- Email: [email protected]
Dedifferentiation of iris epithelial cells
DEVELOPMENTAL
BIOLOGY
29,
385-401
(1972)
Dedifferentiation
of Iris Epithelial
JAMES N. DUMONT AND Biology
Division,
Oak Ridge
National
YAMADA
TUNEO
Laboratory,
Cells’
Oak
Ridge,
Tennessee
37830
Electron and phase-contrast microscopic observations demonstrated that depigmentation of iris epithelial cells in vivo after lentectomy is preceded by alterations of cell shape and increases in microfilaments and microtubules in the periphery of the cell. Extensive cell processes are formed, with tips branching into fine strands that contain melanosomes. The macrophages invading the iris epithelium incorporate pieces of such strands, which are composed of cell membrane, cytoplasmic matrix, and melanosomes. Individual melanosomes are also taken up by macrophages. Some of the strands of iris cell processes seem to degenerate within the intercellular space. Thus, depigmentation of iris epithelial cells is accompanied by loss of a substantial part of the cell surface and cytoplasmic matrix of iris epithelial cells. Measurements of the absolute volume of whole iris epithelial cells, their cytoplasm, and their nuclei were conducted at various stages of depigmentation. These measurements reveal an extensive increase in the volume of the cytoplasm preceding activation of the cell surface and a significant reduction in cytoplasmic volume during the phase of extensive depigmentation. In the case of the nuclear volume, the extensive increase which occurs in parallel with that of the cytoplasmic volume is not followed by a significant change during the depigmentation phase. INTRODUCTION
Stepwise tracing of labeled cells in the Wolffian lens-regenerating system in the adult newt indicates that the fully differentiated iris epithelial cells located in the mid-dorsal margin of the iris epithelium are converted into lens cells after passing through two or more induced cell divisions (Eisenberg and Yamada, 1966; Reyer, 1971; Zalik and Scott, 1971; Yamada and Roesel, unpublished). During this transformation, activated by lentectomy, the cells go through the following steps: (1) nucleolar activation (Reese et al., 1969; Dumont et al., 1970), (2) induction of DNA replication and mitosis (Eisenberg and Yamada, 1966; Yamada and Roesel, 1969, 1971; Reyer, 1971), (3) depigmentation (Eguchi, 1963; Karasaki, 1964; Yamada and Dumont, 1972), and (4) appearance of lens-specific antigens associated with lens morphogenesis (Takata et al., 1964, 1966; Yamada, 1966, 1967). This sequence of events suggests that in step 3, during which the whole population of melanosomes is removed from the cytoplasm, the ‘Research Commission Corporation.
sponsored by under contract
U. S. Atomic with Union
Energy Carbide 385
Copyright All rights
0 1972 hy Academic Press. Inc. of reproduction in any form reserved.
iris epithelial cell loses not only its morphological identity but also its developmental specificity, and that it becomes reprogrammed in a new differentiation pathway in step 4. In other words, reversion of morphological differentiation, which is coupled with recovery of a developmentally less committed status, seems to occur in step 3. Such a reversion, not simple loss of overt differentiation, will be referred to as dedifferentiation. A basically similar terminology was used by Wolff (1895) when he discussed for the first time the depigmentation of iris epithelial cells. On the basis of the above facts and ideas, one would expect information concerning dedifferentiation of iris epithelial cells to afford an insight into the cellular control mechanism of differentiation. We owe to Eguchi (1963) the first electron microscopic studies of the depigmentation process. The work so far published from our laboratory on the same subject is in good conformity with Eguchi’s data (Karasaki, 1964; Yamada and Dumont, 1972), except that we identify the major cell type involved in phagocytic uptake of melanosomes as the macrophage, while Eguchi called it a special amoeboid cell. Further studies of the depigmentation process car-
386
DEVELOPMENTAL.
BIOLOGY
ried out in our laboratory have revealed new aspects that appear important for understanding dedifferentiat,ion. This paper describes morphological studies of those aspects. MATERIALS
AND
METHODS
Electron microscopy. Dorsal pupillary margins from normal irises and from irises 4, 6, 8, 13, and 15 days after lentectomy were surgically removed, fixed for 2 hr in cold 3% glutaraldehyde buffered with 0.1 M phosphate buffer at pH 7.4 and washed in several changes of the same buffer. They were then bisected dorsoventrally, placed in cold 1% osmium tetroxide in 0.1 M phosphate buffer, dehydrated, infiltrated, and embedded in Epon in flat embedding molds so that sections could be obtained through the dorsolateral and medioventral axes as previously described (Dumont et al., 1970). Thin sections cut with a PorterBlum ultramicrotome equipped with a diamond knife were placed on Formvarcoated, carbon-stabilized grids, stained with uranyl acetate and lead citrate, and examined in a Hitachi 11-E electron microscope operated at 75 V. Cell volume determinations. Total cell volume, as well as the volumes of the cytoplasmic and nuclear compartments, was determined from 0.25- and 0.5-p serial Epon sections. Only clearly delimited cells entirely contained within the serial sections were selected. These cells were photographed with a light microscope, photographically enlarged, and measured with an Ott compensating polar planimeter to determine total cell and nuclear areas (cytoplasmic area equals total cell area minus nuclear area). The actual volume of each cellular compartment was then calculated from the measured areas, photographic enlargement factor, and section thickness. Because of the complex surface configuration of cells in regenerating tissue, the number of cells which could be completely followed in serial sections was small.
VOLUME
29. 1972
Owing to the small number of samples measured (Table l), the confidence limits were calculated by pooling estimates of variation about each mean. Since the standard deviation decreased linearly on each day following lentectomy, a regression relating standard deviation to days after lentectomy was used. The resulting confidence limits were thus calculated for each mean based on an estimate of the standard deviation using all of the values for days 8, 13, and 15. RESULTS
Electron
Microscopic
Study
Normal iris epithelium. The iris epithelium is continuous with the ciliary epithelium and is composed of inner and outer laminae which merge at the pupillary margin. More proximally, the inner lamina of the ciliary epithelium borders the neural retina, while the outer lamina borders the pigmented retina. The iris epithelium is composed of heavily pigmented cuboidal cells (Fig. 1). The external lamina of the epithelium is partially covered by stroma which supports iridophores, connective tissue cells, and small blood vessels. Care was taken to ensure that all cells except those at the dorsal pupillary margin of the iris epithelium were excluded from our samples. Thus, as in a previous study (Dumont et al., 1970), the present report deals only with the 20-25 cells extending upward from the pupillary margin of the dorsal iris epithelium when viewed in a section sagittal to the eye ball. The basal epithelial cell surface, i.e., the cell surface toward the outside of each lamina rests on a basement membrane. The nuclei are spherical or slightly elongate. Mitochondria, endoplasmic reticulum, and ribosomes are present in scant amounts (Fig. 2). Desmosomal junctions are present on the lateral surfaces of adjacent cells. Subjacent to the apical plasmalemma are 45- to 50-A microfilaments. Iris epithelium 4 days after lentectomy.
