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Neural crest cells organize the eye via TGF-β and canonical Wnt signalling.

Grocott T, Johnson S, Bailey AP, Streit A - Nat Commun (2011)

Bottom Line: In vertebrates, the lens and retina arise from different embryonic tissues raising the question of how they are aligned to form a functional eye.Here we show, using the chick as a model system, that neural crest-derived transforming growth factor-βs activate both Smad3 and canonical Wnt signalling in the adjacent ectoderm to position the lens next to the retina.They do so by controlling Pax6 activity: although Smad3 may inhibit Pax6 protein function, its sustained downregulation requires transcriptional repression by Wnt-initiated β-catenin.

View Article: PubMed Central - PubMed

Affiliation: Department of Craniofacial Development, King's College London, Guy's Campus, London SE1 9RT, UK.

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Wnt mediates lens repression by NCCs and TGF-β.(a–c) Wnt2b (blue) gene expression during neural crest cell (NCC) migration: (a) Whole-mount embryo showing the forebrain section used in panel b and the midbrains section used in panel c. (b) Forebrain level section stained for Wnt2b; (c) midbrain level section stained for Wnt2b. (d) Forebrain level section stained for Axin2 gene expression (blue). (e) Midbrain level section showing Axin2 expression. All sections are immunostained for NCC-specific HNK1 (brown). Arrowheads indicate gene expression in non-lens ectoderm. (f–h) Presumtive lens ectoderm (PLE) explants were cultured alone or in combination with NCC or SB431542 and assayed for Wnt2b gene expression (blue) and HNK1 (brown): (f) PLE alone; (g) PLE+NCC; (h) PLE+NCC+SB431542. (i, j) PLE explants were cultured alone or with Activin A and assayed for Wnt2b gene expression: (i) PLE alone; (j) PLE+Activin A. (k, l) Wnt2b gene expression (blue) following in ovo ablation of premigratory NCCs: (k) control embryo; (l) NCC ablated embryo. Compare Wnt2b expression in the brain (white arrowheads) with non-lens surface ectoderm (yellow arrowheads). (m, n) Wnt2b gene expression (blue) following in ovo electroporation of expression vectors encoding green fluorescent protein (GFP) or Smad7+GFP: (m) GFP; (n) Smad7+GFP. (o–q) PLE explants were cultured alone or in combination with Activin A or N-Fz8 and assayed for Pax6 gene expression (blue): (o) PLE alone; (p) PLE+Activin A; (q) PLE+Activin A+N-Fz8. (r) Proposed molecular model to explain TGF-β- and Wnt-mediated lens restriction. Broken lines: interactions inferred from the literature. (s) Proposed embryological model summarizing how NCCs organize the eye: NCCs (blue) secrete TGF-βs, which signal to the non-lens ectoderm and dorsal optic vesicle. As a consequence, Wnt2b (red) is induced, and together they repress lens formation in the non-lens ectoderm. This leads to the alignment of Pax6 expression in the future lens and neural retina (grey). Scale bars: a, 500 μm; b, 50 μm for panels b–e; f, 50 μm for panels f–j, o–q; k, 250 μm for panels k, l; m, 250 μm for panels m, n.
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f3: Wnt mediates lens repression by NCCs and TGF-β.(a–c) Wnt2b (blue) gene expression during neural crest cell (NCC) migration: (a) Whole-mount embryo showing the forebrain section used in panel b and the midbrains section used in panel c. (b) Forebrain level section stained for Wnt2b; (c) midbrain level section stained for Wnt2b. (d) Forebrain level section stained for Axin2 gene expression (blue). (e) Midbrain level section showing Axin2 expression. All sections are immunostained for NCC-specific HNK1 (brown). Arrowheads indicate gene expression in non-lens ectoderm. (f–h) Presumtive lens ectoderm (PLE) explants were cultured alone or in combination with NCC or SB431542 and assayed for Wnt2b gene expression (blue) and HNK1 (brown): (f) PLE alone; (g) PLE+NCC; (h) PLE+NCC+SB431542. (i, j) PLE explants were cultured alone or with Activin A and assayed for Wnt2b gene expression: (i) PLE alone; (j) PLE+Activin A. (k, l) Wnt2b gene expression (blue) following in ovo ablation of premigratory NCCs: (k) control embryo; (l) NCC ablated embryo. Compare Wnt2b expression in the brain (white arrowheads) with non-lens surface ectoderm (yellow arrowheads). (m, n) Wnt2b gene expression (blue) following in ovo electroporation of expression vectors encoding green fluorescent protein (GFP) or Smad7+GFP: (m) GFP; (n) Smad7+GFP. (o–q) PLE explants were cultured alone or in combination with Activin A or N-Fz8 and assayed for Pax6 gene expression (blue): (o) PLE alone; (p) PLE+Activin A; (q) PLE+Activin A+N-Fz8. (r) Proposed molecular model to explain TGF-β- and Wnt-mediated lens restriction. Broken lines: interactions inferred from the literature. (s) Proposed embryological model summarizing how NCCs organize the eye: NCCs (blue) secrete TGF-βs, which signal to the non-lens ectoderm and dorsal optic vesicle. As a consequence, Wnt2b (red) is induced, and together they repress lens formation in the non-lens ectoderm. This leads to the alignment of Pax6 expression in the future lens and neural retina (grey). Scale bars: a, 500 μm; b, 50 μm for panels b–e; f, 50 μm for panels f–j, o–q; k, 250 μm for panels k, l; m, 250 μm for panels m, n.

