Mouse Epidermal Development- Effects of Retinoic Acid Exposure in Utero (Pages 36–44)

8/9/2019 Mouse Epidermal Development- Effects of Retinoic Acid Exposure in Utero (Pages 36–44)

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36 © 2006 The Authors. Journal compilation © 2006 European Society of Veterinary Dermatology

Veterinary Dermatology 2006, 17, 36–44

BlackwellPublishingLtd

Mouse epidermal development: effects ofretinoic acid exposure in utero

ROSA A. GARCÍA-FERNÁNDEZ*, CLAUDIA PÉ REZ-MARTÍNEZ†, JAVIER ESPINOSAÁLVAREZ †, ALEX J. DURÁN NAVARRET E† and MARIA J. GARCÍA-I GLE SIAS†

*Histology and Pathological Anatomy Section, Department of Animal Medicine and Surgery, Faculty ofVeterinary Science, University of Madrid (UCM), Madrid, Spain, †Histology and Pathological Anatomy

Section, Department of Animal Pathology, Animal Medicine, Faculty of Veterinary Science,University of León, León, Spain

(Received 20 May 2005; accepted 24 November 2005)

Abstract Epidermal morphogenesis was studied in vivo following prenatal exposure to retinoic acid (RA). Inpregnant mice, a single oral dose of RA on day 11.5 of gestation failed to induce histological changes in fetalepidermal development except in epidermal thickness. Epidermal thickness increased from 16.5 days post-coitum(dpc) onwards, and temporal and spatial epidermal modifications in keratins K5 and K14 related to proliferative

activity of keratinocytes were observed. An RA effect on cell proliferation was supported by a statisticallysignificant increase in the number of epidermal S-phase cells, containing BrdU–incorporated DNA in RA-exposed mice compared with nonexposed animals. The prolonged in utero action of RA on epidermal prolifer-ative activity in fetuses and newborns suggests a long-term RA effect that may play a role on the developmentand evolution of diseases in adult skin.

INTRODUCTION

Retinoic acid (RA), the most biologically active naturalmetabolite of vitamin A, plays a fundamental role in

embryogenesis and in the differentiation of tissues, includ-ing the skin, and is involved in the control of epithelialcell growth.1

RA is used therapeutically for dermatological2 andneoplastic diseases,3 although the clinical usefulness of retinoids is limited by their teratogenic potential.4–6

Postnatal studies on the effects of retinoids in dermato-logy and oncology have been undertaken in animals7,8

and humans,9,10 following topical application or oraladministration, but knowledge of the in vivo effects onskin exposed to RA in utero is limited to mouse vibrissal11

and pelage hair follicle12 development. The physiological

retinoid, RA, mediates in a wide variety of activities suchas the proliferation of epithelial cells.13 It plays a role inthe expression of keratin (K) proteins in keratinocytes,14

which express different types of RA receptors.15 Theseintermediate filaments have therefore been used as toolsto investigate cutaneous morphogenesis and differenti-ation.16 Thus, the present aim was to determine thein vivo effects of prenatal RA exposure on epidermaldevelopment by analysing changes in morphology,keratin expression and proliferative activity in NavyMarine Research Institute (NMRI) mice, therebycomplementing previous studies on the effects of this

retinoid on vibrissal11 and pelage hair follicles12 andwidening the knowledge of its local, systemic andtemporal actions on the skin.

METHODS

Animals and treatmentSix- to 8-week-old albino NMRI mice (n = 90), weighing30–35 g, were obtained from Antibióticos LaboratoriesS.A. (León, Spain). All were maintained in a cycle of 12 h light and 12 h dark at a controlled temperature(21 ± 1 °C) and relative humidity (55 ± 10%) with freeaccess to food and water. All experiments were carriedout following the guidelines of the European Law on theProtection of Animals.17 To obtain timed pregnancies,

individual female mice were placed randomly withindividual male mice at 20.00 h and inspected dailythereafter at 09.00 h for the appearance of a vaginalplug, which indicated mating. Appearance of the plugwas designated day 0.5 of pregnancy.

