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

2. Colin Jamora
IFOM-inStem Joint Research Laboratory
Centre for Inflammation and Tissue Homeostasis
Institute for Stem Cell Biology and Regenerative Medicine
Organ Development and Regeneration:
A Hairy Situation
University of Salento, Nov. 2

3. How do organs form?
1. Break it down into simple “morphometric modules” and identify the
developmental 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

4. Examples of Morphometric Modules Studied During Organogenesis
Tubulogenesis
Budding morphogenesis
Branching morphogenesis

5. Embryonic Development

6. 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 animal
Mice are viable until birth in the absence of epidermis
Loss 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:

7. Embryonic Development

8. 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

9. The mammalian skin as a model system to study lineage determination
epidermal ectoderm
basement membrane
mesenchyme

10. Cellular Adhesion Molecules Maintain Epidermal Organization
Vaezi et al., Dev Cell 2002
E-cadherin/actin
Basal layer
Spinous layer
Granular layer
Stratum corneum

11. Budding morphogenesis initiates hair follicle development
E-cadherin
Adapted from S. Millar J. Invest. Derm. 2002
P-cadherin
Cadherin switch
Development: Gastrulation
Neural Crest Formation
Genital Ridge Population
Disease: Metastasis

12. hair
Morphogenesis
Adult organ
liver lung
mammary
gland
tooth
Bud
Undifferentiated
epithelium
Adapted from Pispa & Thesleff Dev. Bio. 2003

13. Morphogen(s)/Extracellular Signal(s)
Transcription Factor
Target Gene
Phenotype/Cell Behavior
Strategy for elucidating the mechanisms regulating hair bud morphogenesis

14. TOP = Tcf/Lef Optimal Promoter
Lef1/ß-catenin Transcription Factor Is Active in the Hair Bud

16. Canonical Wnt Signaling Pathway

17. Bone Morphogenic Protein (BMP) Signaling Pathway Regulates Lef Expression
(e.g. noggin, chordin, gremlin) Lef expressing cell

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

19. Wnt & Noggin produce a transcriptionally competent Lef1/ß-cat complex
TOP c-fos luciferaseTOP TOP
ß-cat
Lef-1
* * *

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

21. Morphogen
Transcription Factor
Target Gene ?

22. The Hair Cycle

23. (from DP)
(from bulge)
Hair regeneration = Hair morphogenesis

24. Coupling Cell Fate Determination with Changes in Cell Morphology
Is E-cadherin a target of the Lef1/ß-catenin transcription complex ?

25. TOP = Tcf/Lef Optimal Promoter
Cadherin Dynamics Occur at Sites of Lef1/ß-catenin Activity

26. Lef1 is required for E-cadherin downregulation in vivo

27. ß-catenin is necessary but not sufficient for E-cadherin repression
K14-ΔNßcat
transgene
Tg
Wt

28. Regulation of Cadherin Expression
Skin sections: 16.5d embryo

29. Possible Mechanisms for Lef1/ß-cat-mediated E-cadherin Repression
• Lef1/ß-cat functions as a transcriptional activator
ß-cat
Lef1 Repressor
Repressor
E-cadherin
ß-cat
Lef1 E-cadherin
• Lef1/ß-cat functions as a transcriptional repressor

30. Lef1/ß-cat transcriptionally downregulates E-cadherin
ß-gal

31. Wnt and Noggin downregulate E-cadherin promoter activity

32. Noggin is required for E-cadherin downregulation in vivo

33. (epi) (mes)

34. Morphogen
Transcription Factor
Target Gene
?
?
Effector(s)
What is the Physiological Relevance of E-cadherin Dynamics?
(mes) (epi)

35. Overexpression of E-cadherin inhibits hair follicle formation

36. E-cadherin overexpression retards follicle morphogenesis

37. Loss of E-cadherin gradient in transgenic epidermis
K14-Ecad(HA)
Stratum corneum
Basement membrane

38. E-cadherin overexpression promotes epidermal differentiation
E-cadherin conditional null:
C. Tinkle, T. Lechler, & E. Fuchs
PNAS 2004
(laminin)
(K5)
wt Tg
K5

40. Dual Functions of Adhesion Proteins Implicate E-cadherin
Dynamics in a Variety of Processes

41. 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

42. Common Themes in Mammalian Stem Cell Systems
epidermal intestinal
bone marrow
From Moore and Lemischka
Science 2006

43. Acknowledgements
Rolf Kemler
Ralf Paus
Andrew McMahon
Rudolf Grosschedl
Richard Harland
Antonio Garcia de Herreros
Hans Clevers
Masatoshi Takeichi
Shinji Takada
Jamora Lab
Luke Denly
Pedro Lee
Joanne Lee
Carol Chan
John Gibson
Samuel Lasse
Fuchs Lab
Elaine Fuchs
Ram Dasgupta
Pawel Kocienski
Members of the Fuchs lab