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

Swiss-Japanese workshop 2008

Unexpected consequences of loss of Notch signaling in the skin

Undergo replenishment or repair from SC throughout life

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Tumorigenesis

Transient amplifying cells Transient amplifying cells

• Diff. cells
• Diff. cells

Stem cells Stem cells

no division no division « work work »

Cancer is a disease of uncontrolled self-renewal

Cancer Initiating Cell

• r

Cancer Stem Cells

De-regulated self-renewal Re-acquirement of long term self-renewal capacity

Dll1 & 4 Dll3 Jagged1& 2 Delta Serrate dNotch Notch1 Notch2 Notch3 Notch4

EGF-like repeats LIN ANK R TAD P NLS

TAD

TAD

EGF DSL CR

Normal wing “Notched” Wing

PM PM

Thomas Hunt Morgan 1917

Hes

LT-HSC ST-HSC MPP CLP CMP T progenitor B progenitor NK progenitor T cell B cell NK cell Monocyte Eosinophil Basophil Neutrophil Megakaryocyte Erythrocyte Erythroblast GMP MEP Granulocyte

Hematopoiesis Tumorigenesis Intestine Skin and hair follicle homeostasis

Lineage specifier

• ncogene

Progenitor gate keeper

Cornea

Corneal conjunctivalization Aniridia and Bitot’s spots. Limbal dermoide

The corneal phenotype can change!

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

Majo and Barrandon

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 4. Cells can be cultured and transplanted to improve visison.

Notch1-DAPI

Ctrl

E S S

Epithelium Stroma Endothelium

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Sophie Vauclair

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K14-DAPI E S S E

Yann Barrandon Francois Majo

Ctrl

A

N1 K14Cre N1 K5CreERT

B C

(2) Inactivation of Notch1 in the corneal epithelium induces its differentiation into epidermis.

K1/K10 Involucrin Filaggrin Loricrin K5/K14 Ctrl N1 K14Cre K14 LORICRIN K1

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

REGULAR STROMA NEO STROMA

Time course

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

Which growth factors induce neovascularization of the dermis?

Notch1loxlox Notch1loxlox&K14Cre Notch1loxlox&K5CreERT eye

Meibomian gland

HE SB K14 K1

A

Ctrl N1 K14Cre N1 K5CreERT

B C D

Notch1-/- Meibomian glands form cysts

closed eye

• pen eye

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

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

Hypothesis

Control wounded N1 lox/lox K14 Cre wounded (<2month)

Notch1 deficient mice develop a vascularised corneal plaque after 3 consecutive woundings

K14

Control (wounded) N1 lox/lox K14 Cre (wounded)

K1 Loricrin

Differentiation of the cornea into an epidermis-like structure after 3 surgical woundings

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

N1 K14Cre limbus cornea

• 1. Burned

limbus N1 K14Cre

C D B

Ctrl N1 K14Cre

Notch1-/- corneal stem cells within the central cornea can repair a wounded cornea only into skin-like epidermis

?

N1 K14Cre R26R Nude

• 1. Graft

isolation

• 2. Graft

transplantation and wounding

• 3. Do grafted

cells migrate? limbus cornea Nude

E F

Ctrl N1 K14Cre Ctrl

Notch1-/- corneal stem cells are still migration competent

Lac-Z stop

Xerophtalmia

Due to vitamin A deficiency!

Retinol CRBP1 cellular retinol binding protein

C

N1 K14Cre CRBP1-DAPI

Ctrl

Loss of CRBP1 expression in the Notch1 deficient corneal epithelium

Notch1-/-

Putative RBP-Jk binding sites

+1

• 3794
• 3756
• 3321
• 1704

TTCCCAC TTCCCAC TTCCCAC GTGGGAA ATG

luciferase

1 2 3 4 5 6 7

CRBP1-luc + N1IC CRBP1-lucmut3 + N1IC CRBP1-lucmut1&2 + N1IC

Relative Luciferase Activity

n=2 X X X

n=3

Notch1 IC activates the CRBP1 promoter

Andre Durham

Tear film Ep S En keratocyte corneal keratinocyte stem cell fibers S S fibroblast

• A. With Notch1
• B. Without Notch1

bFGF

Corneal cell fate is maintained during repair by Notch1 signaling via regulation of Vitamin A metabolism Andre-Dante Durham, Poster 21

Fuchs & Raghavan Nature Reviews Genetics Vol. 8 March 2002

The skin and its appendages

EPIDERMIS

Suprabasal layers

Notch in the skin

Notch1lox/lox Notch1lox/loK14 Cre Notch1lox/loK5 CreERT

Notch1 is necessary for the maintenance of skin appendage: eg. hair, sweat and Meibomian glands Notch1 deficient cornea adopts an epidermal cell fate upon repair

Notch1l/l K5CreERT

Notch1 deficient mice develop BCC- like tumors

Loss of Notch1 fascilitates chemically induced carcinogenesis

20 40 60 80 100 10 20

Weeks

Control KO

Percentage of mice with tumors

2 4 6 8 10 12 14 10 20

Weeks

Control KO

A v e r a g e n u m b e r

• f

l e s i

• n

s p e r m

• u

s e

Notch1-/- Control

Notch1 lox/lox K5 CreERT

Tumor suppressor function of Notch1 in the mouse skin

p63