DUMONT
AND
Dedifferentiution
YAMADA
TABLE CELL VOLUME
Source of cells Normal
CHANGES
Volume (~‘1 Total cell
3
4
Mean Day-13 regenerate
5
Mean Day-15 regenerate
2 Mean
Four days after lens removal, the epithelial cells of the dorsal margin of the iris enlarge and elongate in the apicobasal axis; thus, the melanosomes are not as densely packed as in normal cells. As in normal cells, there is a paucity of cytoplasmic organelles and membrane systems. Desmosomal junctions with associated microfilaments and microtubules remain between adjacent cells (Fig. 3). In some areas intercellular spaces become enlarged. The basement membrane remains on the basal cell surface, but where adjacent cells have separated it may indent into the intercellular space. Iris epithelium 6-8 days after lentectomy. At 6-8 days after lentectomy, the epithelial cells have elongated further, and pseudopod-like extensions, which contain concentrations of melanosomes, have formed at the basal end of the cell (Figs. 4 and 5). Mitochondria and some microtubules oriented parallel to the long axis are
387
Cells
CELLS DURING LENS REGENERATION
Mean Day-8 regenerate
Epitheliul
1
IN IRIS EPITHELIAL
No. cells measured
of Iris
Cytoplasm
Nucleus
986 948 898
635 578 545
351 370 353
944
586
358
3688 3485 2100 3481
1314 2401 1342 2245
2374 1083 759 1236
3190
1826
1363
2966 2421 2327 1829 2063
1465 1141 1298 755 944
1501 1280 1028 1075 1069
2321
1131
1191
1620 1954
494
734
1126 1220
1787
614
1173
present in the pseudopods. Many of these extensions are attenuated; they are very thin and contain only a few aligned melanosomes. The cytoplasm of these attenuated processes often appears more dense than that of other areas of the cell (Fig. 5). These pseudopodial extensions of the iris epithelial cells are easily distinguished from the extensions and infoldings of the ciliary epithelium, which are normal characteristics of the cell surface. The intercellular spaces become prominent, although many cell contacts are maintained. Other cytoplasmic organelles also become altered at 6 days. For example, some cisternae of rough endoplasmic reticulum and Golgi complexes are now present along with increasing numbers of polyribosomes and mitochondria. Microfilaments and microtubules also appear more abundant in the periphery of the cells than in earlier stages of lens regeneration. The microfilaments lie in a band subjacent and parallel
388
390
DEVELOPMENTAL
BIOLOGY
to the plasmalemma and the long axis of the pseudopodial extensions. The band of microfilaments appears to be slightly thicker at the tips of the pseudopodial extensions than along the lateral margins. Microtubules are more centrally located in the cytoplasm of the pseudopodial processes. Six to eight days after lentectomy, there appears on the surface of the iris epithelial cells, especially on the pseudopodia and to some extent within the cytoplasm, highly dense, usually spherical condensations or concretions ranging from 200 to 600 A (Figs. 4 and 6). They are present in specimens fixed either in glutaraldehyde or osmium or in both, in unstained as well as single- or double-stained (uranyl acetate and/or lead citrate) sections, and are not removed by en bloc treatment of the tissue with EDTA or sodium citrate. In these respects they appear unlike the micropapillae described by Clawson and Good (1971) on the surface of cultured cells. The concretions are normally present on the surface of iris epithelial cells, but not on macrophages or mast cells. Occasionally some appear in the iris cell cytoplasm, where they are associated with melanosomes. One interpretation is that they represent the breakdown of melanosomes; however, we have no evidence for this other than their proximity to melanosomes and a density similar to that of melanin. These concretions are also associated with macrophage phagosomes that contain fragments of iris epithelial cells and melanosomes.
VOLUME
29. 1972
Depigmentation becomes evident during this 6- to a-day period following lentectomy. In the early phases of depigmentation, individual or small groups of melanosomes are lost and are occasionally encountered lying within the intercellular spaces (Figs. 7 and 8). In some cases,as in Fig. 7, these extracellular melanosomes appear to be surrounded by membranous material which has some concretions associated with it. In other cases, as in Fig. 9, melanosomes are enclosed, along with cytoplasm, by a concretion-studded membrane. Because of the size of the cells and their highly pleomorphic shapes it is impossible to determine, without inordinate numbers of serial sections, whether such configurations are separated portions of iris epithelial cell cytoplasm containing melanosomes or are still connected to the epithelial cell by a thin cytoplasmic strand such as is shown in Fig. 6. Macrophage processes, however, frequently circumscribe and appear to engulf such structures (Fig. 10). In either case, materials engulfed by macrophages become isolated in phagosomes that contain melanosomes, membranes, and concretions (Figs. 11 and 12). Iris epithelium 13-15 days after lentectomy. By 13-15 days following lentectomy, many of the formerly pigmented cells have lost most of their melanosomes. The nuclei of these cells have become enlarged, and are surrounded only by a thin rim of cytoplasm (Fig. 13). The cytoplasm of the pseudopodia is now even more richly pop-
FIG. 1. A survey micrograph of the pupillary margin showing The stroma contains iridophores. x 2300.
the two laminae
of the dorsal
iris epithelium.
Fro. 2. A portion of an iris epithelial cell cytoplasm from a normal iris. The cytoplasm contains large quantities of melanosomes but indicates a paucity of other organelles. Subjacent to the plasmalemma, at the apical end of the cell, is a network of fine filaments (F). A continuous basement membrane (G) covers the bases of all epithelial cells. (N); the nucleus. x 27,300. FIG. 3. High magnification of a desmosomal junction between adjacent epithelial cells. x 23,000. FIG. 4. Cells from a day-8 regenerate. The formerly cuboidal cells elongate, and many of the melanosomes are lost. The cytoplasm becomes richer in mitochondria and ribosomes. The plasmalemma acquires dense concretions (arrows). x 10,000.
DUMONT AND YAMADA
Dedifferentiution
ulated with rough endoplasmic reticulum, polyribosomes, mitochondria, microtubules, and microfilaments (Figs. 13-15). The dense concretions of the plasmalemma, so prominent at 6 and 8 days, have essentially disappeared (Fig. 11). Light microscopic study. Phase-contrast observations of serial sections of Eponembedded normal iris and irises 4, 6, 8, 13, and 15 days after lentectomy confirm the changes of shape of epithelial cells and the intercellular spaces between them detected by electron microscopic studies. Diagrams based on light microscopic observations illustrating changes in the shape of dorsal iris epithelial cells at various stages of depigmentation are reproduced in Fig. 16.