The pregnant mice were treated by oral administrationwith 30 mg kg−1 body weight of RA (Sigma ChemicalCo., St Louis, MO, USA) in corn oil on day 11.5 of gestation. This procedure was carried out in the darkunder dim yellow light to retard photodegradation.Two control groups were established at the same gesta-tion time by treating pregnant mice with an identicalvolume of the corn oil vehicle and by leaving one group

untreated. Five embryos across different litters from eachgroup (RA-exposed, vehicle-exposed and nonexposed)were obtained daily from the mothers post-mortembeginning at gestational day 12.5 until day 18.5. In

Correspondence: Dr Rosa Garcia-Fernandez, Histology and Patho-logical Anatomy Section, Department of Animal Pathology, AnimalMedicine and Surgery, Faculty of Veterinary Science, Universityof Madrid (UCM), Madrid, Spain. E-mail: [email protected]



© 2006 The Authors. Journal compilation © 2006 European Society of Veterinary Dermatology

Mouse epidermal development after RA exposure 37

addition, three to seven pregnant mice from eachgroup were allowed to give birth, and their offspringwere euthanized at birth (0) and at 1 day of age. In eachgroup, five animals from different litters were analysedfor each age.

To assess epidermal cell proliferation, 2 h beforeembryo and offspring collection, all pregnant mice andoffspring were given an intraperitoneal injection of 5-bromo-2′-deoxyuridine (BrdU) (Sigma Chemical Co.)(1 mg per 20 g body weight in 150 µL of saline solution).BrdU is incorporated into newly synthesized strands of DNA of S-phase cells in vivo.

HistologyFetuses and newborn animals were fixed in Bouin’ssolution or in 70% ethanol and processed routinely to

paraffin wax. Sections (3 µm) were cut at three differ-ent levels in each instance: one midsagittal and twoparasagittal (one when an eye was seen and the otherwhen the stomach lumen was observed), and stainedwith haematoxylin and eosin (H&E) and Masson’strichrome stains.

ImmunohistochemistryFrom day 12.5 of gestation to 1 day of age, an immuno-histochemical study was carried out on epidermisfrom the seven body regions cited previously in themorphometric study. Midsagittal sections, mounted on

poly

-lysine-coated slides, were subjected to immuno-histochemical staining using the avidin–biotin–peroxidasecomplex (ABC) method (Peroxidase Standard, Vectastain,ABC Kit, Vector Laboratories, Burlingame, CA, USA).Table 1 shows the working dilution and the character-istics of the primary antibodies used. The immuno-reactivity of masked antigens was restored by treatingsections with a solution of 0.1% trypsin (Sigma ChemicalCo.) for monoclonal mouse anti-BrdU antibody or withheat18 for the other antibodies used. After the sequentialincubations with the primary and secondary anti-bodies and the avidin–biotin–peroxidase complex, theimmunoreactivity was demonstrated using diaminoben-

zidine (DAB, substrate kit for peroxidase, VectorLaboratories) as chromogen to produce a brown stainsuitable for light microscopy, and the nuclei werecounterstained with haematoxylin.

Negative and positive control sections for the primaryand secondary antibodies were included. Adult mouseepidermis was used as positive control for all antikeratinantibodies except anti-K6 and anti-K8. A hyperplasicmouse epidermis and mouse hepatic bile ducts were usedas positive controls for anti-K6 and anti-K8 antibodies,respectively. A mouse previously inoculated with BrdUwas used as positive control for anti-BrdU antibody;for corresponding negative control sections, the primaryantibody was replaced with Tris-buffered saline (TBS).

Morphometric study: epidermal thicknessFrom day 12.5 of gestation to 1 day of age, livingepidermal thickness (i.e. without stratum corneum)was measured in the midsagittal tissue section of eachanimal using an ocular micrometre (PK 12.5 × m) (C.

Richert, Austria) calibrated with a stage micrometre.Ten measures on three different sections in each animalwere recorded in each of seven different body regions:cranium, back, abdomen, thorax, neck, mandible andmuzzle, except in the back where 20 were made.

