Printed in Great Britain Glandular metaplasia of hair ... · Glandular metaplasia of hair follicles and other responses to vitamin A excess in cultures of rodent skin By MARGARET

/. Embryol. exp. Morph. Vol. 19, 2, pp. 157-80, April 1968 \ 57With 4 platesPrinted in Great Britain

Glandular metaplasiaof hair follicles and other responses to vitamin A

excess in cultures of rodent skin

By MARGARET H. HARDY1

From the Department of Anatomy, College of Physicians and Surgeons,Columbia University, New York

INTRODUCTION

The demonstration of mucous metaplasia in chicken embryonic epidermisexposed to an excess of vitamin A (Fell & Mellanby, 1953) stimulated manyfurther investigations (reviewed by Fell, 1964; Fell & Rinaldini, 1965; Dingle &Lucy, 1965), leading to new insights into control of differentiation and functionat the cellular level.

The object of the present experiments was to study the effects of excess vitaminA on another keratin-producing system, the developing hair follicle, in organo-typic cultures. Hairs and their follicles have received little attention from otherinvestigators of vitamin A effects in vitro, although Fell & Mellanby (1953)referred to ' evidence of an inhibitory effect on hair formation' in some pre-liminary experiments with mouse embryonic skin, and New (1963) reportedthat 'usually the development of hair follicles was suppressed' in cultures ofembryonic skin from the rat and mouse, both in the presence and absence ofexcess vitamin A.

Some unexpected and dramatic metaplastic changes in hair follicles exposedto an excess of vitamin A are described in this paper. New information obtainedin the course of the same experiments about the effects of vitamin A on mam-malian epidermis (Hardy, 1965, 1967 a) and tissues of the oral cavity (Hardy,1967 c) are reported elsewhere.

MATERIALS AND METHODS

The mouse embryos used and the four experiments performed have alreadybeen described in the paper dealing with the epidermis (Hardy, 1967 a). Theexperiments are summarized in Table 1. Briefly, organotypic cultures of abdomi-nal skin, upper lip and lower jaw were grown in a solid medium consisting of

1 Author's address: Department of Anatomy, University of Guelph, Guelph, Ontario,Canada.

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158 M. H. HARDY

3 drops of fowl plasma and 1 drop of chicken embryo extract on coverslipsinverted over Maximow slides and incubated at 35-5 °C. In each experiment theeffect of adding certain concentrations of vitamin A was tested, and in Exp. 4,hydrocortisone was added to two of the four treatment groups to see whethervitamin A effects were suppressed. The explants were transferred to fresh clotsevery 3 or 4 days and subdivided when necessary to meet nutritional requirements.

Table 1. Summary of experiments and numbers of explants

Number of explants with additions to basic medium

Age of Dura- EthExp. embryos tion Tissue Eth Eth Eth 12-5A Eth Ethno. (days) (days) explanted None Eth HC 6-2A* 12-5A HC 25-OA 37-5A

1 15 11 Trunk skin 8 8 — — 8 — 8 —2 13 | 11 Trunk skin — 8 — 4 4 — 4 4

Upper lip — 1 — — 1 — — —Half lower — 1 — — 1 — — —jaw

3 14 14 Upper lip — 4 — — 4 _ _ _Half lower — 4 — — 4 — — —jaw

4 15 11 Half upper — 6 6 — 6 6 — —lip

Total explants for each treatment 8 32 6 4 28 6 12 4

Eth = ethanol (00015 ml./ml. final medium); A = vitamin A; HC = hydrocortisone(7-5 /tg/ml. final medium).

* The numbers preceding 'A ' denote concentration expressed as i.u./ml. of final medium.

All explants were fixed after 4-14 days, a few in buffered 2 % osmic acid for1 /i sections stained with toluidine blue, and the remainder in Zenker's fluid forcomplete serial paraffin sections at 8/t. The latter were stained (see Hardy,1967 a) with Mayer's haemalum, eosin and picric acid (HEP), Mayer's haema-lum and Alcian blue (HAB), or by the periodic acid Schiff procedure (PAS) inthe presence or absence of diastase digestion and of counterstaining by Mayer'shaemalum.

The explants of skin from the trunk were either devoid of hair follicles(13^-day embryos) or contained pelage hair follicles in early stages of develop-ment (14-, 15-day embryos). Each explant of upper lip in Exps. 2 and 3 containedall the five rows of mystacial vibrissal follicles from one side of the face (Text-fig. 1) and each 'half upper lip' explant in Exp. 4 contained two or three rows.The lower jaws were bisected medially to provide explants of 'half lower jaw',of which those in Exp. 2 contained interramal vibrissal follicles while those inExp. 4 contained submental follicles of a type intermediate between vibrissaland pelage.

The use of Maximow slides permitted daily microscopic examination of

Skin response to vitamin A excess 159living explants and recording of the development stages of many follicles fromday to day. Other follicles, however, were obscured. The stage of the mostadvanced follicle which was clearly visible in the living explant each day wasarbitrarily chosen to record the stage of the explant, since there are widevariations in follicle development times and rates within one skin region both

'A Interramal

Text-fig. 1. Head of mouse showing the main groups of vibrissae.

Table 2. Summary of stages of development of pelageand vibrissal hair follicles in the mouse

Stage no.

0123a

3b3c

45678

No folliclesFollicle placode or plugPre-papillaDermal papilla invaginationbegun (depth < width)

Dermal papilla depth = widthDermal papilla fully invaginated(depth > width)

Hair coneHair canalFully keratinized hair formedTip of hair in canalTip of hair emerged

Modified from Davidson & Hardy

Average age atearliest appearance

(days from conception)

Pelage folliclesdorsal and

lateral trunk

<14141516

1718

1920212223

(1952).

Mystacialfollicles

<12121313*

1414

14|151617171

160 M. H. HARDY

in vivo and in vitro. In a fixed explant the stage recorded was that of the mostadvanced follicle found in the complete serial sections, and there was goodagreement between observations on living and fixed tissue. Stages 1-8 in hairfollicle development in the mouse as originally defined for the pelage hairs byHardy (1949), and a later subdivision of stage 3 (Hardy, 1967b), were used.Table 2 summarizes the criteria for recognizing these stages and the embryonicage at which each was first observed in sections of skin from the dorsolateraltrunk (pelage) and the upper lip (vibrissae) respectively. The distinguishingmorphological features of vibrissal follicles at all stages of development andtheir faster rate of development have been described previously (Davidson &Hardy, 1952).

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Days from conception

Text-fig. 2. Graph showing rates of development of pelage follicles from 15-dayembryos, x • • • x , in trunk skin in situ; • • , in explant showing maximumprogress obtained in control medium (Exp. 1); O - - O , in explant showing themaximum progress obtained in vitamin A medium (25-0 i.u./ml.). The meaning of theshaded bands in this and subsequent text-figs, is explained in the Discussion, p. 176.

RESULTS

To understand the peculiar orientation of mouse embryonic skin with develop-ing hair follicles in coverslip cultures, the reader should consult earlier papers(Hardy, 1949, 1951). The overall behaviour of explants and the histology ofthe dermis and epidermis in the present series of experiments has already beendescribed (Hardy, 1967 a).