of Iris
Epitheliul
Cells
391
cated in the mid-dorsal marginal iris epithelium and were expected to become lens cells of the regenerate. There is an increase in total cell volume from day 0 to day 8 and a decrease in volume from day 8 to day 15. The nuclear volume increases from day 0 to day 8, then does not change. All of these volume changes are statistically significant. DISCUSSION
The light and electron microscopic observations presented indicate that depigmentation of the iris epithelial cells is accompanied by extensive alterations in the topography of the cell surface. Elongation of the cell along the apicobasal axis is followed by formation of cell processes which branch into thin cytoplasmic strands. The melanosomes, uniformly distributed Changes in the Volume of Iris Epithelial throughout the cytoplasm in the normal Cells before and during Depigmentation condition, are displaced into these cell Microscopic observations implied the processes. These cellular changes are assoloss of cytoplasmic materials, so efforts ciated not only with an increase in cell surwere made to determine whether this loss face but also with an expansion of the inof material is reflected by a reduction in tercellular space and partial obliteration of the volume of the cytoplasm after depigintercellular junctions including desmomentation. The usual methods of cytologisomes. These observations imply that the cal measurement turned out to be too in- quiescent condition of the cell surface, i.e., accurate for this purpose because of the the plasmalemma and the cytoplasm imvery irregular shape of the cells. Measuremediately subjacent to it, characteristic of ments on dissociated cells were precluded normal iris epithelial cells is replaced by a by the extensive changes in volume caused more mobile condition after lentectomy. by dissociation. Finally, the very time-conThat the cells are highly mobile at the time suming method described in the Materials of depigmentation has been confirmed by and Methods sections was adopted. The observation of iris epithelial cells depigmethod depends on, among other factors, menting in vitro (J. Ortiz, unpublished). the clear delineation of the cell boundary in The cellular projections formed are not the enlarged photographs. The number of static structures but dynamic formations cells satisfying the condition was very of the cell surface in constant movement. small, especially in day-15 samples. Thus, Involvement of microfilaments in the the limited number of measurements made cellular changes is suggested by the apnecessary a careful statistical check, as in- pearance of an oriented network of microdicated in the Methods section. filaments 45-50 A in diameter in the cortiThe data obtained for the total cell, cy- cal layer of the cell surface. A population toplasmic, and nuclear volumes of iris epi- of microtubules is also present in the cell thelial cells of day 0 (control), and day 8, projections. Unpublished results of in vitro 13, and 15 series are presented in Table 1 tests (carried out by J. Ortiz in our laboraand Fig. 17. All cells measured were lo- tory) indicate that cellular morphogenesis
DUMONT
AND
YAMADA
Dedifferentiation
of Iris
Epitheliul
Cells
393
is suppressed by cytochalasin B, a drug melanosomes after phagocytosis. (3) The which is believed to modify the assembly observations that the projections of iris epithelial cells which contain melanosomes or stability of microfilaments in cellular extensions (Wessells et al., 1971). These are wrapped by pseudopodia of the macrophages and that the composition of some observations suggest that microtubules and microfilaments play a role in affecting phagosomes corresponds to that of iris cell shape and motility. epithelial cell projections support the view In confirmation of earlier publications that the transfer occurs in the form of a (Wolff, 1895; Fischel, 1900; Eguchi, 1963; package of melanosomes and cortical cyYamada and Dumont, 1972), the present toplasm covered by cell membrane. This data show that almost all melanosomes mode of transfer of melanosomes from iris originally present in the iris epithelial cells epithelial cells to macrophages is compaare transferred to phagocytes now identirable to that described for the transfer of fied as macrophages of monocyte origin. melanosomes from melanocytes to epitheAccording to Eguchi (1963), melanosomes lial cells in hair (Mottaz and Zelickson, are either transferred directly from the 1967) and feather (Ruprecht, 1971). Howiris epithelial cells to phagocytes or dis- ever, the subsequent fate of transferred charged in a large mass without the coop- melanosomes is entirely different in our eration of phagocytes. The present data system. emphasize the following points concerning Our observations also suggest that melthe transfer mechanism, none of which has anosomes may be lost by two additional been previously discussed in the literature: modes, both of which appear to involve (1) A significant part of melanosome trans- separation of melanosomes from the cytofer occurs through projections or pseudo- plasm. The first mode involves more-orpodial extensions of iris epithelial cells, less direct transfer of melanosomes from which are formed as the result of cell sur- iris epithelial cells to macrophages and face activation induced by lentectomy. occurs when epithelial cell projections (2) The majority of phagosomes present in come into contact with, but are not enmacrophages in the vicinity of iris epithegulfed by, macrophages. In such cases the lial cells contain fragments of cell memcell membrane and subjacent cytoplasm of brane and cortical cytoplasm as well as the epithelial cell appear to disintegrate, melanosomes. The presence of dense con- perhaps in response to enzymatic action of cretions in such phagosomes, which mark the macrophage, and melanosomes are lost specifically the cell membrane of the iris and immediately incorporated by the macepithelial cells, supports the possibility rophages. This mode of transfer probably that melanosomes are transferred together results in the formation of phagosomes with the cell membranes and cortical cyto- composed only of melanosomes. The secplasm of the projections. The absence of ond mode apparently involves the loss of dense concretions in phagosomes commelanosomes without the mediation of posed purely of melanosomes suggests that macrophages and is less frequent. In this those concretions are not produced by instance melanosomes are expelled from FIG. 5. A portion of the dorsal pupillary margin of a day-8 regenerate. Note the long slender extensions of iris epithelial cells (IE), which contain dense populations of melanosomes. The distal tips of some of these extensions become attenuated and appear to be attached only by a thin strand of cytoplasm (arrows). An invading macrophage with villuslike surface extensions lies in a space surrounded by iris epithelial cells. x 7200. FIG. 6. Portions of the plasmalemmae of three iris epithelial cells from a day 8 regenerate. Note that some of the concretions are located on the cytoplasmic side of the membrane (*) while others appear to be attached to its external surface (0). Microfilaments (MF); microtubules (MT). x 42,300.
FIGS. 7-9. This series singly or in small groups FIG. 7. A melanosome plasm. x 38,700. FIGS. 8 and 9. Groups cellular spaces. Fig. 8, x
of electron micrographs from day-8 regenerates illustrates the loss of melanosomes from iris epithelium. apparently being extruded. Note the microtubules and microfilaments in the cytoof melanosomes, surrounded 14,200; Fig. 9, x 31,200.
by membranous 394
remnants
and concretions
in the inter-
FIG. 12. A phagosome of a macrophage which dense concretions, melanosomes, and membranous
has engulfed many melanosomes. remnants. x 31,300.
FIG. 10. A macrophage whose processes encompass a portion ing melanosomes and fine concretions. x 14,200. FIG. 11. A macrophage phagosome containing melanosomes, membranes. x 39,600. 395
of iris epithelial several
The phagosome
cell cytoplasm
dense concretions
contains
(I@ contain.
and degenerating
----.--...-”
.--_“““--
FIG. 13. An iris epithelial melanosomes. The nucleus x 14,800.
l_-_-_..
--.-_---.“““~-II
cell 15 days after is now surrounded
--
-.-“_l-__”
.---
lentectomy. The long, pseudopod-like by only a thin rim of cytoplasm,
1
“--
-“”
--_I___(_____--
extension contains some which is rich in ribosomes.
FIG. 14. High magnification of an iris epithelial cell. The plane of sectioning is slightly below the plasmalemma, revealing an extensive array of microfilaments. x 47,600. FIG. 15. A portion of the cytoplasm of the pseudopod-like extension of an iris epithelial cell at the end of depigmentation 15 days after lentectomy. Note the polyribosomes, microtubules (arrows), and microfilaments (MO beneath the plasmalemma. x 19,800. 396
DEVELOPMENTAL
BIOLOGY
VOLUME
29, 1972
FIG. 16. Diagrams of the general structure of marginal dorsal iris epithelial cells before, during, and after depigmentation. (a) normal, (b) 4 days, (c, d) 8 days, (e) 13 days, (fj 15 days after lentectomy. The basement membrane is indicated by a curved line and marks the basal end of the cell.
-C‘
(TOPLASM
- NI JCLEUS
I' 1 0
Fro 17. Changes in total 90% confidence limits.
5
10
cell, cytoplasmic,
15
0 DAYS
5 AFTER
and nuclear
IO 15 0 LENTECTOMY
volumes
following
5
10
lentectomy.
15
The bars represent
DUMONT AND YAMADA
Dedifferentiution
the epithelial cells and become free within the intercellular spaces both inside and outside the epithelium. There is no reason to doubt that such free melanosomes can also be incorporated when encountered by macrophages. Although the discharge of cytoplasmic components, including melanosomes, occurs in uiuo in close association with macrophages, the iris epithelial cells may have an inherent tendency for such discharge. This is demonstrated by the observation that iris epithelial cells shed cytoplasmic fragments containing melanosomes during depigmentation in vitro in the absence of macrophages (J. R. Ortiz, unpublished). Almost all melanosomes observed in the present material are judged to be mature according to morphological criteria. However, some premelanosomes are present, although rarely, in day-8 iris epithelial cells in the process of depigmentation. On the other hand, after discharge of the mature melanosomes from cells of the mid-dorsal margin of the iris, premelanosomes do not appear. Thus a feedback-control mechanism of melanosome synthesis can be excluded in those cells. On the other hand, other iris epithelial cells only partially depigment and later recover the original level of pigmentation. Recent cell electrophoretic studies of Zalik and Scott (1972) demonstrate a series of changes in surface charge density of iris cells after lentectomy and suggest a loss of negatively charged groups from the cell surface as cells go through dedifferentiation. The morphological observations reported in this paper may be directly related to those changes. The measurement of cell volume reported here indicates that mobilization of the cell surface is preceded by an intensive increase in the cytoplasmic volume, and that depigmentation of the iris epithelial cell is accompanied by a decrease in the cytoplasmic volume. By 15 days after lentectomy, the volume of cytoplasm is reduced to 34% of its day-8 value (Table 1).
