artificial metaplasia of pigmented epithelium into retina ... · artificial metaplasia of pigmented...

/ . Embryol. exp. Morph. Vol. 28, 3, pp. 521-546, 1972 5 2 1

Printed in Great Britain

Artificial metaplasia of pigmented epithelium intoretina in tadpoles and adult frogs

By G. V. LOPASHOV1 AND ALLA A. SOLOGUB1

From the Institute of Developmental Biology,Academy of Sciences of USSR, Moscow

SUMMARYThe present work was aimed at investigating the possibility and conditions necessary for

the artificial transformation of one tissue into another. Experiments were carried out with thepigmented epithelium of the eye in tadpoles and adult frogs oiRana temporaria. Following theremoval of the mesenchyme envelopes (or their exfoliation during the experiment), pigmentedepithelium transformed into retina under the influence of retina from tadpoles of the samespecies. This phenomenon was observed both under the cultivation of a piece of retina in asandwich of pigmented epithelium and the transplantation of pigmented epithelium layersinto the eye cavity of tadpoles. Such transformation did not occur in the absence of retinalinfluence. Metaplasia requires the removal of the mesenchyme envelopes, the action of theretinal agent, as well as preservation of the integrity of the pigmented epithelium layer andsubsequent proliferation of its cells. The character of general control mechanisms both main-taining the stability of cell types and leading to their transformation into other cell types isdiscussed.

INTRODUCTION

The problem of stability of tissue differentiation remains one of the little-studied problems of developmental biology. Having attained a certain level ofdifferentiation, cells of different tissues of vertebrates steadily keep this differ-entiated state. Differentiating and dying cells are only replaced by descendantsof progenitor cells such as epidermal or pancreatic cells (Wessels, 1964), or elsesome reversible changes of differentiation occur with respect to the environmentin vitro (Coon & Cahn, 1966; Cahn, 1968). But these changes are limited by celltype. Very few reliable cases of artificial metaplasia (Fell, 1961) do not suggest anydefinite rule. Apart from the phenomena of stable differentiation, only far-reaching tissue transformation proceeds during regeneration in urodelanamphibians. This ability of true metaplasia as well as its absence in othervertebrates should be based on some general laws which can be studied inexperiments on artificial metaplasia.

The pigmented epithelium of vertebrate eyes appears to be the most suitabletissue for such studies. Following the removal of the retina in newts, a new retina

1 Authors' address: Institute of Developmental Biology, Academy of Sciences of the USSR,Vavilov Street 26, Moscow 117334, USSR.

522 G. V. LOPASHOV AND A. A. SOLOGUB

is regenerated from the pigmented epithelium (Colucci, 1891; Wachs, 1920;Stone, 1950; Hasegawa, 1958; Mitashov, 1968, 1969; Reyer, 1971), but itsdifferentiation is stable in all other vertebrates, including adult anuran amphib-ians (Stroeva, 1956). It has been clearly shown that the pigmented epitheliumchanges its differentiation (pigmentation, cell shape) in vitro with respect to someenvironmental conditions but, given the initial conditions, returns to itsoriginal type (Doljanski, 1930; Cahn & Cahn, 1966; Cahn, 1968; Whittaker,1968). The transformation of pigmented epithelium into retina has an advantage:these tissues differ sharply from each other in their differentiation, thus allowingthe exact identification of developing changes.

Our attempts to produce artificial metaplasia were based on the followinggeneral prerequisites. The process of differentiation during development is duenot only to the suppression of alternative ways or activation of one particularway of differentiation but to both of these phenomena (Lopashov, 1968;Nieuwkoop, 1968). At the same time influences stimulating one way of differ-entiation can suppress other ones. To obtain the transformation of pigmentedepithelium into retina, we considered it necessary, at least: (a) to remove fromthe pigmented layer the mesenchyme envelopes which can fix its flattened stateand, hence, its differentiation; (b) to introduce in its cells a lacking factor ofretinal differentiation which is most probably accumulated in retina by the timeof its transition to the final differentiation.

Some data have already suggested the possibility of transformation of pig-mented epithelium into retina in other than urodelan amphibians. Followingthe removal of retina and iris in tadpoles of Bufo viridis, pigmented epithelium isnot usually transformed into retina; but if it comes to lie between the margins ofregenerating retina it is transformed into retina (Lopashov, 1949). Following theremoval of retina in 4-day chick embryos, it is not restored from the pigmentedepithelium, but if a piece of retina from a chick embryo or a mouse embryo of thesame age is transplanted into the cavity of such an eye, islets of retina arise inthe pigmented epithelium (Coulombre & Coulombre, 1965, 1970). While inter-preting these results a possibility to be excluded is that at these stages the pig-mented epithelium can transform into retina simply as a result of accumulationof its cells due to delamination of mesenchyme envelopes; retina can arise fromthe outer eye layer in 4-day chick embryos both during its in vitro cultivation(Dorris, 1938) and in vivo following the exfoliation of mesenchyme envelopesunder oxygen deficiency (Mushett, 1953).

We investigated the possibility of artificially stimulating the transformation ofpigmented epithelium into retina at two stages of development in the commonfrog Rana temporaria, early tadpole and adult frog, by combining the removal ofmesenchyme envelopes and the effect of retina from a tadpole at the stage whenit has just attained the differentiated state. Some of these results have alreadybeen published elsewhere (Sologub, 1968; Lopashov & Sologub, 1970).

Artificial metaplasia 523

MATERIALS AND METHODS

Experiments were performed on eyes of tadpoles (stage 41 after Cambar &Marrot, 1954; stage III-IV after Taylor & Kollros, 1946; Rugh, 1962) andadult frogs of Rana temporaria. The following experiments were carried outto study the potencies of pigmented epithelium (Fig. 1):

I. Cultivation of pigmented epithelium of tadpoles and adult frogs in the emptyorbit of a tadpole eye.

II. Cultivation of a sandwich of pigmented epithelium of tadpoles and adultfrogs with a piece of tadpole retina inside within the empty orbit of a tadpoleeye.

