Vibrissal Follicle Development Following Exposure to Retinoic Acid
198
papilla\ known as the hair matrix[ In this stage\ the K expression
was identical to that described for stage 4 with negative reaction
in IRS and hair "Table 1#[ From day 03[4 of gestation\ blood
cells were seen within the dermal sheath which surrounded
the bottom!developing vibrissal follicle\ representing the initial
stage of blood sinus which appeared well di}erentiated 3 days
later[
was similar to that described previously in hair follicles[ The
conservation of the pattern of K expression during the vibrissal
di}erentiation in RA!treated embryos is in contrast to the
changes observed in the epidermis both of in vitro RA!exposure
mice "Lenoir!Viale et al[\ 0882# and of long!term topical RA
treatment in humans "Eichner et al[\ 0881#[ As the RA!treated
group did not show any changes in the patterns of expression
of K4 and K03 proteins related to the proliferative activity of
the epithelial cells "Byrne et al[\ 0883# when compared with the
control group\ our data may indicate that the protocol used in
this study did not increase the hair growth\ in contrast to the
stimulating e}ect attributed to topical RA treatment in humans
"Bazzano et al[\ 0875#[
In conclusion\ these preliminary results are encouraging con!
cerning the possibility of using a non!teratogenic protocol of
pre!natal RA!exposure\ which induces no adverse e}ects on the
morphogenesis of the skin and its hair follicles[ Thus\ it may
represent a valuable protocol and a promising tool for use in
studies on the protection a}orded by pre!natal RA!exposure on
the development of experimental skin tumours in the exposed
o}spring^ a protective e}ect previously demonstrated after
post!natal RA!administration in mice "Chen et al[\ 0883#[ The
relevance for RA exposure in utero compared with other routes
of administration used during the post!natal period is to
improve the knowledge of the protective e}ect of this retinoid
on skin carcinogenesis with respect to its local:systemic or tem!
poral actions[ Further studies into the e}ects of RA exposure
in utero on mouse pelage hair follicles and the epidermis should
be carried out to con_rm these _ndings[
At day 04[4 of gestation\ the hair canals began to open due
to the keratinization of cells of hair canal "stage 5b or opening
of hair canals# and they began to show a K5!positive companion
layer "CL# made up by a single!cell layer of elongated cells
which rested against the outermost face of the IRS[ The CL
was negative for K5 in the region of the hair bulb and the
follicle above the sebaceous gland[ The companion cells were
also positive for K03 and K4 "Table 1#[ At stage 6 "hair in hair
canals#\ the hair pierced the IRS and to reach the open hair
canal[ From day 04[4 of gestation onward\ a few cells in the
upper third of the vibrissal Outer Root Sheath "ORS# and
epidermal basal cells close to these follicles showed positive
reaction for K7\ being identi_ed as Merkel cells[ At 05[4 day of
gestation\ sebaceous gland cells were seen for the _rst time\
showing a positive immunoreactivity for K03[ One day later\
the hair emerged above the skin surface\ being the last stage of
vibrissal development "stage 7 or emerged hair#[ At stages 6 and
7\ the K expression pattern was similar to that described for
stage 5b "Table 1#[ All embryonic vibrissae showed negative
immunolabelling for K0 and K09 in the three groups examined[
Discussion
Con~icting results on the e}ects of RA have been described in
the literature because the extent of retinoid!induced damage
depends on dosage\ route of administration\ the stage of devel!
opment "Kalter and Warkany\ 0850# and vehicle used "Lehman
et al[\ 0877#[ The variety of results on RA!induced teratogenicity
is even found when our data are compared with those obtained
by other authors "Kochhar et al[\ 0885# who described several
types of malformations in mice\ using a similar protocol to that
used in this study[ Taking into account that the objective of
this study was to obtain a potentially non!teratogenic dosage
without adverse e}ects on skin and its appendages\ our exper!