Epidermal cell kineticsFrom day 12.5 of gestation to 1 day of age, the numberof BrdU-positive nuclei per area (µm2) was counted infive ×400 fields of the epidermis of a midsagittal tissuesection of each animal using an image analysis system( version 3.0, Soft Imaging System, Münster,

Germany). Quantification was undertaken on threedifferent body regions: cranial (frontal bone–atlas length),dorsal (atlas–rump length) and ventral (abdomen– thorax–neck length). Cells that had incorporatedBrdU were identified by the precipitation of a brownsubstance in their nuclei.

Statistical analysisOne-way

19 was used for each age (fetuses andnewborns) to test whether the mean of epidermalthickness and BrdU-positive nuclei per area (µm2) of epidermis differed among the groups examined (RA-exposed, vehicle-exposed and nonexposed). As no

significant difference was found between the meansof the two control groups (vehicle-exposed andnonexposed), the average value of both groups was usedfor comparison with the mean of RA-exposed group.

Table 1. Primary antibodies used for immunohistochemical labelling

Clone Character/species Specificity Dilution† Source

TROMA 1* mo/R K8 ud R. Kemler‡AF 109 po/Rb K1 1 in 2 × 105 BabCO, CA, USAAF 138 po/Rb K5 1 in 105 BabCO, CA, USAMK6 po/Rb K6 1 in 105 BabCO, CA, USA

MK10 po/Rb K10 1 in 4 × 10

5

BabCO, CA, USAAF 64 po/Rb K14 1 in 105 BabCO, CA, USAZBU 30 mo/M S-phase cells 1 in 2 × 102 Zymed Laboratories, San Francisco, CA, USA

mo, monoclonal; po, polyclonal; K, keratin; M, mouse; R, rat; Rb, rabbit; ud, undiluted.*70% ethanol-fixed tissues fetuses.†µL in µL (microlitre in microlitre).‡Dr R. Kemler, Max Planck Institute, Freiburg, Germany.



38 RA García-Fernández et al .


Multifactor 19 was carried out to analyse epidermalthickness, using body region as covariate. Differenceswere considered statistically significant at P < 0.05. Datawere expressed as mean ± standard error of the mean(SEM). Analysis was carried out using

version 2.0.

RESULTS

Histogenesis and immunohistochemistryThe main histological features of epidermal develop-ment could be broadly divided into four stages, and thesequence did not differ between control and treatedgroups.

Stage 1. Twelve and a half to 14.5 days post-coitum

(dpc). During this period, the cranium epidermis wasa single layer of ectoderm (Fig. 1) consisting of flattenedor cuboidal cells that expressed K8 keratin. The controlgroup expressed K14 at 13.5 and 14.5 dpc and K5 at 14.5dpc. RA induced an earlier expression of K5 at 13.5 dpc.

In other regions, the epidermis developed into astratum basale (SB) covered by periderm (P): a singlelayer of cuboidal cells, with clear, spheric nuclei andscant basophilic cytoplasm covered by a single layerof flattened cells with dark ovoid nuclei (Fig. 2). Incontrol animals, the SB expressed at 12.5 dpc, K14 (inmandible, neck, thorax and back) and K5 (in mandibleand neck). K14 in the muzzle and abdomen, and K5in the thorax and back first appeared at 13.5 dpc. RAtreatment induced an earlier expression of K5 in the SBof the thorax and back (at 12.5 dpc) and in the muzzleand abdomen (at 13.5 dpc). Likewise, an earlier RA-induced K14 expression was observed in the muzzle at

12.5 dpc (Fig. 3).At 12.5 and 13.5 dpc, the periderm expressed K8 in

both groups but only in the neck and thorax.

Stage 2. Fourteen and a half to 15.5 dpc. During thisperiod, the epidermis was now developing into a three-layered structure – a stratum basale, a stratum inter-medium (SI) and an outer periderm. The new layer

(the SI) was composed of polygonal cells each with anovoid nucleus containing one or two nucleoli (Fig. 4).