of Iris
Epithelial
Cells
399
One possible interpretation of the volume decrease is that it directly reflects the loss of cytoplasm implied in our present observations. In contrast to the cytoplasmic volume, in which a decrease occurs during the depigmentation phase, the nuclear volume does not show a significant change during the same phase. However, an increase in the nuclear volume occurs in parallel with the increase in the cytoplasmic volume in the earlier phase. The change of nuclear volume is correlated with ultrastructural changes of chromatin; the chromatin is very condensed in the normal condition, becomes dispersed after lentectomy, and remains so during depigmentation. Further, the nuclear volume changes can also be correlated with the synthetic activities of the nucleus and nucleolus; during the early phase, ribosomal RNA synthesis is activated (Reese et al., 1969; Dumont et al., 1970) and DNA replication is induced (Eisenberg and Yamada, 1966; Reyer, 1971; Yamada and Roesel, 1969). Those data agree well with information obtained by transplantation of tissue cell nuclei into amphibian egg cytoplasm (Gurdon, 1968) and by hybridizing metabolically repressed cells with metabolically active cells (Harris, 1967; Sidebottom, 1969). In both casesexperimentally induced enhancement of incorporation of labeled precursors into nucleic acids is correlated with enlargement of nuclear volume. Reexamination of earlier autoradiographic data on incorporation of 13H] cytidine into nuclear RNA of cells engaged in Wolffian lens regeneration in uiuo (Yamada, 1966) shows that a strong enhancement of the incorporation is associated with depigmentation. The ratio between the grain count per unit nuclear area of the dorsal iris epithelial cells in regeneration and that of the normal (control) increases from 3.3 to 7.1 during depigmentation and reaches 9.0-10.6 after completion of depigmentation. This ratio is increased if we consider the enlargement of the nucleus.
400
DEVELOPMENTAL
BIOLOGY
On the other hand, those iris epithelial cells which only partially depigment and retain their iris specificity show values ranging from 2.1 to 4.3 during the corresponding period. The increase in incorporation by the completely depigmented cells correlates with the increase in the number of ribosomes per unit area of cytoplasmic matrix (Karasaki, 1964; Eguchi, 1964). Hence the increase in incorporation may reflect an increase in RNA synthesis instead of a decrease in the size of the precursor pool. Other autoradiographic studies on the incorporation of leucine into the same cells (Yamada and Takata, 1963) also indicated an enhancement of incorporation after complete depigmentation. However, in this case there is a lag period. These data, combined with the present data, suggest the possibility that the loss of cytoplasm and cytoplasmic components, contributes to an enhancement of transcription in the iris epithelial cell, which is later reflected in an enhancement of protein synthesis. It should be pointed out that control of nuclear activity by cytoplasmic loss, as proposed here, is exerted in this case on a cell which already has an enhanced level of activity over the normal cell and possesses an enlarged nucleus, as discussed above. The authors wish to express their thanks to Dr. David G. Gosslee, Statistics Department, Mathematics Division, Oak Ridge National Laboratory, for his statistical analysis of the data and to Drs. R. A. Wallace and J. J. Eppig, Jr., for their helpful comments and criticisms during the preparation of the manuscript. REFERENCES CLAWSON, C. C., and GOOD, R. A. (1971). Micropapillae. A surface specialization of human leukocytes. J. Cell Viol. 48, 207-211. DUMONT, J. N., YAMADA, T., and CONE, M. V. (1970). Alteration of nucleolar ultrastructure in iris epithelial cells during initiation of Wolffian lens regeneration. J. Exp. 2001. 174, 187-204. EGUCHI, G. (1963). Electron microscopic studies on lens regeneration. I. Mechanism of depigmentation of the iris. Embryologia 8, 47-62. EGUCHI, G. (1964). Electron microscopic studies on
VOLUME
29. 1972
lens regeneration. II. Formation and growth of lens vesicle and differentiation of lens fibers. Embryologia 8, 274-287. EISENBERG, S., and YAMADA, T. (1966). A study of DNA synthesis during the transformation of the iris into lens in the lentectomized newt. J. Erp. Zoo&. 162, 353-368. FISCHEL, A. (1900). Uber die Regeneration der Linse. Anut. Hefte 14, l-256. GURDON, J. B. (1968). Changes in somatic cell nuclei inserted into growing and maturing amphibian oocytes. J. Embryol. Exp. Morphol. 20, 401-414. HARRIS, H. (1967). The reactivation of the red cell nucleus. J. Cell Sci. 2,23-32. KARASAKI, S. (1964). An electron microscopic study of Wolffian lens regeneration in the adult newt. J. Ultmstruct. Res. 11, 246-273. MOTTAZ, J. H., and ZELICKSON, A. S. (1967). Melanin transfer: a possible phagocytic process. J. Inuest. Dermatol. 49, 605-610. REESE, D. H., PUCCIA, E., and YAMADA, T. (1969). Activation of ribosomal RNA synthesis in initiation of Wolffian lens regeneration. J. Exp. Zool. 170, 259-268. REYER, R. W. (1971). DNA synthesis and the incorporation of labeled iris cells into the lens during lens regeneration in adult newts. Deuelop. Biol. 24, 533-558. RUPRECHT, K. W. (1971). Pigmentierung der Dunenfelder von Gallus domesticus L. Lichtund elektronenmikroskopische Untersuchungen xur Melanosomeniibertragung. Z. Zellforsch. Mikrosk. Anat. 112, 396-413. SIDEBOTTOM, E. (1969). The function of the nucleolus in the expression of genetic information: a study with hybrid animal cells. In “Problems in Biology RNA in Development” (E. W. Hanly, ed.), pp. 3349. University of Utah Press, Salt Lake City. TAKATA, C., ALBRIGHT, J. F., and YAMADA, T. (1964). Lens antigens in a lens-regenerating system studied by the immunofluorescent technique. Deuelop. Eiol. 9,385-397. TAKATA, C., ALBRIGHT, J. F., and YAMADA, T. (1966). Gamma crystallins in Wolffian lens regeneration demonstrated by immunofluorescence. Deuelop. Viol. 14, 382-400. WESSELLS, N. K., SPOONER, B. S., ASH, J. F., BRADLEY, M. O., LUDUENA, M. A. TAYLOR, E. L., WRENN, J. T., and YAMADA, K. M. (1971). Microfilaments in cellular and developmental processes. Science 171, 135143. WOLFF, F. (1895). Entwickelungsphysiologische Studien. Arch. Entwickelungsmech. Omgnismen 1, 380-390. YAMADA, T. (1966). Control of tissue specificity: the pattern of cellular synthetic activities in tissue transformation. Amer. Zool. 6, 21-31. YAMADA, T. (1967). Cellular and subcellular events in
DUMONT
AND YAMADA
Dediffei
Wolffian lens regeneration. Curr. Top. Develop. Biol. 2, 247-283. YAMADA, T., and DUMONT, J. N. (1972). Macrophage activity in Wolffian lens regeneration. J. Morphol. 136, 367-384. YAMADA, T., and ROESEL, M. E. (1969). Activation of DNA replication in the iris epithelium by lens removal. J. Exp. Zool. 171, 425-432. YAMADA, T., and ROESEL, M. (1971). Control of mitotic activity in Wolffian lens regeneration. J. Exp. Zool. 177,119-128.
rentiation
of Iris
Epitheliul
Cells
401
YAMADA, T., and TAKATA, C. (1963). An autoradiographic study of protein synthesis in regenerative tissue transformation of iris into lens in the newt. Develop. Biol. 8, 358-369. ZALIK, S. E., and SCOTT, V. (1971). Development of the H3-thymidine-labeled iris in the optic chamber of lentectomized newts. Exp. Cell Res. 66,446-448. ZALIK, S. E., and Scorr, V. (1972). Cell surface changes during dedifferentiation in the metaplastic transformation of iris into lens. J. Cell Biol. 55, 1344146.