Skin response to vitamin A excess 161

(1) Pelage hair follicles from the trunk of 15-day embryos (Exp. 1)

At the time of explanation, the skin contained the first-formed pelage folliclesat the beginning of stage 2, evenly spaced over the skin surface. A few lessdeveloped follicles (stage 1) had appeared between them.

Observations on living explants and sections

During the first 4 days in vitro some normal follicle development took placein all treatment groups, and at least six explants reached stage 4, the normalstage for 19 days in vivo (Text-fig. 2). Some new follicles were initiated in most

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Text-fig. 3. Graph showing rate of development of pelage follicles from 13J-dayembryos, x • • • x, in trunk skin in situ; O - - O , in explant (Exp. 2) showing themaximum progress obtained in vitamin A medium (6-2 i.u./ml.).

explants. Thereafter differences were noted in the living explants between controland vitamin-treated groups. Because of the curling of the epidermis around thedermis which frequently occurs in 15-day explants with the culture techniqueused, many follicles were prevented by poor nutrition from continuing todevelop at the normal rate. However, in three of the eight control explantswhich were cultivated for 8 or more days, normal keratinized hairs were formed(Text-fig. 2), and six out of eight explants reached stage 4 at least. There was nodevelopment beyond stage 4 in either of the vitamin A-treated groups, and mostof the follicles which could be distinguished in the living explants were seen to

162 M. H. HARDY

regress to earlier stages—3 a, 2 or 1. Many follicles disappeared completely.After 8 or more days of cultivation, none of the vitamin-treated explants wereas advanced as stage 4. In one the follicles were observed regressing fromstage 4 at 4 days to stage 2 at 8 days and then partially recovering, to stage 3 cafter 11 days (Text-fig. 2).

(2) Pelage hair follicles from the trunk ofYh\-day embryos (Exp. 2)Two control groups (combined in Table 1, but actually in media containing

plasma from different batches) were compared with four groups receivingdifferent levels of vitamin A. At the beginning of the experiment there were nosigns of follicle development.

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Text-fig. 4. Graph showing rates of development of vibrissal follicles from 13J-dayembryo, x • • • x, in upper lip in situ; • • , in explant of upper lip (Exp. 2)in control medium; O - - O, in explant of upper lip in vitamin A medium (12-5 i.u./ml.) (the upper line represents maximum progress and the lower line minimumprogress of those mystacial follicles which were most advanced at the beginning ofthe experiment); • 1, in explant of lower jaw (interramal follicles) in controlmedium; • - - • , in explant of lower jaw (interramal follicle) in vitamin A medium(12-5 i.u./ml.).

Observations on living explants and sections

The first follicles did not appear for 3 or 4 days, more slowly than in vivo.Once they began, some follicles proceeded to develop at about the normal rate,but when the experiment was terminated at 11 days the most advanced follicles

Skin response to vitamin A excess 163were still only at stage 5 (Text-fig. 3). The numbers of successful explants in thisexperiment were too small for an evaluation of different treatments, but thefollowing tentative conclusions were drawn: (a) the 37-5 i.u./ml. level of vitaminA, which was toxic to the skin (Hardy, 1967 a,) was probably also inhibitory tothe hair follicles (e.g. only one explant in four reached stage 2); (b) at all otherlevels of vitamin A (25-0, 12-5, 6-2 i.u./ml.) the development of hair follicles,though limited, was no more limited than in the control explants; (c) the mostadvanced hair follicles in the vitamin A-containing media appeared to beprogressing normally (Plate 1, fig. A), except that in the living state they fre-quently had a swollen appearance or an irregular outline.

(3) Vibrissal follicles in the upper lip of a \3\-day embryo (Exp. 2)

At the beginning of the experiment most of the largest mystacial vibrissalfollicles in the two explants were at stage 3 a (Plate 1, fig. B), but an occasionalone was at stage 3b. Many smaller follicles were at stages 2 and 1. The dermalpapillae of most follicles were still shallow, and the hour-glass shape which ischaracteristic of the largest 14-day vibrissal follicles (Plate 1, figs. C, D) hadnot yet appeared.

Observations on living explants

After 2 days the control explant of upper lip reached stage 5 (Text-fig. 4) andthe refractile cones of Henle's layer of the inner root sheath were clearly visible.After 3 days it was at stage 6 and after 6 days at stage 8. The shape of the follicleswas preserved, and well-developed hairs were seen. The vitamin-treated explantreached stage 4 after 2 days, when the follicles appeared swollen and flaccid.After 3 days, swellings were noted on the outer root sheaths of a few of thefollicles (stage 3 c) and by 4 days these were well-defined buds. Elongation ofthe buds was observed in the remaining 3 days of culture, and the refractilecones of Henle's layer were not seen in these follicles. Follicle differentiationremained 2 or 3 days behind that of the control explant, but continued untilfixation at 7 days. Keratinization of the epidermis was delayed by 2 days in thevitamin-treated explant, but in other respects the structure of the skin appearedsimilar in the two cultures.

Observations on histological sections

After 7 days in vitro the control explants contained many relatively thick(10-12 ft) keratinized hairs in follicles at stage 8 (Plate 2, figs. A-C). The vitaminA-treated explant had only a few very fine keratinized hairs (maximum diameter4-5 /*) in follicles at stage 6, although all tissues were healthy and the dimensionsof follicles and root sheaths were similar to those in the control explant. In thetreated explant, three less advanced follicles (two at stage 5 and one at stage 4),which in the living state had shown lateral budding, were traced through serialsections. The main part of these follicles and the surrounding tissues were

164 M. H. HARDY

healthy and differentiating normally (Plate 2, figs. D-G). The buds were at firstsolid cylindrical outgrowths from the outer root sheath (Plate 2, fig. D) con-sisting of cuboidal epithelial cells. One had a terminal swelling (Plate 2, fig. G)and the beginning of a lumen (Plate 2, fig. F).

Several other glandular structures not seen in the living explants which werereceiving excess vitamin A were discovered in the fixed tissue and two weretraced through serial sections. They were both branching epithelial down-growths from the epidermis. One was mainly solid, with two unusually largesebaceous glands budding from it. The other had a duct with a long narrowlumen, a very small sebaceous gland and a solid terminal swelling with manymitoses. No trace of similar structures was found in the control explant. Thesignificance of these epidermal appendages will be referred to in the discussion.

(4) Vibrissal follicles in the lower jaw of a \3\-day embryo (Exp. 2)

At 13 -̂ days the three interramal follicles were at stage 1 in a papilla-likeelevation of the skin in the mid-ventral line (Text-fig. 1). The development of twointerramal follicles very close together was observed in the living control explantconsisting of one half of the jaw, and sections after 11 days of cultivationshowed one follicle at stage 5 (Text-fig. 4) and a second follicle base at stage 3 cjoining the side of the first follicle. A third follicle close by, probably a pelagefollicle, was observed first at 9 days and reached stage 3 a at 11 days. In thevitamin A-treated explant a single follicle, which was probably interramal,reached stage 2 at 2 days and stage 3 a at 4 days, but when fixed at 11 days wasatrophic and had only reached stage 3 c. A few other atrophic follicles at stages1 and 2 which were seen only in sections may have been submental vibrissal orpelage follicles. No budding from follicles was observed in the lower jaw explants.