The SB in control animals still expressed K5 andK14 in all body regions. The periderm now containedK6 as well as K8, and this was more constantly presentfrom 15.5 dpc onwards. The new SI layer had no keratinexpression at 14.5 dpc, but stained for K1 and K10 at15.5 dpc.

With RA treatment, the SI of the muzzle, neck andthorax expressed K1 earlier (14.5 dpc) (Fig. 5, contrastcontrol group in Fig. 6). Other RA-induced differenceswere found in the periderm where K8 expression wasseen in the mandible at 14.5 dpc. K6 was absent from

the neck at 14.5 dpc and the cranium, muzzle, mandibleand back at 15.5 dpc.

Stage 3. Sixteen and a half to 18.5 dpc. Basophilic granulesappeared in the most superficial layer of stratum inter-medium from 16.5 dpc onwards, and scattered strandsof stratum corneum (SC) were seen at 18.5 dpc (Fig. 7).The stratum intermedium was then referred to as the

gure . tage . cto erm: t e pattern oectodermal keratin expression is shown.

Figure 2. Stage 1: Stratum basale covered byperiderm illustrating the pattern of keratinexpression.

Figure 4. Stage 2: Stratum basale plusstratum intermedium plus periderm showingthe pattern of keratin expression.

Figure 3. Skin (muzzle). RA-exposed fetus. Day 12.5 of gestation.K14 expression in the stratum basale (SB). Avidin–biotin– peroxidase complex (brown); Harris haematoxylin (blue);P, periderm; SB, stratum basale; D, dermis. Light microscope:Olympus Provis AX 70 (Tokyo, Japan).





suprabasal layer or stratum suprabasale (SS). Peridermwas only observed when there was no stratum corneum.In controls, the SB continued to express K5 and K14

and the SI K1 and K10. Periderm still contained K8and K6, whereas the stratum corneum always remainedunstained.

Stage 4. Postnatal 0–1 day of age. The postnatal epider-mis consisted of basal and suprabasal layers, where amorphological difference between the stratum spinosumand granulosum was clear, and a stratum corneum waspresent (Fig. 8).

As in the prenatal situation, the SS still expressed K1

and K10 and the SB K5 and K14 in all body regions.Scattered K6 expression in the SS was seen at day 0 of age (neck, thorax and abdomen) and at 1 day of age(cranium, neck, thorax, abdomen and back). There wasno expression in the stratum corneum.

RA treatment caused expression of K14 and K5 inthe suprabasal layer (Fig. 9) in addition to the normalK1 and K10 expression. The first K6-positive suprabasalcells in RA-exposed mice were seen on 1 day of age infewer body regions than in control groups.

In summary, during prenatal development, RAtreatment induced an earlier expression of K5 andK14 than in controls, and these keratins persisted inthe SS after birth. RA treatment also caused an earlierexpression of K1 and decreased K6 expression inthe SS.

Morphometric study: epidermal thicknessTable 2 shows the mean epidermal thickness in controland RA-exposed groups. One-way for each bodyregion revealed a significant epidermal increase at 12.5dpc (back), 17.5 dpc (cranium, mandible, neck, thoraxand abdomen) and 0 day of age (cranium and thorax),whereas a significant decrease was shown at 14.5 dpc(mandible, thorax and back) and 18.5 dpc (back), both

in RA-exposed animals. When body region was used ascovariate (multifactor), a statistically significantincrease was demonstrated in all skin examined inRA-exposed mice at 16.5 and 17.5 dpc and 0 day of age, whereas a significant decrease was only shown at14.5 dpc.

Epidermal cell kineticsMore BrdU-positive cells were seen in RA-exposedanimals (Figs 10 and 11), demonstrating an increase in S-phase cells and hence indicating proliferative activity.The means of BrdU-positive epidermal cells in RA-

exposed and nonexposed mice were used to comparethe proliferative activity in both groups (Table 3). Astatistically significant increase in proliferative activitywas found in the epidermis from all body regions andin both the prenatal and the postnatal periods follow-ing RA exposure.

Figure 5. Skin (muzzle). RA-exposed fetus. Day 14.5 of gestationshowing K1 expression in the stratum intermedium. Avidin–biotin– peroxidase complex; Harris haematoxylin. P, periderm; SI, stratum

intermedium; SB, stratum basale; D, dermis.