BIOLOGY
29,
385-401
(1972)
Dedifferentiation
of Iris Epithelial
JAMES N. DUMONT AND Biology
Division,
Oak Ridge
National
YAMADA
TUNEO
Laboratory,
Cells’
Oak
Ridge,
Tennessee
37830
Electron and phase-contrast microscopic observations demonstrated that depigmentation of iris epithelial cells in vivo after lentectomy is preceded by alterations of cell shape and increases in microfilaments and microtubules in the periphery of the cell. Extensive cell processes are formed, with tips branching into fine strands that contain melanosomes. The macrophages invading the iris epithelium incorporate pieces of such strands, which are composed of cell membrane, cytoplasmic matrix, and melanosomes. Individual melanosomes are also taken up by macrophages. Some of the strands of iris cell processes seem to degenerate within the intercellular space. Thus, depigmentation of iris epithelial cells is accompanied by loss of a substantial part of the cell surface and cytoplasmic matrix of iris epithelial cells. Measurements of the absolute volume of whole iris epithelial cells, their cytoplasm, and their nuclei were conducted at various stages of depigmentation. These measurements reveal an extensive increase in the volume of the cytoplasm preceding activation of the cell surface and a significant reduction in cytoplasmic volume during the phase of extensive depigmentation. In the case of the nuclear volume, the extensive increase which occurs in parallel with that of the cytoplasmic volume is not followed by a significant change during the depigmentation phase. INTRODUCTION
Stepwise tracing of labeled cells in the Wolffian lens-regenerating system in the adult newt indicates that the fully differentiated iris epithelial cells located in the mid-dorsal margin of the iris epithelium are converted into lens cells after passing through two or more induced cell divisions (Eisenberg and Yamada, 1966; Reyer, 1971; Zalik and Scott, 1971; Yamada and Roesel, unpublished). During this transformation, activated by lentectomy, the cells go through the following steps: (1) nucleolar activation (Reese et al., 1969; Dumont et al., 1970), (2) induction of DNA replication and mitosis (Eisenberg and Yamada, 1966; Yamada and Roesel, 1969, 1971; Reyer, 1971), (3) depigmentation (Eguchi, 1963; Karasaki, 1964; Yamada and Dumont, 1972), and (4) appearance of lens-specific antigens associated with lens morphogenesis (Takata et al., 1964, 1966; Yamada, 1966, 1967). This sequence of events suggests that in step 3, during which the whole population of melanosomes is removed from the cytoplasm, the ‘Research Commission Corporation.
sponsored by under contract
U. S. Atomic with Union
Energy Carbide 385
Copyright All rights
0 1972 hy Academic Press. Inc. of reproduction in any form reserved.
iris epithelial cell loses not only its morphological identity but also its developmental specificity, and that it becomes reprogrammed in a new differentiation pathway in step 4. In other words, reversion of morphological differentiation, which is coupled with recovery of a developmentally less committed status, seems to occur in step 3. Such a reversion, not simple loss of overt differentiation, will be referred to as dedifferentiation. A basically similar terminology was used by Wolff (1895) when he discussed for the first time the depigmentation of iris epithelial cells. On the basis of the above facts and ideas, one would expect information concerning dedifferentiation of iris epithelial cells to afford an insight into the cellular control mechanism of differentiation. We owe to Eguchi (1963) the first electron microscopic studies of the depigmentation process. The work so far published from our laboratory on the same subject is in good conformity with Eguchi’s data (Karasaki, 1964; Yamada and Dumont, 1972), except that we identify the major cell type involved in phagocytic uptake of melanosomes as the macrophage, while Eguchi called it a special amoeboid cell. Further studies of the depigmentation process car-
386
DEVELOPMENTAL.
BIOLOGY
ried out in our laboratory have revealed new aspects that appear important for understanding dedifferentiat,ion. This paper describes morphological studies of those aspects. MATERIALS
AND
METHODS
Electron microscopy. Dorsal pupillary margins from normal irises and from irises 4, 6, 8, 13, and 15 days after lentectomy were surgically removed, fixed for 2 hr in cold 3% glutaraldehyde buffered with 0.1 M phosphate buffer at pH 7.4 and washed in several changes of the same buffer. They were then bisected dorsoventrally, placed in cold 1% osmium tetroxide in 0.1 M phosphate buffer, dehydrated, infiltrated, and embedded in Epon in flat embedding molds so that sections could be obtained through the dorsolateral and medioventral axes as previously described (Dumont et al., 1970). Thin sections cut with a PorterBlum ultramicrotome equipped with a diamond knife were placed on Formvarcoated, carbon-stabilized grids, stained with uranyl acetate and lead citrate, and examined in a Hitachi 11-E electron microscope operated at 75 V. Cell volume determinations. Total cell volume, as well as the volumes of the cytoplasmic and nuclear compartments, was determined from 0.25- and 0.5-p serial Epon sections. Only clearly delimited cells entirely contained within the serial sections were selected. These cells were photographed with a light microscope, photographically enlarged, and measured with an Ott compensating polar planimeter to determine total cell and nuclear areas (cytoplasmic area equals total cell area minus nuclear area). The actual volume of each cellular compartment was then calculated from the measured areas, photographic enlargement factor, and section thickness. Because of the complex surface configuration of cells in regenerating tissue, the number of cells which could be completely followed in serial sections was small.
VOLUME
29. 1972
Owing to the small number of samples measured (Table l), the confidence limits were calculated by pooling estimates of variation about each mean. Since the standard deviation decreased linearly on each day following lentectomy, a regression relating standard deviation to days after lentectomy was used. The resulting confidence limits were thus calculated for each mean based on an estimate of the standard deviation using all of the values for days 8, 13, and 15. RESULTS
Electron
Microscopic
Study
Normal iris epithelium. The iris epithelium is continuous with the ciliary epithelium and is composed of inner and outer laminae which merge at the pupillary margin. More proximally, the inner lamina of the ciliary epithelium borders the neural retina, while the outer lamina borders the pigmented retina. The iris epithelium is composed of heavily pigmented cuboidal cells (Fig. 1). The external lamina of the epithelium is partially covered by stroma which supports iridophores, connective tissue cells, and small blood vessels. Care was taken to ensure that all cells except those at the dorsal pupillary margin of the iris epithelium were excluded from our samples. Thus, as in a previous study (Dumont et al., 1970), the present report deals only with the 20-25 cells extending upward from the pupillary margin of the dorsal iris epithelium when viewed in a section sagittal to the eye ball. The basal epithelial cell surface, i.e., the cell surface toward the outside of each lamina rests on a basement membrane. The nuclei are spherical or slightly elongate. Mitochondria, endoplasmic reticulum, and ribosomes are present in scant amounts (Fig. 2). Desmosomal junctions are present on the lateral surfaces of adjacent cells. Subjacent to the apical plasmalemma are 45- to 50-A microfilaments. Iris epithelium 4 days after lentectomy.
