Cell Biology International Reports, Vol. 75, No. 5, 199 I 389

RETINOIC ACID DOES NOT INDUCE FORMATION OF CILIA ON THE SURFACE OF WOUND EPITHELIAL CELLS IN AXOLOTLS

Koussoulakos, S.*, Matzke, R.**, Anton, H.J.**

* Zoological Laboratory, University of Athens, Panepistimiopolis 157 84, Athens, Greece

** Institute of Zoology, University of Cologne, 5 Cologne 41, Weyertal 119, Germany

* The stay of Dr. S. Koussoulakos at the University of Cologne was financially supported by a Heinrich-Herzt-Stiftung fellowship.

* Corresponding author

ABSTRACT

It has been reported that vitamin A palmitate induces the production of cilia on the epidermal cells of the regenerating axolotl limb, and the formation of crevices in the epidermal surface. The aim of the present investigation was to reexamine under well defined conditions the potential of retinoids to evoke the above described metaplastic changes. In order to achieve our purpose we administered. axolotls with retinoic acid for 2, 4, 6, 8, and 10 days after limb amputation. The young regenerates were inspected by scanning electron microscopy (SEM). The data obtained showed that the external layer of the wound epithelium and of the stump epidermis as well was quite normal without any sign of cilia formation. In some cases, crevices were observed even in control animals.

INTRODUCTION

Amputation of a urodele amphibian limb at any level along its proximodistal axis results in the regeneration of those structures which, on the intact limb, lie distal to that level. Imme- diately after limb removal cells from the adjucent stump epidermis start migrating over the wound surface and produce the so called wound epithelium which is indespensable for further limb development (Singer and Salpeter, 1961). Limb regeneration is initiated by dedifferentiation of stump tissues and formation of a mass of mesenchyme-like cells, the mesodermal blastema.

Whereas embryonalization of stump mesodermal cells is well established (Hay and Fischmann, 1961), the evidence for transformation of mature epidermal cells to embryonic epithelial ones is weak (Ide-Rozas, 1936). On the contrary, there is convincing evidence that the wound epithelium is different from the embryonic limb bud skin. The limb buds of Ambysroma mexicanurn, just like the entire embryo at the developmental stage 37 (Harrison, 1969), are endowed with many ciliated cells. These cells soon disappear (Billet and Courteny, 1973), a fact which implies a rapid cessation in the activity of the corresponding genes. The most striking difference between the epidermis of embryonic and regenerating limbs is the occurence of the ciliated cells seen on the embryo. The epithelium of the regeneration blastema does not posses ciliated cells (Tank et al., 1977; Geraudie and Singer, 1981).

0309-l 651/91/050389-7/$03.00/O 0 1991 Academic Press Ltd

Ceil Biology international Reports, Vol. 75, No. 5, 1991

Identifying similarities and/or differences between embryonic and regenerating epithelial cells might reveal clues to the role of the wound epithelium during regeneration, and help to clarify the general rules governing developmental phenomena. Therefore, it was highly interesting when Scadding (1989) reported a case of overt wound epithelial cell embryonalization. He administered vitamin A to amputated limbs of larval axolotls and observed the development of cilia on some of the epidermal cells, but the information provided is insufficient and leaves a lot of questions open. Scadding’s work (1989) is the first, and thus far the single one reporting such metaplastic changes on the wound epithelial ceils.

Our interest in clarifying this issue motivated the present investigation. The experiments described here were designed to answer the following questions: Does retinoic acid induce the reappearance of cilia on the surface of wound epithelial cells? Does retinoic acid modulate shape and size of these cells?

MATERIALS AND METHODS

20 young Ambystoma mexicanum specimens, ranging in body length between 6 and 9 cm, raized ab ovo in our laboratory were used. Both forelimbs were amputated at the wrist joint by a sharp razor blade under MS 222 (I:3 000) anesthesia. Immediately after surgery the animals were returned to individual plastic boxes containing a 5 mM aqueous solution of retinoic acid (all trans retinoic acid, Sigma, type XX). No bleeding neither bone protrusion occurred. The medium was changed every day to replace the precipitated and inactivated retinoic acid molecules. The animals remained under these conditions for 2, 4, 6, 8, and 10 days, 4 animals for each case.

