Functions of Wnt/beta-catenin signalling in skin Laura Perks Shabina Begum Simon Bishop Baldness: A wonder cure?
Hair Morphogenesis & Follicle Cycling <ul><li>Begins with three signals: mesenchymal signal to ectoderm (forms hair germ);...
 
De Novo Hair Follicle Morphogenesis and Hair Tumors in Mice Expressing a Truncated ß-Catenin in Skin  Gat et al. (1998) Ce...
<ul><li>Results and Conclusions </li></ul><ul><li>Day 18: Epithelial invaginations in interfollicular epidermis </li></ul>...
Pilomatricomas <ul><li>Common benign adnexal neoplasms with follicular differentiation towards matrical and hair cortical ...
Method <ul><li>Microdissection of formalin-fixed, paraffin-embedded sections of pilomatricoma biopsies from 16 different h...
Results <ul><li>Direct sequencing of the N-terminal segment showed a single nucleotide substitution in the DNA from 12 of ...
Results <ul><li>The 12 nucleotide alterations all encode putative missense mutations in the N-terminal segment of  β -cate...
Conclusions <ul><li>Differences between  CTNNB1  mutations in pilomatricomas and those in other tumours lies  not  in thei...
β -catenin signaling can initiate feather bud development  – Noramly, Freeman, Morgan (Development 126 1999) <ul><li>Prima...
Methods  -  <ul><li>Immunohistochemistry </li></ul><ul><li>Skin from dorsal pterylae & femoral tracts taken from white leg...
Results -   <ul><li>Localisation of  β -catenin signalling </li></ul><ul><li>Signalling Dorsal – lateral </li></ul><ul><li...
Conclusions <ul><li>β -catenin is likely to have a role in dense dermin formation </li></ul><ul><li>Several phenotypes in ...
<ul><li>Other growth factors involved in hair signalling: IGF1, FGFR7, FGF5 and EGFR </li></ul><ul><li>Control of growth c...
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Topic 11 Slides On Gat Paper

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Topic 11 Slides On Gat Paper

  1. 1. Functions of Wnt/beta-catenin signalling in skin Laura Perks Shabina Begum Simon Bishop Baldness: A wonder cure?
  2. 2. Hair Morphogenesis & Follicle Cycling <ul><li>Begins with three signals: mesenchymal signal to ectoderm (forms hair germ); ectodermal signal to mesenchyme (forms dermal papilla); dermal signal to epithelial component (follicle and sebaceous gland) </li></ul><ul><li>Once developed undergoes cycling: Anagen (Growth), Catagen (Regression) and Telogen (Rest) </li></ul><ul><li>Possible role for Lef-1/Tcf family of DNA-binding proteins </li></ul><ul><li>Lef-1 null mice display a reduction in follicle number and lack whiskers </li></ul>
  3. 4. De Novo Hair Follicle Morphogenesis and Hair Tumors in Mice Expressing a Truncated ß-Catenin in Skin Gat et al. (1998) Cell, 95, 605-614 <ul><li>Methods </li></ul><ul><li>Wnt signalling via ß-catenin cascade </li></ul><ul><li>K14-ΔN87ßcat: </li></ul><ul><ul><li>N-terminally truncated ß-catenin: constitutively stabilized </li></ul></ul><ul><ul><li>Cannot be targeted for ubiquitin-mediated degradation as a result of phosphorylation by GSK3 kinase </li></ul></ul><ul><li>Expression vector in keratinocytes & Luciferase reporter genes </li></ul><ul><li>RT-PCR (tail DNA) and immunoblot analyses (epidermal proteins) to check transgenic mouse </li></ul><ul><li>Anti-ßcatenin detection in transgenic epidermis, but not dermis or wild type epidermis </li></ul>
  4. 5. <ul><li>Results and Conclusions </li></ul><ul><li>Day 18: Epithelial invaginations in interfollicular epidermis </li></ul><ul><li>Invaginations proportional to high K14 activity </li></ul><ul><li>Invaginations coincide with hair cycle initiation </li></ul><ul><li>Day 21: Embryonic hair germs arise </li></ul><ul><li>Day 24: Evidence of hair shafts </li></ul><ul><li>Apolarisation of Shh and subsequent misangling of hair follicle </li></ul><ul><li>De novo genesis of dermal papillae and sebaceous glands </li></ul><ul><li>Is ß-catenin the long-sought after “epidermal message”? </li></ul><ul><li>Pilomatricomas observed over time </li></ul>De Novo Hair Follicle Morphogenesis and Hair Tumors in Mice Expressing a Truncated ß-Catenin in Skin Gat et al. (1998) Cell, 95, 605-614