DUMONT
AND
Dedifferentiution
YAMADA
TABLE CELL VOLUME
Source of cells Normal
CHANGES
Volume (~‘1 Total cell
3
4
Mean Day-13 regenerate
5
Mean Day-15 regenerate
2 Mean
Four days after lens removal, the epithelial cells of the dorsal margin of the iris enlarge and elongate in the apicobasal axis; thus, the melanosomes are not as densely packed as in normal cells. As in normal cells, there is a paucity of cytoplasmic organelles and membrane systems. Desmosomal junctions with associated microfilaments and microtubules remain between adjacent cells (Fig. 3). In some areas intercellular spaces become enlarged. The basement membrane remains on the basal cell surface, but where adjacent cells have separated it may indent into the intercellular space. Iris epithelium 6-8 days after lentectomy. At 6-8 days after lentectomy, the epithelial cells have elongated further, and pseudopod-like extensions, which contain concentrations of melanosomes, have formed at the basal end of the cell (Figs. 4 and 5). Mitochondria and some microtubules oriented parallel to the long axis are
387
Cells
CELLS DURING LENS REGENERATION
Mean Day-8 regenerate
Epitheliul
1
IN IRIS EPITHELIAL
No. cells measured
of Iris
Cytoplasm
Nucleus
986 948 898
635 578 545
351 370 353
944
586
358
3688 3485 2100 3481
1314 2401 1342 2245
2374 1083 759 1236
3190
1826
1363
2966 2421 2327 1829 2063
1465 1141 1298 755 944
1501 1280 1028 1075 1069
2321
1131
1191
1620 1954
494
734
1126 1220
1787
614
1173
present in the pseudopods. Many of these extensions are attenuated; they are very thin and contain only a few aligned melanosomes. The cytoplasm of these attenuated processes often appears more dense than that of other areas of the cell (Fig. 5). These pseudopodial extensions of the iris epithelial cells are easily distinguished from the extensions and infoldings of the ciliary epithelium, which are normal characteristics of the cell surface. The intercellular spaces become prominent, although many cell contacts are maintained. Other cytoplasmic organelles also become altered at 6 days. For example, some cisternae of rough endoplasmic reticulum and Golgi complexes are now present along with increasing numbers of polyribosomes and mitochondria. Microfilaments and microtubules also appear more abundant in the periphery of the cells than in earlier stages of lens regeneration. The microfilaments lie in a band subjacent and parallel
388
390
DEVELOPMENTAL
BIOLOGY
to the plasmalemma and the long axis of the pseudopodial extensions. The band of microfilaments appears to be slightly thicker at the tips of the pseudopodial extensions than along the lateral margins. Microtubules are more centrally located in the cytoplasm of the pseudopodial processes. Six to eight days after lentectomy, there appears on the surface of the iris epithelial cells, especially on the pseudopodia and to some extent within the cytoplasm, highly dense, usually spherical condensations or concretions ranging from 200 to 600 A (Figs. 4 and 6). They are present in specimens fixed either in glutaraldehyde or osmium or in both, in unstained as well as single- or double-stained (uranyl acetate and/or lead citrate) sections, and are not removed by en bloc treatment of the tissue with EDTA or sodium citrate. In these respects they appear unlike the micropapillae described by Clawson and Good (1971) on the surface of cultured cells. The concretions are normally present on the surface of iris epithelial cells, but not on macrophages or mast cells. Occasionally some appear in the iris cell cytoplasm, where they are associated with melanosomes. One interpretation is that they represent the breakdown of melanosomes; however, we have no evidence for this other than their proximity to melanosomes and a density similar to that of melanin. These concretions are also associated with macrophage phagosomes that contain fragments of iris epithelial cells and melanosomes.
VOLUME
29. 1972
Depigmentation becomes evident during this 6- to a-day period following lentectomy. In the early phases of depigmentation, individual or small groups of melanosomes are lost and are occasionally encountered lying within the intercellular spaces (Figs. 7 and 8). In some cases,as in Fig. 7, these extracellular melanosomes appear to be surrounded by membranous material which has some concretions associated with it. In other cases, as in Fig. 9, melanosomes are enclosed, along with cytoplasm, by a concretion-studded membrane. Because of the size of the cells and their highly pleomorphic shapes it is impossible to determine, without inordinate numbers of serial sections, whether such configurations are separated portions of iris epithelial cell cytoplasm containing melanosomes or are still connected to the epithelial cell by a thin cytoplasmic strand such as is shown in Fig. 6. Macrophage processes, however, frequently circumscribe and appear to engulf such structures (Fig. 10). In either case, materials engulfed by macrophages become isolated in phagosomes that contain melanosomes, membranes, and concretions (Figs. 11 and 12). Iris epithelium 13-15 days after lentectomy. By 13-15 days following lentectomy, many of the formerly pigmented cells have lost most of their melanosomes. The nuclei of these cells have become enlarged, and are surrounded only by a thin rim of cytoplasm (Fig. 13). The cytoplasm of the pseudopodia is now even more richly pop-
FIG. 1. A survey micrograph of the pupillary margin showing The stroma contains iridophores. x 2300.
the two laminae
of the dorsal
iris epithelium.
Fro. 2. A portion of an iris epithelial cell cytoplasm from a normal iris. The cytoplasm contains large quantities of melanosomes but indicates a paucity of other organelles. Subjacent to the plasmalemma, at the apical end of the cell, is a network of fine filaments (F). A continuous basement membrane (G) covers the bases of all epithelial cells. (N); the nucleus. x 27,300. FIG. 3. High magnification of a desmosomal junction between adjacent epithelial cells. x 23,000. FIG. 4. Cells from a day-8 regenerate. The formerly cuboidal cells elongate, and many of the melanosomes are lost. The cytoplasm becomes richer in mitochondria and ribosomes. The plasmalemma acquires dense concretions (arrows). x 10,000.
DUMONT AND YAMADA
Dedifferentiution
ulated with rough endoplasmic reticulum, polyribosomes, mitochondria, microtubules, and microfilaments (Figs. 13-15). The dense concretions of the plasmalemma, so prominent at 6 and 8 days, have essentially disappeared (Fig. 11). Light microscopic study. Phase-contrast observations of serial sections of Eponembedded normal iris and irises 4, 6, 8, 13, and 15 days after lentectomy confirm the changes of shape of epithelial cells and the intercellular spaces between them detected by electron microscopic studies. Diagrams based on light microscopic observations illustrating changes in the shape of dorsal iris epithelial cells at various stages of depigmentation are reproduced in Fig. 16.
