, muscular process of palatoquadrate; pq, palatoquadrate;
	Cranial nerves in Xenopus embryo, at NF stage 48. B: Ventral view, D:Dorsal view on wild‐type embryo. Key: fa, nervus facialis [facial nerve]; fo, nervus frontalis; hg, nervus hypoglossus [hypoglossal nerve]; max, nervus maxillare; md, nervus mandibulare; nc, nervus nasociliaris. Roman numbers indicate the cranial nerves: cranial nerve II [optic nerve]), cranial nerve III (oculomotorius) [oculomotor nerve], and cranial nerve V (trigeminus) [trigeminal nerve].
	Analysis of cranial crest cell migration during embryogenesis. Embryos were injected with 200 pg EosFP RNA and 13 ng FoxN3‐MO into each blastomere at 2‐cell stage. At stage 26, cells of the mandibular arch were photoconverted. A: Lateral view of FoxN3‐MO‐injected embryo, stage 26. B: Lateral view of Co‐MO‐injected embryo, stage 26. C: Schematic representation of cranial crest migration at stage 26 (lateral view, converted area is shown as dashed circle). D: Embryo shown in A at stage 46, ventral view. The FoxN3‐MO injected embryos show an unshaped structure of Meckel's cartilage. E: Embryo shown in B at stage 46, ventral view. The Co‐MO‐injected embryos revealed a normal structure of Meckel's cartilage. The dotted red cells in D and the red cells at the anterior front of the embryo in E represent converted epidermal cells. F: Cartilage structures (ventral view) at stage 48 (modified from: Sadaghiani and Thiebaud, 1987; Baltzinger et al., 2005). The rectangle in F denotes the area seen in D and E. 1, mandibular arch (blue); 2, hyoid arch (yellow); 3, anterior branchial arch (green); 4, posterior branchial arch (brown). ba, branchial arch; ch, ceratohyale; l, lens; mc, Meckel's cartilage; pq, palatoquadrate.
	Schematic representation of cranial neural crest cell migration pathways during embryogenesis (C) at stage 26, and (F) tissue to which they contribute. Cartilage structures (ventral view) at stage 48 (modified from: Sadaghiani and Thiebaud, 1987; Baltzinger et al., 2005). The rectangle in F denotes the area seen in D and E. 1, mandibular arch (blue); 2, hyoid arch (yellow); 3, anterior branchial arch (green); 4, posterior branchial arch (brown). ba, branchial arch; ch, ceratohyale; l, lens; mc, Meckel's cartilage; pq, palatoquadrate. Image extracted from Figure 7, XB-ART-34864.
	Figure 8. Analysis of apoptosis and cell proliferation in FoxN3‐depleted embryos. Embryos were co‐injected with 13 ng FoxN3‐MO and 200 pg GFP RNA into one blastomere at 2‐cell stage. An asterisk denotes the injected side of embryos. A, A': Left and right view on an unilaterally FoxN3‐MO‐injected and TUNEL‐stained embryo at stage 24. A' shows the injected side. Almost no apoptotic cells can be observed on either side. B, B': Left and right view on the head of an unilaterally FoxN3‐MO‐injected and TUNEL‐stained embryo stage 32. The injected side (B') shows a higher rate of apoptosis than the uninjected side (B). C: Section of an unilaterally FoxN3‐MO‐injected embryo at stage 41. An increased number of apoptotic cells is found on the injected side, especially within the eye. D, E: Tissue sections of the eye of a unilaterally injected embryo at stage 41. The eye of the injected side (E) shows an increase of apoptosis in the retina as compared to the uninjected side (D). F–H: Tissue sections of unilaterally injected embryos analysed by BrdU immunohistochemistry. F: Section of the head of an embryo at stage 39/40. No difference in the proliferation rate of the injected side in comparison to the uninjected side can be observed. G, H: Sections of BrdU stained eyes of an unilaterally injected embryo at stage 45. The labeled cells of the retina indicate the ciliary marginal zone (arrowheads), but are equally stained in the uninjected (G) and in the FoxN3‐MO‐injected side (H).
	Figure 9. FoxN3 interacts with components of the histone deacetylase complex (HDAC). A: Scheme of FoxN3 deletion constructs used for GST‐pull down assays. The fork head/winged helix domain is indicated by a black box. B: [35S] methionine‐labeled Sin3 protein was incubated with GST‐fusions of the two isoforms of FoxN3 or with GST‐FoxN2, respectively. The pull‐down reactions were analysed using a 10% SDS‐PAGE. C: Pull‐down analysis of GST‐FoxN3 deletion mutants with [35S] methionine‐labeled Sin3 protein. Sin3 requires for binding the C‐terminus of FoxN3 but not the N‐terminus. D: [35S] methionine‐labeled RPD3 protein was incubated with GST‐FoxN3 mutants. RPD3 binds to N‐terminal, but not to C‐terminal region of FoxN3 deletion constructs. E: Alignment of putative Sin3 binding sites of FoxN2, FoxN3, and human Mad1 (see: van Ingen et al., 2004). Conserved amino acids are underlined and shown in grey.