At the end of each time-period all 4 animals had their forelimbs amputated at the middle of the stylopodium. The amputates were fixed for 24 h in Bouin’s fixative made up in a neutral amphibian Ringer solution. Fixed limbs were dehydrated in alcoholic series and acetone, critical point dried, sputter coated and examined at 10 KV in a Hitachi S-250 SEM. Limb epidermis and wound epithelium were examined at anterior, posterior, dorsal and ventral sides.

RESULTS

Under our experimental conditions, results obtained from parallel experimental series have shown that animals treated for 2 days with retinoic acid, and then returned to tap water and remained intact, regenerated normal appendages. A 4-day treatment induces mainly changes in the number and shape of the wrist skeletal elements. Zeugopodial and stylopodial duplications were evoked after 6- and 8-day treatment, whereas, the majority of the animals kept for 10 days in the retinoic acid solution displayed regeneration arrest (Fig. 1).

Ceil Biology International Reports, Vol. 15, No. 5, 1991

Fig. 1. Proximodistal duplication (A) and regeneration arrest (6) in axolotl forelimbs treated with retinoic acid for 6 and 10 days, respectively. Arrows indicate level of amputation,

All 40 regenerates, treated as described in the section ‘Materials and Methods”, developed a well formed wound epithelium covered externally by flattened, polyhedral, normally appearing cells. These cells did not differ from those of the limb epidermis, and are characterized by complete lack of embryonic cilia. The main results of our investigation are displayed in figures 2 through 7.

Fig. 2. Wound epithelial cells 2 days after limb amputation. T=vitamin A-treated limbs, C=control limbs. The absence of cilia, and the similarity in size and shape between the ceils is obvious.

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Figs. 3, 4, 5. Wound epithelial cells 4, 6, and 6 days after limb amputation, respectively. Indications as in figure 2.

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Fig. 6. Wound epithelial cells 10 days after limb amputation. Indications as in the previc IUS

figures.

Fig. 7. Wound epithelial cells 2(A) and 4(B) days after control limb amputation. Even in non-treated limbs crevices are present at the top of the regenerate.

DISCUSSION

The potential of retinoids in modulating phenotypic expression of various epithelial cell lines is well documented (Mori, 1922; Fell and Mellanby, 1958; Shapiro, 1985; Stadler et al., 1987; Asselineau et al., 1989). Among the earliest and most impressive manifestations of retinoid imbalance are the keratinization of normally nonkeratinizing epithelia (Wolbach and Howe, 1925) the induction of glandular structures from rodent hair follicles (Hardy, 1968), and the metaplastic changes of scale forming integument to feather forming one (Dhouailly and Hardy, 1978). These observations have been repeatedly documented and helped elucidating, with a certain degree of clarity, the mode of action of retinoids at the cellullar and molecular level.

Recently, Scadding (1989) reported the formation of cilia on axolotl epidermal cells treated with retinol palmitate for 14 days, a time-period sufficient to inhibit limb regeneration. How- ever, it is not clearly stated in his report whether the ciliated cells are located on the stump limb epidermis and / or on the wound epithelium. If the ciliated cells are actually wound

394 CellBiology International Reports, Vol. 75, No. 5, 1991

epithelial cells, then their appearance, in combination with limb regeneration arrest has a great theoretical importance in the light of the significance of the wound epithelium for limb regeneration and morphogenesis (Stocum, 1985).

Previous studies performed in our laboratory on Trirurus alpestris (Koussoulakos et al., 1990 and unpublished results) did not disclose production of cilia by wound epithelial cells as a result of retinoid treatment. Therefore, we decided to perform the experiments on axolotls by exploiting the higher activity of retinoic acid at time-windows which induce the whole spectrum of corresponding phenotypes (Maden, 1983).

Our present results do not support the findings of Scadding (1989). The morphology of the wound epithelial cells, as well as that of the stump epidermal cells was quite normal, not differing from that observed on non-treated animals. Apart from that, these cells were not induced to produce cilia in response to retinoid treatment.

