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Planar polarization in embryonic epidermis orchestrates global asymmetric morphogenesis of hair follicles.

Devenport D, Fuchs E - Nat. Cell Biol. (2008)

Bottom Line: Moreover, loss-of-function mutations in Vangl2 and Celsr1 show that they have an essential role in hair follicle polarization and orientation, which develop in part through non-autonomous mechanisms.Vangl2 and Celsr1 are both required for their planar localization in vivo, and physically associate in a complex in vitro.Finally, we provide in vitro evidence that homotypic intracellular interactions of Celsr1 are required to recruit Vangl2 and Fzd6 to sites of cell-cell contact.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Laboratory of Mammalian Cell Biology & Development, The Rockefeller University, New York, NY 10065, USA.

ABSTRACT
Mammalian body hairs align along the anterior-posterior (A-P) axis and offer a striking but poorly understood example of global cell polarization, a phenomenon known as planar cell polarity (PCP). We have discovered that during embryogenesis, marked changes in cell shape and cytoskeletal polarization occur as nascent hair follicles become anteriorly angled, morphologically polarized and molecularly compartmentalized along the A-P axis. Hair follicle initiation coincides with asymmetric redistribution of Vangl2, Celsr1 and Fzd6 within the embryonic epidermal basal layer. Moreover, loss-of-function mutations in Vangl2 and Celsr1 show that they have an essential role in hair follicle polarization and orientation, which develop in part through non-autonomous mechanisms. Vangl2 and Celsr1 are both required for their planar localization in vivo, and physically associate in a complex in vitro. Finally, we provide in vitro evidence that homotypic intracellular interactions of Celsr1 are required to recruit Vangl2 and Fzd6 to sites of cell-cell contact.

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Evidence for interactions between Celsr1 and Vangl2 in vivo and in vitro(a–f) In vivo studies. Planar confocal sections through the basal layer of whole mount skin from E15.5 WT, Lp/Lp, and Crsh/Crsh embryos, labelled with Celsr1 (green), Vangl2 (red) and DAPI (blue). Note that in Lp/Lp mutants, Celsr1’s A–P polarity is lost. In Crsh/Crsh mutants both Celsr1 and Vangl2 lose A–P polarity, and Vangl2 is punctate and discontinuous at cell borders. Boxed areas are magnified in insets. (g–t) In vitro studies. Cultured keratinocytes expressing fluorescently tagged Celsr1, Vangl2 or their mutant versions as indicated. Confluent monolayers were shifted to high Ca2+ medium to induce the formation of intercellular junctions, except in (i) where cells were kept in low Ca+2. Each image shown is representative of ≥ 50 examples. Boxed areas are magnified, and red and green fluorescence is separated for additional clarity. Scale bars 10µm. Schematics depict domain structure of WT and mutant proteins. (g–l) Celsr1 data. Note that Celsr1WTGFP shifts its localization to cell-cell borders in a calcium-dependent but α-catenin (adherens junction) independent fashion when two transfected cells come into direct contact. Note also that the Celsr1Crsh mutant and Celsr1 lacking the C-terminal cytoplasmic tail (Δcyto) are compromised in this ability. (m–t) Vangl2 and co-expression data. Note that Vangl2 is unable to localize to cell-cell borders on its own. In the presence of Celsr1WTGFP, Cherry-Vangl2 shifts its localization to cell-cell borders only when two Celsr1-expressing cells are in direct contact (p), and mutant versions of Celsr1 are compromised in this ability (q–r). Cherry-Vangl2 mutants lacking the C-terminal cytoplasmic tail or carrying the Lp point mutation fail to be recruited by Celsr1 to cell contacts. (u) Surface biotinylation assay. Biotinylated surface proteins from keratinocytes expressing Flag-Vangl2WT or Flag-Vangl2Lp were recovered with streptavidin-coated sepharose beads and analyzed by Western blot to show that the Lp Vangl2 mutation does not appear to compromise the relative stability and/or cell surface localization of Vangl2. (v) Protein extracts from keratinocytes expressing Flag-Vangl2 ± Celsr1-Myc as indicated were immunoprecipitated with anti-Myc Abs. Flag-Vangl2WT but not Flag-Vangl2Lp coimmunoprecipitated with Celsr1-Myc.
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Figure 7: Evidence for interactions between Celsr1 and Vangl2 in vivo and in vitro(a–f) In vivo studies. Planar confocal sections through the basal layer of whole mount skin from E15.5 WT, Lp/Lp, and Crsh/Crsh embryos, labelled with Celsr1 (green), Vangl2 (red) and DAPI (blue). Note that in Lp/Lp mutants, Celsr1’s A–P polarity is lost. In Crsh/Crsh mutants both Celsr1 and Vangl2 lose A–P polarity, and Vangl2 is punctate and discontinuous at cell borders. Boxed areas are magnified in insets. (g–t) In vitro studies. Cultured keratinocytes expressing fluorescently tagged Celsr1, Vangl2 or their mutant versions as indicated. Confluent monolayers were shifted to high Ca2+ medium to induce the formation of intercellular junctions, except in (i) where cells were kept in low Ca+2. Each image shown is representative of ≥ 50 examples. Boxed areas are magnified, and red and green fluorescence is separated for additional clarity. Scale bars 10µm. Schematics depict domain structure of WT and mutant proteins. (g–l) Celsr1 data. Note that Celsr1WTGFP shifts its localization to cell-cell borders in a calcium-dependent but α-catenin (adherens junction) independent fashion when two transfected cells come into direct contact. Note also that the Celsr1Crsh mutant and Celsr1 lacking the C-terminal cytoplasmic tail (Δcyto) are compromised in this ability. (m–t) Vangl2 and co-expression data. Note that Vangl2 is unable to localize to cell-cell borders on its own. In the presence of Celsr1WTGFP, Cherry-Vangl2 shifts its localization to cell-cell borders only when two Celsr1-expressing cells are in direct contact (p), and mutant versions of Celsr1 are compromised in this ability (q–r). Cherry-Vangl2 mutants lacking the C-terminal cytoplasmic tail or carrying the Lp point mutation fail to be recruited by Celsr1 to cell contacts. (u) Surface biotinylation assay. Biotinylated surface proteins from keratinocytes expressing Flag-Vangl2WT or Flag-Vangl2Lp were recovered with streptavidin-coated sepharose beads and analyzed by Western blot to show that the Lp Vangl2 mutation does not appear to compromise the relative stability and/or cell surface localization of Vangl2. (v) Protein extracts from keratinocytes expressing Flag-Vangl2 ± Celsr1-Myc as indicated were immunoprecipitated with anti-Myc Abs. Flag-Vangl2WT but not Flag-Vangl2Lp coimmunoprecipitated with Celsr1-Myc.

