THE JouR AL OF INVESTIGATIVE DERMATOLOGY Vol. 55, No.

6
Copyright © 1970 by The Williams & Wilkins Co. Printed in U.S.A.

THE INDUCTION OF HAIR FOLLICLES BY

EMBRYONIC DERMAL PAPILLAE*
EDWARD J. KOLLAR, PH.D .

AB TRACT
Pelage and vibri sal follicle development was examined in experimental combinations
of embryonic epithelial and mesodermal tissues isolated from the snout, dorsum and
plantar surfaces of the foot plate. Following separation of the tissues by cold tryptic
digestion, experimental and control explants were grafted to the anterior chambers of
homologous adult eyes. When mesodermal tissues from follicle-bearing areas are
combined with glabrous epithelium from the foot plate, hair follicles and sebaceous
glands are induced. Complete and harmonius development occurs in the intraocular
grafts. Experimental exchanges of tissues between the two hair-bearing regions indicated
that epithelium from the snout and dorsum can participate with homologous and
heterologous follicle-bearing mesoderm to produce hair follicle . These data proYide con-
clusive evidence for the inductive role of the dermal papilla during the ontogeny of
pelage and vibrissae follicles.

Investigations of the factors responsible for
the initiation and maintenance of hair growth
generally support the notion that dermal con-
den ations and dermal papillae are obligatory
determinants of development (1-6) . The view
that the dermal papilla induces skin differentia-
tion i a derivative of the classic work of Lillie
and Wang (7, 8), Wang (9), Cairns and Saun-
ders (10), and others (11, 12) who have demon-
trated and defined the tissue interactions oper-
ating in the embryonic avian integument.
Briefly, thi notion suggests that the initiation
of pelage and vibrissae primordia i the result of
inductive epithelia-mesenchymal interactions,
and that in post natal organism hair follicles
are maintained by the continued, if poorly un-
derstood, activities of dermal papillae.
In an earlier report (13), the advantages of
attacking this problem in embryonic skin were
di cus ed. The po sibility of obtaining uncon-
taminated beets of epithelium and mesoderm
containing the anlage of hair is increased during
the early stages of development. In addition,
embryonic rudiment can be cultured in vitro or
can be grafted to advantageous explanation
ite . Finally, following separation, epithelial and
. Received June 29, 1970; accepted for publica-
twn July 20, 1970.
This study was supported by American Cancer
Society Institutional Grant In-41 J.
The author thanks Grace R. Baird for her com-
p tent assistance.
*¥rom th~ Departl'!lent of Anatomy, The Uni-
ver 1ty of Chicago, ChiCago, lllinois 60637.
374
me odermal components from various body sites
can be experimentally recombined to test the
developmental capabilities of the tissues. Thus,
when embryonic snout epithelium was combined
with heterologous mesoderm from the dorsum,
follicle developed. However, the reciprocal com-
bination did not result in hair formation al-
though the epithelium remained viable. More-
over, the follicle structure formed were vibris-
ae-like and suggested that the specificity for the
integumental derivative resides in the snout epi-
thelium, at the ages tested. Furthermore, the e
follicles were produced in response to the induc-
tive stimuli of dermal condensations in the men-
oderm of the dorsum.
These suggestive data notwithstanding, the
experimental design did not provide an unequiv-
ocal demonstration of the inductive nature of
the dermal condensations present in the meso-
dermal components. The most serious shortcom-
ing wa the choice of epithelia and mesoderm
from two follicle-bearing body sites for the re-
ciprocal experimental combinations. Rather, ti -
sue interactions between glabrous skin compo-
nent and haired skin provide the ideal experi-
mental approach analogou ~ to the demonstration
that dermal condensations of embryonic feather
follicles can initiate feather development in ap-
terous regions of the avian integument.
Thi paper describes the results of experimen-
tally exchanging the tissue components of gla-
brou plantar surface skin with the component
 HAIR FOLLICLE INDUCTION
from the nout and dorsum of embryonic mice.
Hair follicles are induced by the dermal conden-
sations in the mesoderm from haired regions in-
dicating that these condensations, the anlage of
the dermal papillae, act inductively and elicit
new developmental expres ion from hairless epi-
thelium.
MATERIALS AND METHODS
Embryonic C5d 10 mice were used. Develop-
mental ages were determined by the appearance
of a vaginal plug and the staging criteria of Grune-