of Iris
Epitheliul
Cells
391
cated in the mid-dorsal marginal iris epithelium and were expected to become lens cells of the regenerate. There is an increase in total cell volume from day 0 to day 8 and a decrease in volume from day 8 to day 15. The nuclear volume increases from day 0 to day 8, then does not change. All of these volume changes are statistically significant. DISCUSSION
The light and electron microscopic observations presented indicate that depigmentation of the iris epithelial cells is accompanied by extensive alterations in the topography of the cell surface. Elongation of the cell along the apicobasal axis is followed by formation of cell processes which branch into thin cytoplasmic strands. The melanosomes, uniformly distributed Changes in the Volume of Iris Epithelial throughout the cytoplasm in the normal Cells before and during Depigmentation condition, are displaced into these cell Microscopic observations implied the processes. These cellular changes are assoloss of cytoplasmic materials, so efforts ciated not only with an increase in cell surwere made to determine whether this loss face but also with an expansion of the inof material is reflected by a reduction in tercellular space and partial obliteration of the volume of the cytoplasm after depigintercellular junctions including desmomentation. The usual methods of cytologisomes. These observations imply that the cal measurement turned out to be too in- quiescent condition of the cell surface, i.e., accurate for this purpose because of the the plasmalemma and the cytoplasm imvery irregular shape of the cells. Measuremediately subjacent to it, characteristic of ments on dissociated cells were precluded normal iris epithelial cells is replaced by a by the extensive changes in volume caused more mobile condition after lentectomy. by dissociation. Finally, the very time-conThat the cells are highly mobile at the time suming method described in the Materials of depigmentation has been confirmed by and Methods sections was adopted. The observation of iris epithelial cells depigmethod depends on, among other factors, menting in vitro (J. Ortiz, unpublished). the clear delineation of the cell boundary in The cellular projections formed are not the enlarged photographs. The number of static structures but dynamic formations cells satisfying the condition was very of the cell surface in constant movement. small, especially in day-15 samples. Thus, Involvement of microfilaments in the the limited number of measurements made cellular changes is suggested by the apnecessary a careful statistical check, as in- pearance of an oriented network of microdicated in the Methods section. filaments 45-50 A in diameter in the cortiThe data obtained for the total cell, cy- cal layer of the cell surface. A population toplasmic, and nuclear volumes of iris epi- of microtubules is also present in the cell thelial cells of day 0 (control), and day 8, projections. Unpublished results of in vitro 13, and 15 series are presented in Table 1 tests (carried out by J. Ortiz in our laboraand Fig. 17. All cells measured were lo- tory) indicate that cellular morphogenesis
DUMONT
AND
YAMADA
Dedifferentiation
of Iris
Epitheliul
Cells
393
is suppressed by cytochalasin B, a drug melanosomes after phagocytosis. (3) The which is believed to modify the assembly observations that the projections of iris epithelial cells which contain melanosomes or stability of microfilaments in cellular extensions (Wessells et al., 1971). These are wrapped by pseudopodia of the macrophages and that the composition of some observations suggest that microtubules and microfilaments play a role in affecting phagosomes corresponds to that of iris cell shape and motility. epithelial cell projections support the view In confirmation of earlier publications that the transfer occurs in the form of a (Wolff, 1895; Fischel, 1900; Eguchi, 1963; package of melanosomes and cortical cyYamada and Dumont, 1972), the present toplasm covered by cell membrane. This data show that almost all melanosomes mode of transfer of melanosomes from iris originally present in the iris epithelial cells epithelial cells to macrophages is compaare transferred to phagocytes now identirable to that described for the transfer of fied as macrophages of monocyte origin. melanosomes from melanocytes to epitheAccording to Eguchi (1963), melanosomes lial cells in hair (Mottaz and Zelickson, are either transferred directly from the 1967) and feather (Ruprecht, 1971). Howiris epithelial cells to phagocytes or dis- ever, the subsequent fate of transferred charged in a large mass without the coop- melanosomes is entirely different in our eration of phagocytes. The present data system. emphasize the following points concerning Our observations also suggest that melthe transfer mechanism, none of which has anosomes may be lost by two additional been previously discussed in the literature: modes, both of which appear to involve (1) A significant part of melanosome trans- separation of melanosomes from the cytofer occurs through projections or pseudo- plasm. The first mode involves more-orpodial extensions of iris epithelial cells, less direct transfer of melanosomes from which are formed as the result of cell sur- iris epithelial cells to macrophages and face activation induced by lentectomy. occurs when epithelial cell projections (2) The majority of phagosomes present in come into contact with, but are not enmacrophages in the vicinity of iris epithegulfed by, macrophages. In such cases the lial cells contain fragments of cell memcell membrane and subjacent cytoplasm of brane and cortical cytoplasm as well as the epithelial cell appear to disintegrate, melanosomes. The presence of dense con- perhaps in response to enzymatic action of cretions in such phagosomes, which mark the macrophage, and melanosomes are lost specifically the cell membrane of the iris and immediately incorporated by the macepithelial cells, supports the possibility rophages. This mode of transfer probably that melanosomes are transferred together results in the formation of phagosomes with the cell membranes and cortical cyto- composed only of melanosomes. The secplasm of the projections. The absence of ond mode apparently involves the loss of dense concretions in phagosomes commelanosomes without the mediation of posed purely of melanosomes suggests that macrophages and is less frequent. In this those concretions are not produced by instance melanosomes are expelled from FIG. 5. A portion of the dorsal pupillary margin of a day-8 regenerate. Note the long slender extensions of iris epithelial cells (IE), which contain dense populations of melanosomes. The distal tips of some of these extensions become attenuated and appear to be attached only by a thin strand of cytoplasm (arrows). An invading macrophage with villuslike surface extensions lies in a space surrounded by iris epithelial cells. x 7200. FIG. 6. Portions of the plasmalemmae of three iris epithelial cells from a day 8 regenerate. Note that some of the concretions are located on the cytoplasmic side of the membrane (*) while others appear to be attached to its external surface (0). Microfilaments (MF); microtubules (MT). x 42,300.
FIGS. 7-9. This series singly or in small groups FIG. 7. A melanosome plasm. x 38,700. FIGS. 8 and 9. Groups cellular spaces. Fig. 8, x
of electron micrographs from day-8 regenerates illustrates the loss of melanosomes from iris epithelium. apparently being extruded. Note the microtubules and microfilaments in the cytoof melanosomes, surrounded 14,200; Fig. 9, x 31,200.
by membranous 394
remnants
and concretions
in the inter-
FIG. 12. A phagosome of a macrophage which dense concretions, melanosomes, and membranous
has engulfed many melanosomes. remnants. x 31,300.
FIG. 10. A macrophage whose processes encompass a portion ing melanosomes and fine concretions. x 14,200. FIG. 11. A macrophage phagosome containing melanosomes, membranes. x 39,600. 395
of iris epithelial several
The phagosome
cell cytoplasm
dense concretions
contains
(I@ contain.
and degenerating
----.--...-”
.--_“““--
FIG. 13. An iris epithelial melanosomes. The nucleus x 14,800.
l_-_-_..
--.-_---.“““~-II
cell 15 days after is now surrounded
--
-.-“_l-__”
.---
lentectomy. The long, pseudopod-like by only a thin rim of cytoplasm,
1
“--
-“”
--_I___(_____--
extension contains some which is rich in ribosomes.
FIG. 14. High magnification of an iris epithelial cell. The plane of sectioning is slightly below the plasmalemma, revealing an extensive array of microfilaments. x 47,600. FIG. 15. A portion of the cytoplasm of the pseudopod-like extension of an iris epithelial cell at the end of depigmentation 15 days after lentectomy. Note the polyribosomes, microtubules (arrows), and microfilaments (MO beneath the plasmalemma. x 19,800. 396
DEVELOPMENTAL
BIOLOGY
VOLUME
29, 1972
FIG. 16. Diagrams of the general structure of marginal dorsal iris epithelial cells before, during, and after depigmentation. (a) normal, (b) 4 days, (c, d) 8 days, (e) 13 days, (fj 15 days after lentectomy. The basement membrane is indicated by a curved line and marks the basal end of the cell.
-C‘
(TOPLASM
- NI JCLEUS
I' 1 0
Fro 17. Changes in total 90% confidence limits.
5
10
cell, cytoplasmic,
15
0 DAYS
5 AFTER
and nuclear
IO 15 0 LENTECTOMY
volumes
following
5
10
lentectomy.
15
The bars represent
DUMONT AND YAMADA
Dedifferentiution
the epithelial cells and become free within the intercellular spaces both inside and outside the epithelium. There is no reason to doubt that such free melanosomes can also be incorporated when encountered by macrophages. Although the discharge of cytoplasmic components, including melanosomes, occurs in uiuo in close association with macrophages, the iris epithelial cells may have an inherent tendency for such discharge. This is demonstrated by the observation that iris epithelial cells shed cytoplasmic fragments containing melanosomes during depigmentation in vitro in the absence of macrophages (J. R. Ortiz, unpublished). Almost all melanosomes observed in the present material are judged to be mature according to morphological criteria. However, some premelanosomes are present, although rarely, in day-8 iris epithelial cells in the process of depigmentation. On the other hand, after discharge of the mature melanosomes from cells of the mid-dorsal margin of the iris, premelanosomes do not appear. Thus a feedback-control mechanism of melanosome synthesis can be excluded in those cells. On the other hand, other iris epithelial cells only partially depigment and later recover the original level of pigmentation. Recent cell electrophoretic studies of Zalik and Scott (1972) demonstrate a series of changes in surface charge density of iris cells after lentectomy and suggest a loss of negatively charged groups from the cell surface as cells go through dedifferentiation. The morphological observations reported in this paper may be directly related to those changes. The measurement of cell volume reported here indicates that mobilization of the cell surface is preceded by an intensive increase in the cytoplasmic volume, and that depigmentation of the iris epithelial cell is accompanied by a decrease in the cytoplasmic volume. By 15 days after lentectomy, the volume of cytoplasm is reduced to 34% of its day-8 value (Table 1).