Mentions: In a survey of Wnt expression, we did not find any Wnt transcripts present in migrating NCCs. However, we did find a strong correlation between NCC migration and Wnt2b expression in the ectoderm: following contact with migrating NCCs, Wnt2b began to be expressed in the neighbouring ectoderm but remained absent from the prospective lens territory (Fig. 3a–c). The highest levels of Axin2 expression (a read-out of canonical Wnt activity) were present in ectoderm that had been in contact with NCCs for longest duration (Fig. 3d,e, arrowheads; compare midbrain and forebrain levels). These observations suggest that NCCs initiate Wnt2b expression in the ectoderm in which lens formation is normally inhibited. Indeed, migratory NCCs strongly induced Wnt2b in presumptive lens explants, which normally do not express the transcript (Fig. 3f,g), suggesting that TGF-β may be the NCC signal responsible for ectodermal Wnt2b. Indeed, in vitro, Wnt2b induction by NCCs failed when TGF-β signalling was inhibited (Fig. 3h), whereas Activin A strongly induced Wnt2b in presumptive lens explants (Fig. 3i,j). Similarly, in vivo, ectodermal Wnt2b expression depended on TGF-β signalling from NCCs: NCC ablation (Fig. 3k,l) and misexpression of the inhibitory Smad7 (Fig. 3m,n; Smad7: 2/6 Wnt2b+, green fluorescent protein: 5/5 Wnt2b+) caused downregulation of Wnt2b transcripts in the non-lens ectoderm. Together, these results show that TGF-β signalling from NCCs induces Wnt2b expression in the non-lens ectoderm.


Neural crest cells organize the eye via TGF-β and canonical Wnt signalling.

Grocott T, Johnson S, Bailey AP, Streit A - Nat Commun (2011)

