/. Embryol. exp. Morph. 94,113-119 (1986)
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Induction of hair follicles in mouse skin by rat vibrissa
dermal papillae
P. PISANSARAKIT AND G. P. M. MOORE
CSIRO Division of Animal Production, PO Box 239, Blacktown, NSW2148,
Australia
SUMMARY
Rat vibrissa dermal papillae were transplanted between the epidermis and dermis of isolated
embryonic mouse skin and then grafted onto nude mice. The papillae induced the formation of
hair follicles which were larger than those of the host skin but smaller than vibrissa follicles. The
potential of isolated dermal papillae to induce follicles with characteristics of those from which
the papillae originated is discussed. One of the major factors affecting the sizes of induced
follicles may have been related to the splitting of the papilla mass and dispersal of the cells by
invading cords of epidermal cells from the host skin during induction.
INTRODUCTION
The initiation and development of hair follicles occur as a consequence of
interactions between the epidermis and dermis during foetal life. Although the
nature of this association has not been determined, Dhouailly (1977) established
that the types of follicles initiated in embryonic skin grafts and their distribution
pattern are governed by the regional origin of the dermis. Subsequent development of initiated follicles occurs under the influence of condensations of dermal
cells associated with each of the growing follicular plugs (Kollar, 1970). Each
dermal condensation eventually becomes incorporated into a pocket at the base of
the follicle, forming the dermal papilla.
During the anagen phase of the hair growth cycle in the adult animal, a close
association is maintained between the papilla and the proximal bulb region of the
follicle, suggesting that interactions between the cellular components may be a
necessary requirement for fibre production. A structural feature of this dermal/
epidermal association has been identified by Rudall (1956) and Ibrahim & Wright
(1982) who showed correlations between the size of the papillae and the sizes of
the hair follicle and fibre.
A definitive demonstration of the role of the dermal papilla in epidermal cell
differentiation and follicle morphogenesis has been provided by transplantation
experiments. Cohen (1964, 1965) showed that insertion of isolated papillae into
rat skin resulted, in some instances, in the development of follicular structures. However, the sizes of the dermal papillae were not maintained in these
Key words: dermal papilla, follicle induction, nude mouse, hair, vibrissa follicle, rat.
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P. PISANSARAKIT AND G. P. M. MOORE
new associations. Transplantation of large dermal papillae from mature vibrissa
follicles into adult ear skin induced much smaller structures in association with the
host epidermis and only fine hairs were produced (Cohen, 1965). Subsequently,
Oliver (1969, 1970) also using adult skin as host tissue but employing a somewhat
different procedure, observed shrinkage of the implanted papillae, although not to
the sizes of those within the surrounding host skin. Follicles with large bulbs
developed, some of which grew either fine, non-medullated fibres (Oliver, 1970)
or thick whisker-like hairs (Oliver, 1973). However, the sites of origin of the
epidermis (ear, scrotal sac) or epithelium (labial) were found to be crucial to the
type of follicular structure which developed. The foregoing experiments clearly
demonstrated that the inductive activity of the dermal papilla of a mature follicle
was retained. The development of structures showing different degrees of follicular organization also indicated that the differentiated tissues of the adult host may
either modulate, or have a reduced capacity to respond to new induction signals.
Thus, it remained to be established whether the dermal papilla retained the
potential exhibited during follicle development in the foetus. We have therefore
investigated this aspect by implanting dermal papillae from mature rat vibrissa
follicles between the epidermis and dermis of flank skin from mouse embryos at
the stage of hair follicle initiation.
MATERIALS AND METHODS
Isolation of dermal papillae
Vibrissa dermal papillae were obtained from 4- to 5-month-old Sprague-Dawley rats. The
animals were killed and the upper lips were clipped and excised under sterile conditions. The
tissues were quickly washed several times with 70 % alcohol before being transferred into a
solution of phosphate-buffered saline (PBS) supplemented with 2-5 % antibiotic-antimycotic
(AA) solution (Gibco). Follicles were isolated under a binocular microscope and cleaned of the
adhering dermal tissues. The collagenous capsule was punctured with watchmaker's forceps and
peeled over the bulb. The lower portion of follicle bulb was cut open to release the dermal
papilla which at this stage was still attached to the dermal sheath. All of the epidermal matrix
components were removed from the dermal papilla which was then detached from the dermal
sheath. The isolated dermal papillae were collected in PBS+2-5% AA and kept chilled for
4-5 h at 4°C before being implanted in the skin.