berg (14).
Skin containing the mystacial vibrissae pri-
mordia was excised from the snouts of 11- to 13-
day old embryos. The mid-dorsal skin was re-
moved from 13- to 15-day old embryos. Plantar
surfaces of the posterior foot plates were dissected
from 14- and 15-day old embryonic mice. Posterior
foot plates were used exclusively in the experi-
ments reported here because pilot experiments in-
dicated that plantar skin isolated from anterior
limbs was easily contaminated with hair-bearing
skin from the lateral aspects of the foo t plate.
Careful trimming of the plantar skin and selection
of the central portions of the posterior foot plate
resulted in uniformly follicle-free control explants.
Th e method of stripping epithelium from meso-
derm in these tissue fragments has been described
elsewhere in detail ( 13, 15, 16). The tissue was
treated with a 1% solution of trypsin (Bacto-
Difco, 1 :250) for 1-2 hours at 4° C. Following this
t reatment, the tissues were separated and stored
until recombinations were constructed.
Early attempts to culture recombinations in
conventional culture systems such as plasma clots,
roller tube cultures, agar-solidified complex me-
dium, etc., did not yield consistently satisfactory
results. Instead, the intraocular anterior chamber
was used as an explantation site for long term
culture.
Experimental and control ti sue combinations
were prepared and cultured for 1 or 2 days on me-
dium ( 17) composed of Eagle's basal medium sup-
plemented with 10 % fetal bovine erum, 1% gluta-
mine, penicillin-streptomycin (50 units each / ml) ,
and 0.4 % agar (Grand Island Biological Com-
pany). Recombinants were allowed to cohere on
this medium in a humidified atmo phere of 5%
C02 in air at 37° C. Following thi incubation pe-
riod, the explants could be tran ferred safely tc
the anterior chamber of homologou adult mice.
Four categories of explants were constructed:
(i) control explants of homologous tissues recom-
bined immediately following trypsinization and
separation; (ii) experimental heterologous recom-
binations of mesoderm from he hair-bearing snout
or dorsum with epithelium from the plantar sur-
face; (iii) reciprocal experimental recombinations
of mesoderm from the plantar surface with epi-
thelium from the snout or dorsum; and (iv) re-
375
ciprocal combinations of epithelium and me oderm
derived from the snout and dorsum.
The explants were harvested after 1-2 week in
the intraocular site. The tissues were fixed in
Zenker's-acet ic acid, serially sectioned, and stained
with hematm:ylin and eosin.
RESULTS
The development of pelage and vibrissa! hair
follicles has been described in several recent
publications (13, 15, 18, 19). Despite minor dif-
ference , the two follicle types share similar on-
togenetic sequences. The epithelium invades the
mesenchyme in association with an aggregation
of mesodermal cells, the dermal condensation,
and eventually incorporates these mesodermal
cell as a dermal papilla into the base of the
elongated epithelial follicle. Cytodifferentiation
results in the formation of a keratinized hair
haft. This typical association of epithelium and
mesenchyme is particularly obvious in snout
kin following trypsinization (Fig. 1). The activ-
ity of the trypsin permits separation of the two
tissue components at the level of the basement
membrane.
In contrast, the plantar surface of the foot
does not develop follicle downgrowths into the
me enchyme. Rather a thickened, heavily kera-
tinizing, glabrous epithelium develops on the
plantar surface. The foot pads are specialized
regions characteristic of the plantar surface
(Fig. 2).
The isolated epithelium and mesodermal com-
ponents do not develop independently; the iso-
lated ti sues must be as ociated with each other
if typical morphogenesis is to proceed (13, 15).
On the other hand, trypsin mediated separation
does not severely impair development; when the
two component are re-associated before cultur-
ing, pelage and vibrissae follicles (Fig. 3) de-
velop in control recombinant of homologously
derived component from the same body Eite
(13). Similarly, plantar epithelium and meso-
derm continue to develop (Fig. 4). In this case,
however, the epithelium of these grafts keratin-
izes heavily as is characteristic of this epithelium
in situ; hair follicles do not develop in homol-
ogous control explants derived from the plantar
surface of the posterior foot plate.
Plantar epithelium al o keratiniz.es heavily
when experimentally confronted with mesoderm
from the hair-bearing snout or dorsum. In addi-
tion, however, in the presence of thi. mesoderm
376 THE JO RNAL OF INVESTIGATIVE DERMATOLOGY