(5) Vibrissal follicles in the upper lip of 14-day embryos (Exp. 3)

The observations on budding from follicles made in Exp. 2 led to the designof Exp. 3, in which four pairs of carefully matched explants from the upper lipand lower jaw were compared. Some of the rapidly enlarging explants were

PLATE 1

Fig. A. Section through explant of trunk skin from 13^-day embryo after 11 days in 6-2 i.u./ml. vitamin A medium (Exp. 2), showing pelage follicle developed normally to stage 5.d.p. = dermal papilla; o. = outer root sheath; He. = Henle's layer; Hu. = Huxley's layer;cu.i. = cuticle of inner root sheath; cu. = cuticle of developing hair; co. = cortex of de-veloping hair (not yet keratinized). HEP, x 250.Fig. B. Section through upper lip of 13^-day embryo (control to Exp. 2) showing vibrissalfollicle at advanced stage 3 a. Note shallow dome-shaped dermal papilla (d.p.), and beginningof condensation of mesenchyme cells to form connective tissue sheath (c.t.). HEP, x 250.Fig. C. Section through upper lip of 14-day embryo (control to Exp. 3) showing vibrissalfollicle at stage 3 b with dermal papilla (d.p.), hour-glass shape and connective tissue sheath(c.t.). HEP, x 100.Fig. D. Vibrissal follicle in fig. C at the same magnification as fig. B to show the greaterfollicle length and deeper dermal papilla invagination. HEP, x 250.

J. Embryol. exp. Morph., Vol. 19, Part 2 PLATE 1

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J. Embryol. exp. Morph., Vol. 19, Part 2 PLATE 4

M. H. HARDY

Skin response to vitamin A excess 165divided after 4 days to ensure adequate nutrition, so that some groups eventuallycontained as many as seven explants.

Immediately after explantation the living mystacial follicles were seen to beat stages up to 3b, and were a little more advanced than those in the 13^-dayembryos. The larger ones had acquired the characteristic hour-glass shape ofvibrissal follicles (Plate 1, fig. D), and a well-defined connective tissue sheath(Plate 1, fig. C).

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Days from conceptionText-fig. 5. Graph showing rates of development of vibrissal follicles from 14-dayembryos, x • • • x, in upper lip in situ; • • , in explants of upper lip (Exp. 3)in control medium; O- - O, in explants of upper lip in vitamin A medium (12-5 i.u./ml.) (the upper line represents maximum progress and the lower line minimumprogress of the mystacial follicles which were most advanced at the beginning of theexperiment); • • , in explants of lower jaw (submental follicles) in controlmedium; • • in explants of lower jaw (submental follicles) in vitamin Amedium (12-5 i.u./ml.).

PLATE 2

Figs. A-C. Sections through explant of upper lip of 13^-day embryo cultivated for 7 days incontrol medium (Exp. 2), showing normal vibrissal follicles at stage 8. Figs. B and C illustratethe middle and lower portions of the same follicle and fig. A the upper portion of a similarfollicle with emerging hair tip (/*.). HEP, x 250.Figs. D-G. Sections through a single vibrissal follicle in explant of upper lip from the sameembryo as figs. A-C but cultivated for 7 days in 12-5 i.u./ml. vitamin A medium (Exp. 2),showing gland-like structure arising, from follicle. Note enlarged outer root sheath butotherwise normal differentiation of follicle layers to stage 5. /. = lumen. HEP, x 250.

166 M. H. HARDY


For the first 2 days in culture the vibrissal follicles developed normallyalthough a little slowly in both treated and control groups (Text-fig. 5). After3 days many follicles in both groups had reached stage 4, but in one explant inthe vitamin A medium the epidermal parts of the follicles had rounded basesinstead of being invaginated by dermal papillae.

At 5 days all of the five control explants containing epidermis had normallyshaped follicles at stages 4, 5, 6 or 7, but no normal follicles were seen in anyof the corresponding five vitamin-treated explants. Most of the follicles in thevitamin A group were long and thin, and had lost their dermal papillae, thickconnective tissue sheaths and internal structure. Their epithelial outlines wereirregular and buds were commencing to form from the outer root sheaths. At7 days the follicles in control explants were still developing normally (stages 6,7 and 8; Plate 3, figs. A, B), and small sebaceous glands with one to threedifferentiated sebaceous cells were observed in two out of five explants. In allof the four treated explants in which details could be observed clearly, most ofthe follicles were quite abnormal, and only one of normal appearance wasobserved, at stage 4. The others had no dermal papilla, and knobs or cylindersof cuboidal cells arose from one or more sites on the outer root sheath (Plate 3,figs. C, D). The thick connective tissue sheaths were absent from these irregularfollicles. There was also an excessive development of sebaceous cells at thistime. In every treated explant many of the follicles showed buds or parts of theoriginal follicles which were filled with differentiated sebaceous cells and darkbrown masses of sebum or sebum-like material.

PLATE 3

Fig. A. Entire living unstained explant from upper lip of 14-day embryo after 7 days in controlmedium (Exp. 3) showing numerous vibrissal follicles at stages 6-8 attached to central massof keratinizing epidermis, d.p. = Dermal papilla; c.t. = connective tissue sheath. x40.Fig. B. A follicle at stage 8 in the upper part of fig. A. d.p. = margin of dermal papilla;o. = outer root sheath; He. = Henle's layer; co. = developing hair cortex (not keratinizedat this level in follicle), x 100.Fig. C. Entire living unstained explant from upper lip of same embryo after 7 days in 12-5 i.u./ml. vitamin A medium, showing loss of normal structure and budding from follicles, x 40.Fig. D. A follicle at lower left of fig. C which consisted only of a budding mass of cuboidalepithelial cells, x 100.Fig. E. Section through entire explant shown in fig. A after 10 days in control medium, showingcentral epidermis and well-developed vibrissal follicles cut at various angles. HAB, x 40.Fig. F. Enlargement of part of field in fig. E to show follicle structure, d.p. = Dermal papilla;c.t. = connective tissue sheath; h. = keratinized hair. HAB, x 100.Fig. G. Section through entire explant shown in fig. C after 10 days in 12-5 i.u./ml. vitamin Amedium, showing central epidermis surrounded by numerous glands and one hair follicle.HAB, x40.Fig. H. Enlargement of part of field in fig. G, showing hair follicle at stage 5 (/.) and branchingtubular glands (g.). HAB, x 100.

Skin response to vitamin A excess 167At 9 days the control explants showed many normal follicles up to stage 8,

but sebaceous glands were no longer seen. Most of the follicles in treated explantswere abnormal, and the single normal follicle previously observed had nowreached stage 7. The buds from other follicles had undergone extensive branch-ing to form complex glandular but mainly non-sebaceous masses. Three outof four treated explants each contained in addition three or more sebaceousglands.

