The Beauty of the Skin
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1 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
2 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
3 Identifying the cues necessary for proper skin development
4 Dermis Epidermis Skin Functions: -Regulation of body temperature -Prevents dehydration -Protects from toxic substances & radiation -Protects against mechanical stress and trauma -Immunological functions -Sensory organ
5 Skin Epidermis Cornified layer Granular layer Loricrin Spinous layer K1/K10 Self-renewing Keratinocytes- major cell type Other cell types: Melanocytes, Langerhans cells Basal layer K5/K14
6 Skin Epidermis Cornified layer Granular layer Loricrin Spinous layer K1/K10 Basal layer K5/K14 Pilosebaceous Unit Hair follicle Sebaceous Gland
7 Epithelial Specification Commitment to Stratification Commitment to Differentiation Commitment to Terminal Differentiation Embryonic Skin Development Ectodermal Progenitor Cells E7.5 B Sp B K8/18 E9.5 K5/14 E13.5 K1/10 E17 Loricrin Gr Sp B K8 K5 K5/K10 K5/Lor
8 Stages of Hair Follicle Development E13.5 E14.5 E16.5 Epidermis Dermis Dermal signals initiate hair follicle formation Epidermal signals organize the mesenchyme Dermal signals coordinate growth and differentiation
9 Hair Follicle Cycling Anagen Catagen Telogen Return to Anagen
10 How Does the Skin Self-renew? Stratum Corneum Granular Layer Terminally Differentiated Keratinocyte Basement Membrane Spinous Layer Basal Layer TA cell TA Cells -Daughter stem cells that divide for a limited number of times and then undergo terminal differentiation SCs Location: Basal Layer -Express Keratin5 and Keratin14 Stem cell Proliferative Potential 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
11 Epithelial Specification Commitment to an epithelial lineage Early Stages of Epithelial Development Mammary Gland Ectodermal Progenitor Cells Skin K8/18 K8 Bladder Ureter
12 Epithelial Specification Commitment to an epithelial lineage Early Stages of Epithelial Development Ectodermal Progenitor Cells E7.5 K8/18 B K5/14 K8 K5
13 Dermis Epidermis p63 is expressed in the proliferating cells of the epidermis and hair follicle p63/dapi Romano et al. Development 2010
14 Transcription factor p63 Belongs to the p53 family of proteins Mutations of p63 associated with several human developmental syndromes: EEC Syndrome (Ectrodactyly, Ectodermal Dysplasia, and cleft lip/palate) AEC Syndrome (Ankyloblepharon, ectodermal defects-cleft lip/palate)
15 The different roles of each p53 family member Growth arrest Apoptosis Morphogenesis, Epithelial development/ differentiation Neurogenesis, Neuronal development/ differentiation
16 p63 P1 (TA) P2( N) p63 genomic locus TAp63 Isoforms DNA Binding Oligo SAM TID DNA Binding Oligo SAM TID Np63 Isoforms
17 Loss of p63: We Have a Major Problem TAp63 ΔNp63 TA DNA binding Oligo DNA binding Oligo p63 +/- p63 -/- p63 +/- p63 -/- Yang et al. Nature 1999 Mills et al. Nature 1999
18 Epidermal Specification Commitment to Stratification Biological Function of p63 +/+ -/- McKeon Lab Roop Lab +/+ +/+ +/+ -/- -/- -/- E17 Lor E17 Lor Self Renewal Terminal Differentiation Ectodermal Progenitor Cells Stem Cell Yang et al. Nature (1999). Mills et al. Nature (1999). K8/K18 K5/K14
19 Loss of p63: We Have a Major Problem TAp63 ΔNp63 TA DNA binding Oligo DNA binding Oligo p63 +/- p63 -/- Which Isoform is Important? p63 +/- p63 -/-
20 Roles for TAp63 TAp63 TA DBD OLIGO SAM In Vitro K14/p63 TAp63 Np63 TAp63 Np63 Ptk2 Cells Roop et al. Genes Dev. 2004
21 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