Figure 6. Skin (muzzle). Control fetus. Day 14.5 of gestation.No K1 expression in the stratum intermedium. Avidin–biotin– peroxidase complex; Harris haematoxylin. P, periderm;SI, stratum intermedium; SB, stratum basale; D, dermis.

Figure 7. Stage 3: Appearance of basophilicgranules in the most superficial layer of the

stratum intermedium (stratum granulosum)from 16.5 days post-coitum (dpc) onwardsand scattered strands of stratum corneum at18.5 dpc. The distribution of the layers andthe pattern of keratin expression are shown.





DISCUSSION

The results show that prenatal exposure of mice to RAon day 11.5 of gestation did not modify histogenesis

timing but did have a stimulatory effect on the prolif-erative activity of the developing epidermis. There wasevidence of epidermal hyperplasia mainly at 16.5 and17.5 dpc and in newborn animals, a feature that hasalso been found during topical treatment with RA20–22

and previously related to RA hyperproliferative action.22

The finding of a significant increase in the mean numberof epidermal S-phase cells in treated group, as evidencedby the incorporation of BrdU into their nuclear DNA,and modifications in the K5/K14 filament system (basalkeratins that are expressed in cells that maintain pro-liferative capacity)23 provide further evidence of RAstimulatory action. These findings corroborate previous

descriptions of the proliferative activity of retinoidsin vitro24 and topical treatments.20,25

Epidermis from animals exposed in utero to RAexpressed K5 and K14 proliferation-related keratins

earlier than nonexposed mice during the fetal periodand, after birth, expressed them in both basal and supra-basal layers of the epidermis. RA-induced migration of keratinocytes to the superficial layers without loss of capacity to synthesize keratins related with prolifera-

tion has previously been described and attributed toretinoid interaction with transforming growth factorbeta (TGFβ).26 However, disturbances in K5 and K14expression were not observed when RA was appliedtopically in normal mouse20 and in human27,28 skin.

The only result that does not seem to support theconclusion of a stimulatory action of this retinoid onproliferative activity was the scant K6 expression inthe epidermis from RA-exposed mice. This keratin hasbeen associated with hyperproliferative processes,29 butK6 expression is apparently not essential as its absencein cultured tumour cells is not associated with decreasedcell proliferation.24 Thus, despite the lack of K6 expres-

sion, RA-exposure in utero can be considered to inducea hyperproliferation of the epidermis.

Retinoids are also reported to inhibit epidermaldifferentiation.30 Some authors have attributed this to

Figure 8. Stage 4: Stratum basale plusstratum suprabasale plus stratum corneum.The terminal differentiation of the epidermisand the pattern of keratin expression at birthand at 1 day of age are shown.

Figure 9. Skin (back). RA-exposed fetus. Day 1 of age. Note the K14expression in the basal and suprabasal layers (no expression instratum corneum). Avidin–biotin–peroxidase complex; Harrishaematoxylin. SC, stratum corneum. SS, stratum corneum;SB, stratum basale; D, dermis.

Figure 10. Skin (back). Control fetus. Day 18.5 of gestation. Only asmall number of cells react to anti-BrdU antibody. Avidin–biotin– peroxidase complex; Harris haematoxylin. SC, stratum corneum;*, granular layer of stratum intermedium; SI, stratum intermedium;SB, stratum basale; D, dermis.



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Table 2. Comparison of epidermal thickness between NMRI control and RA-exposed mice during the prenatal and postnatal periods

Bodyregion Groups

Days of gestation

12.5 13.5 14.5 15.5 16.5 17.5 1

Cranium C – 7.95 ± 3.18 6.90 ± 0.31 10.06 ± 0.73 20.39 ± 1.77 17.57 ± 2.13 2RA – 2.80 ± 2.84 5.83 ± 0.57 9.67 ± 1.27 16.38 ± 2.65 26.38 ± 2.82† 2