III. Cultivation of pigmented epithelium of tadpoles and adult frogs in thelensless tadpole eye.

Prior to the operation, tadpoles were anaesthetized by 1 % urethane; inadult frogs, eyes were removed without narcosis. To remove the eye from theorbit in tadpoles, the anterior margin of the outer cornea was cut through, theeye was pressed out by needles and the nerve and vessels cut by iridectomyscissors. All manipulations were performed in double Holtfreter solution withantibiotics. Mesenchyme envelopes of the eye (scleral and choroid) were removedwith steel needles and a sharp-edged knife starting at the posterior eye pole.Remnants of the choroid coat were removed by rolling the eye on to Milliporefilter HA.

In the present work an attempt was undertaken to remove the mesenchymeenvelopes and Bruch's membrane completely. However, the cells of the pig-mented epithelium in tadpoles are so tightly connected with Bruch's membranethat we rarely succeeded in obtaining the intact cell layer following the removalof Bruch's membrane. It can only be successfully removed in adult frogs, inwhich it is not so tightly connected with cells of the pigmented epithelium.Attempts to remove this membrane by chemical methods proved to be un-successful (Sologub, 1968; Lopashov & Sologub, 1970), that is why it wasremoved surgically. With this aim after the removal of the sclera and cornea theeye knife was inserted between Bruch's membrane and the cells of pigmentedepithelium; Bruch's membrane was then drawn off by forceps and torn, thusreleasing a layer of pigmented epithelium.

In the first series of experiments the pigmented epithelium with or without theBruch's membrane was implanted into the empty eye orbit of tadpoles forcultivation (Fig. 1A).

In the second series of experiments, with sandwiches, only retina fromtadpoles (at the above-mentioned stage) was used. With the mesenchymeenvelopes intact, the eye was cut through around the limbus of cornea andthe whole retina was removed. A piece with all layers (0-25 mm x 0-25 mm to0-5 mm x 0-5 mm) was then cut out from its posterior pole. This piece was put ina sandwich of pigmented epithelium and placed through the cut into the orbit of


: rv^^^t"^^^'^'

B

Selera

ChoroidcoatBruch'smembrane

Pigmentedepithelium

Retina

Fig. 1. Schemes of operations. (A) Isolation of pigmented epithelium with or withoutthe Bruch's membrane, wrapping up a piece of retina into it and its subsequentimplantation into the empty eye orbit of a tadpole. (B) Implantation of pigmentedepithelium with or without the Bruch's membrane in the anterior chamber, pupil orposterior cavity of the tadpole lensless eye.


Table 1. Transformation of pigmented epithelium of tadpoles andadult frogs into retina

ResultsTotal no. , * »of fixed % of trans-

Series Type of experiment animals + / ~ formations

I Pigmented epithelium of tadpoles 84 0/84 0with mesenchyme envelopes orwithout them in the orbit of tadpoles

II(a) Pigmented epithelium of tadpoles 90/74* 51/23 68-9with a piece of retina in the orbitof tadpoles

III (a) Pigmented epithelium of tadpoles 92/87 * 81/6 93-1in the lensless eye of tadpoles

I Pigmented epithelium of adult frogs 35 0/35 0without mesenchyme envelopes andBruch's membrane in the orbit oftadpoles

II(b) Pigmented epithelium of adult frogs 90/75* 28/47 37-4without mesenchyme envelopes andBruch's membrane and with a pieceof retina in the orbit of tadpoles

III(b) Pigmented epithelium of adult frogs 169/99* 46/53 46-5in the eye of tadpoles

Pigmented epithelium was taken from two stages of development (tadpoles - stage 41- andadult frogs), retina was taken from tadpoles (stage 41).

+ Transformation of pigmented epithelium into retina.— No transformation.* Of the total number of fixed animals, only those were taken into account in the Results

section which were fixed at the time when metaplasia became possible (see text).

a tadpole from which the eye had been removed (Fig. 1 A). The empty orbit wasused as a chamber for cultivation of implants.

In the third series of experiments with lensless eyes, an eye of a tadpole waspressed out from the orbit through a cut along the anterior margin in the outercornea, nerve and vessels being intact. After zonules had been carefully cut, thelens was removed through the cut in the inner cornea. A layer of pigmentedepithelium taken from another tadpole or adult frog was implanted in the anteriorchamber, pupil, or the posterior cavity (vitreous chamber) of the lensless eyeby means of a needle and a knife (Fig. 1B). The eye with its implant was thencautiously pushed back under the outer cornea, which healed very rapidly.

Operated animals were fixed at intervals of 1 h, 1, 3, 5, 7, 10, 15, 20 and 30days following the operation by the Bouin's fluid. Sections 6/tm thick werestained by azocarmine after Heidenhain.


Fig. 2. Pigmented epithelium of the tadpole eye 15 days after theimplantation in the eye orbit.

RESULTS

/. Cultivation of pigmented epithelium of tadpoles and adult frogs in the orbit oftadpoles

A sheet of pigmented epithelium of tadpoles implanted in the orbit did nottransform into retina even after a long period of cultivation - 15-20 days afteroperation (Table 1). Cells of pigmented epithelium remained densely pigmentedduring the whole period of cultivation (Fig. 2). The layer of pigmented epitheliumin most cases closed and it could be suggested that no metaplasia occurredbecause it was closed. To keep the margins of the pigmented epithelium free, weplaced into it a disc cut out of the polyester film 'Melinex O \ However, in thistype of experiment all cells of pigmented epithelium remained unchanged aswell.

When cultivated in the orbit without mesenchyme envelopes and Bruch'smembrane, the pigmented epithelium of adult frogs did not change its differ-entiation. Tn most cases, the layer became dissociated into individual cellsdispersed in the orbit. In others, pigmented epithelium remained in the form ofsheet or gave rise to groups of cells (Fig. 3). No cases of transformation intoretina were recorded (Table 1).


Fig. 3. Pigmented epithelium of the adult frog eye without the mesenchyme envelopesand the Bruch's membrane in the orbit, .15 days after the implantation.

Fig. 4. Pigmented epithelium of the tadpole completely closed around the piece ofretina in the orbit. Ten days after the implantation. No changes in the pigmentedepithelium, ir, Implanted retina.