22 Both TAp63 and Np63 Induce K14 Expression DAPI HA K14 MERGE DNA binding domain Oligo SAM HA-TAp63 DNA binding domain Oligo SAM HA- Np63 DNA binding domain Oligo HA- Np63 DNA binding domain Oligo HA- Np63 (R249W) DNA binding domain Oligo SAM HA- Np63MT Romano, R.A. et al. JID (2007) Ptk2 Cells
23 Epithelial Specification Commitment to an epithelial lineage Early Stages of Epithelial Development Ectodermal Progenitor Cells E7.5 K8/18 B K5/14 K8 K5
24 Tet-inducible Expression of Np63 in Transgenic Mice Keratin 5 Promoter tta TRE HA Np63 Tet driver Tet responder K5 Bitransgenic Mouse + Dox - Dox TRE HA Np63 TRE HA Np63
25 Phenotype of Np63 Expressing Mice BG (P30)
26 Phenotype of Np63 Expressing Mice Epidermal Hyperplasia Hypertrophic Hair Follicles
27 Follicular Transformation K14/K10/DAPI Hair Shaft Epidermis Sebaceous Gland Bulge ORS- K14 +,K10 - IRS Matrix Derma Papilla WT BG (P28) Romano, et al. Development, 2010
28 Follicular Transformation K14/Fil/DAPI Hair Shaft Epidermis Sebaceous Gland Bulge ORS- K14 +, Fil - IRS Matrix Derma Papilla WT BG (P28) Romano, et al. Development, 2010
29 Expression of Np63 in Lung Epithelium Np63 Bronchi p63 (null) K5 (null) K14 (null) Human SPC Promoter tta TRE HA Np63 Tet driver Tet responder Bitransgenic Mouse (BG)
30 Expression of Np63 in Lung Epithelium Human SPC Promoter tta TRE HA Np63 Tet driver Tet responder Bitransgenic Mouse (BG) WT BG
31 Np63 Can Direct Cell Lineage Commitment and Induce Stratification SPC Promoter tta TRE HA Np63 Bronchi Np63 WT Np63 (BG) WT Np63 (BG) DAPI DAPI K1/HA K1/HA Np63 (BG) Np63 (BG) WT Np63 (BG) K5/HA K14/HA K10/HA K10/HA
32 p63 and Epithelial Cell Fate Ectodermal Progenitor Cells Epithelial Specification Commitment to Stratification K8/18 K5/14 E7.5 E9.5 Np63 Np63 regulates basal keratinocyte-specific gene expression Np63 Bronchi Np63 directs epithelial cell fate K5/HA K14/HA How Can We Confirm This?
33 Np63GFP Knock-in mice: P1 Isoform Specific Knockout P2 p63 genomic locus P1 (TAp63) P2 ( Np63) P1 (TAp63) P2 ( Np63) p63 gene egfp Np63GFP
34 P1 Validation P2 p63 genomic locus Np63 TAp63 GAPDH /+ +/- -/- Np63GFP +/- Np63GFP -/- Np63/K5/DAPI Np63/K5/DAPI Np63eGFP +/- (E14.5) Day 4
35 Loss of Np63: Major Problems Np63 +/+ Np63 gfp/gfp Np63 +/+ (E18.5) Np63 gfp/gfp (E18.5) E18.5 Romano, et al. Development, 2012
36 Np63 Knockout Mice Np63GFP +/- Np63GFP -/- Np63GFP +/+ Np63GFP -/- Tongue Palate E18.5 E18.5 Buccal Mucosa
37 Keratinocytes lacking Np63: Should I Commit? Epithelial Specification Commitment to Stratification Np63 E7.5 K8/18 E9.5 K5/14 Romano, et al. Development, 2012
38 Loss of Np63 Results in Premature Keratinocyte Differentiation Np63 +/+ Np63 gfp/gfp Np63 +/+ Np63 gfp/gfp E15.5 K5/DAPI K5/DAPI K1/DAPI K1/DAPI K10/DAPI K10/DAPI Lor/DAPI Lor/DAPI Fil/DAPI Fil/DAPI Romano, et al. Development, 2012
39 Compromised Basement Membrane BASAL CELL Np63 +/+ Np63 gfp/gfp DERMIS Romano, et al. Development, 2012
40 Compromised Integrin Expression Np63 +/+ Np63 gfp/gfp BASAL CELL 6/K5/DAPI 6/K5/DAPI 4/K5/DAPI 4/K5/DAPI DERMIS 1/K5/DAPI 1/K5/DAPI Romano, et al. Development, 2012
41 Compromised Cell Adhesion Np63 +/+ Np63 gfp/gfp Dsg1/K5/DAPI Dsg1/K5/DAPI Dsg2/K5/DAPI Dsg2/K5/DAPI Pkp2/K5/DAPI Pkp2/K5/DAPI
42 Loss of Np63: Major Issues K8/18 K5/14 Block in Squamous Switch Altered Differentiation Cell-Cell & Cell-Basement Membrane Contacts
43 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
44 How are Knockout Mice Made? Pick your favourite gene What genomic region to target? LoxP sites 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 pseudopregnant foster mouse to generate chimera mice Breed mice with Cre Recombinase mice DNA between LoxP sites is removed in cells expressing Cre, generating KO mice
45
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