of Iris
Epithelial
Cells
399
One possible interpretation of the volume decrease is that it directly reflects the loss of cytoplasm implied in our present observations. In contrast to the cytoplasmic volume, in which a decrease occurs during the depigmentation phase, the nuclear volume does not show a significant change during the same phase. However, an increase in the nuclear volume occurs in parallel with the increase in the cytoplasmic volume in the earlier phase. The change of nuclear volume is correlated with ultrastructural changes of chromatin; the chromatin is very condensed in the normal condition, becomes dispersed after lentectomy, and remains so during depigmentation. Further, the nuclear volume changes can also be correlated with the synthetic activities of the nucleus and nucleolus; during the early phase, ribosomal RNA synthesis is activated (Reese et al., 1969; Dumont et al., 1970) and DNA replication is induced (Eisenberg and Yamada, 1966; Reyer, 1971; Yamada and Roesel, 1969). Those data agree well with information obtained by transplantation of tissue cell nuclei into amphibian egg cytoplasm (Gurdon, 1968) and by hybridizing metabolically repressed cells with metabolically active cells (Harris, 1967; Sidebottom, 1969). In both casesexperimentally induced enhancement of incorporation of labeled precursors into nucleic acids is correlated with enlargement of nuclear volume. Reexamination of earlier autoradiographic data on incorporation of 13H] cytidine into nuclear RNA of cells engaged in Wolffian lens regeneration in uiuo (Yamada, 1966) shows that a strong enhancement of the incorporation is associated with depigmentation. The ratio between the grain count per unit nuclear area of the dorsal iris epithelial cells in regeneration and that of the normal (control) increases from 3.3 to 7.1 during depigmentation and reaches 9.0-10.6 after completion of depigmentation. This ratio is increased if we consider the enlargement of the nucleus.
400
DEVELOPMENTAL
BIOLOGY
On the other hand, those iris epithelial cells which only partially depigment and retain their iris specificity show values ranging from 2.1 to 4.3 during the corresponding period. The increase in incorporation by the completely depigmented cells correlates with the increase in the number of ribosomes per unit area of cytoplasmic matrix (Karasaki, 1964; Eguchi, 1964). Hence the increase in incorporation may reflect an increase in RNA synthesis instead of a decrease in the size of the precursor pool. Other autoradiographic studies on the incorporation of leucine into the same cells (Yamada and Takata, 1963) also indicated an enhancement of incorporation after complete depigmentation. However, in this case there is a lag period. These data, combined with the present data, suggest the possibility that the loss of cytoplasm and cytoplasmic components, contributes to an enhancement of transcription in the iris epithelial cell, which is later reflected in an enhancement of protein synthesis. It should be pointed out that control of nuclear activity by cytoplasmic loss, as proposed here, is exerted in this case on a cell which already has an enhanced level of activity over the normal cell and possesses an enlarged nucleus, as discussed above. The authors wish to express their thanks to Dr. David G. Gosslee, Statistics Department, Mathematics Division, Oak Ridge National Laboratory, for his statistical analysis of the data and to Drs. R. A. Wallace and J. J. Eppig, Jr., for their helpful comments and criticisms during the preparation of the manuscript. REFERENCES CLAWSON, C. C., and GOOD, R. A. (1971). Micropapillae. A surface specialization of human leukocytes. J. Cell Viol. 48, 207-211. DUMONT, J. N., YAMADA, T., and CONE, M. V. (1970). Alteration of nucleolar ultrastructure in iris epithelial cells during initiation of Wolffian lens regeneration. J. Exp. 2001. 174, 187-204. EGUCHI, G. (1963). Electron microscopic studies on lens regeneration. I. Mechanism of depigmentation of the iris. Embryologia 8, 47-62. EGUCHI, G. (1964). Electron microscopic studies on
VOLUME
29. 1972
lens regeneration. II. Formation and growth of lens vesicle and differentiation of lens fibers. Embryologia 8, 274-287. EISENBERG, S., and YAMADA, T. (1966). A study of DNA synthesis during the transformation of the iris into lens in the lentectomized newt. J. Erp. Zoo&. 162, 353-368. FISCHEL, A. (1900). Uber die Regeneration der Linse. Anut. Hefte 14, l-256. GURDON, J. B. (1968). Changes in somatic cell nuclei inserted into growing and maturing amphibian oocytes. J. Embryol. Exp. Morphol. 20, 401-414. HARRIS, H. (1967). The reactivation of the red cell nucleus. J. Cell Sci. 2,23-32. KARASAKI, S. (1964). An electron microscopic study of Wolffian lens regeneration in the adult newt. J. Ultmstruct. Res. 11, 246-273. MOTTAZ, J. H., and ZELICKSON, A. S. (1967). Melanin transfer: a possible phagocytic process. J. Inuest. Dermatol. 49, 605-610. REESE, D. H., PUCCIA, E., and YAMADA, T. (1969). Activation of ribosomal RNA synthesis in initiation of Wolffian lens regeneration. J. Exp. Zool. 170, 259-268. REYER, R. W. (1971). DNA synthesis and the incorporation of labeled iris cells into the lens during lens regeneration in adult newts. Deuelop. Biol. 24, 533-558. RUPRECHT, K. W. (1971). Pigmentierung der Dunenfelder von Gallus domesticus L. Lichtund elektronenmikroskopische Untersuchungen xur Melanosomeniibertragung. Z. Zellforsch. Mikrosk. Anat. 112, 396-413. SIDEBOTTOM, E. (1969). The function of the nucleolus in the expression of genetic information: a study with hybrid animal cells. In “Problems in Biology RNA in Development” (E. W. Hanly, ed.), pp. 3349. University of Utah Press, Salt Lake City. TAKATA, C., ALBRIGHT, J. F., and YAMADA, T. (1964). Lens antigens in a lens-regenerating system studied by the immunofluorescent technique. Deuelop. Eiol. 9,385-397. TAKATA, C., ALBRIGHT, J. F., and YAMADA, T. (1966). Gamma crystallins in Wolffian lens regeneration demonstrated by immunofluorescence. Deuelop. Viol. 14, 382-400. WESSELLS, N. K., SPOONER, B. S., ASH, J. F., BRADLEY, M. O., LUDUENA, M. A. TAYLOR, E. L., WRENN, J. T., and YAMADA, K. M. (1971). Microfilaments in cellular and developmental processes. Science 171, 135143. WOLFF, F. (1895). Entwickelungsphysiologische Studien. Arch. Entwickelungsmech. Omgnismen 1, 380-390. YAMADA, T. (1966). Control of tissue specificity: the pattern of cellular synthetic activities in tissue transformation. Amer. Zool. 6, 21-31. YAMADA, T. (1967). Cellular and subcellular events in
DUMONT
AND YAMADA
Dediffei
Wolffian lens regeneration. Curr. Top. Develop. Biol. 2, 247-283. YAMADA, T., and DUMONT, J. N. (1972). Macrophage activity in Wolffian lens regeneration. J. Morphol. 136, 367-384. YAMADA, T., and ROESEL, M. E. (1969). Activation of DNA replication in the iris epithelium by lens removal. J. Exp. Zool. 171, 425-432. YAMADA, T., and ROESEL, M. (1971). Control of mitotic activity in Wolffian lens regeneration. J. Exp. Zool. 177,119-128.
rentiation
of Iris
Epitheliul
Cells
401
YAMADA, T., and TAKATA, C. (1963). An autoradiographic study of protein synthesis in regenerative tissue transformation of iris into lens in the newt. Develop. Biol. 8, 358-369. ZALIK, S. E., and SCOTT, V. (1971). Development of the H3-thymidine-labeled iris in the optic chamber of lentectomized newts. Exp. Cell Res. 66,446-448. ZALIK, S. E., and Scorr, V. (1972). Cell surface changes during dedifferentiation in the metaplastic transformation of iris into lens. J. Cell Biol. 55, 1344146.