the beauty of the skin - university at buffalorgron/bch512/skin.pdf · the beauty of the skin...

The Beauty of the Skin

Rose-Anne Romano, Ph.D Assistant Professor

Department of Oral Biology

School of Dental Medicine

State University of New York at Buffalo

The Big Question…

How do approximately 50 trillion cells, each one with the SAME genetic information, co-ordinate to make all the different tissues of the human body…

Identifying the cues necessary for proper skin development…

Skin

Functions: -Regulation of body temperature

-Prevents dehydration

-Protects from toxic substances & radiation

-Protects against mechanical stress and trauma

-Immunological functions

-Sensory organ

Epiderm

is

Derm

is

Skin Epidermis

• Self-renewing

• Keratinocytes- major cell type

• Other cell types: Melanocytes, Langerhans’ cells

Cornified layer

Granular layer

Spinous layer

Basal layer

K5/K14

K1/K10

Loricrin

Pilosebaceous Unit

Hair follicle

Sebaceous Gland

Skin Epidermis

Cornified layer

Granular layer

Spinous layer

Basal layer

K5/K14

K1/K10

Loricrin

E7.5

B Sp

B

Sp

B

Gr Ectodermal Progenitor

Cells

Epit

he

lial

Spe

cifi

cati

on

Co

mm

itm

en

t to

St

rati

fica

tio

n

Co

mm

itm

en

t to

D

iffe

ren

tiat

ion

Co

mm

itm

en

t to

Te

rmin

al

Dif

fere

nti

atio

n

K8/18 K5/14 K1/10 Loricrin

Embryonic Skin Development…

E9.5 E13.5 E17

K8 K5/Lor K5/K10 K5

Stages of Hair Follicle Development

Epidermal signals

organize the

mesenchyme

Dermal signals

coordinate growth and

differentiation

Dermal signals initiate

hair follicle formation

Epidermis

Dermis

E13.5 E14.5 E16.5

Hair Follicle Cycling

Anagen Catagen Telogen Return to

Anagen

How Does the Skin Self-renew?

Stratum

Corneum

Granular

Layer

Spinous

Layer

Basal

Layer Basement Membrane

TA cell

Terminally

Differentiated

Keratinocyte

Stem cell

Proliferative

Potential

TA Cells

-Daughter stem cells that

divide for a limited number of

times and then undergo

terminal differentiation

Stem Cells (SCs) -potential to develop into many

different cell types in the body

-can divide an unlimited number

of times

-each new cell has the potential

to either remain a stem cell or to

become a Transit Amplifying (TA)

Cell

SCs Location: Basal Layer

-Express Keratin5 and Keratin14

Early Stages of Epithelial Development…

Ectodermal Progenitor

Cells

Epit

he

lial

Spe

cifi

cati

on

Co

mm

itm

en

t

to a

n

ep

ith

elia

l lin

eag

e

K8/18

K8

Mammary Gland

Skin

Bladder

Ureter


E7.5


Cells

Epit

he

lial

Spe

cifi

cati

on

Co

mm

itm

en

t

to a

n

ep

ith

elia

l lin

eag

e

K8/18

K8

B

K5/14

K5

p63 is expressed in the proliferating cells of the epidermis and hair follicle

Epiderm

is

Derm

is

p63/DAPI

Romano et al. Development 2010

Transcription factor

Belongs to the p53 family of proteins

Mutations of p63 associated with several human developmental syndromes:

p63

AEC Syndrome (Ankyloblepharon, ectodermal defects-cleft lip/palate)

EEC Syndrome (Ectrodactyly, Ectodermal Dysplasia, and cleft lip/palate)