We do not yet know the reasons for this discrepancy. From the protocol of Scadding (1989) it is not possible to calculate the number of animals subjected to SEM examination, neither the number of animals exhibiting ciliated epidermal cells. In the light of the above reasoning we believe that some of the animals used in Scadding’s study (1989) were quite small so as to be still endowed with the embryonic cilia.

REFERENCES

Asselineau, D., Bernard, B.A., Bailly, C. and Darmon, M. (1969). Retinoic acid improves epidermal morphogenesis. Dev. Biol. 133: 322335.

Billet, F.S. and Courteny, T.H. (1973). A stereoscan study of the origin of ciliated cells in the embryonic epidermis of Ambystoma mexicanum. J. Fmbrvol. exp. MorDhol. 29: 549-558.

Dhouailly, D. and Hardy, MM. (1978). Retinoic acid causes the development of feathers in the scale-forming integument of the chick embryo. Roux’s Arch. Dev. Biol. 185: 195-200.

Fell, H.B. and Mellanby, F. (1953). Metaplasia produced in cultures of chick ectoderm by high vitamin A. J. Physiol. 119:470-488.

Geraudie. J. and Singer, M., (1981). Scanning electron microscopy of the normal and denervated limb regenerate in the newt Notophthalmus, including observations on embryonic amphibian limb-bud mesenchyme and blastemas of fish-fin regenerates. Am. J. Anat. 162:73-87.

Hay, E.D. and Fischmann, D.A. (1961). Origin of the blastema in regenerating limbs of the newt Triturus viridescens. Dev. Biol, 3:26-59.

Hardy, M.M. (1968). Glandular metaplasia of hair follicles and other responses to vitamin A excess in cultures of rodent skin. J. Embrvol. exp. Morphol. 19:157-180.

Harrison, R.G. (1969). Organization and Development of the Embryo. Yale University Press, New Haven,

Ide-Rozas, A. (1936). Die zytologische Verhaeltnisse bei der Regeneration von Kaulquappenextremitaeten. Arch. Entwmech. Oraan. 135:522-608.

Koussoulakos, S., Sharma, K.K. and Anton, H.J. (1990). Effect of vitamin A on wound epidermis during forelimb regeneration in adult newts. Int. J Dev. BIOI 34:433-439.

Maden, M. (1983). The effect of vitamin A on the regenerating axolotl limb. J. Embrvol. exp. Morohol. 77:273-295.

Mori, S. (1922). The changes in the paraocular glands which follow the administration of diets low in fat-soluble A; with notes of the effect of the same diets on salivary glands and the mucosa of the larynx and trachea. John Hookins HOSD. Bull. 33:357-359.

Cell Biology International Reports, Vol. 15, No. 5, 199 I 395

Scadding, S.R. (1989). Effects of vitamin A on limb regeneration in the larval axolotl, Ambystoma mexicanum, in: Kiortsis, V., Koussoulakos, S. and Wallace, H. (eds). Recent Trends in Regeneration Research. Plenum Press, New York, pp. 341-353.

Shapiro, S.S. (1985). Retinoids and epithelial differentiation, in: Sherman, E.I. (ed.). Retinoids and Cell Differentiation. CRS Press, Boca Raton, F.L., pp. 30-35.

Singer, M. and Salpeter, M.M. (1961). Regeneration in Vertebrates: the role of the wound epithelium, in: Zarrow, M.X. (ed.). Growth in Living Systems. Basic Books Inc., New York, pp. 277-311.

Stadler, R., Mueller, R., Detmar, M. and Orfanos, C.E. (1987). Retinoids and keratinocyte differentiation in vitro. Dermatoloaica 175(suppl.):45-55.

Stocum, D.L. (1985). The role of the skin in urodele limb regeneration, in: Sicard, R.E. (ed.). Regulation of Vertebrate Limb Regeneration. University Press, Oxford, pp. 32-53.

Tank, P.W., Carlson, B.M. and Connelly, T.G. (1977). A scanning electron microscopic comparison of the development of embryonic and regenerating limbs in the axolotl. J. END. Zool. 201:417-430.’

Wollbach, S.B. and Howe, P.R. (1925). Tissue changes following deprivation of fat soluble A vitamin. J. Exp. Med. 42:753-778.

Paper received 04.02.91. Revised paper accepted 01.04.91.