Preparation of embryonic mouse skin
Day-14 Quackenbush mouse embryos were used as skin donors. The age of the embryos was
calculated from the day on which a vaginal plug was found in mated females (day 0). For the
purposes of this study the skin is referred to as host skin. The procedures for the removal and
separation of the dermal and epidermal layers of the embryonic flank skin were those described
by Raphael & Pennycuik (1980). Following separation, one or two vibrissa papillae were placed
on the dermis, and the epidermis replaced. The recombinants were then incubated overnight at
37-5°C in an agar-based culture medium.
Transplantation of host skin grafts
Adult nude mice were used as recipients for the grafts; details of the transplantation
techniques have been previously outlined (Raphael & Pennycuik, 1980). A total of ten grafts
was placed on the backs of six nude mice and harvested 21-42 days later.
The dermal papilla and hair follicle induction
115
Histology
Skin samples which included the whole of the host graft and a peripheral strip of nude skin
were excised, placed in Serra's fixative (60% ethanol, 30% formalin and 10% glacial acetic
acid) for 1 h and processed for paraffin embedding. Serial sections (8 jum) of the entire sample
were cut at right angles to the skin surface and stained with haematoxylin, eosin and picric
acid.
RESULTS
With one exception, all of the grafts were covered with hair at harvest.
Examination of sectioned material revealed that the host skin follicles were
morphologically normal. Fibres in the piliary canals had a mean diameter
measurement of 13-6 ± 0-3 /zm (n = 60). In a number of the grafts, the implanted
dermal papillae had induced the formation of hair follicles, or structures resembling follicles. These were readily distinguishable from host hair follicles
because of their large size and included in some instances thick fibres in the hair
canals. A dermal sheath, inner and outer root sheaths and sebaceous glands were
identified in most of the induced follicles, but the blood sinuses and thick
collagenous capsule characteristic of the vibrissa follicle were absent. In all grafts,
the development of induced follicles was completed by 21 days. No additional
structures were observed in follicles harvested after this period. The numbers of
induced structures varied from 0-2 in each transplant, a total of 7 from the 19
implanted papillae. Most of the induced follicles were adjacent and orientated
approximately parallel to the epidermis.
Two large follicles were found in one graft of nude mouse 1 which received two
dermal papillae as implants. One was located deep in the dermis and was aligned
with the surrounding host follicles (Fig. 1). Apart from its large size, the follicle
had a number of unusual morphological features. In the bulb region, a thick
tongue of matrix cells projected into the papilla (Fig. 2) which had become
U-shaped in transverse section. The innermost region of the tongue had differentiated into a column of inner root sheath-like cells which extended distally from the
base of the bulb. As a consequence, the differentiating cortical cells in the upper
bulb region formed two columns, each of which became incorporated into a
medullated fibre. In the zone of hardening, the fibres appeared to have become
fused, forming a hair with two medullae. This compound fibre had a diameter of
45/on.
The second papilla of this graft was located near the epidermis and had
separated into two cell masses. Epidermal cells (Fig. 3) had invested one of the
masses, forming a follicular structure about four times larger than the hair follicles
of the graft. Sebaceous glands had developed from the distal region of the hair
canal and a stout medullated fibre (approximately 30 /im diameter) had penetrated
the skin surface. The remaining part of the papilla formed a dense mass of cells
nearer the epidermis. The epidermis of the whole region had thickened (Fig. 4),
many mitotic figures were evident and a number of short plug-like downgrowths
were closely apposed to the papilla mass.
P. PlSANSARAKIT AND G. P . M. MOORE
116
*
Figs 1-7. Photomicrographs of sections of mouse skin grafts at various times after
implantation of rat vibrissa dermal papillae between the epidermis and dermis.
Abbreviations: dp, dermal papilla; e, epidermis;/, fibre. Bar, 100jum.
Fig. 1. Vertical section of graft 21 days after implantation of dermal papilla showing
large induced follicle surrounded by small host follicles.
Fig. 2. An adjacent section of the induced follicle in Fig. 1 showing a thick tongue
of matrix cells (arrow) projecting through the dermal papilla to the base of the bulb.
The innermost cells of the tongue have differentiated into inner root sheath-like
components.
The dermal papilla and hair follicle induction
111
Fig. 3. Vertical section of skin graft 21 days after implantation of a dermal papilla
showing a large induced follicle incorporating a proximal mass of dermal papilla cells.
A second mass of isolated papilla cells (arrow) is distally located.
Fig. 4. Thickened epidermis adjacent to the distal papilla cell mass (arrow) shown
in Fig. 3. Plugs of epidermal cells protrude into the dermis adjacent to the papilla
cell mass.
Fig. 5. Vertical section of a skin graft after 26 days showing investment of implanted
papilla by epidermal cells. A short follicle composed of a thin matrix (arrow) and a
somewhat disorganized root sheath has formed.
