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To assess hair follicle density, we measured the distance between
follicles in three different conditions: 1. intact back skin in normal mice, 2. the area of new hair follicle formation
following wounding in normal mice, and 3. the area of new hair follicle formation following wounding in
Wnt7a overexpressing mice. We examined 100 follicles in 3 mice for each condition.
In normal mice, the space between hair follicles is smaller along the sagittal axis (head to tail) compared
to the coronal axis (side to side). For example, the distance between follicles in intact pelage skin of normal
mice was 0.09±0.02 mm sagittally and 0.17±0.04 mm coronally. In contrast, the newly formed follicles in both
control and K14-Wnt7a mice following wounding were spaced with an intermediate distance between them
(0.11±0.03 mm for control and 0.11±0.04 for Wnt7a mice), and there was no apparent difference between coronal
and sagittal distances.SD, standard deviation

Hair type and length: We analyzed
all of the regenerated hairs on 3 mice. All of the regenerated
hairs were of the zig-zag type, which is the most
common type of mouse hair comprising approximately
70% of the pelage hairs. The length of the hair (5.8±0.5
mm in intact skin, 6.0±0.6 mm in regenerated skin [mean
± standard deviation], p=0.27) and spacing of the bends
in these hairs was not different than zig-zag hairs from
intact skin. A representative image of the regenerated hair
is shown.

Hair follicle orientation. To evaluate the
orientation of the newly formed follicles, we examined the direction
of the follicles in 3 mice. Approximately 72-80% of the follicles
were pointing in the correct direction (within approximately
a 30 degree angle). A representative example is shown here in this
K14CreR26R mouse in which the keratinocytes are blue for easier
visualization of the follicles. In this mouse, 31/41 hair follicles
were growing in the correct cephalad orientation. The image is
taken 30 days after wounding.

Hair follicle cycling. To determine the number of times that the new hair follicles
cycled, we counted the number of hair shafts within each follicle 90 days after wounding in one mouse. In
mice, the hair shaft is often retained in the follicle with each cycle, thus the number of hairs per follicle is a
conservative estimate of the number of times the follicle has been through an anagen stage. We found 10 follicles
containing one hair, 14 follicles with two hairs and 6 follicles with 3 hairs, indicating that at least 20%
of the follicles had cycled three times. Interestingly, a normal follicle in a 90-day-old mouse would have also
cycled three times.

Age-related changes in response to wounding. In addition to the time points shown
in the main text, we performed wounding (1.5 x 1.5 cm, full thickness) at 3 (n = 2) and 6 (n = 3) months of
age. 48±13 and 70±15 follicles formed in these wounds, respectively (values are mean±standard deviation). A
representative example of hair follicle neogenesis after wounding in a 10 month old mouse is shown here. The
purple dots represent nascent dermal papilla detected with a stain for alkaline phosphatase activity.