Muzzle C 11.40 ± 0.85 17.04 ± 4.01 19.44 ± 0.81 25.81 ± 0.86 35.86 ± 1.27 33.20 ± 3.95 3RA 11.87 ± 1.20 12.40 ± 4.74 16.67 ± 1.41 25.38 ± 1.21 34.38 ± 1.68 37.67 ± 6.04 3

Mandible C 9.13 ± 0.74 13.71 ± 2.96 20.80 ± 0.58 24.39 ± 1.05 29.67 ± 1.72 27.46 ± 3.14 3RA 9.00 ± 1.05 13.48 ± 3.50 16.75 ± 0.91† 26.00 ± 1.57 33.38 ± 2.58 41.25 ± 4.15† 3

Neck C 10.80 ± 0.94 17.44 ± 2.32 27.60 ± 0.80 29.79 ± 1.27 31.94 ± 2.15 26.94 ± 2.97 3

RA 8.30 ± 1.21 15.98 ± 2.75 25.50 ± 1.47 29.88 ± 1.68 34.13 ± 2.15 39.50 ± 3.93† 3Thorax C 8.67 ± 0.89 8.71 ± 3.66 3.71 ± 0.26 5.42 ± 0.24 6.72 ± 0.22 6.84 ± 0.22

RA 8.53 ± 1.26 4.05 ± 4.84 2.20 ± 0.47* 4.85 ± 0.36 7.03 ± 0.33 8.63 ± 0.29‡Abdomen C 2.73 ± 0.29 9.48 ± 5.08 12.65 ± 1.04 25.33 ± 1.85 34.06 ± 2.58 32.70 ± 3.59 3

RA 3.00 ± 0.46 4.70 ± 7.19 8.50 ± 1.90 18.38 ± 2.77 36.88 ± 3.65 46.63 ± 4.75† 3Back C 2.43 ± 0.22 9.05 ± 4.17 13.55 ± 0.70 22.00 ± 1.25 32.19 ± 1.41 30.11 ± 3.17 3

RA 4.10 ± 0.31† 4.42 ± 4.93 8.42 ± 4.93† 18.42 ± 1.27 35.63 ± 2.12 36.19 ± 4.20 2All skin examined C 7.05 ± 0.22 12.09 ± 3.16 15.60 ± 0.33 22.00 ± 0.85 27.81 ± 0.96 27.52 ± 2.99 3

RA 7.13 ± 0.30 8.95 ± 3.74 13.19 ± 0.52* 21.63 ± 1.27 33.33 ± 1.44† 34.92 ± 3.97* 3

Data are expressed in µm as mean ± SEM. C, control group; RA, RA-exposed group; All skin examined, data obtained when body region was used as covariangroup: *P < 0.05, †P < 0.01; ‡P < 0.001.





RA inhibition of normal regulation of signalling in thebasal layer, associated with RA-induced K6 expressionin epidermis in vivo.24 In this study, reduced K6 expressionfollowing RA treatment indicates that RA does nothave an inhibitory effect on epidermal differentiation.

Changes in keratinocyte maturation have beendemonstrated in previous in vitro studies with RAinducing a decrease in the expression of K131,32 or K1033

or both.24,28,34 This action has also been observed inin vivo studies after topical RA treatment of adult

epidermis.31,35 However, in this study, RA induced anearlier epidermal K1 expression in fetuses. This indi-cates an earlier RA-induced epidermal differentiationin contrast to a delay in the terminal differentiation

of keratinocytes as described previously for in vitro

treatment.36 They also contrast with the absence of aneffect on epidermal differentiation following in vivo RAtreatment in human skin.28,34

In conclusion, RA administered to pregnant miceinduced, in developing skin from their offspring, anearlier epidermal differentiation. It also stimulated cellproliferation both in the epidermis and in the pelage hair

follicle.12 Such prolonged RA action on skin proliferationraises the question as to whether RA-exposure in utero

could induce a long-term effect on adult skin, therebyplaying a role in development, evolution and prognosisof skin diseases, such as neoplasms, in adult animals.Longer-term studies of animals exposed to RA in utero

are needed to determine the long-term action of thisretinoid on adult mouse skin carcinogenesis.

