PROSTAGLANDIN-BI, EMBRYONIC SKIN,

AND THE DERMO-EPIDERMAL JUNCTION

C. WARD KISCHER and JOE S. KEETER. From the Department of Anatomy, University of TexasMedical Branch, Galveston, Texas 77550 . Mr. Keeter's present address is the Department of Biology,North Texas State University, Denton, Texas 76023

INTRODUCTION

In previous studies, prostaglandins have been ap-plied to organ cultures of dorsal skin from the chickembryo (11, 12) . When prostaglandin-B, (PGB,) isadded to the culture medium in which skin piecesare explanted at a time when feather organ lociare present, the latter fail to develop during incu-bation but the skin continues to grow and mature .Control skins demonstrate normal feather develop-ment during 5-6 days of incubation .

We are continuing the effort to explain this ef-fect, especially through examination of the tissuesby electron microscopy . In the course of thesestudies we have observed an unusual defect in thedermo-epidermal junction . Throughout the entirePGB,-treated skin there are numerous areas inwhich the dermo-epidermal junction is discon-tinuous . This effect is attributed directly to treat-ment of the tissue with PGB,. It is repeatable andhas been found as early as the second day ofincubation of the cultured skin . In some casesthese areas of disjunction can be followed by serialsection and are seen to lead into complete perfora-tions of the epidermis, through which mesen-chymal cells migrate or proliferate and coat thefree surface layer (Fig . 1) .

MATERIALS AND METHODS

Dorsal skin of the chick embryo was removed andplaced in organ culture according to procedures pre-viously described (11) . The culture medium fortreated skins contained 50 µg/m1 of crystallizedPGB, . The cultures were fixed at daily intervals for5 days in cold 1 % glutaraldehyde buffered withcacodylate and CaC12 . After a cacodylate bufferwash, they were postfixed in I % osmium tetroxidebuffered with cacodylate and CaC1 2 . The tonicity ofboth fixatives and the buffer wash was adjusted to300 mosmols. Dehydration was accomplished througha graded series of ethanol followed by embedment inEpon 812. Sections were cut on a diamond knifefrom 600 A to 800 A, doubly stained in lead citrateand uranyl acetate, and examined in an RCA EMU3G electron microscope .

An equivalent number of control tissues and PGB,-treated tissues were examined . In addition, becauseof the possibility that the disjunctions and perforationsarose from mechanical probing of the tissue with thedissecting needles during preparation of the skin forculture, a series of control tissues were deliberatelyand repeatedly stabbed with a No . 1 I beading needle .These tissues were cultured, processed, and examinedas above .

RESULTS

Fig. 2 shows a gap in the dermo-epidermal junc-tion. When breaks could be traced through serial

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All figures depict areas of dorsal skin of the chick embryo grown in organ culture from 3 to 5 days in thepresence of 50 µg/ml of PGB I .

FIGURE 1 A photomicrograph of a thick plastic section of dorsal skin showing a complete perforation ofthe epidermis (E) . Mesenchymal cells (M) have formed a plaque atop. X 1200 .

sectioning, it was commonly found that the periph-ery of a break contained a thickened basal lamina(Fig . 3) . In some cases, isolated sections of the basallamina can be found across the break (Fig . 4) .Also, in other instances, cells can be seen wedgedin-between the space of the break (Fig . 5) . Exami-nation of serial sections suggests that these cells areessentially spherical and lack filopodia, whichwould ordinarily indicate motility .

In the case of the control skins which weredeliberately stabbed in order to produce the breaksmechanically, no such disjunctions were found . Forthe period up to 5 days of incubation, the epidermisand basement membrane were observed to beintact . It should be similarly noted that the seniorauthor, who over a period of several years has beenexamining cultured skin prepared in the usual way

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BRIEF NOTES

with beading needles, has never observed breaksin the dermo-epidermal junction such as reportedhere .

DISCUSSION

Several reports in the literature have demonstrateddiscontinuities, or breaks, of the dermo-epidermaljunction . In some cases, the basal epidermal mem-brane and basal lamina have appeared fused andgranular (10) . However, an observation such asthis might be accounted for by tangential section-ing through this area . In other cases, small papil-lary processes of the basal epidermal cells havebeen observed to penetrate into the space of themesenchyme through a small gap in the basallamina (2, 5) . In still others, complete separationsof the basal lamina have been accompanied by

FIGURE 2 A disjunctional area of the dermo-epidermal junction . Basal lamina (B) is separated by in-timate contact between epidermal (E) and mesenchymal (M) cells . X 18,200 .

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FIGURE 3 Periphery of break shown in Fig . 2 . Arrow indicates approximate point where serial sectioningdemonstrates site of dermo-epidermal gap . Basal lamina (B) of basement membrane . Epidermis (E) .Mesenchyme (M) . X 13,200.

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FIGURE 4 An isolated, membrane-bound section of basal lamina (B) is seen within and above the breakin the dermo-epidermal junction (J) . Mesenchyme (M) . Epidermis (E) X 13,600.

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FIGURE 5 A section of a cell (M), presumably from the mesenchyme, is wedged within the area of dis-junction. Isolated sections of the basal lamina (B) lie above the continuous portions of the dermo-epi-dermal junction (J) . Epidermis (E) . X 9200.

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penetration of large processes of the basal epi-dermal cells into the underlying mesenchyme (1, 3,4, 6, 7, 9, 13, 15) . These processes are commonlyseen to be devoid of cellular organelles .However, in none of these reported cases have

mesenchymal or dermal cells, or the processesthereof, been shown to penetrate into the epi-dermis .