  5. 6. Pilomatricomas <ul><li>Common benign adnexal neoplasms with follicular differentiation towards matrical and hair cortical cells </li></ul>A common human skin tumour is caused by activating mutations in β -catenin Chan EF et al. (1999) Nature Genetics 21: 410-413 The group stained human tumour sections with anti LEF-1 antibodies Shadow cells Transitional cells Matrix cells
  6. 7. Method <ul><li>Microdissection of formalin-fixed, paraffin-embedded sections of pilomatricoma biopsies from 16 different human patients </li></ul><ul><li>Isolation of genomic DNA from these samples </li></ul><ul><li>PCR amplification of a 200bp fragment from exon 3 encoding an N-terminal portion of β -catenin </li></ul>A common human skin tumour is caused by activating mutations in β -catenin Chan EF et al. (1999) Nature Genetics 21: 410-413
  7. 8. Results <ul><li>Direct sequencing of the N-terminal segment showed a single nucleotide substitution in the DNA from 12 of the 16 samples </li></ul><ul><ul><li>7 mutations represent alterations in serine 33, serine 37 and/or threonine 41 </li></ul></ul><ul><ul><ul><li>Essential for GSK-3 β -dependent phophorylation </li></ul></ul></ul><ul><ul><li>5 mutations are in the aspartic acid or glycine residues flanking serine 33 </li></ul></ul>A common human skin tumour is caused by activating mutations in β -catenin Chan EF et al. (1999) Nature Genetics 21: 410-413
  8. 9. Results <ul><li>The 12 nucleotide alterations all encode putative missense mutations in the N-terminal segment of β -catennin: </li></ul><ul><ul><li>D32Y (GAC->TAC) </li></ul></ul><ul><ul><li>D32G (GAC->GGC) </li></ul></ul><ul><ul><li>S33F (TCT->TTT; found in 2 pilomatricomas) </li></ul></ul><ul><ul><li>S33Y (TCT->TAT; found in 2) </li></ul></ul><ul><ul><li>G34E (GGA->GAA; found in 3) </li></ul></ul><ul><ul><li>S37C (TCT->TGT) </li></ul></ul><ul><ul><li>S37F (TCT->TTT) </li></ul></ul><ul><ul><li>T41I (ACC->ATC) </li></ul></ul>A common human skin tumour is caused by activating mutations in β -catenin Chan EF et al. (1999) Nature Genetics 21: 410-413
  9. 10. Conclusions <ul><li>Differences between CTNNB1 mutations in pilomatricomas and those in other tumours lies not in their location but in their significantly higher frequency </li></ul><ul><li>Pilomatricomas arise primarily from genetic alterations that act by influencing β -catenin stabilisation </li></ul><ul><li>Results illustrate that: </li></ul><ul><ul><li>At least 75% of the cases had β -catenin-stabilising mutations in CTNNB1 </li></ul></ul><ul><ul><li>Pilomatricomas do arise from hair matrix cells and that they are typified by expression of the β -catenin partner protein LEF-1 </li></ul></ul><ul><ul><li>And, that acquisition of a β -catenin-stabilising mutation in CTNNB1 leads to LEF-1 trans-activation, resulting in the development of human tumours of hair matrix differentiation </li></ul></ul>A common human skin tumour is caused by activating mutations in β -catenin Chan EF et al. (1999) Nature Genetics 21: 410-413
