Ldb 145 Geni Mutanti_2014-11-19 Jamora - mercato biomedico 6

Colin JamoraIFOM-inStem Joint Research Laboratory

Centre for Inflammation and Tissue HomeostasisInstitute for Stem Cell Biology and Regenerative Medicine

Organ Development and Regeneration:A Hairy Situation

University of Salento, Nov. 2014

How do organs form?

1. Break it down into simple “morphometric modules” and identify thedevelopmental signals that guide their formation

2. Describe the changes in cellular anatomy that occur during morphogenesis

3. Elucidate the molecular programs that underlie the changes in cell parameters:Shape, Polarity, Movement, Adhesion, Proliferation

Examples of Morphometric Modules Studied During Organogenesis

Tubulogenesis

Budding morphogenesis

Branching morphogenesis

Embryonic Development

Advantages of the skin/hair as a model organ system

Ability to easily culture adult stem/progenitor cells

Biochemical and cell biological techniques to elucidate intracellular signaling pathways

Ease of scoring defects (both mild and dramatic) in both transgenic and knockout mice

Not critical for the development and viability of the whole animalMice are viable until birth in the absence of epidermisLoss of hair does not impair the health of housed mice

Amenable to genetic manipulation to test in vivo function(s) of genes

In vivo:

In vitro:

Embryonic Development

2012 Nobel Prize: Reprogramming dermal fibroblasts into stem cells

Skin  fibroblast  

Pa/ent  

Reprogramming  Unknown  gene/c    

and  epigene/c  processes  

Pluripotent  stem  cell  

Disease  Models    (chemical  +  gene/c  

screening)  

“Personalized”  Regenera/ve  Medicine    

(no  rejec/on)  

Nanog

Sox 2 Oct 3/4

The mammalian skin as a model system to study lineage determination

epidermal ectoderm

basement membrane

mesenchyme

Cellular Adhesion Molecules Maintain Epidermal Organization

Vaezi et al., Dev Cell 2002

E-cadherin/actin

Basal layer

Spinous layer

Granular layerStratum corneum

Budding morphogenesis initiates hair follicle development

E-cadherin

Adapted from S. Millar J. Invest. Derm. 2002

P-cadherin

Cadherin switchDevelopment: Gastrulation

Neural Crest FormationGenital Ridge Population

Disease: Metastasis

hair

Morphogenesis

Adult organ

liver lung mammarygland

tooth

Bud

Undifferentiatedepithelium

Adapted from Pispa & Thesleff Dev. Bio. 2003

Morphogen(s)/Extracellular Signal(s)

Transcription Factor

Target Gene

Phenotype/Cell Behavior

Strategy for elucidating the mechanisms regulating hair bud morphogenesis

TOP = Tcf/Lef Optimal Promoter

Lef1/ß-catenin Transcription Factor Is Active in the Hair Bud

Canonical Wnt Signaling Pathway

Bone Morphogenic Protein (BMP) Signaling Pathway Regulates Lef Expression

(e.g. noggin, chordin, gremlin) Lef expressing cell

Wnt stabilizes ß-catenin & Noggin induces Lef-1 in keratinocytes

Wnt & Noggin produce a transcriptionally competent Lef1/ß-cat complex

TOP c-fos luciferaseTOP TOP

ß-catLef-1

* * *

Nuclear ß-cat, Lef1 and TOPGAL require noggin in vivo

Morphogen

Transcription Factor

Target Gene ?

The Hair Cycle

(from DP)

(from bulge)

Hair regeneration = Hair morphogenesis

Coupling Cell Fate Determination with Changes in Cell Morphology

Is E-cadherin a target of the Lef1/ß-catenin transcription complex ?

TOP = Tcf/Lef Optimal Promoter

Cadherin Dynamics Occur at Sites of Lef1/ß-catenin Activity

Lef1 is required for E-cadherin downregulation in vivo

ß-catenin is necessary but not sufficient for E-cadherin repression

K14-ΔNßcattransgene

Tg

Wt

Regulation of Cadherin Expression

Skin sections: 16.5d embryo

Possible Mechanisms for Lef1/ß-cat-mediated E-cadherin Repression

•  Lef1/ß-cat functions as a transcriptional activator

ß-catLef1 Repressor

RepressorE-cadherin

ß-catLef1 E-cadherin

•  Lef1/ß-cat functions as a transcriptional repressor

Lef1/ß-cat transcriptionally downregulates E-cadherin

ß-gal

Wnt and Noggin downregulate E-cadherin promoter activity

Noggin is required for E-cadherin downregulation in vivo

(epi) (mes)

Morphogen

Transcription Factor

Target Gene ?

?Effector(s)

What is the Physiological Relevance of E-cadherin Dynamics?

(mes) (epi)

Overexpression of E-cadherin inhibits hair follicle formation

E-cadherin overexpression retards follicle morphogenesis

Loss of E-cadherin gradient in transgenic epidermis

K14-Ecad(HA)

Stratum corneum

Basement membrane

E-cadherin overexpression promotes epidermal differentiation

E-cadherin conditional null:C. Tinkle, T. Lechler, & E. Fuchs

PNAS 2004

(laminin)(K5)

wt Tg

K5

Dual Functions of Adhesion Proteins Implicate E-cadherinDynamics in a Variety of Processes

Budding Morphogenesis is Guided By A Network of Signals and Genes

?Morphogen

Transcription Factor

Effector

Gene

Noggin Wnt

Lef-1 ß-cat

E-cadherin

Polarity Differentiation Proliferation Motility

Snail

TGFß2

?

BM

Common Themes in Mammalian Stem Cell Systems

epidermal intestinal

bone marrow

From Moore and LemischkaScience 2006

Acknowledgements

Rolf Kemler Ralf Paus

Andrew McMahonRudolf GrosschedlRichard Harland

Antonio Garcia de HerrerosHans Clevers

Masatoshi TakeichiShinji Takada

Jamora LabLuke DenlyPedro LeeJoanne LeeCarol ChanJohn GibsonSamuel Lasse

Fuchs LabElaine Fuchs

Ram DasguptaPawel Kocienski

Members of the Fuchs lab