At 10 days all control explants showed a number of normally keratinizedvibrissae, while only one of the vitamin-treated explants showed a follicle atstage 8 (Text-fig. 5). Sebaceous glands were not identified with certainty in anycontrol explant, but were present in three out of four treated explants. Thebranching non-sebaceous glandular masses derived from follicles were seen inall treated but no control explants.

Some explants were fixed after 10 days and the remainder examined at 12and 14 days. The two control tissues changed little during the remaining periodof cultivation but the two vitamin-treated explants showed further proliferation

PLATE 4

Fig. A. Section through explant of upper lip from 14-day embryo after 14 days in 12-5 i.u./ml. vitamin A (Exp. 3), showing large healthy vibrissal follicle (stage 8) cut obliquely andcontaining a minute keratinized hair (/*.). Attached to follicle is a branching tubular glandwith cystic enlargement of some ducts. HAB, x 67.Fig. B. Section through another part of explant shown in fig. A with parts of two branchingtubular glands. Gland on left has a duct opening directly to epidermis. Gland on right has,attached to its duct, an epidermal mass (/.) representing the original hair follicle. HAB, x 67.Fig. C. Section through same explant as in figs. A and B, showing two glandular trees. Noremnants of the original follicle were found in association with the gland on left. HAB, x 67.Fig. D. Section through another explant of upper lip from 14-day embryo after 10 days in12-5 i.u./ml. vitamin A medium (Exp. 3) showing four former hair follicles whose completetransformation into glands was observed in the living state. At left, left centre and rightcentre are three branching tubular glands derived from three follicles, and at the upper rightis the sebaceous gland portion of a glandular structure derived from the fourth follicle.HEP, x67.Fig. E. Section through explant of upper lip from 14-day embryo after 10 days in 12-5 i.u./ml.vitamin A medium (Exp. 3) showing differentiated sebaceous cells (s.c.) at left, and sebaceouscells breaking down to produce sebum (se.) at right. HEP, x 250.Fig. F. Another part of explant shown in figs. A-C showing transverse sections of a largecyst and several small ducts (d.) and terminal portions of branching tubular glands. HEP,x250.Fig. G. Part of section seen in fig. D enlarged to show oblique section of small duct (on left)and two terminal swellings with small lumen (/.) surrounded by cuboidal (?'alveolar') cells.HEP, x250.Fig. H. Another part of explant shown in figs. A-C and F with small gland ducts (d.) and'alveoli', the latter with a very small lumen (/.) of an intercellular canal. HAB, x 250.Fig. I. Duct of tubular gland in same explant showing mucopolysaccharide reaction. The darkshade of the cytoplasm of marked cell (c.) and of strands crossing the duct from this cell aredue entirely to Alcian blue staining. HAB, x 1000.

168 M. H. HARDY

of the non-sebaceous glands, and traces of patent ducts and terminal 'alveoli'.The sebaceous secretion was also still very evident. The hair follicle in thetreated explant which was previously observed at stage 8 was still visible, andtwo other follicles in the same explant reached stage 5. A second treated explantnow had several normal-looking follicles at stage 4. Some of these 'recovering'follicles had acquired a fairly well developed connective tissue sheath, but stillhad an irregular epithelial outline and lacked the typical hour-glass shape.


Three of the four control explants fixed after 10 days and the two fixed at14 days showed essentially normal vibrissal follicles at stage 8, with relativelythick and normally keratinized hairs (Plate 3, figs. E, F). Some assumed dis-torted shapes, probably due to abnormal tensions in the explants, but noneshowed budding from the outer root sheath or abnormal gland formation. Thesebaceous glands, which in vivo remain very small in vibrissal follicles, haddisappeared from these explants. In each follicle the dermal papilla remainedfully invaginated and was usually healthy and the thick connective tissue sheathwas still present (Plate 3, fig. F). The fourth control explant fixed at 10 dayswas derived by subdivision at 4 days when it consisted of epidermis and dermiswith no hair follicles. Newly initiated follicles, which were observed in theliving explant 3 days later (i.e. after 7 days in vitro), were at stage 3 b at 10 days,and also appeared normal. The latter were probably pelage follicles, althoughit was not possible to establish this from morphological criteria. The sectionsstained in HAB showed alcian-positive reactions only in the locations wherethey are found regularly in normal active hair follicles in vivo (Braun-Falco,1958).

By contrast, the vitamin-treated explants sectioned after 10 and 14 daysshowed a wide spectrum of hair follicle and gland differentiation. Follicleswere found in various stages of departure from normal (Plate 3, figs. G, H;Text-fig. 5), and only one explant contained hairs. The three minute hair tipswere found in follicles which had normal dermal papillae and root sheaths, andalso some large sebaceous glands and extensively branching tubular glands(Plate 4, fig. A). Several other hair follicles had normal inner root sheath cones,sebaceous glands and branching tubular glands, but no hairs. Some had bothtypes of glands and shrunken dermal papillae, but no root sheaths. The majorityof follicles originally present were represented only by a solid downgrowthfrom the epidermis, from which the sebaceous and non-sebaceous glands werederived (Plate 4, fig. B), but some were lacking even this remnant of the originalstructure (Plate 4, fig. C). In two explants all the follicles originally present werecompletely transformed into sebaceous glands and tubular branching glands(Plate 4, fig. D; Text-fig. 5). There were also a few small follicles which, likethose in a control explant referred to above, were initiated in vivo, and weredeveloping normally up to stage 4 without any buds, but all the vibrissal

Skin response to vitamin A excess 169follicles originally present showed some deviations from the behaviour of thosein control explants.

Nearly all the vitamin-treated explants had many differentiated sebaceouscells and some sebum-like secretory masses produced by the characteristicholocrine process (Plate 4, fig. E).

The non-sebaceous branching tubular glands were the most prominentfeature of the vitamin-treated explants, and in many instances by repeatedbranching they grew as large as the follicles in control medium. In nearly allexplants there were some patent ducts opening either directly on to the surfaceof the epidermis (Plate 4, fig. B), or leading to a cyst (Plate 4, fig. F) whichusually communicated by another duct with the skin surface. In the glands whichwere best differentiated the collecting ducts had a wide lumen and a two-layeredwall of compact cuboidal or columnar cells with a smooth inner border (Plate4, fig. B). Terminal bars and terminal webs were recognized in some ducts. Thecysts (Plate 4, fig. F) were lined for the most part by a single layer of flattenedcells which sometimes had an irregular border. The smaller ducts derived bybranching had a single layer of cuboidal cells with a smooth border (Plate 4,fig. F). Most of the glands had some terminal swellings or 'alveoli' containingcuboidal cells (Plate 4, fig. G) which could sometimes be distinguished by palernuclei and a more eosinophilic cytoplasm. The nuclei were central or slightlybasal. Many terminal swellings did not show any central lumen in the ratherthick sections but the majority had a few very small and irregular intercellularcanals (Plate 4, figs. G, H). Although most of the cells in terminal swellings didnot stain with Alcian blue, both of the explants grown for 14 days had occasionalcells of the same morphological type whose cytoplasm was filled with Alcian-positive and therefore presumably mucous granules. One of the explants fixedafter 10 days had Alcian blue staining material in some lining cells and in thelumen of several ducts (Plate 4, fig. I).