  10. 11. β -catenin signaling can initiate feather bud development – Noramly, Freeman, Morgan (Development 126 1999) <ul><li>Primary inductive signals in chick tract development is </li></ul><ul><li>not known </li></ul><ul><li>Wnt signalling has been implicated </li></ul><ul><li>– Wnt 7a expressed in epidermal placodes </li></ul><ul><li>– Wnt 11 localised to dermis </li></ul><ul><li>Wnt receptor Frizzled expressed in chick skin at time of tract development </li></ul><ul><li>Frizzled activation = stabilised β -catenin </li></ul><ul><li>β -catenin enters nucleus where it can alter gene expression via TCF/lef1 </li></ul><ul><li>This paper looks at β -catenin as a positive initiator of feather formation </li></ul>Introduction
  11. 12. Methods - <ul><li>Immunohistochemistry </li></ul><ul><li>Skin from dorsal pterylae & femoral tracts taken from white leghorns </li></ul><ul><li>Samples disected, fixed, dehydrated through graded sucrose & frozen </li></ul><ul><li>Monoclonal antibody (15b8 sigma) used to detect β -catenin </li></ul><ul><li>Secondary detection by fluorescein-conjugated anti-mouse IgG </li></ul><ul><li>Counterstained with 7-amino-actinomy cin D/TOPRO3 </li></ul><ul><li>Infected cells detected with polyclonal antisera </li></ul>β -catenin signaling can initiate feather bud development – Noramly, Freeman, Morgan (Development 126 1999) <ul><li>Forced expression of stabilised β -catenin </li></ul><ul><li>RCAS β -catenin retrovirus injected into embryos </li></ul><ul><li>Expression of viral transcripts examined in embryos harvested at day 6,7,8 and 9 </li></ul><ul><li>Effect of exogenous β -catenin analysed by looking for BMP-2, Shh, BMP-4, Wnt 7a, TCF-1 </li></ul><ul><li>-all were examined for viral transcripts </li></ul>
  12. 13. Results - <ul><li>Localisation of β -catenin signalling </li></ul><ul><li>Signalling Dorsal – lateral </li></ul><ul><li>β -catenin observed in ectodermal surface but not nucleus or cytoplasm </li></ul><ul><li>Nuclar β -catenin in dermal cells adjacent to ectoderm when dense dermin is formed </li></ul><ul><li>β -catenin expressed in lateral ectoderm </li></ul><ul><li>Becomes up regulated in placodes as they are formed </li></ul>β -catenin signaling can initiate feather bud development – Noramly, Freeman, Morgan (Development 126 1999) <ul><li>Retrovirus forced expression of β -catenin </li></ul><ul><li>Small patches of viral transcript detected at day 6 </li></ul><ul><li>Larger area of infection in placodes at day 7 </li></ul><ul><li>Feather fields are well established by day 8 </li></ul><ul><li>Feather Bud initiation </li></ul><ul><li>With forced β -catenin expression - ectopic expression of BMP-2, BMP-7 </li></ul><ul><li>Ectoderm assumes the morphology of epidermal placode cells </li></ul>
  13. 14. Conclusions <ul><li>β -catenin is likely to have a role in dense dermin formation </li></ul><ul><li>Several phenotypes in common between forced β -catenin expression in chick and mouse experiments using Keratin-14-promotor </li></ul><ul><li>Interactions of β -catenin with - BMP </li></ul><ul><li> - FGF </li></ul><ul><li> - Notch </li></ul><ul><li>There were differences between mouse and chick </li></ul><ul><li>- No effects in embryonic mouse skin </li></ul><ul><li>These differences may be accounted for due to experimental varience </li></ul><ul><li>- β -catenin contructs </li></ul><ul><li>- K-14 expression </li></ul>β -catenin signaling can initiate feather bud development – Noramly, Freeman, Morgan (Development 126 1999)
  14. 15. <ul><li>Other growth factors involved in hair signalling: IGF1, FGFR7, FGF5 and EGFR </li></ul><ul><li>Control of growth cycle phases </li></ul><ul><ul><li>Decrease the number of follicles in telogen or catagen </li></ul></ul><ul><ul><li>Delay telogen onset </li></ul></ul><ul><ul><li>Hair cycle is potentially reversible </li></ul></ul><ul><li>Substance P, corticotropin, capsaicin </li></ul><ul><li>Androgens </li></ul><ul><ul><li>Finasteride, minoxidil </li></ul></ul>(If only we could get rid of those pesky tumours!) Baldness: A wonder cure?

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