The different roles of each p53 family member…

Morphogenesis, Epithelial development/ differentiation

Neurogenesis, Neuronal development/ differentiation

Growth arrest Apoptosis

p63

1 2 3 3’ 4 5 6 7 8 9 10 11 12 13 14 15

P1 (TA) P2(N)

p63 genomic

locus

TAp63

Isoforms

Np63

Isoforms

DNA Binding Oligo SAM TID DNA Binding Oligo SAM TID

Loss of p63: We Have a Major Problem…

Yang et al. Nature 1999 Mills et al. Nature 1999

p63 +/- p63-/-

p63 +/- p63-/-

TA DNA binding Oligo TAp63

DNA binding Oligo ΔNp63

Biological Function of p63

+/+ -/-

E17 Lor E17 Lor

Stem Cell

Self

Renewal

Terminal

Differentiation

McKeon Lab

Yang et al. Nature (1999). Mills et al. Nature (1999).

Roop Lab

+/+ -/-

K5/K14 K8/K18

Ep

iderm

al

Sp

ecif

icati

on

Co

mm

itm

en

t

to

Str

ati

ficati

on

Ectodermal

Progenitor

Cells

+/+ +/+

-/- -/-

Loss of p63: We Have a Major Problem…

p63 +/- p63-/-

p63 +/- p63-/-

TA DNA binding Oligo TAp63

DNA binding Oligo ΔNp63

Which Isoform is Important?

Roles for TAp63

TAp63 TA DBD OLIGO SAM

Roop et al. Genes Dev. 2004

Ptk2 Cells

In Vitro

TAp63

TAp63

Np63

Np63

K14/p63

Which p63 Isoform Is Critical In the Skin?

H&E Np63 p63 K5 K14 TAp63

E10.5 E11.5 E14.5 E18.5

Romano et al. PLoS One 2009

Ptk2 Cells Romano, R.A. et al. JID (2007)

Both TAp63 and Np63 Induce K14 Expression

HA-Np63

HA-Np63

HA-Np63

HA-Np63MT

DNA binding domain Oligo SAM

DNA binding domain Oligo

DNA binding domain Oligo

DNA binding domain Oligo SAM

(R249W)

DAPI HA K14 MERGE

DNA binding domain Oligo SAM HA-TAp63


E7.5


Cells

Epit

he

lial

Spe

cifi

cati

on

Co

mm

itm

en

t

to a

n

ep

ith

elia

l lin

eag

e

K8/18

K8

B

K5/14

K5

tTA Keratin 5 Promoter

Tet driver

Np63 TRE HA

Tet responder

Bitransgenic Mouse

Np63 TRE HA Np63 TRE HA

- Dox + Dox

Tet-inducible Expression of Np63 in Transgenic Mice

K5

BG (P30)

Phenotype of Np63 Expressing Mice…

Phenotype of Np63 Expressing Mice…

Epidermal Hyperplasia

Hypertrophic Hair Follicles

Epidermis

Hair Shaft

Sebaceous Gland

Bulge

ORS- K14+,K10-

IRS

Matrix

Derma Papilla

Follicular Transformation

K14/K10/DAPI

WT (P28)

K14/K10/DAPI

BG (P28)

Romano, et al. Development, 2010

Epidermis

Hair Shaft

Sebaceous Gland

Bulge

ORS- K14+, Fil-

IRS

Matrix

Derma Papilla

Follicular Transformation

K14/Fil/DAPI

WT (P28)

K14/Fil/DAPI

BG (P28)


Np63

Bronchi

tTA Human SPC Promoter

Tet driver

Np63 TRE HA

Tet responder

Bitransgenic Mouse (BG)

p63 (null) K5 (null) K14 (null)

Expression of Np63 in Lung Epithelium…

tTA Human SPC Promoter

Tet driver

Np63 TRE HA

Tet responder

Bitransgenic Mouse (BG)

Expression of Np63 in Lung Epithelium…

WT BG

Np63 Can Direct Cell Lineage Commitment and Induce Stratification…

Np63

Bronchi

K1/HA K1/HA

K10/HA K10/HA

WT Np63 (BG)

K5/HA K14/HA

DAPI DAPI

WT

Np63 (BG) Np63 (BG)

Np63 (BG)

WT Np63 (BG)

tTA SPC Promoter Np63 TRE HA

p63 and Epithelial Cell Fate…

E7.5 E9.5


Cells

Epithelial Specification

Commitment to Stratification

K8/18 K5/14

Np63

Np63 regulates basal keratinocyte-specific gene

expression

Np63

Bronchi

K5/HA K14/HA

Np63 directs epithelial cell fate

How Can We Confirm This?