(6) Vibrissal follicles in the lower jaw of 14-day embryos {Exp. 3)

The submental group of follicles, of intermediate type between pelage andvibrissal follicles, were at stage 1 or stage 2 when the experiment began. Con-ditions were not very favourable for the growth of hair folhcles in the lowerjaw explants, but there were some indications of a difference between control andtreated groups. In the control group, follicles were observed at various stages inliving explants (Text-fig. 5), and after fixation at 10 or 14 days, eight normalfollicles at stage 8 were found in the sections from two explants and somefollicles at earlier stages in the sections from two more. In the vitamin-treatedgroup, follicles were identified in only one of the living explants, and after 10or 14 days only one normal follicle at stage 8 and a few other follicles at earlierstages were found in sections from three explants. There was no sign of abnormalbudding or gland development from these follicles.

170 M. H. HARDY

(7) Vibrissalfollicles in the upper lip of\5-day embryos {Exp. 4)

Each explant contained two or three rows of follicles of which the mostadvanced was at stage 3 c, 4 or 5 and the least advanced at stage 1 (Text-fig. 6).Some of the follicles at stage 5 had a small swelling at the side to mark theposition of the future sebaceous gland, although differentiated sebaceous cellsdo not appear in the embryo until about a day later, when the hairs begin toform.

12 13 14 15 16 17 18 19 20 21 22 23 24 25


Text-fig. 6. Graph showing rates of development of vibrissal follicles from 15-dayembryos, x • • • x , in upper lip in situ; • • , in explants of upper lip (Exp. 4)in control medium (initially most advanced follicles); O — O, in explants of upperlip in vitamin A medium (12-5 i.u./ml.) (initially most advanced follicles); A A,in explants of upper lip in control medium (initially least advanced follicles);A - - A , in explants of upper lip in vitamin A medium (12-5 i.u./ml.) (initiallyleast advanced follicles).


After 3 days many follicles identified in the original explants had progressedto stage 6, 7 or 8 (Text-fig. 6), and the extent of progress was similar in alltreatment groups. Sebaceous cells were recognized in about half of the explants.After 4 days there was further development of hair follicles in all groups, andmore differentiated sebaceous cells were observed. Although some effects ofvitamin A on the epidermis were apparent at 3 and 4 days (Hardy, 1967 a), nosignificant difference was found between treatment groups with respect to hair

Skin response to vitamin A excess 111follicles. At 6 days, however, some differences showed up in the follicles. In thecontrol group all 6 explants had reached stage 8 in follicle development and inthe hydrocortisone (HC) group five out of six explants were at this stage, but inthe vitamin A group only one explant and in the A + HC group only three ex-plants out of six were at stage 8. Some follicles in one explant in the A grouphad lost their well-defined refractile Henle's layer. The number of keratinizedhairs observed after 6 and 7 days in vitro in the two groups receiving vitamin Awas only about half as great as in the control groups, while the number in theHC group was greater.

Table 3. Numbers of keratinized hairs observed in explantsof upper lip of 15-day embryos (Exp. 4)

Additions to basic medium

None A A,HC HC

No. of keratinized hairs visible in six living explants after 29 16 15 396 days in vitro

No. of keratinized hairs visible in the same living 29 18 17 48explants after 7 days in vitro

No. of keratinized hairs in complete serial sections from 77 58 62 80the same explants fixed after 7 or 11 days in vitro

No. of above hairs which were perfectly keratinized 50 13 12 35Percentage of perfectly keratinized hairs 65% 22% 19% 44%

A = vitamin A; HC = hydrocortisone.


Explants sectioned after 7 and 11 days revealed additional differences betweenthe treated and control groups. While the majority of explants in all groups hadsome follicles which had reached stage 7 or 8, there were differences in thenumbers and the quality of hairs produced. Table 3 shows that each of the twogroups receiving vitamin A had about three-quarters as many stage 8 folliclesas the control group, but only one-third as many perfectly keratinized fibres(which stain bright yellow in HEP and remain unstained in HAB). The remain-ing hairs in all groups were pink, or pink and yellow, in HEP, and deep tur-quoise blue in HAB, and showed an unusual amount of bending, irregularoutlines, imperfect cuticular scales, swollen hair cortex, loss of birefringence orsome combination of these features denoting faulty keratinization, even thoughmany of them had emerged beyond the skin surface and grown considerablyin length.

The four treatment groups were similar with respect to the development ofsebaceous glands. Differentiated sebaceous cells were identified in sections fromone, two or three of the six explants in each group.

Although the formation of non-sebaceous glandular buds from hair follicles

172 M. H. HARDY

was not nearly as extensive in the 15-day upper lip skin as in the 14-day skin,there were indications that the same tendency to gland formation existed. In thevitamin A group, three of the four explants fixed after 11 days showed buddingfrom a few of the smallest and least advanced vibrissal follicles (Text-fig. 6).One explant had a well-developed branching gland-type structure with a solidterminal bud, and a duct with a wall three cells thick and a lumen opening atthe skin surface. A second explant had three budding structures derived fromhair follicles which were identified in living explants—(a) a solid downgrowthfrom the epidermis with several buds but no dermal papilla, (b) a solid down-growth with some sebaceous secretion, and (c) a bilobed gland with a duct lumenopening at the skin surface and the beginning of lumina in the two lobes(Text-fig. 6). The third explant had a follicle, whose dermal papilla had beenlost, with a lateral bud containing some sebaceous secretion. In the A + HCgroup one of the four explants fixed after 11 days had a follicle with an elongatedlateral bud, and a second explant had buds beginning to form from an outerroot sheath. Neither the control group nor the one receiving hydrocortisonealone showed any of these budding or glandular structures, and all of theirvibrissal follicles differentiated in a more normal fashion (Text-fig. 6).

DISCUSSION

Metaplasia of vibrissal follicles

The most radical changes in response to vitamin A occurred in the vibrissalfollicles of the upper lip in Exp. 3. This appears to be a true metaplasia, in whichcells of the outer root sheath or less differentiated follicle cells changed thedirection of their development and formed branching tubular glands. Sometimesan entire hair follicle was transformed into an entire gland, and it seemed thatmost of the follicle cells of epidermal origin were involved, so that a potentiallycomplex keratin-forming organ changed into an equally complex secretory typeof organ. Other hair follicles were able to pursue the two developmental path-ways simultaneously, and produce both a keratinized hair and a secretory-typegland. In Exps. 2 and 4 a few follicles from the upper lip underwent similarchanges, and it is possible that the glandular structures which were attacheddirectly to the epidermis in these experiments were also derived originally fromhair follicle rudiments.

The exact nature of the glands formed remains an open question. They aremorphologically distinct from sebaceous glands, which are usually simplesaccular glands in the mouse, and even in their more elaborate forms such asthe human Meibomian glands are branched saccular but not tubular glands.Furthermore, the metaplastic glands were not undergoing holocrine secretionor producing fatty substances. They could also be distinguished from the eccrinesweat glands of mammals, which are unbranched coiled tubular glands arisingfrom the epidermis independently, and which are absent from the hair-bearing


areas in the mouse. They differ also from the apocrine sweat glands, the un-branched coiled tubular glands with wide terminal lumina which are attachedto the hair follicles in many species but not in the mouse. In species possessingapocrine sweat glands a single gland begins as a bud from the ental side of a hairfollicle (i.e. in the obtuse angle between follicle and skin surface) above the neckof the sebaceous gland. The metaplastic glands, on the other hand, arose bybudding at one or more points from any level of the follicle.