imental design was established using a lower dosage than the
Acknowledgements
This work was supported in part by grants from the Junta de
ꢁ
Castilla y Leon "LE93:83#[ The authors thank Dr Miguel Angel
ꢁ
Vidal Caballero of the Centro de Investigaciones Biologicas
"Madrid\ Spain# for supplying the anti!K7 antibody used in this
work[
References
Banks!Schlegel\ S[\ 0871] Keratin alterations during embryonic epi!
dermal di}erentiation] a presage of adult epidermal maturation[ J[
Cell[ Biol[ 82\ 440Ð448[
39 mg:kg dose employed by other studies which investigated Bazzano\ G[ S[\ N[ Terezakis\ and W[ Galen\ 0875] Topical tretinoin
for hair growth promotion[ J[ Am[ Acad[ Dermatol[ 04\ 779Ð789[
Byrne\ C[\ M[ Tainsky\ and E[ Fuchs\ 0883] Programming gene
expression in developing epidermis[ Development 019\ 1258Ð1272[
Chen\ L[ C[\ L[ Sly\ and L[ M[ De Luca\ 0883] High dietary retinoic
acid prevents malignant conversion of skin papillomas induced by a
two!stage carcinogenesis protocol in female SENCAR mice[ Car!
cinogenesis 04\ 1272Ð1275[
Council Directive 75:598 EEC\ 0875] Council Directive on the Approxi!
mation of Laws\ Regulation and Administrative Previsions of the
Member States Regarding the Protection of Animals used for Exper!
imental and other Scienti_c Purposes "75:598:EEC#[
the teratogenic activity in mice "Yasuda et al[\ 0876\ 0878#[
In contrast to in vitro studies which showed that RA led to
the development of glomerular glands instead of hair vibrissal
follicles "Dhouailly\ 0882^ Viallet and Dhouailly\ 0883#\ the
absence of RA!induced morphological changes using our pro!
tocol agrees with the results obtained in hair pelage follicles
both in humans after topical application "Eichner et al[\ 0881#
and in mouse teguments cultured in vitro with RA "Dhouailly\
0882^ Viallet and Dhouailly\ 0883#[ Particular attention was
paid to the K expression during the development of mouse
vibrissal follicle because\ to the authors| knowledge\ it has never
been reported in the literature[ As reported for the epidermis
and hair pelage in previous researches "Banks!Schlegel\ 0871^
Fuchs\ 0884#\ our data also indicate that during embryonic
development the vibrissae undergo a programme of di}er!
entiation involving changes in the distribution of K proteins[
Although in our case negative K0 and K09 expression was
found in IRS\ which di}ers from other results concerning hair
pelage follicles of di}erent species "Heid et al[\ 0877#\ in general
the pattern of K expression during the vibrissal di}erentiation
Crave\ N[ M[\ and C[ E[ M[ Gri.ths\ 0885] Topical retinoids and
cutaneous biology[ Clin[ Exp[ Dermatol[ 10\ 0Ð09[
Davidson\ P[\ and M[ H[ Hardy\ 0841] The development of mouse
vibrissae in vivo and in vitro[ J[ Anat[ 75\ 231Ð245[
De Luca\ L[ M[\ L[ Sly\ C[ S[ Jones\ and L[ C[ Chen\ 0882] E}ects of
dietary retinoic acid on skin papilloma and carcinoma formation in
female SENCAR mice[ Carcinogenesis 03\ 428Ð431[
ꢁ
Á
Dhouailly\ D[\ 0882] Expression genique et morphogenese de la peau
ꢁ
ꢁ
ꢁ ꢁ
des vertebres[ Ann[ Genet[ 25\ 36Ð44[
Eichner\ R[\ M[ Kahn\ R[ J[ Capetole\ G[ J[ Gendimenico\ and J[ A[
Mezik\ 0881] E}ects of topical retinoids on cytoskeletal proteins]
Weidenhagen