The fact that, in many sections, isolated portionsof the basal lamina can be observed, which, whenfollowed serially are found to fuse together, leadus to believe that, in these cases, the periphery ofthe gap in the basal lamina must appear somewhatscalloped. Perhaps, then, the wider or more ex-pansive breaks have been formed from fusion ofseveral distinct holes . Further, in cross-sectionstudy, the isolated portions of basal lamina lieabove the continuous portion . This suggests dis-placement from below, perhaps as a result ofmesenchymal cells pushing upward in their ad-vance through the epidermis . What relationshipthis geometric configuration might have with themode of action of PGB, within the skin, or spe-cifically on the basal lamina, remains to be under-stood .

The question arises, can these breaks be analo-gous to skin lesions involving ulceration? Hansenet al . (8) demonstrated disruption of the basementmembrane of skin of dogs fed a diet deficient inessential fatty acids. These areas were traced tocomplete perforations of the epidermis .' In a re-cent report by Menton (14), who studied adultmice deprived of essential fatty acids in the diet,no defects in the basement membrane were cited .However, he did report that he observed wideintercellular spaces in the Malpighian layer of theepidermis, and eventual loss of hair . It is interestingto note that the prostaglandins are related to theessential fatty acids, in that some of the latter serveas the precursors for the synthesis of the former .

It cannot be determined at this time whetherthe disjunction phenomenon is directly related tothe failure of the down feather to develop whenskin is treated with PGBI . If this were the case, onemight expect to see disjunctional areas strategicallylocated in the treated skin, e.g ., in presumptivefeather sites . Such a possibility must still be con-sidered .

Whether or not PGB,-induced breaks in thedermo-epidermal junction are peculiar to embry-

1J. G . Sinclair. Personal communication .

onic skin remains to be demonstrated . However, itwould be of special interest to attempt to duplicatethe effect in human adult skin . If this were possible,a metabolic explanation for the development ofcertain skin lesions might be closer at hand .

SUMMARY

The down feather organ derived from the dorsalskin of the chick embryo fails to develop from pre-sumptive sites when treated in vitro with pros-taglandin-B, (PGBI ) . Examination of the culturedtissues by electron microscopy reveals many breaksor gaps in the dermo-epidermal junction . In somecases, these areas of disjunction are continuouswith complete perforations of the epidermis .Mesenchymal cells fill the perforated area andform plaques on the dorsal surface of the skin .

It has yet to be determined whether or not thisphenomenon is directly related to the morphoge-netic block of the feather organ produced byPGB, .

This work was supported by a grant from the Depart-ment of Health, Education, and Welfare, NationalInstitute of Health, 5-R01 AM 13530-01 .PGBI was supplied through the courtesy of Dr .

John Pike, The Upjohn Company .Received for publication 17 March 1970, and in revisedform 27 April 1970 .

REFERENCES

1 . ASHWORTH, C. T ., V . A. STEMBRIDGE, and F . J .LUIBEL . 1961 . A study of basement membranesof normal epithelium, carcinoma in situ andinvasive carcinoma of uterine cervix utilizingelectron microscopy and histochemicalmethods . Acta Cytol . 5 :369.

2 . BREATHNACH, A. S., and J . SMITH . 1968. Finestructure of the early hair germ and dermalpapilla in the human foetus . J. Anat . 102 :511 .

3 . BRODY, I . 1962. The ultrastructure of the epi-dermis in psoriasis vulgaris as revealed byelectron microscopy . 1 . The dermo-epidermaljunction and the stratum basale in parakerato-sis without keratohyalin . J. Ultrastruct . Res .6 :304 .

4. Cox, A . J . 1969 . The dermal-epidermal junctionin psoriasis . J. Invest. Dermatol. 53:428 .

5 . FARBMAN, A. I . 1968 . Electron microscope studyof palate fusion in mouse embryos . Develop .Biol . 18:93.

6 . FREI, J. V . 1962 . The fine structure of the base-

BRIEF NOTES

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ment membrane in epidermal tumors . J. CellBiol. 15 :335.

7 . FRY, L., and F . R . JOHNSON . 1969 . Electronmicroscopic study of dermatitis herpetiformis .Brit. J. Dermatol . 81 :44 .

8 . HANSEN, A. E ., J . G. SINCLAIR, and H . F . WIESE .1954 . Sequence of histologic changes in skinof dogs in relation to dietary fat . J. Nutr.52:541 .

9. HINGLAIS-GUILLAUD, N ., R . MORICARD, and W.BERNHARD . 1961 . Ultrastructure des cancerspavimenteux invasifs due col uterin chez lafemme. Ass . Fr . pour l'etude du cancer . 48 :283 .

10 . JURAND, A. 1965 . Ultrastructural aspects of earlydevelopment of the fore-limb buds in the chickand the mouse . Proc . Roy . Soc . Ser . B. 162:387 .

11 . KISCHER, C . W. 1967 . Effects of prostaglandins on

development of chick embryo skin and downfeather organ, in-vitro . Develop. Biol . 16 :203 .

12 . KISCHER, C. W. 1969 . Accelerated maturation ofchick embryo skin treated with a prostaglandin(PGB,) : An electron microscopic study . Amer .J. Anat. 124:491 .

13. LUIBEL, F. J ., E. SANDERS, and C . T . ASHWORTH .1960 . An electron microscopic study of cancerin-situ and invasive carcinoma of the cervixuteri . Cancer Res . 20 :357 .

14 . MENTON, D. W. 1968 . The effects of essentialfatty acid deficiency on the skin of the mouse .Amer. J. Anat . 122 :337.

15 . WOODS, D . A., and C . J . SMITH. 1969 . Ultra-structure of the dermal-epidermal junction inexperimentally induced tumors and humanoral lesions. J. Invest . Dermatol . 52 :259.

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