The origin of the metaplastic glands differed in several ways from that ofmammary glands, the only remaining major group of glands derived from themammalian epidermis. The mammary glands of the mouse develop from largerounded epidermal buds after a lag period of about 5 days (Balinsky, 1950<z, b)with a primary sprout from which secondary and later branches are derived.Mammary glands are characterized both in vivo and in vitro by early duct lumenformation and the absence of terminal swellings or alveoli during the earlyweeks of development (Hardy, 1950) unless suitable hormones are added tothose already present in the usual biological media (Lasfargues & Murray,1959).

Caution must be exercised in interpreting the nature of the secretory processin glands which are obviously immature. Certainly the metaplastic glandsshowed no signs of holocrine activity, and, in the absence of any evidence ofapocrine secretion, they would be classified as merocrine or eccrine skin glands.The cells composing the terminal swellings corresponded morphologically tothe 'special serous' (i.e. non-zymogenic) type discussed by Jacoby & Leeson(1959), but a few of these cells were filled with mucous granules and thusresembled the occasional 'mucoserous' cells of human mixed salivary glands(Bloom & Fawcett, 1962). One may therefore refer to the glandular metaplasiaof hair follicles but there is as yet insufficient evidence for regarding this as aspecifically mucous metaplasia such as is found in chick epidermis (Fell &Mellanby, 1953).

The vitamin A-induced glands bore a surprising resemblance to some struc-tures of ectodermal but not epidermal origin—the developing salivary glands.The vitamin A-induced glands were compared with the developing mousesalivary glands as studied both in vivo and in vitro by Borghese (1950), and withthe salivary glands which developed in the explants of lower jaw in the presentexperiments (Hardy, 1967 c). (The metaplastic glands were found only in theupper lip explants while the salivary glands were found only in the lower jawexplants, so there was no possibility of confusing them). The metaplastic glandsresembled the submandibular rather than the sublingual glands in their generalaborizing form, the histology of large and small ducts and the development ofcysts from ducts in vitro, the histology of terminal swellings, and even theoccasional appearance of morphologically' serous' but histochemically' mucous'cells. Since both the metaplastic glands and the salivary glands (Jacoby, 1959;Jacoby & Leeson, 1959) were still immature when the tissues were fixed, it is

12 JEEM 19

174 M. H. HARDY

not known whether the subsequent differentiation of the two groups of glandswould show a divergence.

Most of the 'specialized' glands in mammalian skin are merely elaborationsof typical sebaceous and sweat glands. However, Lyne, Molyneux, Myktowycz& Parakkal (1964) found an exception in the chin-gland of the rabbit, whichoriginated as an apocrine sweat gland from a hair follicle, but resembled themetaplastic glands of the mouse in two departures from the usual pattern. Itunderwent extensive branching to form a system of ducts, and it formed someeccrine secretory lobules as well as the usual apocrine ones.

Another transformation of hair follicles was described by Dawe (1963) inmice affected by polyoma virus. The sequence of events could only be inferredfrom a series of autopsy specimens, but there were some striking similarities.to the changes actually observed in the present study in response to vitamin A.Extensively branching tubular secretory glands and branching sebaceous glandsdeveloped from the side of hair follicles.

Effect of vitamin A excess on sebaceous glands

In Exp. 3 the glandular metaplasia in excess vitamin A was accompanied bya marked increase in the differentiation and secretion of sebaceous cells, but thesebaceous glands of control explants either did not form at all or began todevelop and then disappeared. In Exps. 2 and 4 the majority of follicles invitamin A-treated groups did not undergo metaplasia and their sebaceousglands were similar to those in the control groups. However, in the few meta-plastic follicles produced by vitamin A excess, sebaceous differentiation andsecretion were also greater than that in the non-metaplastic follicles of thecontrol group. In all three experiments, differentiated sebaceous cells werelocated mostly in the usual position at the upper part of the follicle neck, butoccasionally, like the non-sebaceous buds, they appeared at other levels alongthe follicle. Thus it seems that vitamin A may have a stimulating effect onsebaceous glands, at least under the conditions in which it also produces aglandular metaplasia of a non-sebaceous type. Sebaceous glands were apparentlystimulated to differentiate and secrete rather than to grow faster or to undergobranching.

Interference with hair follicle development and hairdifferentiation caused by excess vitamin A

A number of effects of vitamin A excess on hair follicles were inhibitory tothe follicles without adversely affecting the rest of the skin. The metaplasticchanges in vibrissal follicles from the upper lip in Exps. 2-4 were all accom-panied by some inhibition of normal follicle development. Even the follicleswhich achieved some normal development as well as glandular metaplasiashowed a delay in follicle differentiation, and their hairs were either abnormallyfine or absent. The majority of mystacial follicles from 15-day embryos (Exp. 4)differentiated normally and did not undergo glandular metaplasia, but the large

Skin response to vitamin A excess 175number of imperfectly keratinized hairs can be attributed to the hypervitamino-sis. A small number of observations on vibrissal follicles from the lower jaw inExps. 2 and 3 suggested a similar delay or inhibition of follicle development inthe presence of excess vitamin A without any inhibition of epidermal growth ordifferentiation. Pelage follicle development also was inhibited in Exp. 1, andmost of the follicles regressed. While the regression in 25-0 i.u./ml. vitamin Amight perhaps be attributed to a toxic effect of the medium, since there werealso a few degenerative changes in the epidermis and dermis (Hardy, 1967 a),this is unlikely to be the explanation for follicle regression in 12-5 i.u./ml., whenthe skin was just as healthy and vigorously growing as that in control medium.

Failure of hydrocortisone to affect hair follicles

Very little change in epidermal differentiation (Hardy, 1967 a) or in hairfollicle development was brought about by adding hydrocortisone to eithercontrol medium or vitamin A medium in Exp. 4. This was unexpected, sincehydrocortisone in similar concentrations antagonized the action of vitamin Ain many organ culture systems (Lasnitzki, 1965), and also when administeredalone affected rat skin in vitro (Weissmann & Fell, 1962). It is necessary to checkthe activity of the hydrocortisone preparation and to do further experimentsbefore drawing any conclusions. The observations are included in this papermerely to provide complete data on vitamin A effects, since experimental workhad to be discontinued for several years.

The 'recovery' of some follicles affected by vitamin A

Text-figures 2 and 5 and many reported observations on individual explantssuggest that follicles which are severely affected by vitamin A after 4 to 6 daysin culture may later recover some capacity for normal differentiation even thoughthey are still in vitamin A medium and might have attached metaplastic glands.One would first suspect that this might be due to a falling off of vitamin Aconcentration in the medium, but from published analyses of similar media(Fell & Mellanby, 1953) it seems unlikely that the concentration would fallbelow an effective dose. In Exp. 3, the introduction of fresh vitamin A solutionson the 9th day failed to check this 'recovery' effect. Furthermore, other vitaminA responses continued in full force in the same explants (Hardy, 1967 a, c), andthe metaplastic glands, once initiated, did not show any signs of regressiontowards the end of the experiments. Probably there is a real recovery of theabihty of hair follicles to differentiate normally after some time in the highvitamin medium.