Fro. 1. Section of two vibrissae follicles in the process of being freed from the mesenchy-
mal bed of 13-d.ay embryonic snout skin. The tissue was subjected to cold trypsinization
and fixed before complete separation of the epithelium and mesenchyme occurred. X 382.
Fro. 2. Epithelium released from a posterior foot plate of a 15-day embryo following
trypsinization. X 153.
Fro. 3. Advanced follicles in an explant of snout epithelium recombined with homologous
snout mesoderm immediately after separation of the two tissues. This explant was harvested
after two weeks in the anterior chamber. X 382.
 HAIR FOLLICLE INDUCTION 377
containing dermal condensations, posterior foot condensations in the mesoderm. The initiation
plate epithelium participates in hair follicle de- and maintenance of advanced stages of hair fol-
velopment. Large numbers of histotypically per- licle and sebaceous gland differentiation from the
fect follicles are formed in these combinations. usually glabrous plantar epithelium provides an
Advanced states of hair shaft formation and se- unequivocal demonstration of the inductive role
baceous gland development are characteristic of of the dermal papilla.
these explants (Figs. 5 and 6). These data are supported by a similar finding
The reciprocal experimental combinations of in experimental confrontations between snout
epithelium derived from the hair-bearing regions mesoderm and the enamel organs of embryonic
and mesoderm from the plantar surface never mouse tooth germs (20). The enamel organ, a
participate in hair follicle development. The derivative of the oral ectoderm, is a follicle-like
basal layer of the epithelium is thrown into folds structure responsible for enamel secretion during
and large amounts of keratin are formed. These tooth differentiation. When the isolated enamel
observations are typical of homologous or heter- organ is experimentally combined with heterolo-
ologous epithelia supported by mesoderm from gous snout mesoderm, regulation occurs and the
the plantar region. Significantly, follicle and dental epithelium develops as a keratinizing epi-
glandular elements are consistently absent (Fig. thelium resembling surface epithelium. Hair fol-
7). licles and sebaceous glands are induced in the
Finally, heterologous combinations of snout reorganized epithelium. Similarly, follicles are
and dorsum components were of interest since obtained when mesoderm from the dorsum is
these explants were grown in the nutritionally used. Thus, epithelia obtained from very dif-
advantageous explantation site provided by the ferent sources and with seemingly disparate onto-
anterior chamber. Previous data from an organ genetic repertoires can respond to the inductive
culture study (13) indicated that snout epithe- cues provided by dermal papillae of snout and
lium and dorsum mesoderm were able to partici- dorsum mesoderm. Of course, the manner in
pate in hair follicle morphogenesis. The present which the dermal condensations are formed,
study confirms these data. Snout epidermis from their source, and unique properties are of special
the vibrissa! area produced large, well-formed, interest (21, 22). For example, it is not clear
follicles and associated sebaceous glands after whether the inductive cells present when the
one week of explantation (Figs. 8 and 9). Con- mesoderm is isolated function or whether new
trary to our earlier finding, hair follicles did cells arise de novo after recombination of the
develop in reciprocal combinations composed of isolated tissue components. These intriguing
snout mesoderm and epithelium from the dor- problems await future examination.
sum. However, fewer follicles developed in these Nevertheless, it is clear that the epithelium
explants especially when the dorsum epithelium remains responsive to the inductive cues late
was isolated from 15-day old embryos (Fig. 10). into the murine fetal period. The ability of the
plantar epithelium to respond to dermal papillae
DISCUSSION
reported here is supported by our recent finding
Hair follicles develop as a result of inductive (20) that this epithelium can be induced to form
interactions between the epithelium and dermal histotypically normal teeth. Plantar epithelium
FIG. 4. Epithelial keratinization in a control recombinant of plantar epithelium and
plantar mesoderm after one week in the anterior chamber. X 153.
FIG. 5. Follicles (F) and a sebaceous gland (SG) induced from 15-day embryonic plantar
epithelium by 12-day embryonic snout mesoderm after one week in the anterior chamber.
X 382.
FIG. 6. Follicles induced from 15-day embryonic plantar epithelium by 14-day embryonic
mesoderm from the dorsum after ten days in the eye. X 382.
FIG. 7. Heavily keratinized epithelium devoid of follicle or glandular elements in an
explant of dorsal epithelium from a 14-day embryo and mesoderm from 15-day embryonic
foot plate. X 382.
FIG. 8. Ten days after explantation, follicles are developing in a recombinant of 12-day
embryonic snout epithelium and 13-day embryonic mesoderm from the dorsum. X 382.
FIG. 9. Well developed follicles in another explant of 12-day embryonic snout epithelium
combined with 13-day embryonic dorsal mesoderm. X 153.
FIG. 10. Follicles developing in an explant composed of 15-day embryonic epithelium
from the dorsum and 12-day embryonic snout mesoderm after one week in the eye. X 382.
 378 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
from 15-day old embryos are induced to deposit
enamel in response to the dental papilla and to
participate in tooth morphogenesis.
Finally, these data clarify a difficulty raised
by our earlier examination of hair development;
that is, follicles did not develop when epithelium
from the dorsum was combined with snout mes-
oderm (13). Similar combinations examined in
this study, however, did result in follicle devel-
opment. From the general appearance of the
grafts, this experimental combination appears to
be more refractory to inductive stimuli than
other combinations tested; but, nonetheless,
follicles do develop in the intraocular grafts. The
uperior milieu provided by the anterior cham-
ber apparently permits the epithelium to re-
spond to the inductive cues resident in the mes-
odermal tissue.
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