Np63GFP Knock-in mice: Isoform Specific Knockout

1 2 3 3’ 4 5 6 7 8 9 10 11 12 13 14 15

P1 P2

p63 genomic locus

P1 (TAp63) P2 (Np63)

P1 (TAp63) P2 (Np63)

eGFP

p63 gene

Np63GFP

1 2 3

+/+ +/- -/-

Np63

TAp63

GAPDH

Np63eGFP +/- (E14.5)

Np63/K5/DAPI Np63/K5/DAPI

Np63GFP +/- Np63GFP -/-

Day 4

1 2 3 3’ 4 5 6 7 8 9 10 11 12 13 14 15

P1 P2

p63 genomic locus

Validation…

Np63gfp/gfp (E18.5) Np63+/+ (E18.5) Np63+/+ Np63 gfp/gfp

E18.5

Loss of Np63: Major Problems…


Np63 Knockout Mice…

Tongue

Palate

Buccal Mucosa

Np63GFP +/+ Np63GFP -/- Np63GFP +/- Np63GFP -/-

E18.5 E18.5

Keratinocytes lacking Np63: Should I Commit?

E7.5 E9.5 K8/18 K5/14

Epithelial Specification

Commitment to Stratification


Np63

K5/DAPI

K1/DAPI

K5/DAPI

K1/DAPI K10/DAPI K10/DAPI

E15.5

Fil/DAPI Fil/DAPI Lor/DAPI Lor/DAPI

Loss of Np63 Results in Premature Keratinocyte Differentiation

Np63gfp/gfp Np63+/+ Np63gfp/gfp Np63+/+



Compromised Basement Membrane…

Np63gfp/gfp Np63+/+

BASAL CELL

DERMIS

6/K5/DAPI 6/K5/DAPI

4/K5/DAPI 4/K5/DAPI

1/K5/DAPI 1/K5/DAPI

Np63+/+ Np63gfp/gfp

Compromised Integrin Expression…


BASAL CELL

DERMIS

Compromised Cell Adhesion…

Np63+/+ Np63gfp/gfp

Dsg1/K5/DAPI Dsg1/K5/DAPI

Dsg2/K5/DAPI Dsg2/K5/DAPI

Pkp2/K5/DAPI Pkp2/K5/DAPI

Loss of Np63: Major Issues…

K8/18 K5/14

Block in Squamous Switch

Altered Differentiation

Cell-Cell & Cell-Basement Membrane Contacts

Knockout Mice…

• Mice in which researchers have disrupted or “knocked out” an existing gene by replacing it with a foreign piece of DNA.

• The loss of the gene often causes a phenotypic change in the mouse which includes appearance or behaviour

How are Knockout Mice Made? •Pick your favourite gene

•What genomic region to target?

•Design a targeting vector

•Must have 5’ and 3’ homologous (identical)

sequences and selection marker (Neo)

•Targeting vector is electroporated into

Embryonic Stem (ES) cells

•Can select for positive clones that are Neo

resistant

•Positive ES cells are then injected into a

blastocyst and transferred into a pseudo-

pregnant foster mouse to generate chimera

mice

LoxP sites

•Breed mice with Cre Recombinase mice

•DNA between LoxP sites is removed in

cells expressing Cre, generating KO mice

the beauty of the skin - university at buffalorgron/bch512/skin.pdf · the beauty of the skin...

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