Transient expression of simple epithelial keratins by mesenchymal cells of regenerating newt limb

DEVELOPMENTAL

b’

Transient

‘LOGY

133,415-424

(1989)

Expression of Simple Epithelial Keratins by Mesenchymal Cells of Regenerating Newt Limb P. FERRETTI,*

*MRC

D.M.

FEKETE,*

M. PATTERSON,* AND E.B.LANE~

Cell Biophysics Unit, 26-29 Drury Lane, London WC2B 5RL, United Kingdom, and L&wig Institute 91 Riding House Street, London WIP 8BT, United Kingdom; and TICRF Glare Hall Laboratories, South Mimms, Potter’s Bar, Hertfordshire EN6 SLD, United Kingdom Accepted

November

for Cancer Research, Blanche Lane,

3, 1988

Structural proteins of the intermediate filament family are an early indicator of differentiation before organogenesis becomes apparent. Keratin intermediate filaments are characteristically expressed only by epithelial and not by mesenchymal cells. Here we show, using monoclonal antibodies, a transient expression of the keratin pair 8 and 18 in a population of mesenchymal cells in the regenerating newt limb, specifically in the undifferentiated progenitor cells (blastemal cells) which give rise to the new tissues. These keratins are also expressed in cultured limb cells that can differentiate into muscle. In contrast no reactivity with anti-keratin 8 and 18 antibodies was observed in the newt limb bud at an early stage of development, indicating a molecular difference between the developing and regenerating limb. The molecular weights of the newt proteins detected by these antibodies are very similar to those of human keratins 8 and 18, further supporting the immunocytochemical evidence that the newt homologs of these keratins are expressed in blastemal cells. This is the first demonstration of keratin expression in mesenchymal progenitor cells in an adult animal. 0 1989 Academic Press, Inc. INTRODUCTION

Urodele amphibians are the only vertebrates that can regenerate their limbs and tails as adults. After amputation epithelial cells migrate to cover the wound and form a specialized epithelium (wound epithelium) that lacks a basal lamina and is thicker than the normal epidermis (Thornton, 1968). Undifferentiated mesenchymal cells (blastemal cells) accumulate under the wound epithelium, divide rapidly under control of the nervous system and subsequently differentiate into the various tissues of the regenerated limb (Thornton, 1968; Wallace, 1981). At early stages of regeneration, when blastemal cell division is under nerve control, these cells can be identified by the monoclonal antibody 22/18 (Kintner and Brockes, 1985). Although it is not fully understood how blastemal cells originate, it is widely accepted that the mesodermal tissues of the stump, but not the epidermis, can contribute cells to the blastema through a process of dedifferentiation (Wallace, 1981, Kintner and Brockes, 1985). Muscle is at present the only tissue of the newt limb for which the existence of reserve cells, called postsatellite cells, has been suggested (Cameron et ah, 1986). Expression of specific subclasses of intermediate filaments is currently considered one of the best indicators of tissue differentiation, although it is not fully understood why this diversity is required or what role it plays in cell and tissue physiology. Keratin intermediate filaments are a major cytoskeletal component of epithelial 415

cells and are considered specific for this cell type, although apparent expression of keratins in “atypical” locations such as mesenchymal tumors (Ramadan and Goudie, 1986; Gusterson, 1987), mesenchymal cells of human amniochorion and chorionic villi (Khong et al., 1986), muscle cells (Huitfeldt and Brandtzaeg, 1985; Jahn et al., 1987), and a subclass of astrocytes and Purkinjie cells (Frank0 et ah, 1987) has been recently reported. Keratins represent the most complex group of proteins of the intermediate filament family with more than 20 different polypeptides identified so far (Moll et al., 1982, Osborn and Weber, 1986). They are encoded by two distinct gene families, type I (acidic) and type II (neutral to basic), that have been highly conserved through evolution and appear closely related among vertebrates (Fuchs and Marchuk, 1983; Hoffmann and Franz, 1984; Franz and Franke, 1986; Fuchs et ab, 1987). The expression of at least one keratin of each type is necessary for filament assembly and specific “pairs” appear to be associated with different epithelia and different stages of differentiation and development (Moll et al., 1982; Sun et ah, 1984; Lane et al, 1985; Osborn and Weber, 1986; O’Guin et al., 1987). Monoclonal antibodies have been widely used to characterize different types of keratins and their pattern of distribution in normal and pathological conditions (Lane et al., 1985; Osborn and Weber, 1986; Broers et ah, 1986; Morgan et al., 1987). We have assayed the reactivity of many anti-keratin monoclonal antibodies on early limb regenerates (blas0012.1606/89 Copyright All rights

$3.00

9 1989 by Academic Press, Inc. of reproduction in any form reserved.