Mentions: Vangl2 and Celsr1 have been genetically implicated in PCP, but their functional relationship remains poorly understood. Lp harbours an S464N mutation in Vangl2’s C-terminal cytoplasmic domain4, resulting in dramatically reduced Vangl2 at skin cell borders (Fig. 7a–b). Despite the reduction of Vangl2 in Lp/Lp epidermis, Celsr1 still localized to cell borders. However, rather than distributing asymmetrically as in WT, Celsr1 was distributed uniformly at membrane junctions (Fig. 7d–e)21.


Planar polarization in embryonic epidermis orchestrates global asymmetric morphogenesis of hair follicles.

Devenport D, Fuchs E - Nat. Cell Biol. (2008)

Evidence for interactions between Celsr1 and Vangl2 in vivo and in vitro(a–f) In vivo studies. Planar confocal sections through the basal layer of whole mount skin from E15.5 WT, Lp/Lp, and Crsh/Crsh embryos, labelled with Celsr1 (green), Vangl2 (red) and DAPI (blue). Note that in Lp/Lp mutants, Celsr1’s A–P polarity is lost. In Crsh/Crsh mutants both Celsr1 and Vangl2 lose A–P polarity, and Vangl2 is punctate and discontinuous at cell borders. Boxed areas are magnified in insets. (g–t) In vitro studies. Cultured keratinocytes expressing fluorescently tagged Celsr1, Vangl2 or their mutant versions as indicated. Confluent monolayers were shifted to high Ca2+ medium to induce the formation of intercellular junctions, except in (i) where cells were kept in low Ca+2. Each image shown is representative of ≥ 50 examples. Boxed areas are magnified, and red and green fluorescence is separated for additional clarity. Scale bars 10µm. Schematics depict domain structure of WT and mutant proteins. (g–l) Celsr1 data. Note that Celsr1WTGFP shifts its localization to cell-cell borders in a calcium-dependent but α-catenin (adherens junction) independent fashion when two transfected cells come into direct contact. Note also that the Celsr1Crsh mutant and Celsr1 lacking the C-terminal cytoplasmic tail (Δcyto) are compromised in this ability. (m–t) Vangl2 and co-expression data. Note that Vangl2 is unable to localize to cell-cell borders on its own. In the presence of Celsr1WTGFP, Cherry-Vangl2 shifts its localization to cell-cell borders only when two Celsr1-expressing cells are in direct contact (p), and mutant versions of Celsr1 are compromised in this ability (q–r). Cherry-Vangl2 mutants lacking the C-terminal cytoplasmic tail or carrying the Lp point mutation fail to be recruited by Celsr1 to cell contacts. (u) Surface biotinylation assay. Biotinylated surface proteins from keratinocytes expressing Flag-Vangl2WT or Flag-Vangl2Lp were recovered with streptavidin-coated sepharose beads and analyzed by Western blot to show that the Lp Vangl2 mutation does not appear to compromise the relative stability and/or cell surface localization of Vangl2. (v) Protein extracts from keratinocytes expressing Flag-Vangl2 ± Celsr1-Myc as indicated were immunoprecipitated with anti-Myc Abs. Flag-Vangl2WT but not Flag-Vangl2Lp coimmunoprecipitated with Celsr1-Myc.
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Figure 7: Evidence for interactions between Celsr1 and Vangl2 in vivo and in vitro(a–f) In vivo studies. Planar confocal sections through the basal layer of whole mount skin from E15.5 WT, Lp/Lp, and Crsh/Crsh embryos, labelled with Celsr1 (green), Vangl2 (red) and DAPI (blue). Note that in Lp/Lp mutants, Celsr1’s A–P polarity is lost. In Crsh/Crsh mutants both Celsr1 and Vangl2 lose A–P polarity, and Vangl2 is punctate and discontinuous at cell borders. Boxed areas are magnified in insets. (g–t) In vitro studies. Cultured keratinocytes expressing fluorescently tagged Celsr1, Vangl2 or their mutant versions as indicated. Confluent monolayers were shifted to high Ca2+ medium to induce the formation of intercellular junctions, except in (i) where cells were