Is there a critical period in follicle development for themetaplastic action of vitamin A ?

It was surprising that the extensive metaplasia of mystacial follicles producedby vitamin A which was so widespread in all the 14-day explants was less

176 M. H. HARDY

common in a 13^-day explant, infrequent in the 15-day explants and absentfrom the vibrissal follicles of the lower jaw. Perhaps there is not only a criticaltime interval between the administration of vitamin A and the appearance of amorphological change, but also a critical period in follicle development atwhich the metaplastic action of vitamin A can operate. In many investigationsby Fell and her colleagues (see Fell & Rinaldini, 1965) and in the present seriesof experiments (Hardy, 1967 a, c) most of the histological changes in skin andother epithelial tissues began after 3 or 4 days of vitamin A treatment. Whateverthe earlier critical time and critical stage may be for the significant changes atthe biochemical level, what the histologist observes is a budding from vibrissalfollicles if they are at the later dermal papilla or early hair cone stage (stages 3 c,4) after about 3 days in excess vitamin A. To illustrate the hypothesis of acritical stage, in Text-figs. 2-6 the supposed 'histological critical stage' and'histological critical time' are indicated by shaded bands. The majority ofmystacial follicles from 14-day embryos reached stage 3 c or 4 at the criticaltime (Text-fig. 5), and so metaplasia was widespread. New follicles (not indicatedin the figure) arising in vitro many days later were unaffected. The submentalfollicles from 14-day embryos treated with vitamin A reached stage 4 severaldays too late and did not undergo metaplasia. The largest mystacial folliclesfrom a 13^-day embryo for technical reasons developed more rapidly than thosefrom 14-day embryos and reached stage 4 just before the critical time (Text-fig. 4). These were not greatly affected, but a few of the next largest folliclesunderwent a moderate degree of metaplasia. The majority of mystacial folliclesdeveloped a little later and were unaltered. The interramal follicles, developingeven later, were also unaffected. The largest and medium-sized upper lip folliclesfrom 15-day embryos were well past stage 4 at the critical time (Text-fig. 6) anddid not show metaplasia, but some of the latest vibrissal follicles to develop inthese explants met the conditions for the metaplastic change.

It is not known whether pelage follicles would undergo metaplasia given theright conditions, but it may be significant that those which reached stage 4 justafter the postulated critical time (Text-fig. 2) underwent extensive regression,while those which were not initiated until after the critical time (Text-fig. 3)proceeded to develop normally. On the other hand, the failure of pelage folliclesto undergo metaplasia may be another instance of the lower responsiveness tovitamin A of skin on the trunk as compared with skin on the extremities (Hardy,1967a). This and many other questions, in particular several relating to the roleof the mesenchyme, await further experimentation.

SUMMARY

1. This paper reports the effects of excess vitamin A on the development ofhair follicles in organotypic cultures of embryonic mouse skin. Pieces of trunkskin, upper lip and lower jaw were cultivated on the surface of a clot of adult


cock plasma and chicken embryo extract in slide preparations for 11-14 days.Changes in individual living follicles were observed daily and the findingsconfirmed from complete serial sections.

2. In skin from the trunk of 13^-day embryos the pelage follicles wereinitiated and underwent some normal differentiation, both in the presence andabsence of added vitamin A (6-2-37-5 i.u./ml.).

3. In skin from the trunk of 15-day embryos the pelage follicles differentiatednormally for 4 days in both treated and untreated groups but then regressed inthe vitamin-treated groups (12-5, 25-0 i.u./ml.), while normal differentiationcontinued and some keratinized hairs were formed in the control groups.

4. Vibrissal follicles in the upper lip from a 13^-day embryo differentiatednormally for 4 days in excess vitamin A (12-5 i.u./ml.), and then some folliclesbecame misshapen and developed gland-like lateral buds, while follicles in thecontrol explant continued normal development and produced many keratinizedhairs.

5. All the vibrissal follicles originally present in four explants of upper lipfrom 14-day embryos differentiated normally for about 3 days in excess vitaminA (12-5 i.u./ml.) and then showed metaplastic changes. Many of them under-went a complete glandular metaplasia, being transformed into compoundtubular glands with branching duct systems, terminal 'alveolar' swellings andoccasional signs of mucous secretion. Some follicles retained traces of theoriginal structure, and in a few the follicle eventually recovered its capacity fornormal differentiation, producing simultaneously a small keratinized hair anda vigorously growing gland. The metaplastic glands were unlike any normalmammalian skin glands but resembled immature salivary glands. At the time ofinitiation of the metaplasia there was also an increase in the secretion of sebumor sebum-like material in the same hair follicles. In the control group withoutexcess vitamin A the follicles continued normal differentiation and hair pro-duction until the experiment was terminated, and there was no metaplasia.

6. Most of the vibrissal follicles in the explants of upper lip from 15-dayembryos continued normal differentiation in the presence of excess vitamin A(12-5 i.u./ml.), although a higher proportion of the hairs were abnormal instructure than in the control group. In excess vitamin A a few of the initiallyleast advanced follicles underwent glandular metaplasia, but no metaplasiawas observed in the control group.

7. Some of the differences between the behaviour of explants from embryosof different ages could be explained by the hypothesis that there is only a relativelyshort critical stage in follicle development during which vitamin A is capableof inducing metaplasia.

178 M. H. HARDY

RESUME

Metaplasie glandulaire des follicules pileux et autres reactions a un excesde vitamine A dans des cultures de peau de Rongeur

1. Cet article rapporte les effets d'un exces de vitamine A sur le developpementdes follicules pileux dans des cultures organotypiques de peau embryonnaire deSouris. Des morceaux de peau du tronc, de la levre superieure et de la machoireinferieure sont cultives a la surface d'un coagulum de plasma de Coq adulteet d'extrait embryonnaire de Poulet, pendant l l a 14 jours. Les transformationsdans les follicules vivants sont observees chaque jour et les resultats confirmespar des coupes seriees completes.

2. Dans la peau du tronc d'embryons de 13,5 jours, les follicules du pelageapparaissent et commencent une differentiation normale; a la fois en presenceet en absence de vitamine A (6,2-37,5 u.i./ml).

3. Dans la peau du tronc d'embryons de 15 jours, les follicules du pelage sedifferencient normalement pendant 4 jours dans les groupes traites et non traitesmais regressent ensuite dans les groupes qui recoivent de la vitamine (12,5, 25,0u.i./ml), tandis que la differenciation normale continue et que des poils kerati-nises se forment dans les groupes temoins.

4. Des follicules de vibrisses dans la levre superieure d'un embryon de 13,5jours se differencient normalement pendant 4 jours en presence d'un exces devitamine A (12,5 u.i./ml.), puis certains follicules deviennent malformes et pro-duisent des bourgeons lateraux d'apparence glandulaire; les follicules dansl'explant temoin poursuivent un developpement normal et produisent denombreux poils keratinises.

