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…
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
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
Early Stages of Epithelial Development…
E7.5
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
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
Early Stages of Epithelial Development…
E7.5
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
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
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)
Romano, et al. Development, 2010
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
Ectodermal Progenitor
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…
Romano, et al. Development, 2012
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
Romano, et al. Development, 2012
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+/+
Romano, et al. Development, 2012
Romano, et al. Development, 2012
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…
Romano, et al. Development, 2012
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