kept in low Ca+2. Each image shown is representative of ≥ 50 examples. Boxed areas are magnified, and red and green fluorescence is separated for additional clarity. Scale bars 10µm. Schematics depict domain structure of WT and mutant proteins. (g–l) Celsr1 data. Note that Celsr1WTGFP shifts its localization to cell-cell borders in a calcium-dependent but α-catenin (adherens junction) independent fashion when two transfected cells come into direct contact. Note also that the Celsr1Crsh mutant and Celsr1 lacking the C-terminal cytoplasmic tail (Δcyto) are compromised in this ability. (m–t) Vangl2 and co-expression data. Note that Vangl2 is unable to localize to cell-cell borders on its own. In the presence of Celsr1WTGFP, Cherry-Vangl2 shifts its localization to cell-cell borders only when two Celsr1-expressing cells are in direct contact (p), and mutant versions of Celsr1 are compromised in this ability (q–r). Cherry-Vangl2 mutants lacking the C-terminal cytoplasmic tail or carrying the Lp point mutation fail to be recruited by Celsr1 to cell contacts. (u) Surface biotinylation assay. Biotinylated surface proteins from keratinocytes expressing Flag-Vangl2WT or Flag-Vangl2Lp were recovered with streptavidin-coated sepharose beads and analyzed by Western blot to show that the Lp Vangl2 mutation does not appear to compromise the relative stability and/or cell surface localization of Vangl2. (v) Protein extracts from keratinocytes expressing Flag-Vangl2 ± Celsr1-Myc as indicated were immunoprecipitated with anti-Myc Abs. Flag-Vangl2WT but not Flag-Vangl2Lp coimmunoprecipitated with Celsr1-Myc.
Mentions: Vangl2 and Celsr1 have been genetically implicated in PCP, but their functional relationship remains poorly understood. Lp harbours an S464N mutation in Vangl2’s C-terminal cytoplasmic domain4, resulting in dramatically reduced Vangl2 at skin cell borders (Fig. 7a–b). Despite the reduction of Vangl2 in Lp/Lp epidermis, Celsr1 still localized to cell borders. However, rather than distributing asymmetrically as in WT, Celsr1 was distributed uniformly at membrane junctions (Fig. 7d–e)21.

Bottom Line: Moreover, loss-of-function mutations in Vangl2 and Celsr1 show that they have an essential role in hair follicle polarization and orientation, which develop in part through non-autonomous mechanisms.Vangl2 and Celsr1 are both required for their planar localization in vivo, and physically associate in a complex in vitro.Finally, we provide in vitro evidence that homotypic intracellular interactions of Celsr1 are required to recruit Vangl2 and Fzd6 to sites of cell-cell contact.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Laboratory of Mammalian Cell Biology & Development, The Rockefeller University, New York, NY 10065, USA.

ABSTRACT
Mammalian body hairs align along the anterior-posterior (A-P) axis and offer a striking but poorly understood example of global cell polarization, a phenomenon known as planar cell polarity (PCP). We have discovered that during embryogenesis, marked changes in cell shape and cytoskeletal polarization occur as nascent hair follicles become anteriorly angled, morphologically polarized and molecularly compartmentalized along the A-P axis. Hair follicle initiation coincides with asymmetric redistribution of Vangl2, Celsr1 and Fzd6 within the embryonic epidermal basal layer. Moreover, loss-of-function mutations in Vangl2 and Celsr1 show that they have an essential role in hair follicle polarization and orientation, which develop in part through non-autonomous mechanisms. Vangl2 and Celsr1 are both required for their planar localization in vivo, and physically associate in a complex in vitro. Finally, we provide in vitro evidence that homotypic intracellular interactions of Celsr1 are required to recruit Vangl2 and Fzd6 to sites of cell-cell contact.

Show MeSH