5. Tous les follicules de vibrisses presents au depart dans 4 explants de levresuperieure d'embryons de 14 jours se differencient normalement pendant en-viron 3 jours, en presence d'un exces de vitamine A (12,5 u.i./ml.), puis presententdes alterations metaplasiques. Beaucoup d'entre eux subissent une metaplasieglandulaire complete, et sont transformers en glandes tubulaires composees,avec un systeme de canaux ramifies, des gonflements 'alveolaires' terminauxet a l'occasion des signes de secretion muqueuse. Certains follicules gardent destraces de leur structure originelle, et quelques-uns recuperent eventuellementleur capacite de differenciation normale; ceux-ci produisent alors simultanementun petit poil keratinise et une glande a groissance vigoureuse. Les glandesmetaplasiques ne ressemblent a aucune glande normale de la peau des Mammi-feres, mais ressemblent a des glandes salivaires immatures. Au moment ou lametaplasie apparait, il y a egalement une augmentation de la secretion de sebum,ou de materiel analogue a du sebum, dans le follicule pileux. Dans le groupetemoin non soumis a la vitamine A en exces, les follicules continuent la differen-ciation normale et la formation de poils jusqu'au terme de l'experience et aucunemetaplasie ne se manifeste.

6. La plupart des follicules de vibrisses dans les explants de levre superieure


d'embryons de 15 jours continuent la differentiation normale en presence d'unexces de vitamine A (12,5 u.i./ml.), pourtant une proportion plus importantedes poils presentent un structure anormale que dans le groupe temoin. Avec unexces de vitamine A quelques-uns des follicules les moins avances au departsubissent la metaplasie glandulaire; aucune metaplasie n'apparait dans legroupe temoin.

7. Certaines des differences de comportement entre les explants provenantd'embryons d'ages differents pourraient etre expliquees par l'hypothese de labrievete du stade critique du developpement folliculaire pendant lequel lavitamine A peut induire la metaplasie.

The author is grateful to Mr Vincent Blancuzzi for careful and patient assistance in thepreparation of thousands of serial sections and to Mrs Joline Spivack and others for technicalhelp. Dr J. P. W. Gilman of the University of Guelph kindly read the manuscript. ProfessorMargaret R. Murray of Columbia University College of Physicians and Surgeons andProfessor Dame Honor Fell, D.B.E., F.R.S., of the Strangeways Research Laboratory gaveencouragement in many ways, and Dr Richard P. Bunge provided in his laboratory anexcellent medium for the growth and differentiation of research scientists. This research wassupported by U.S.P.H.S. N.I.H. Grant NB 04235 and National Multiple Sclerosis SocietyGrant 328 administered by Dr Richard P. Bunge.

REFERENCES

BALINSKY, B. I. (1950a). On the prenatal growth of the mammary gland rudiment in themouse. / . Anat. 84, 227-35.

BALINSKY, B. I. (19506). On the developmental processes in mammary glands and otherepidermal structures. Trans. R. Soc. Edinb. 62, 1-31.

BLOOM, W. & FAWCETT, D. (1962). A Textbook of Histology. London and Philadelphia:W. B. Saunders.

BORGHESE, E. (1950). The development in vitro of the submandibular and sublingual glands ofMus musculus. J. Anat. 84, 287-318.

BRAUN-FALCO, O. (1958). The histochemistry of the hair follicle. In The Biology of HairGrowth, pp. 65-90. London and New York: Academic Press.

DAVIDSON, P. & HARDY, M. H. (1952). The development of mouse vibrissae in vivo andin vitro. J. Anat. 86, 342-56.

DAWE, C. J. (1963). Skin-appendage tumors induced by polyoma virus in mice. Natn.Cancer Inst. Monogr. 10, 459-88.

DINGLE, J. T. & LUCY, J. A. (1965). Vitamin A, carotenoids and cell function. Biol. Rev. 40,422-61.

FELL, H. B. (1964). The experimental study of keratinization in organ culture. In The Epi-dermis, pp. 61-81. New York: Academic Press.

FELL, H. B. & MELLANBY, E. (1953). Metaplasia produced in cultures of chick ectoderm byhigh vitamin A. / . Physiol., Lond. 119, 470-88.

FELL, H. B. & RINALDINI, L. M. (1965). The effects of vitamins A and C on cells and tissuesin culture. In Cells and Tissues in Culture, 1, pp. 659-99. London and New York: AcademicPress.

HARDY, M. H. (1949). The development of mouse hair in vitro with some observations onpigmentation. J. Anat. 83, 364-84.

HARDY, M. H. (1950). The development in vitro of the mammary glands of the mouse. / . Anat.84, 388-93.

HARDY, M. H. (1951). The development of pelage hairs and vibrissae from skin in tissueculture. Ann. N.Y. Acad. Sci. 53, 546-51.

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HARDY, M. H. (1965). Effect of excess vitamin A on rodent epidermis and developing hairfolilcles in vitro. Excerpta Medica, 19, Section 1, no. 10: xrr.

HARDY, M. H. (1967 a). Responses in embryonic mouse skin to excess vitamin A in organo-typic cultures from the trunk, upper lip and lower jaw. Exp. Cell. Res. 46, 367-84.

HARDY, M. H. (1967b). The differentiation of hair follicles and hairs in organ culture.Advances in Biology of Skin, 9 (in the Press).

HARDY, M. H. (1967 C). Responses in oral epithelium and tissues of the tongue and lowerjaw of the embryonic mouse to excess vitamin A in organotypic cultures (in preparation).

JACOBY, F. (1959). Observations on the post-natal development of the mouse submaxillarygland. / . Anat. 93, 579.

JACOBY, F. & LEESON, C. R. (1959). The post-natal development of the rat submaxillarygland. / . Anat. 93, 201-16.

LASFARGUES, E. Y. & MURRAY, M. R. (1959). Hormonal influences on the differentiation andgrowth of embryonic mouse mammary glands in organ culture. Devi. Biol. 1, 413-35.

LASNITZKI, I. (1965). The action of hormones on cell and organ cultures. In Cells and Tissuesin Culture, 1, pp. 591-658. London and New York: Academic Press.

LYNE, A. G., MOLYNEUX, G. S., MYKTOWYCZ, R. & PARAKKAL, P. F. (1964). The develop-ment, structure and function of the submandibular cutaneous (chin) glands of the rabbit.Aust. J. Zool. 12, 340-8.

NEW, D. A. T. (1963). Effects of excess vitamin A on cultures of skin and buccal epitheliumof the embryonic rat and mouse. Br. J. Derm. 75, 320-5.

WEISSMANN, G. & FELL, H. B. (1962). The effect of hydrocortisone on the response of fetalrat skin in culture to ultra-violet irradiation. J. exp. Med. 116, 365-80.

{Manuscript received 8 August 1967)

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Printed in Great Britain Glandular metaplasia of hair ... · Glandular metaplasia of hair follicles and other responses to vitamin A excess in cultures of rodent skin By MARGARET