Fig. 5. Pigmented epithelium of the tadpole with the piece of retina inside. Threedays after implantation, dpe, Depigmented cells of pigmented epithelium; />,implanted retina.

II(a). Cultivation of pigmented epithelium as a sandwich with a piece of retinainside the tadpole eye orbit

The transformation of the pigmented epithelium of tadpoles into retinainvolves depigmentation of its cells, their proliferation and, then, formation oflayers in the newly formed retina. If one follows the course of metaplasia,characteristic sources of retina formation are disclosed. Transformation of pig-mented epithelial cells begins either on its free margins or in breaks through thesheet. When the sheet of pigmented epithelium closed around the piece of retina,no new retina formed (Fig. 4). In other cases, beginning from the 3rd day after theoperation, cells of pigmented epithelium depigmented and the flattened elipsoidcell nuclei became rounded, with a distinct nucleolus (Fig. 5). On the 5-7th dayafter the operation iris-like regions arose and their intensive proliferation pro-ceeded with the formation of retina (Fig. 6A). In the iris-like region, cells hadlarge light-stained nuclei and one or two nucleoli brightly stained (Fig. 6B). By10-15 days after the operation, large masses of newly formed retina appeared.Later (25 days after the operation) the newly formed retina became subdividedinto main layers: ganglionar and inner nuclear ones, layer of visual cells andreticular layers (Fig. 7). The frequency of transformation of the pigmentedepithelium of tadpoles into retina amounted to 68-9 % (Table 1).


B

Fig. 6. Pigmented epithelium of the tadpole eye regenerating into retina on the 7thday after the implantation into the orbit. (A) General view. (B) A region of A at agreater magnification. Cells of pigmented epithelium are depigmented simultaneouslyin two regions on both sides of the break in it. dir, Depigmented iris-like regions;epg, extruded pigment granules; m, mitoses in developing retina; pe, pigmentedepithelium; n, nucleoli.

II(b). Cultivation of the pigmented epithelium of adult frogs without Bruch'smembrane together with a piece of retina in the orbit of tadpoles

A sandwich of the pigmented epithelium of adult frogs with the retina oftadpoles implanted in the orbit broke rather frequently in many places and inthese cases a layer of pigmented epithelium was dissociated into individual cellsnot surrounding the piece of retina and the cells of the pigmented epitheliumremained unchanged. Only in 28 cases (Table 1) did the sandwich remainintact, and the transformation of pigmented epithelium into retina proceeded inthe same way as in the experiments where the pigmented epithelium and retinaof tadpoles were combined. Later (13-15 days after the operation, Fig. 8) onecould see the transformation into retina of nearly half of the pigmented epitheliumlayer (Fig. 9). A large number of mitosing cells could be seen in the newlyforming retina (Fig. 10). Metaplasia of pigmented epithelium in these experimentsoccurred in only 37-4 % of cases (Table 1), due to the wide separation of cellsdevoid of connecting envelopes.

34- E M B 28


Fig. 7. Pigmented epithelium of the tadpole eye with the retina completely re-generated from it. Twenty-five days after the implantation in the orbit, nr, Newlyformed retina; ir, implanted retina; mz, zone of mitoses; pe, pigmented epithelium.

pe

Fig. 8. Pigmented epithelium of the adult frog without mesenchyme envelopes andthe Bruch's membrane 15 days after the implantation in the orbit with a piece ofretina, nr, Newly formed retina; ir, implanted retina; pe, pigmented epithelium.


Fig. 9. Retina developed from the pigmented epithelium of the adult frog eye.Twenty days after the implantation in the orbit, nr, Newly formed retina.

Fig. 10. A region of retina developed from the pigmented epithelium of the adult frogeye. Twenty days after the implantation. Many mitosing cells are seen.

34-2


Fig. 11. Pigmented epithelium of the tadpole in the anterior chamber of lensless eye.Twenty days after the implantation. No changes in differentiation, ic, Inner cornea;hir, host iris; pe, pigmented epithelium.

Fig. 12. Pigmented epithelium of the tadpole in the posterior cavity of the lenslesseye. Three days after the implantation. Onset of transformation into retina, dpe,Depigmented cells of pigmented epithelium.


Fig. 13. Pigmented epithelium of the tadpole in the pupil of the lensless eye. Tendays after the implantation, pe, Pigmented epithelium; nr, newly formed retina, notdivided into layers; ilm, membrane on its surface corresponding to the inner limitingmembrane; hir, host iris.

Ill (a). Cultivation of pigmented epithelium of tadpoles in the lensless eye oftadpoles

These experiments were performed by the method used by Ikeda (1935),Mikami (1941), Sato (1953), Reyer (1956), Sologub (1972) and others: the lenswas removed from the eye and, instead of iris, a layer of pigmented epitheliumwas implanted in the lensless eye. In this series of experiments an isolated sheetof pigmented epithelium stuck to the outer cornea and remained in the anteriorchamber or it came to lie in the pupil or in the posterior cavity of the eye. Notransformation of pigmented epithelium into retina proceeded in the anteriorchamber (Fig. 11). In those cases where the pigmented epithelium remained inthe pupil, the whole process of metaplasia of the pigmented epithelium could befollowed. Depigmentation of cells began on the 3rd day after the operation(Fig. 12) followed by an intense proliferation of depigmented cells. Nucleolibecame distinct. On the 7th day after the operation the region of epitheliumadjacent to the posterior cavity was already two- or three-layered rather thanmonolayered. Depigmentation and proliferation of cells of the pigmentedepithelium began in the area where Bruch's membrane was absent or broken. Onthe 10th day after the operation a mass of newly formed retina appeared whichwas surrounded by a membrane corresponding to the inner limiting membraneof normal retina (Fig. 13). By 25-30 days after the operation the formation ofthe main reticular and nuclear layers began. On the surface of the new retina


Fig. 14. Retina developed from the pigmented epithelium of the tadpole in theposterior cavity of the lensless eye. Twenty days after the implantation, pe, Pig-mented epithelium of the implant; nr, newly formed retina; ph.r., developing photo-receptors; ic, inner cornea.

adjacent to the pigmented epithelium and corresponding to the outer nuclearlayer, visual cells began to form (Fig. 14). In some cases three layers ofpigmented epithelium with different degrees of transformation into retina couldbe seen in the pupil. The upper layer (1st layer) adjacent to the anterior chamberdoes not develop into retina; the layer of pigmented epithelium adjacent to theposterior cavity (2nd layer) transforms into retina partially; and the portionswhich moved in the posterior chamber (3rd layer) transformed into retinacompletely (Fig. 15).