Fig. 6. Section of two induced follicles 35 days after implantation of papillae. One
follicle has been cut longitudinally and shows a bulb matrix enclosing one papilla cell
mass and a distorted fibre. The second follicle has been sectioned transversely through
the piliary canal showing a hardened fibre (arrow) surrounded by root sheath cells.
Fig. 7. An adjacent section of the follicles of Fig. 6 showing a remnant of the bulb
region of the first follicle (arrow) and the upper bulb region of the second follicle
surrounded by a halo of papilla cells.
Only one of the four papilla implants was located in the two grafts of nude mouse
2 after 26 days. The cell mass was positioned directly under the epidermis and had
become almost completely surrounded by cells derived from this layer (Fig. 5).
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P. PlSANSARAKIT AND G. P. M. MOORE
A bulb region formed by the innermost cells was discernible adjacent to the
papilla. The follicle had produced a somewhat distorted fibre approximately 40 pm
in diameter which projected above the skin surface. The cortical zone of the fibre
had hardened completely but the medullary region contained a disorganized
cluster of cells which had not differentiated fully.
Two fibre-producing follicles were found near the surface of one of the two
grafts harvested from nude mouse 4, 35 days after transplantation. The epidermis
was thickened in the region of the follicles which were in very close proximity.
The two follicles were associated with a large group of papilla cells which were
probably an aggregation derived from the original implants. One follicle consisted
of a bulb region partially surrounding a cluster of dermal papilla cells (Fig. 6)
and a distorted fibre had penetrated the skin surface. The other follicle had
produced a fully-formed fibre 25 pm in diameter which projected from a mass of
differentiating epidermal cells (Fig. 7). Surprisingly, the usual configuration of
the epidermal and papilla cells found in the normal follicle was reversed, the
epidermal cells being surrounded by several layers of papilla cells.
Three implanted papillae were located in two grafts 42 days after transplantation (nude mouse numbers 5 and 6). One appeared as a large compact mass
of cells located close to the epidermis. No follicular structures were associated with
the implant. The epidermis was slightly thickened but there were no mitotic cells
in the basal layer. The remaining two papillae had induced the formation of
apparently normal follicles, one of which had produced a fibre measuring 30 fjan in
diameter. Both induced structures were larger than the surrounding host skin
follicles which were all in telogen. However, the papillae were smaller than those
originally implanted.
DISCUSSION
The question we have attempted to answer was whether the dermal papilla of a
mature follicle would retain its inductive potential such that, upon transplantation
in association with embryonic epidermis, a follicle commensurate with that from
which the papilla was isolated would form. Our observations show that the dermal
papillae that were located in skin grafts were, with one exception, reduced in size.
Although the induced follicles and their fibres were larger than those of the host
skin, they did not attain the dimensions of rat vibrissa follicles. Mouse skin may
simply have been unable to support the formation of a structure as large as a rat
vibrissa follicle. However, the implantation procedure itself may have affected
follicle development since reimplantation of dermal papillae or dermal papilla
cells into the bases of vibrissa follicles did not result in the growth of fibres of
normal dimensions (Oliver, 1967; Ibraham & Wright, 1977; Jahoda, Home &
Oliver, 1984).
After implantation, papilla cells may be utilized in the formation of the dermal
sheath around the new structure (Oliver, 1970). The sheath cells have a similar
origin to those of the papilla since their recruitment appears to be the mechanism
The dermal papilla and hair follicle induction
119
by which a new papilla is regenerated in vibrissa follicles from which the proximal
end has been removed (Oliver, 1966). Notwithstanding these effects on papilla cell
number, a factor affecting the inductive potential of implanted dermal papillae in
the current study was the loss of integrity of the papilla structure. Although
the papilla cells may simply migrate out of the implant, some unusual induced
structures observed in the transplants suggest that dispersal of the papilla cell mass
may have resulted from invasion by epidermal plugs. Examples of this include the
presence of a thick layer of cells of epidermal origin in the follicle bulb of Fig. 1,
the divided papilla of Fig. 3 and the fibre-producing plug of epidermal cells
surrounded by a halo of papilla cells of Fig. 7. It is speculated that the influence of
an introduced papilla was so intense as to cause a burst of rapid and relatively
uncontrolled epidermal cell growth. This would be in contrast to the more gradual
inductive influences exerted by the dermis during normal follicle initiation in
embryonic skin as mesenchymal cells form focal condensations adjacent to the
epidermal basement membrane.
We are indebted to Dr K. A. Raphael who performed the skin transplantations of this study.
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