Wnt mediates lens repression by NCCs and TGF-β.(a–c) Wnt2b (blue) gene expression during neural crest cell (NCC) migration: (a) Whole-mount embryo showing the forebrain section used in panel b and the midbrains section used in panel c. (b) Forebrain level section stained for Wnt2b; (c) midbrain level section stained for Wnt2b. (d) Forebrain level section stained for Axin2 gene expression (blue). (e) Midbrain level section showing Axin2 expression. All sections are immunostained for NCC-specific HNK1 (brown). Arrowheads indicate gene expression in non-lens ectoderm. (f–h) Presumtive lens ectoderm (PLE) explants were cultured alone or in combination with NCC or SB431542 and assayed for Wnt2b gene expression (blue) and HNK1 (brown): (f) PLE alone; (g) PLE+NCC; (h) PLE+NCC+SB431542. (i, j) PLE explants were cultured alone or with Activin A and assayed for Wnt2b gene expression: (i) PLE alone; (j) PLE+Activin A. (k, l) Wnt2b gene expression (blue) following in ovo ablation of premigratory NCCs: (k) control embryo; (l) NCC ablated embryo. Compare Wnt2b expression in the brain (white arrowheads) with non-lens surface ectoderm (yellow arrowheads). (m, n) Wnt2b gene expression (blue) following in ovo electroporation of expression vectors encoding green fluorescent protein (GFP) or Smad7+GFP: (m) GFP; (n) Smad7+GFP. (o–q) PLE explants were cultured alone or in combination with Activin A or N-Fz8 and assayed for Pax6 gene expression (blue): (o) PLE alone; (p) PLE+Activin A; (q) PLE+Activin A+N-Fz8. (r) Proposed molecular model to explain TGF-β- and Wnt-mediated lens restriction. Broken lines: interactions inferred from the literature. (s) Proposed embryological model summarizing how NCCs organize the eye: NCCs (blue) secrete TGF-βs, which signal to the non-lens ectoderm and dorsal optic vesicle. As a consequence, Wnt2b (red) is induced, and together they repress lens formation in the non-lens ectoderm. This leads to the alignment of Pax6 expression in the future lens and neural retina (grey). Scale bars: a, 500 μm; b, 50 μm for panels b–e; f, 50 μm for panels f–j, o–q; k, 250 μm for panels k, l; m, 250 μm for panels m, n.
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f3: Wnt mediates lens repression by NCCs and TGF-β.(a–c) Wnt2b (blue) gene expression during neural crest cell (NCC) migration: (a) Whole-mount embryo showing the forebrain section used in panel b and the midbrains section used in panel c. (b) Forebrain level section stained for Wnt2b; (c) midbrain level section stained for Wnt2b. (d) Forebrain level section stained for Axin2 gene expression (blue). (e) Midbrain level section showing Axin2 expression. All sections are immunostained for NCC-specific HNK1 (brown). Arrowheads indicate gene expression in non-lens ectoderm. (f–h) Presumtive lens ectoderm (PLE) explants were cultured alone or in combination with NCC or SB431542 and assayed for Wnt2b gene expression (blue) and HNK1 (brown): (f) PLE alone; (g) PLE+NCC; (h) PLE+NCC+SB431542. (i, j) PLE explants were cultured alone or with Activin A and assayed for Wnt2b gene expression: (i) PLE alone; (j) PLE+Activin A. (k, l) Wnt2b gene expression (blue) following in ovo ablation of premigratory NCCs: (k) control embryo; (l) NCC ablated embryo. Compare Wnt2b expression in the brain (white arrowheads) with non-lens surface ectoderm (yellow arrowheads). (m, n) Wnt2b gene expression (blue) following in ovo electroporation of expression vectors encoding green fluorescent protein (GFP) or Smad7+GFP: (m) GFP; (n) Smad7+GFP. (o–q) PLE explants were cultured alone or in combination with Activin A or N-Fz8 and assayed for Pax6 gene expression (blue): (o) PLE alone; (p) PLE+Activin A; (q) PLE+Activin A+N-Fz8. (r) Proposed molecular model to explain TGF-β- and Wnt-mediated lens restriction. Broken lines: interactions inferred from the literature. (s) Proposed embryological model summarizing how NCCs organize the eye: NCCs (blue) secrete TGF-βs, which signal to the non-lens ectoderm and dorsal optic vesicle. As a consequence, Wnt2b (red) is induced, and together they repress lens formation in the non-lens ectoderm. This leads to the alignment of Pax6 expression in the future lens and neural retina (grey). Scale bars: a, 500 μm; b, 50 μm for panels b–e; f, 50 μm for panels f–j, o–q; k, 250 μm for panels k, l; m, 250 μm for panels m, n.
Mentions: In a survey of Wnt expression, we did not find any Wnt transcripts present in migrating NCCs. However, we did find a strong correlation between NCC migration and Wnt2b expression in the ectoderm: following contact with migrating NCCs, Wnt2b began to be expressed in the neighbouring ectoderm but remained absent from the prospective lens territory (Fig. 3a–c). The highest levels of Axin2 expression (a read-out of canonical Wnt activity) were present in ectoderm that had been in contact with NCCs for longest duration (Fig. 3d,e, arrowheads; compare midbrain and forebrain levels). These observations suggest that NCCs initiate Wnt2b expression in the ectoderm in which lens formation is normally inhibited. Indeed, migratory NCCs strongly induced Wnt2b in presumptive lens explants, which normally do not express the transcript (Fig. 3f,g), suggesting that TGF-β may be the NCC signal responsible for ectodermal Wnt2b. Indeed, in vitro, Wnt2b induction by NCCs failed when TGF-β signalling was inhibited (Fig. 3h), whereas Activin A strongly induced Wnt2b in presumptive lens explants (Fig. 3i,j). Similarly, in vivo, ectodermal Wnt2b expression depended on TGF-β signalling from NCCs: NCC ablation (Fig. 3k,l) and misexpression of the inhibitory Smad7 (Fig. 3m,n; Smad7: 2/6 Wnt2b+, green fluorescent protein: 5/5 Wnt2b+) caused downregulation of Wnt2b transcripts in the non-lens ectoderm. Together, these results show that TGF-β signalling from NCCs induces Wnt2b expression in the non-lens ectoderm.

Bottom Line: In vertebrates, the lens and retina arise from different embryonic tissues raising the question of how they are aligned to form a functional eye.Here we show, using the chick as a model system, that neural crest-derived transforming growth factor-βs activate both Smad3 and canonical Wnt signalling in the adjacent ectoderm to position the lens next to the retina.They do so by controlling Pax6 activity: although Smad3 may inhibit Pax6 protein function, its sustained downregulation requires transcriptional repression by Wnt-initiated β-catenin.

View Article: PubMed Central - PubMed

Affiliation: Department of Craniofacial Development, King's College London, Guy's Campus, London SE1 9RT, UK.

Show MeSH
Related in: MedlinePlus