In the posterior cavity, cells of the pigmented epithelium began to depigmenton the 3rd day after the operation. By 15-20 days the layer of pigmentedepithelium is completely transformed into retina. By 25-30 days a large mass ofnewly formed retina developed which sometimes filled the whole posteriorcavity (Fig. 16). The frequency of transformation of pigmented epithelium oftadpoles in the lensless eye amounted to 93-1 % (Table 1).

IH{b). Cultivation of the pigmented epithelium of adult frogs in eyes of tadpoles

Cells of the pigmented epithelium without Bruch's membrane dispersed in theposterior cavity of the lensless eye did not transform into retina. They remaineddensely pigmented even when cultivated for a long time (Fig. 17). When a sheetof pigmented epithelium without Bruch's membrane remained intact, depigmen-tation began on the 5—7th day after the operation (Fig. 18) followed by prolifera-

Artificial metaplasia

n >535

Fig. 15. Pigmented epithelium of the tadpole in the pupil of the lensless eye sub-divided into three zones (1, 2, 3) with different degrees of transformation into retina.Twenty days after the implantation.

tion of the depigmented cells. If the layer of pigmented epithelium remained inthe pupil, metaplasia began in the region adjacent to the posterior cavity. Whenthe layer moved to the posterior cavity by 25-30 days after operation, the com-plete transformation of pigmented epithelium into retina with the formation ofvisual cells on the surface of its inner cavities could be seen (Fig. 19).

To preserve the integrity of the sheet of pigmented epithelium, in a series ofexperiments Bruch's membrane was not removed. Being not so tightly connectedwith the cells of pigmented epithelium as in tadpoles, Bruch's membrane separa-ted from the sheet of pigmented epithelium on the 3rd day after the operation(Fig. 20); the latter preserved its integrity and transformation into retina pro-ceeded more frequently than in the preceding series. Some depigmented cellswere seen in the implant on the 7th day after the operation and by 15 days largeareas of depigmented cells proceeded to proliferation. The transformation ofthe whole layer of pigmented epithelium into retina was completed by 20-30 daysafter the operation (Fig. 21).

In seven cases the lenses were left in the eyes of tadpoles and the pigmented


Fig. 16. Complete transformation of the tadpole pigmented epithelium into retina inthe posterior cavity of the lensless eye. Twenty-five days after the operation.Mitotic divisions can be seen in the mass of newly formed retina, hr, Host retina;hir, host iris; nr, newly formed retina; m, mitoses.

Fig. 17. Separated cells of pigmented epithelium of the adult frog in the posteriorcavity of the lensless eye. Fifteen days after the operation. No changes, pe, Pigmentedepithelium; hir, host iris.


Fig. 18. Pigmented epithelium of the adult frog in the region of pupil of the lens-less eye. Onset of transformation. Five days after the implantation, dpe, Depigment-ing cells of pigmented epithelium.

Fig. 19. Retina developed from the adult frog pigmented epithelium in the cavity ofthe lensless eye. Twenty-five days after the operation, nr, Newly formed retina;hr, host retina; ph.r., rosette with photoreceptors.

epithelium of adult frogs, without Bruch's membrane, was implanted under thelens inside the eye. By 25 days after the operation the transformation of pig-mented epithelium into retina could be seen: its part adjacent to the normalretina had the retinal structure whereas that adjacent to the lens preserved itsinitial pigmentation (Fig. 22).


Fig. 20. Pigmented epithelium of the adult frog with the Bruch's membrane in thepupil of the lensless eye. Three days after the implantation, bm, Separated Bruch'smembrane; dpe, depigmenting cells; hir, host iris.

Fig. 21. Retina developed from the adult frog pigmented epithelium in the eyecavity. Twenty days after the operation, nr, Newly formed retina with mitoses; hr,host retina.


m»*^r t^h

Fig. 22. Transformation of the adult frog pigmented epithelium in the posteriorcavity under the lens. Twenty-five days after the implantation, nr, Newly formedretina; hr, host retina; />, iris-like regions; pe, preserved pigmented epithelium;/, host lens.

In all experiments with implantation of the pigmented epithelium of adultfrogs into the eye of tadpoles, metaplasia into retina was observed, on theaverage, in 46-5 % of cases (Table 1).

DISCUSSION

1. Significance of me sen chyme envelopes and lens to differentiationand morphogenesis of newly forming retina

At first sight, the role of mesenchyme envelopes in the development of retinamight appear unambiguous and purely negative. When mesenchyme envelopessurround the surface of the eye rudiment its cells develop into pigmentedepithelium rather than retina (Lopashov, 1963; Lopashov & Stroeva, 1963). Butunder close examination their role proves to be more complicated. The pigmentedlayer develops from the cells of the eye rudiment when they are arranged in onethin layer as well (Lopashov & Hoperskaya, 1967). At the same time the mesen-chyme envelopes (1) together with the enrolling and expanding retina promotethe stretching and flattening of cells of the outer eye layer, and (2) becomingattached to the pigmented layer fix its differentiation. Consequently the forma-tion of retina in the outer layer is possible after separation of mesenchymeenvelopes at embryonic stages of all vertebrates. Artificial metaplasia at laterstages of development is possible, as follows from our experiments, as a result of


the removal of envelopes or in those cases when mesenchyme envelopes becomeseparated from the pigmented epithelium or slip down from its margins.

