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First published online 31 July 2007
doi: 10.1242/jcs.016253

Journal of Cell Science 120, 2944-2952 (2007)
Published by The Company of Biologists 2007
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Syntenin mediates Delta1-induced cohesiveness of epidermal stem cells in culture

Soline Estrach1, James Legg2 and Fiona M. Watt1,3,*

1 Wellcome Trust Centre for Stem Cell Research, Tennis Court Road, Cambridge, CB2 1QR, UK
2 Cambridge Antibody Technology, Milstein Building, Granta Park, Cambridge, CB1 6GH, UK
3 CR-UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK


Figure 1
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  		Fig. 1. Effects of Delta1 overexpression on Notch activation and keratinocyte differentiation. (A) Schematic representation of the Delta constructs. (B) Anti-DeltaD immunofluorescence staining of human keratinocytes transduced with DeltaFl. Bar, 20 µm. (C) Hes1 luciferase reporter activity (upper panel) in J2-3T3 cells transduced with empty vector (ev), or Delta constructs. Fold induction relative to Renilla control is shown. Mean ± s.e.m. of four experiments. Lower panels show western blot of lysates of J2-3T3 cells used in the luciferase assay, probed with anti-DeltaD and anti-actin (loading control) antibodies. (D) Clonal growth of keratinocytes transduced with empty vector or Delta constructs. (E) Clonal growth of WT keratinocytes on a feeder layer of J2-3T3 cells transduced with empty vector (ev) or Delta constructs. (F-H) Epidermis reconstituted on DED by keratinocytes transduced with empty vector (ev; F,I), DeltaFl (G,J) or DeltaDS (H,K). Sections were stained with H&E (F-H) or anti-keratin 10 antibody (green) with DAPI nuclear counterstain (blue) (I-K). Bars, 50 µm.

 

Figure 2
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  		Fig. 2. Effects of Delta overexpression on differentiation of reconstituted human epidermis. Keratinocytes transduced with empty vector (ev,A,D,G,J), DeltaFl (B,E,H,K) or DeltaDS (C,F,I,L) were grown on DED and stained with antibodies to the proteins shown (green). DAPI was used as a nuclear counterstain (blue). Arrows indicate Ki67-positive cells in the epidermal basal layer. Inserts in A-C represent Ki67 staining only. Bars, 50 µm.

 

Figure 3
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  		Fig. 3. The Delta1 PDZ domain binds syntenin and promotes keratinocyte cohesiveness. (A) GFP-labelled clones formed by keratinocytes expressing the Delta constructs shown 5 days after seeding within a confluent sheet of unlabelled WT keratinocytes. Bar, 20 µm. (B) Percentage of cohesive colonies. Data are mean ± s.d. of five experiments. Values significantly different from WT control are marked with asterisks (Student's t-test, ***P<0.001 or **P<0.05). (C) Western blots of A431 cells transfected with empty vector (ev) or the Delta constructs shown following pull down with GST (control; right panel) or GST-syntenin (left panel). Blots were probed with anti-ZfDelta or anti-GST antibodies. (D) Western blot of untransfected A431 cell lysates following pull down with GST or GST-syntenin (GST-syn). Blot was probed with antibody to human Delta1. Total cell lysates (Lys) served as a positive control. (E) A431 cell lysates immunoprecipitated with antibodies against syntenin ({alpha}-syn) or laminin5 ({alpha}-Lam) followed by western blotting with anti-Delta1 antibody. Lys, lysate subjected to western blotting without prior immunoprecipitation. (F) Immunofluorescence staining of human primary keratinocytes with anti-syntenin antibody (green). DAPI was used as a nuclear counterstain (blue). Bar, 10 µm. (G) Immunofluorescence staining of human interfollicular epidermis with an anti syntenin antibody. Horizontal lines mark position of putative stem cell clusters. Bar, 50 µm.

 

Figure 4
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  		Fig. 4. Functional consequences of syntenin knockdown. (A) Western blot of keratinocytes transduced with empty vector (ev) or syntenin siRNA (Rnai1) probed with anti-syntenin (a-syn) or, as a loading control, anti-tubulin (a-tub). (B) Hes1 luciferase reporter activity in J2-3T3 cells transduced with empty vector (ev) or the Delta constructs shown, in the presence or absence of syntenin siRNA. Fold induction relative to Renilla control is shown. Mean ± s.e.m. of three experiments. Values significantly different from ev control or Fl are marked with asterisks (Student's t-test: ***P<0.001 or **P<0.05). (C) Clonal growth of keratinocytes transduced with DeltaFl or Delta DS and control (Ctrl) or syntenin Rnai1. (D-G) GFP-labelled clones formed by human keratinocytes expressing DeltaFl alone (Fl; D) or in combination with syntenin Rnai1(E), or formed by mouse keratinocytes with endogenous Delta1 levels (Dll1flox/flox; F) or lacking Delta1 (K5Cre x Dll1flox/flox; G). Clones were photographed 5 days after seeding within a confluent sheet of unlabelled WT keratinocytes (human or mouse). Bars, 100 µm. (H) Percentage of cohesive colonies formed by GFP-labelled keratinocytes. Data are mean ± s.d. of three experiments (Student's t-test, ***P<0.001 compared with % clones in WT).

 

Figure 5
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  		Fig. 5. Effects of syntenin knockdown or Delta1 mutation on cell surface expression of Delta mutants. (A-F) Keratinocytes transduced with DeltaFl and pRetrosuper control vector (A-C) or syntenin siRNA (Rnai1; D-F) were double labelled with anti-DeltaD (green A,D) and anti-syntenin (red B,E) antibodies. (C,F) Merged images of middle and left panels. Bar, 20 µm. (G-O) Primary human keratinocytes transduced with DeltaFl, DeltaDS or DeltaVA were incubated with an anti-Delta antibody at 4°C (t=0), then transferred to fresh medium at 37°C for the times indicated. Bars, 50 µm. (P) Percentage of cells with Delta antibody at the plasma membrane. Data are means ± s.e.m. of three independent experiments. 100 cells of each type were counted per experiment. (Q) Western blot of keratinocytes transduced with ev, DeltaFl, DeltaDS and DeltaVA constructs, probed with antibodies to Zf-Delta, syntenin and actin.

 

Figure 6
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  		Fig. 6. Summary of experimental results. Keratinocytes are shown as wild type (WT) or expressing DeltaFl (Fl), DeltaDS (DS) or DeltaVA (VA). Black arrows represents signal from Delta-expressing cell to WT cell; Double-headed arrow represents reciprocal signalling between two Delta-expressing cells. The differentiation signal is represented by three sizes of blue arrow, the thinnest representing no enhanced differentiation, the thickest representing a strongly increased signal, and the medium arrow differentiation that is slightly enhanced. Intercellular adhesion (cohesion) is shown as being promoted (pink arrow) or not (crossed through arrow).

 




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