First published online 22 October 2003
doi: 10.1242/dev.00837
Role of melanoma chondroitin sulphate proteoglycan in patterning stem cells in human interfollicular epidermis
James Legg1,*,
Uffe B. Jensen2,
Simon Broad1,
Irene Leigh3 and
Fiona M. Watt1,
1 Keratinocyte Laboratory, Cancer Research UK London Research Institute, 44
Lincoln's Inn Fields, London, WC2A 3PX, UK
2 Institute of Human Genetics, The Bartholin Building, University of Aarhus,
DK-8000 Aarhus C, Denmark
3 Cancer Research UK Skin Tumour Laboratory, The Royal London Hospital, 2 Newark
Street, London, E1 2AT, UK
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Fig. 1. Expression of MCSP in human interfollicular epidermis. (A,B) Frozen
sections of human scalp labelled with mAb 9.2.27 to MCSP (green in A; red in
B) alone (A) or in combination with an anti-ß1 integrin mAb (green in B).
MCSP staining of outermost epidermal layers is non-specific. The pseudo colour
image in (A) gives an indication of the level of MCSP fluorescence, ranging
from red (highest) to blue (lowest). bl, basal layer of epidermis; Derm,
dermis; Epid, epidermis. (C,D) Whole mounts of human breast interfollicular
epidermis double labelled for MCSP (green) and ß1 integrins (red in C) or
Ki67 (red in D). The MCSP and integrin fluorescence are shown both separately
(C, top) and merged (C, bottom).
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Fig. 2. MCSP expression by cultured human epidermal keratinocytes. (A-C) Abortive
(B) or actively growing (A,C) clones of primary human keratinocytes double
labelled for MCSP and ß1 integrins. (D) Double labelling of MCSP and
actin in a single keratinocyte, demonstrating that MCSP does not localize with
the termini of actin filaments and so is not a component of focal adhesions.
The MCSP and actin labelling are shown separately (top) and merged (bottom).
(E) Immunofluorescence labelling of SCC4 squamous cell carcinoma keratinocytes
showing the expression of MCSP at cell-cell borders and on apical microvilli.
Scale bars: 10 µm (D) and 20 µm (E).
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Fig. 3. Comparison of surface levels of MCSP and ß1 integrins in primary human
keratinocytes by flow cytometry. Profiles of basal keratinocytes are shown
following gating out of differentiated cells. (A) Dot plot of total basal
keratinocyte population. Vertical and horizontal lines represent the
fluorescence of single antibody controls. Boxes represent cells selected on
the basis of highest or lowest 20% of expression of ß1 integrins (FL-1)
or MCSP (FL-3). (B) MCSP fluorescence of total basal keratinocytes (Total) or
20% of cells with highest ß1-integrin expression (ß1-High) or 20% of
cells with lowest ß1-integrin expression (ß1-Low). (C)
ß1-Integrin fluorescence of total basal keratinocytes (Total) or 20% of
cells with highest MCSP expression (MCSP-High) or 20% of cells with lowest
MCSP expression (MCSP-Low). (B,C) Dotted lines show the fluorescence of cells
labelled with secondary antibody alone.
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Fig. 4. Clonal growth of keratinocytes selected for high or low MCSP and ß1
integrin expression. (A) FACS profiles of ß1 integrin and MCSP expression
in the four populations of primary human keratinocytes used for the
clonogenicity assays in (B). Suprabasal cells were gated out on the basis of
forward and side scatter. (B) FACS-sorted keratinocyte populations shown in
(A) were plated at clonal density in triplicate onto mitotically inactivated
fibroblast feeders and cultured for 10-14 days. The proportion of plated cells
that formed clones of any size (%CFE) and the proportion of clones that were
abortive, consisting of terminally differentiated cells (%Ab), were
determined.
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Fig. 5. Expression of CD8/MCSP in primary human keratinocytes. (A) Amino acid
sequence alignment of the cytoplasmic domain of human MCSP with the mouse and
rat forms of the proteoglycan (AN2 and NG2, respectively). Boxes indicate
amino acids completely conserved between all three species. Amino acid
numbering is indicated for the human sequence. (B) Diagrammatic representation
of CD8/MCSP and CD8 constructs. TM, transmembrane. (C,D) Flow cytometry of
single cell suspensions of primary human keratinocytes (C) or SCC4 (D)
transduced with empty vector, CD8 or CD8/MCSP and labelled with anti-CD8
(solid lines in C), anti-MCSP (black lines in D) or secondary antibody alone
(dotted lines in C; grey lines in D).