But the role of mesenchyme envelopes in the formation of the retina is notsimply negative. If the retina develops de novo in the absence of the mesenchymeenvelopes - in the eye cavity (Results, §§ IIl(a), (b)) or in a sandwich of pigmentedepithelium with a piece of retina (Results, § lib) - it contains typical cellconstituents but does not acquire a cup-like shape and regular ratio of its layers(Figs. 9, 16, 19). The cup-like shape of the retina (although without the pupil)arises only in those cases when it is formed from the margins of pigmentedepithelium surrounded by the mesenchyme envelopes (Fig. 7); such a shapenever arises in the absence of mesenchyme envelopes. Metaplasia gives rise onlyto the transformation of pigmented epithelium into retina at the cellular levelbut does not provide the formation of its functional cup-like shape and regularratio of its constituents.

The role of the lens is also evident in the formation of the retina. Duringembryogenesis it provides the development of the initial cup-like shape of theretina (Lopashov, 1963). Later, together with the retina, it gives rise to intra-ocular pressure stretching the retina, pigmented epithelium and mesenchymeenvelopes (Coulombre & Coulombre, 1964) and providing their normal morpho-genesis. During regeneration of the eye the normal morphogenesis of the retinawith the formation of a pupil proceeds only in the presence of a lens (Lopashov,1949; Lopashov & Stroeva, 1963).

The role of the lens is also unambiguous. In the zone of direct contact with thecells of the pigmented epithelium, it prevents their transformation into retina(Results, § III(b), Fig. 22); they remain monolayered and preserve their pig-mentation. Here, the role of the lens is similar to that in differentiation of theinner layer of secondary iris and ciliary body (Stroeva, 1963, 1967), wherecontact with the lens leads to flattening and pigmentation of the former.

Thus, one of the necessary conditions for the initiation of metaplasia ofpigmented epithelium into retina is its liberation from the mesenchyme envelopesstabilizing its differentiation.

2. Role of retinal agent in the transformation of pigmentedepithelium into retina

During development the retina induces the formation of the lens from ecto-derm, thus suggesting the presence of a lens-forming agent, but it could beexpected that the development of the retina itself is directed by another agentas well (Lopashov, 1963). Indeed, at early stages of development the retinal Anlageis able to induce both the formation of retina and lens in gastrula ectoderm,depending on the type of contact (Lopashov & Hoperskaya, 1970). In this caseit is probable that a similar retinal agent is concentrated in the retinal Anlage inthe phase of its homotypic induction, its maximal concentration being attainedby the beginning of its differentiation. It is possible that such an agent disappears

Artificial metaplasia 541from the pigmented epithelium during the development of animals incapable ofregeneration. In this case the introduction of this agent from the retina wouldallow the metaplasia of pigmented epithelium into retina. The results of ourexperiments (Results, §§ II(a), III(b)) have shown that pigmented epithelium didtransform into retina under the influence of retina, thus providing the secondprincipal proof of the presence of a retinal agent. Since the direct contact ofcells is not necessary for its effect, it appears to be an inducing substance.

The retinal (R) agent is not the only agent formed by the retina. It forms a'retina factor' as well which is necessary for regeneration of lenses from thenewt iris (Stone, 1958; Reyer, 1962) and which we have designated as Lr-agent(Lopashov & Sologub, 1970). These agents differ from each other in some otherpeculiarities of their effects as well: R-agent induces the metaplasia of pigmentedepithelium, exerting its effect both from the whole retina (Results, §§///(«),(b)) and its pieces (Results, §§ H(a), (b)), whereas lens regeneration can bestimulated by the whole closed retina rather than by its pieces (Eguchi, 1967;Eisenberg-Zalik & Scott, 1969). Thus, the whole retina can stimulate both lensregeneration and metaplasia of pigmented epithelium into retina. Therefore wecan conclude that the transformation of the pigmented layer of the tadpole eyesinto retina in the posterior cavity of their eyes described by Sato (1953) is due tothe effect of R-agent.

It is known that the primary lens-inducing influence of eye rudiments which iscapable of inducing lens formation from the ectoderm of early embryos is notidentical with the latest Lr-agent. In newts the lens-inducing ability of the retinadecreases during the process of its differentiation and disappears at the time thatthe iris acquires its capacity for lens regeneration (Reyer, 1950, 1954; Takeuchi,1963). The second step of the effect of the retina on lens-forming epithelium,following the initial induction of a lens, involves the stimulation of crystallinformation rather than polarization of lens-forming cells under the influence ofmesenchyme (Muthukkaruppan, 1965; Eguchi, 1967; Philpott & Coulombre,1968). Bearing in mind these changes in time, one can conclude that R-agents inthe retinal Anlage and the differentiated retina are probably not identical. In anyevent, it is likely that the concentration of this agent increases with time since thedifferentiated retina of tadpoles can exert its effect at a distance on a more stablepigmented epithelium and not only on super sensitive gastrula ectoderm.

3. Role of cell proliferation and mass-effect in metaplasiaof pigmented epithelium into retina

Apart from two initial factors which we varied in these experiments - removalof mesenchyme envelopes and introduction of retinal agent - two other factorswere found to be important. First of all, cell proliferation. In its absence, notransformation of the cells of pigmented epithelium proceeded in experiments invitro (unpublished), in spite of the fact that they surrounded a piece of retina.The importance of the second of these factors, mass-effect, is due to the fact


that the cells of pigmented epithelium dispersed in the posterior eye cavity neverchange their differentiation (Results, § IHb). A certain minimal mass is necessaryfor the initiation of transformation into retina.

Does the effect of R-agent involve the stimulation of cell proliferation in thepigmented epithelium? A comparison of the behaviour of cells of pigmentedepithelium in the anterior chamber and the posterior cavity makes this suggestionvery unlikely. Both cavities may serve as good chambers for the cultivation ofvarious tissues in vivo involving intensive processes of proliferation (eye tissues -Royo & Quay, 1959; Stroeva, 1960, 1967; ovaries - Noyes, Clewe & Yamate,1961; nephrogenic mesenchyme - Grobstein & Parker, 1958, etc.). But themetaplasia of pigmented epithelium into retina proceeds only in the posteriorcavity which is surrounded by retina (Results, §§ III(a), (b)). Proliferation of thepigmented epithelium itself in vitro (Cahn, 1968) does not result in such trans-formations.