ACKNOWLEDMENTS

This work was supported by grants from the Junta deCastilla y León (España) (LE04/94). The authors thankDr Miguel Angel Vidal Caballero of the Centro deInvestigaciones Biológicas (Madrid, Spain) for supplyingthe anti-K8 antibody and Dr Juan García Vieitez of theDepartment of Pharmacology, Toxicology and Nursery(University of León, Spain) for advice on RA dosage.

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Figure 11. Skin (back). RA-exposed fetus. Day 18.5 of gestation.Many more cells react positively for anti-BrdU than in controlanimals. Avidin–biotin–peroxidase complex; Harris haematoxylin.SC, stratum corneum; *, granular layer of stratum intermedium;SI, stratum intermedium; SB, stratum basale; D, dermis.

Table 3. BrdU-positive nuclei per µm of epidermis from NMRI control and RA-exposed mice during prenatal and postnatal periods

Age (days) Groups

BrdU-positive nuclei per µm2 according to body region

Cranial Dorsal Ventral

12.5 dpc C 1.7 × 10−2 ± 1.6 × 10−2 2.6 × 10−2 ± 1.2 × 10−2 2.6 × 10−2 ± 1.2 × 10−2

RA 8.5 × 10−2 ± 1.5 × 10−2* 8.3 × 10−2 ± 0.6 × 10−2† 14 × 10−2 ± 1.5 × 10−2†13.5 dpc C 2.5 × 10−2 ± 0.5 × 10−2 3.3 × 10−2 ± 0.5 × 10−2 3.5 × 10−2 ± 0.8 × 10−2

RA 4.2 × 10−2 ± 0.4 × 10−2* 7.1 × 10−2 ± 0.4 × 10−2‡ 10 × 10−2 ± 0.7 × 10−2‡14.5 dpc C 2.4 × 10−2 ± 0.3 × 10−2 2.3 × 10−2 ± 0.5 × 10−2 4.7 × 10−2 ± 0.7 × 10−2

RA 6.4 × 10−2 ± 0.4 × 10−2‡ 10.2 × 10−2 ± 0.6 × 10−2‡ 10.4 × 10−2 ± 0.7 × 10−2‡15.5 dpc C 3.2 × 10−2 ± 0.2 × 10−2 4.3 × 10−2 ± 0.3 × 10−2 5.5 × 10−2 ± 0.6 × 10−2

RA 7.0 × 10−2 ± 0.2 × 10−2† 7.9 × 10−2 ± 0.3 × 10−2† 11 × 10−2 ± 0.6 × 10−2†16.5 dpc C 2.2 × 10−2 ± 0.3 × 10−2 3.0 × 10−2 ± 0.3 × 10−2 3.7 × 10−2 ± 0.4 × 10−2

RA 4.1 × 10−2 ± 0.2 × 10−2† 5.1 × 10−2 ± 0.2 × 10−2† 6.2 × 10−2 ± 0.3 × 10−2†17.5 dpc C 1.8 × 10−2 ± 0.2 × 10−2 1.9 × 10−2 ± 0.2 × 10−2 3.3 × 10−2 ± 0.3 × 10−2

RA 3.8 × 10−2 ± 0.2 × 10−2† 4.1 × 10−2 ± 0.2 × 10−2† 6.1 × 10−2 ± 0.3 × 10−2†18.5 dpc C 2.0 × 10−2 ± 0.2 × 10−2 1.9 × 10−2 ± 0.2 × 10−2 3.0 × 10−2 ± 0.3 × 10−2

RA 3.0 × 10−2 ± 0.2 × 10−2* 2.9 × 10−2 ± 0.2 × 10−2† 4.0 × 10−2 ± 0.3 × 10−2*0 C 0.7 × 10−2 ± 0.1 × 10−2 1.2 × 10−2 ± 0.1 × 10−2 1.7 × 10−2 ± 0.2 × 10−2

RA 1.8 × 10−2 ± 0.1 × 10−2‡ 1.7 × 10−2 ± 0.1 × 10−2* 2.7 × 10−2 ± 0.2 × 10−2‡1 C 1.2 × 10−2 ± 0.2 × 10−2 1.1 × 10−2 ± 0.1 × 10−2 1.8 × 10−2 ± 0.2 × 10−2