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Fig. 6. Lack of any effect of CD8/MCSP on terminal differentiation or clonal growth
of primary human keratinocytes. (A) Flow cytometry of single cell suspensions
of primary human keratinocytes expressing empty retroviral vector, CD8 or
CD8/MCSP labelled with anti-involucrin antibody. The proportion of
involucrin-positive cells (indicated by the M1 marker) is indicated. (B)
Keratinocytes were plated in triplicate at clonal density onto mitotically
inactivated fibroblast feeders and cultured for 10-14 days. The proportion of
plated cells that formed clones of any size (%CFE) and the proportion of
clones that were abortive, consisting of terminally differentiated cells
(%Ab), were determined.
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Fig. 7. Effects of CD8/MCSP on keratinocyte colony morphology and motility (see
Movies 1 and 2 at
http://dev.biologists.org/supplemental/).
(A) Primary human keratinocytes expressing vector alone, CD8 or CD8/MCSP were
plated at clonal density onto mitotically inactivated fibroblast feeders and
cultured for 4 days. Phase-contrast pictures were collected using a digital
camera connected to an inverted microscope. (B) Primary keratinocytes
expressing the constructs were plated sparsely onto dishes coated with type-I
collagen and analysed by time-lapse video microscopy. Data on the motility of
cells that were not in contact with other cells were compiled from four
independent experiments, a total of 110-130 cells for each construct. Analysis
of variance showed that there was no significant difference between data sets.
Means of the average speeds of cells in four independent experiments are shown
with standard errors.
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Fig. 8. Effect of CD8/MCSP on intercellular adhesion. Colonies of primary human
keratinocytes expressing CD8/MCSP, CD8 or vector alone were fixed,
permeabilized and labelled with anti-CD8 (A), anti-E-cadherin (B) or
TRITC-conjugated phalloidin (C) and viewed by confocal microscopy. Regions
demarcated by boxes are shown at higher magnification on the right.
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Fig. 9. Effects of pharmacological inhibitors (A) and antibody crosslinking (B) on
the actin cytoskeleton of keratinocytes expressing CD8 or CD8/MCSP. (A)
Keratinocytes were incubated in medium alone (control) or supplemented with C3
toxin, Y-27632 or LY294002 to inhibit Rho, ROCK and phosphatidylinositol
3-kinase, respectively. (B) Keratinocytes were incubated with OKT8 alone
(cont) or OKT8 in combination with anti-mouse IgG (X link) to induce
crosslinking of the CD8 extracellular domain. Cells were labelled with
TRITC-conjugated phalloidin. Scale bar, 40 µm.
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Fig. 10. Role of MCSP in intercellular adhesion. (A) SCC4 cells were incubated in
low calcium medium to inhibit intercellular adhesion (–Ca), then
transferred to standard medium for 2 hours (+Ca) in the presence or absence of
antibodies to E-cadherin (+Ecad) and MCSP (+MCSP), alone or in combination.
Cells were labelled with TRITC-conjugated phalloidin. Scale bar, 50 µm.
(B,C) Effect of CD8/MCSP on the cohesiveness of keratinocyte clones growing
within intact sheets of cultured epidermis. (B) Keratinocytes expressing
CD8/MCSP or CD8 were mixed with an excess of keratinocytes transduced with
vector alone and allowed to grow to confluence. Cells were then labelled with
anti-CD8 followed by Alexa-488-conjugated anti-mouse antibody. Fluorescent
images of CD8-expressing clones growing amongst unlabelled neighbours and
phase-contrast images of the same cells were collected using a digital camera
connected to a fluorescence inverted microscope. Examples of two
CD8/MCSP-expressing and two CD8-expressing clones are shown. Scale bar, 40
µm. (C) Clones were scored for cohesiveness. The mean proportions of
cohesive colonies from two independent experiments are shown. Error bars
represent standard deviation. The total numbers of clones scored in each assay
were 78 (CD8) and 53 (CD8/MCSP).
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© The Company of Biologists Ltd 2003