However, this does not exclude the proliferation from playing a part in themetaplasia of pigmented epithelium into retina, although it has to be combinedwith the effect of the specific R-agent. Processes of embryonic induction develop incell populations characterized by mitotic activity (gastrula ectoderm - Flickinger,Freedman & Stambrook 1967; epithelium of pancreatic rudiment - Wessels &Cohen, 1967; Rutter et al. 1968; chondrogenic cells of somites - Holtzer, 1968).The metaplasia of iris into lens in newts also involves the stimulation of the wholecomplex of phenomena of the cell cycle (Eisenberg & Yamada, 1966; Eisenberg-Zalik & Yamada, 1967; Yamada, 1967; Dumont, Yamada & Cone, 1970); thesame is true for the metaplasia of pigmented epithelium into retina in newts(Mitashov, 1969). The phenomena of artificial stimulation of the transformationof pigmented epithelium into retina described here involve, besides intensivecell divisions, a steady increase in the size of nucleoli (Figs. 6B, 13).

Proliferation appears to be indispensable for the formation or transformationof cell types because the effect of corresponding agents can be applied only tocells at a certain phase of the cell cycle. This viewpoint (Lopashov & Sologub,1970) agrees with the ideas of Gurdon & Woodland (1968, 1970), according towhom cytoplasmic proteins responsible for long-term changes of cell differentia-tion can enter the nuclei only at certain phases of their cycle. The similaritybetween the most carefully studied inducing agents (Tiedemann, 1968) and non-histone proteins which is due to the derepression of specific DNA sequences innuclear chromatin (Paul, 1971) allows one to suggest that similar agents maydetermine stable cell differentiation in all these cases. The phenomena of arti-ficial metaplasia appear to be based on a similar mechanism which, in the case ofinduction, involves not only exchange of proteins between the nucleus and thecytoplasm but also between different cells. If so, the occurrence of inductivephenomena would depend on the longevity of the corresponding phase of cellcycle and the specificity of cell differentiation on the agents participating in theprocess of induction.


But the mass-effect should precede cell proliferation and therefore cannot bereduced to its consequences. Mitoses resulting in the formation of the mass ofretina in amphibians (Hollyfield, 1968; Straznicky & Gaze, 1971) can proceedonly in its Anlage, which has a certain minimal mass, whereas they do notproceed in the pigmented epithelium (Lopashov, 1963; Coulombre, Steinberg &Coulombre, 1963; Sologub, 1968). The most likely significance of mass-effectto the transformation of cells of the pigmented epithelium consists in the factthat, following the effect of R-agent, subsequent processes of homotypic in-duction occurring in the mass of homogenous cells cannot occur in isolated cells.Meanwhile, it is in this phase (Discussion, § 2) that one-sided reproduction andconcentration of R-agent can proceed, thus providing one-sided differentiationof retina and the development of its ability to induce the formation of retina inthe gastrula ectoderm and the pigmented epithelium. The development of otherAnlagen has similar phases, as witnessed by Cooper (1965) who has shown thatcartilage at a certain period is able to induce the formation of cartilage. Pheno-mena of homotypic induction occurring at the phase of one-sided differentiationcan be found in other Anlagen as well and the agents concentrating at this phasecan be used to obtained directed differentiation and artificial metaplasia in someother tissues and Anlagen.

4. Control circuits of metaplasia and its stimulationin non-regenerating species

In those cases when animals are adapted to regeneration such as the ability toregenerate the eye in newts, an impression can be created that the damage of theeye or its parts with their subsequent destruction results itself in all other pro-cesses of regeneration acting as a trigger. But the experiments with artificialstimulation of metaplasia have clearly shown that for this a definite set of eventshas to be started. Thus, what looks at first sight as the 'stimulation' of meta-plasia deserves in fact more thorough comprehension. Such stimulation requiresa re-arrangement of the whole control circuit of the given process rather than anyparticular treatment.

To obtain metaplasia of the pigmented epithelium in frogs, a number ofconstituents of this circuit is to be reproduced: (1) liberation of pigmentedepithelium from Bruch's membrane; (2) effect of R-agent which appears to actlike inducing agents switching on (derepressing) certain syntheses which resultin differentiation; (3) mass-effect which reflects the situation that any de-repression in an individual cell leads to nothing for a mass of interconnectedcells is necessary for their differentiation; (4) development of conditions forproliferation, other than the liberation from Bruch's membrane. If duringderepression of certain DNA sequences, phenomena at the molecular level areincluded in the circuit, other constituents of the circuit naturally belong to thesupercellular level. The removal of Bruch's membrane, influence of the R-agentand mass-effect should act together on groups of cells, otherwise they would be


inefficient. Therefore such control circuits should involve phenomena atdifferent levels, otherwise they cannot operate.

It does not mean that the control circuit of retinal regeneration can be limitedby the constituents of the control circuit of metaplasia. The formation of retinade novo requires some other above-mentioned factors (Discussion, § 1): thepresence of mesenchyme envelopes, lens and intra-ocular pressure. Metaplasiaand regeneration in newts are operated essentially by the same circuits, but theadaptivity of the process of regeneration in these species is manifested by the factthat these circuits are switched on automatically and in a certain sequenceleading to the restoration of the eye. A study of the structure of such circuits isnot too complicated and their knowledge does not allow one to reduce theprocesses of metaplasia and regeneration to the effects of a single 'priming'event, such as initial damage, inducing influence, or molecular information.

The authors express their cordial gratitude for technical assistance to Mrs Nina A. Ivanova.

REFERENCES

CAHN, R. D. (1968). Factors affecting inheritance and expression of differentiation: somemethods of analysis. In The Stability of the Differentiated State (ed. H. Ursprung), pp. 58-84. Berlin: Springer.

CAHN, R. D. & CAHN, M. D. (1966). Heritability of cellular differentiation: clonal growthand expression of differentiation in retinal pigment cells in vitro. Proc. natn. Acad. Sci.U.S.A. 55, 106-114.

CAMBAR, R. & MARROT, D. R. (1954). Table chronologique du developpement de la grenouilleagile (Rana dalmatina). Bull. biol. Fr. Belg. 88, 168-174.