RA 2.0 × 10−2 ± 0.2 × 10−2† 1.8 × 10−2 ± 0.2 × 10−2* 2.8 × 10−2 ± 0.3 × 10−2*

Data are expressed as mean ± SEM. dpc, day post-coitum. 0, day of birth; 1, 1 day of age; C, control group; RA, RA-exposed group. Significantlyhigher results were found in the RA group at all ages in the three regions studied (* P < 0.05; †P < 0.001; ‡P < 0.0001).





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Résumé La morphogénèse épidermique a été étudiée in vivo en fonction de l’exposition prénatale à l’aciderétinoïque (RA). Chez les souris gestantes, une seule dose orale de RA au jour 11.5 de la gestation ne provoquaitpas d’anomalie du développement fétal, sauf au niveau de l’épaisseur cutanée. L’épaisseur cutanée a augmentée,de 16.5 jours post coitum (dpc) et par la suite et des modifications de kératines K5 et K14 à relier à la proliférationkératincoytaire ont été observées. Un effet de RA sur la prolifération cellulaire était confirmé par une augmen-tation significative du nombre de cellules épidermiques en phase S-contenant BrdU – incorporated DNA, chezles souris exposées au RA par rapport aux animaux non exposés. L’action prolongéein utero du RA sur l’activité

proliférative chez les foetus et les nouveaux nés suggère un effet à long terme du RA qui pourrait jouer un rôlesur le développement et l’évolution de maladies de la peau de l’adulte.

Resumen La morfogénesis de la epidermis se estudio in vivo en ratones tras ser expuestos durante el períodoprenatal a ácido retinoico. En ratones gestantes una sola dosis de ácido retinoico en el día 11.5 de gestación noprodujo cambios histológicos en el desarrollo fetal de la epidermis a excepción del grosor de la misma. El grosorde la epidermis aumentó desde el día 16 postcopula (dpc) en adelante, y se observaron modificaciones espacialesy temporales en la distribución de las queratinas K5 y K14, en relación con la actividad proliferativa de los que-ratinocitos. El efecto del RA en la proliferación celular en ratones expuestos a RA comparados con animales noexpuestos fue corroborado por un aumento significativo en el número de células epidérmicas en fase S (síntesis),indicado por la presencia de BrdU incorporado en el ADN. La prolongada actividad del RA in utero en laactividad proliferativa de la epidermis en fetos y neonatos sugiere un efecto prolongado del RA que puede jugarun papel relevante en el desarrollo y evolución de enfermedades en la piel de adultos.

Zusammenfassung Die epidermale Morphogenese wurde nach pränataler Exposition zu Retinolsäure (RA)invivo untersucht. Bei tragenden Mäusen konnte eine einzige orale Dosis von RA am 11,5. Tag der Trächtigkeitaußer bei der epidermalen Dicke keine histologischen Veränderungen bei der fötalen epidermalen Entwicklunginduzieren. Die epidermale Dicke nahm ab dem 16,5. Tag post coitum (dpc) zu. Es wurden zeitliche und räumlicheepidermale Modifikationen bei den Keratinen K5 und K14 beobachtet, die auf die proliferative Aktivität derKeratinozyten zurückzuführen waren. Eine Wirkung von RA auf die Zellproliferation bei RA-exponiertenMäusen im Vergleich zu nicht-exponierten Tieren wurde von einer statistisch signifikanten Zunahme der Anzahlan epidermalen S-Phase Zellen, die BrdU-inkorporierte DNA enthielten, unterstützt. Die anhaltende Wirkungvon RA in utero auf die epidermale proliferative Aktivität der Föten und Neugeborenen lässt auf eineLangzeitwirkung von RA schließen, die eine Rolle spielen könnte bei der Entwicklung und Evolution vonKrankheiten der adulten Haut.

Mouse Epidermal Development- Effects of Retinoic Acid Exposure in Utero (Pages 36–44)

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