Unexpected consequences of loss of Notch signaling in the skin - PowerPoint PPT Presentation

Unexpected consequences of loss of Notch signaling in the skin Swiss-Japanese workshop 2008

Undergo replenishment or QuickTime™ et un repair from SC throughout life décompresseur TIFF (LZW) sont requis pour visionner cette image. Tumorigenesis

Cancer is a disease of uncontrolled self-renewal Stem cells Stem cells Transient amplifying cells Transient amplifying cells Diff. cells Diff. cells De-regulated self-renewal Re-acquirement of long term self-renewal capacity Cancer Initiating Cell or no division no division « work work » Cancer Stem Cells

Thomas Hunt Morgan 1917 Normal wing “Notched” Wing LIN DSL EGF CR TAD ANK R EGF-like repeats Serrate dNotch P NLS Delta Notch1 TAD Jagged1& 2 Notch2 TAD Dll1 & 4 Notch3 Dll3 Notch4 PM PM

Hes

Hematopoiesis Intestine T cell T progenitor B cell B progenitor CLP NK cell Lineage specifier NK progenitor Monocyte Progenitor gate keeper Eosinophil oncogene LT-HSC MPP ST-HSC GMP Basophil Granulocyte Neutrophil CMP Megakaryocyte Erythrocyte MEP Erythroblast Skin and hair Tumorigenesis Cornea follicle homeostasis

The corneal phenotype can change! Aniridia and Bitot’s spots. Corneal conjunctivalization Limbal dermoide

Corneal stem cells in the limbus are responsible for renewal of the cornea and wound healing 1. Do not express Keratin 3/12 2. Limbus cells are LRC 3. After severe injuries cells of the limbus migrate towards the wound to repair the injurie Majo and Barrandon 4. Cells can be cultured and transplanted to improve visison.

QuickTime™ and a Photo - JPEG decompressor are needed to see this picture. Epithelium Yann Barrandon Sophie Vauclair Stroma Endothelium Notch1-DAPI Ctrl E S S Ctrl K14-DAPI E E Francois Majo S S

N1 K14Cre N1 K5Cre ERT Ctrl A B C

(2) Inactivation of Notch1 in the corneal epithelium induces its differentiation into epidermis. Ctrl N1 K14Cre Filaggrin LORICRIN Loricrin Involucrin K1/K10 K1 K5/K14 K14

NEO STROMA (1) Notch 1 inactivation in the epithelium modifies the corneal stroma. REGULAR STROMA

Notch1 deficiency in the corneal epithelium leads first to changes in the stroma before the epithelium changes its fate Time course

Which growth factors induce neovascularization of the dermis?

Notch1 loxlox Notch1 loxlox&K5CreERT Notch1 loxlox&K14Cre eye Meibomian gland

K14 K1 HE SB N1 K14Cre Notch1-/- Meibomian glands form cysts N1 K5Cre ERT Ctrl A B C D

Only chronic irritation of the cornea results in differentiation of the Notch1-/- cornea into skin closed eye open eye

Hypothesis Notch1 Corneal epithelium Repeated wounding Precursor or stem cells Epidermis-like Notch1

Notch1 deficient mice develop a vascularised corneal plaque after 3 consecutive woundings Control wounded N1 lox/lox K14 Cre wounded (<2month)

Differentiation of the cornea into an epidermis-like structure after 3 surgical woundings Control (wounded) N1 lox/lox K14 Cre (wounded) K14 K1 Loricrin

Corneal stem cells in the limbus are responsible for renewal of the cornea and wound healing

Notch1-/- corneal stem cells within the central cornea can repair a wounded cornea only into skin-like epidermis 1. Burned limbus cornea limbus N1 K14Cre N1 K14Cre Ctrl N1 K14Cre B C D

Notch1-/- corneal stem cells are still migration competent Lac-Z stop E 1. Graft 2. Graft 3. Do grafted isolation transplantation cells migrate? and wounding cornea limbus ? N1 K14Cre Nude Nude R26R F Ctrl Ctrl N1 K14Cre

Xerophtalmia Due to vitamin A deficiency! CRBP1 cellular retinol binding protein Retinol

N1 K14Cre Loss of CRBP1 expression in the Notch1 deficient corneal epithelium Ctrl Notch1-/- C CRBP1-DAPI

Notch1 IC activates the CRBP1 promoter -3794 -3756 -3321 -1704 +1 luciferase TTCCCAC GTGGGAA TTCCCAC TTCCCAC ATG Putative RBP-Jk binding sites 7 n=3 Relative Luciferase Activity 6 n=2 5 4 Andre Durham 3 2 + N1IC + N1IC + N1IC 1 QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. 0 CRBP1-luc CRBP1-luc mut3 CRBP1-luc mut1&2 X X X

Corneal cell fate is maintained during repair by Notch1 signaling via regulation of Vitamin A metabolism A. With Notch1 B. Without Notch1 Tear film bFGF Ep S S En S stem cell keratocyte fibers corneal keratinocyte fibroblast Andre-Dante Durham, Poster 21

The skin and its appendages Fuchs & Raghavan Nature Reviews Genetics Vol. 8 March 2002

Notch in the skin layers Suprabasal EPIDERMIS

Notch1 is necessary for the maintenance of skin Notch1 deficient mice develop BCC- appendage: eg. hair, sweat and Meibomian like tumors glands Notch1 l/l K5Cre ERT Notch1 lox/lox Notch1 lox/lo K14 Cre Notch1 lox/lo K5 CreERT Notch1 deficient cornea adopts an epidermal cell fate upon repair

Loss of Notch1 fascilitates chemically induced carcinogenesis Control Percentage of mice 100 Control KO with tumors 80 60 40 20 0 0 10 20 Notch1-/- Weeks f e o s r u e o 14 b Control m m KO 12 r u e 10 n p e 8 s g n a 6 o r i e s 4 v e A l 2 0 0 10 20 Weeks

p63 Tumor suppressor function of Notch1 in the mouse skin Notch1 lox/lox K5 CreERT