COLUCCI, V. (1891). Sulla rigenerazione parziale dell'occhio nei tritoni. Studio sperimentale.Mem. R. Accad. Sci. 1st. Bologna, Sez. sci. natur. (Ser. 5) 1, 167-203.

COON, H. G. & CAHN, R. D. (1966). Differentiation in vitro: effect of Sephadex fractions ofchick embryo extract. Science, N. Y. 153, 1116-1119.

COOPER, G. W. (1965). Induction of somite chondrogenesis by cartilage and notochord: acorrelation between inductive activity and specific stages of cytodifferentiation. Devi Biol.12, 185-212.

COULOMBRE, A. J. & COULOMBRE, J. L. (1964). Lens development. I. Role of the lens in eyegrowth. /. exp. Zool: 156, 39-47.

COULOMBRE, J. L. & COULOMBRE, A. J. (1965). Regeneration of neural retina from thepigmented epithelium in the chick embryo. Devi Biol. 12, 79-92.

COULOMBRE, J. L. & COULOMBRE, A. J. (1970). Influence of mouse retina on regeneration ofchick neural retina from chick embryonic pigmented epithelium. Nature, Lond. 228,559-560.

COULOMBRE, A. J., STEINBERG, S. N. & COULOMBRE, J. L. (1963). The role of intraocularpressure in the development of the chick eye. V. Pigmented epithelium. Invest. Ophthalmol.2, 83-89.

DOLJANSKI, L. (1930). Sur le rapport entre la proliferation et l'activite pigmentogene dans lescultures de l'epithelium de l'iris. C r. Seanc. Soc. Biol. 105, 343-345.

DORRIS, F. (1938). Differentiation of the chick eye in vitro. J. exp. Zool. 78, 385—415.DUMONT, J. N., YAMADA, T. & CONE, M. V. (1970). Alteration of nucleolar ultrastructure in

iris epithelial cells during initiation of Wolffian lens regeneration. / . exp. Zool. 174,187-204.

EGUCHI, G. (1967). In vitro analyses of Wolffian lens regeneration: differentiation of theregenerating lens rudiment of the newt, Triturus pyrrhogaster. Embryologia 9, 246-266.

EISENBERG, S. & YAMADA, T. (1966). A study of DNA synthesis during the transformation ofthe iris into lens in the lentectomized newt. /. exp. Zool. 162, 353-368.

Artificial metaplasia 545EISENBERG-ZALIK, S. & SCOTT, V. (1969). In vitro development of the regenerating lens.

Devi Biol. 19, 368-379.EISENBERG-ZALIK, S. & YAMADA, T. (J 967). The cell cycle during lens regeneration. /. exp.

Zool. 165, 385-393.FELL, H. B. (1961). Changes in syntheses induced in organ culture. In Synthesis of Molecular

and Cellular Structure (ed. D. Rudnick), pp. J 39-160. New York: Ronald Press.FLICKINGER, R. A., FREEDMAN, M. L. & STAMBROOK, P. J. (1967). Generation times and DNA

replication pattern of cells of developing frog embryos. Devi Biol. 16, 457-473.GROBSTEIN, C. & PARKER, G. (1958). Epithelial tubule formation by mouse metanophrogenic

mesenchyme transplanted in vivo. J. natn. Cancer Inst. 20, 107-119.GURDON, J. B. & WOODLAND, H. R. (1968). The cytoplasmic control of nuclear activity in

animal development. Biol. Rev. 43, 233-267.GURDON, J. B. & WOODLAND, H. R. (1970). On the long-term control of nuclear activity

during cell differentiation. Current Topics Devi Biol. 5, 39-70.HASEGAWA, M. (1958). Restitution of the eye after removal of the retina and lens in the

newt, Triturus pyrrhogaster. Embryologia 4, 1-32.HOLLYFIELD, J. G. (1968). Differential addition of cells to retina in Rana temporaria tadpoles.

Devi Biol. 18, 164-179.HOLTZER, H. (1968). Induction of chondrogenesis: a concept in quest of mechanisms. In

Epithelial-Mesenchymal Interactions (ed. R. F. Fleishmajer & R. E. Billingham), pp. 152-164. Baltimore: Williams & Wilkins.

IKEDA, Y. (1935). Uber die Regeneration von Augenbechern an vershiedenen Korperstellendurch isolierte Irisstiicke. Arb. anat. Inst. Univ. Sendai 17, 11-54.

LOPASHOV, G. V. (1949). On the significance of different processes in the restoration ofamphibian eyes. Dokl. Akad. Nauk SSSR 69, 865-868.

LOPASHOV, G. V. (1963). Developmental Mechanisms of Vertebrate Eye Rudiments. Oxford:Pergamon Press.

LOPASHOV, G. V. (1968). Main Causes of Organism Development. Moscow: Znanie.LOPASHOV, G. V. & HOPERSKAYA, O. A. (1967). On the character of cell interactions during

differentiation of eye rudiments in amphibians. Dokl. Akad. Nauk SSSR 175, 962-965.LOPASHOV, G. V. & HOPERSKAYA, O. A. (1970). Origin and modes of distribution of inducing

agents in development. In Intercellular Interactions in Differentiation and Growth (ed.G. V. Lopashov, N. N. Rott & G. D. Tumanishvili), pp. 52-64. Moscow: Nauka.

LOPASHOV, G. V. & SOLOGUB, A. A. (1970). Stimulation of metaplasia and embryonic in-duction. In Tissue Metaplasia (ed. M. S. Mitskevich, O. G. Stroeva & V. I. Mitashov),pp. 23-44. Moscow: Nauka.

LOPASHOV, G. V. & STROEVA, O. G. (1963). Eye Development in Light of ExperimentalInvestigations. Moscow: Ac. Sci. USSR Press.

MIKAMI, Y. (1941). Experimental analysis of the Wolffian lens regeneration in adult newt,Triturus pyrrhogaster. Jap. J. Zool. 9, 303-319.

MITASHOV, V. 1. (1968). Autoradiographic study of the restoration of the retina in crestednewts {Triturus cristatus). Dokl. Akad. Nauk SSSR 181, 1510-1513.

MITASHOV, V. I. (1969). Dynamics of DNA synthesis in pigmented epithelium cells through-out the eye restitution after a surgical removal of the retina in adult newt Triturus cristatus.Tsitologia 11, 434-446.

MUSHETT, C. W. (1953). Elektive Differenzierungsstorungen des Zentralnervensystems amHiihnchenkein nach kurzfristigem Sauerstoffmangel. Beitr. path. Anat. 113, 367-387.

MUTHUKKARUPPAN, V. (1965). Inductive tissue interaction in the development of the mouselens in vitro. /. exp. Zool. 159, 269-288.

NIEUWKOOP, P. D. (1968). Activation and inhibition as complementary mechanisms inembryonic induction processes. Atti Accad. naz. Lincei Re. 104, 203-208.

NOYES, R. W., CLEWE, T. H. & YAMATE, A.M. (1961). Follicular development, ovularmaturation and ovulation in ovarian tissue transplanted into the eye. In Control of Ovulation(ed. G. A. Villee), pp. 24-34. Oxford: Pergamon Press.

PAUL, J. (1971). DNA/protein interaction in mammalian differentiation. In Intrinsic andExtrinsic Factors in Early Mammalian Development (Adv. in Biosciences 6), pp. 495-505.

35 EMB 28


PHILPOTT, G. W. & COULOMBRE, A. J. (1968). Cytodifferentiation of precultured embryonicchick lens epithelial cells in vitro and in vivo. Expl Cell Res. 52, 140-146.

REYER, R. W. (1950). An experimental study of lens regeneration in Triturus viridiscensviridescens. II. Lens development from the dorsal iris in absence of the embryonic lens./ . exp. Zool. 113, 317-353.

REYER, R. W. (1954). Regeneration of the lens in the amphibian eye. Q. Rev. Biol. 29, 1-46.REYER, R. W. (1956). Lens regeneration from homoplastic and heteroplastic implants of

dorsal iris into the eye chamber of Triturus viridescens and Amblystoma punctatus. J. exp.Zool. 133, 145-189.

REYER, R. W. (1962). Regeneration in the amphibian eye. In Regeneration (ed. D. Rudnick),pp. 211-265. New York: Ronald Press.

REYER, R. W. (1971). The origins of the regenerating neural retina in two species of Urodela.Anat. Rec. 169, 410-411.

ROYO, P. E. & QUAY, W. B. (1959). Retinal transplantation from fetal to maternal mammalianeye. Growth 23, 313-336.

RUGH, R. (1962). Experimental Embryology. Minneapolis: Burgess.RUTTER, W. J., KEMP, J. D., BRADSHAW, W. S., CLARK, W. R., RONZIO, R. A. & SANDERS,

T. G. (1968). Regulation of specific protein synthesis in cytodifferentiation. J. cell Physiol.72 (suppl. 1), 1-18.

SATO, T. (1953). Uber die Ursachen des Ausbleibens der Linsenregeneration and zugleichuber die linsenbildende Fahigkeit des Pigmentepithels bei den Anuren. Wilhelm Roux Arch.EntwMech. Org. 146, 487-514.

SOLOGUB, A. A. (1968). On the capacity of eye pigmented epithelium for transformation intoretina in anuran amphibian tadpoles. Tsitologia 10, 1526-1532.

SOLOGUB, A. A. (1972). Transformation of the tadpole eye pigmented epithelium into theretina under the action of the intact retina. Tsitologia (in the Press).

STONE, L. S. (1950). The role of retinal pigment cells in regenerating neural retinae of adultsalamander eyes. / . exp. Zool. 113, 9-31.

STONE, L. S. (1958). Lens regeneration in adult newt eyes related to retina pigment cells and theneural retina factor. J. exp. Zool. 139, 69-83.

STRAZNICKY, K. & GAZE, R. M. (1971). The growth of the retina in Xenopus laevis: anautoradiographic study. /. Embryol. exp. Morph. 27, 67-79.

STROEVA, O. G. (1956). Studies on the regenerative power of the retina in adult Bombina andrats. Izv. Akad. Nauk SSSR (ser. biol.) 5, 76-84.

STROEVA, O. G. (1960). Experimental analysis of the eye morphogenesis in mammals. / .Embryol. exp. Morph. 8, 349-368.

STROEVA, O. G. (1963). The role of the lens epithelium in the induction of iris and ciliary bodytissue. Dokl. Akad. Nauk SSSR 151, 464-467.

STROEVA, O. G. (1967). The correlation of the processes of proliferation and determination inthe morphogenesis of iris and ciliary body in rat. J. Embryol. exp. Morph. 18, 269-287.

TAKEUCHI, S. (1963). Change in modes of lens formation in the newt during development.Embryologia 8, 21-44.

TAYLOR, A. C. & KOLLROS, J. J. (1946). Stages in the normal development of Rana pipienslarvae. Anat. Rec. 94, 7-24.

TIEDEMANN, H. (1968). Factors determining embryonic differentiation. / . cell Physiol. 72(suppl. 1), 129-144.

WACHS, H. (1920). Restitution des Auges nach Extirpation von Retina und Linse der Tritonen.Wilhelm Roux Arch. EntwMech. Org. 46, 328-390.

WESSELS, N. K. (1964). Tissue interaction and cytodifferentiation. / . exp. Zool. 157, 139-152.WESSELS, N. K. & COHEN, J. H. (1967). Early pancreas organogenesis: morphogenesis,

tissue interaction and mass effect. Devi Biol. 15, 237-270.WHITTAKER, J. R. (1968). The nature and probable cause of modulations in pigment cell

cultures. In The Stability of the Differentiated State (ed. H. Ursprung), pp. 25-36. Berlin:Springer.

YAMADA, T. (1967). Cellular and subcellular events in Wolffian lens regeneration. CurrentTopics Devi Biol. 2, 247-281.

(Manuscript received 28 February 1972)

artificial metaplasia of pigmented epithelium into retina ... · artificial metaplasia of pigmented...

Documents