416

DEVELOPMENTALBIOLOGY

temas) of the newt, Notophthalmus viridescens. Besides a large number of monoclonal antibodies cross-reacting with the newt epidermis, we found monoclonal antibodies that did not stain the epidermis, but reacted with mesenchymal cells of the regenerating limb that express both in vivo and in vitro the regeneration-associated antigen 22/18 (Kintner and Brockes, 1985; Ferretti and Brockes, 1988) and vimentin (Ferretti and Brockes, 1987; Ferretti and Fekete, unpublished observation). It is striking that the only anti-keratin monoclonal antibodies that recognize regeneration-associated filaments in the newt blastema react with keratins 8 and 18: this keratin pair is usually restricted to simple epithelia and represents the earliest keratin pair synthesized during embryogenesis (Jackson et al., 1980; Franz and Franke, 1986; Chisholm and Houliston, 198’7; Semat et ab, 1988; Lehtonen et ah, 1988).

V0~~~~133,1989

PKK3 (Holthcefer et ab, 1984; Virtanen et al., 1984; Labsystems); LE61, LE65, LP34, LP3K, LP5K (Lane et ah, 1985; Boers et ah, 1986) M2C8, M9, M20 (Van Muijen et ab, 1987); RCK102, RCK106 (Ramaekers et al, 1987), CAM5.2 (Makin et al., 1984); TROMA 1 (Kemler et al, 1981); and AE3 (Sun et al, 1984). Anti-keratin 19 antibodies KM4.62 (BioMakor), LPZK, BA16, BA17, and A53-B/A2 (Bartek et al., 1985; Karsten et ah, 1985; Lane et al., 1985) did not stain the blastema. We used 15-day-old blastemas without epidermis, wound epithelium from 15-day-old blastemas, and newt limb cell cultures for biochemical characterization of the antigens recognized by the anti-keratin antibodies. Wound epidermis was removed from blastemas by treatment for lo-15 min with 0.1% EDTA. Tissues were homogenized at 4°C in 10 mM phosphate buffer, pH 7.5, containing 0.12 M NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, and protease inhibitors. The protease inhibitors used were 1 mM phenylmethylsulfonylfluoride MATERIALS AND METHODS (PMSF) and a l/ZOO dilution of a solution of 0.2 mg/ml Adult specimens of the newt Notophthalmus viridesleupeptin, 2 mg/ml Na-benzoyl-L-arginine methyl ester cene (Lee’s Newt Farm, Tennessee) and embryos of the (BAME), 2 mg/ml Na-p-tosyl-L-arginine methyl ester newt Pleurodeles waltl (Xenopus, Ltd., England) were (TAME), 2 mg/ml N-tosyl-L-phenylalanine chloroused in this study. N. viridescens were anesthetized in methyl ketone (TPCK), type II-S soybean, 0.2 mg/ml pepstatin in distilled water. The tissue homogenates 0.1% tricaine (Sigma) and their limbs were bilaterally amputated proximal to the elbow. Regeneration blas- were centrifuged at 15000 rpm for 20 min at 4°C and the temas were harvested 3,7,11,15,22,36, or 42 days after supernatants removed (soluble fraction) and stored at amputation and processed according to Kintner and -70°C until further analysis. The detergent-insoluble Brockes (1985). Long-term cultures of newt limb cells material recovered in the pellet (cytoskeletal fraction) was washed and recentrifuged three times with homogwere obtained from limb explants. The mesenchymal buffer containing 1 M tissue was minced and when cells had migrated out of enizing buffer or homogenizing the explants they were passaged and grown as pre- KC1 (high salt extraction). Cells were removed from the flask by scraping in the presence of cold homogenizing viously described (Ferretti and Brockes, 1988). Unfixed P. waltl embryos staged according to Gallien and Dur- buffer and then processed as for the tissue homogeocher (1957) were processed through graded sucrose so- nates. Three to four micrograms of protein per lane was gel, blotted onto nitrolution, embedded in sucrose-gelatin, and frozen sec- run on 8% SDS-polyacrylamide gel transfer apparatus tioned (Fekete and Brockes, 1987). Sections were per- cellulose using a semi-dry 1984), and stained with either India meabilized and fixed to the slide by dipping in cold (Kyhse-Andersen, ink for total protein (Hancock and Tsang, 1983) or with acid-alcohol for 15 min prior to labeling with antimonoclonal antibodies that were detected by peroxibodies. rabbit anti-mouse immunoglobulin Fifty-five anti-keratin monoclonal antibodies were dase-conjugated (Dako, Denmark). Western blot analysis of the antigens screened by indirect immunofluorescence and immunoreacting with monoclonal antibodies LPlK and LE41 peroxidase on cryostat sections of 15-day-old blastemas was performed after blocking nonspecific binding to niusing undiluted supernatants. The antibodies that trocellulose with 10% fetal calf serum in phosphatecross-reacted with the newt were then assayed on acidalcohol (95% ethanol-5% acetic acid) fixed limb cell buffered saline, while the analysis of RGE53 and CK18.2 reactivity was performed according to Racultures by immunofluorescence. The bound antibodies maekers et aZ. (1984). were detected by peroxidase or rhodamine-conjugated rabbit anti-mouse immunoglobulin (Dako, Denmark). RESULTS Controls in which the incubation with monoclonal antibodies was omitted were run in each experiment. The Fifty-five monoclonal antibodies known to react spemonoclonal antibodies against keratin 8 or keratin 18 cifically with human keratins were screened by imthat did not convincingly react with blastemal cells, munohistochemistry on frozen sections of newt forealthough some reacted with epithelia, are PKKl and limb blastemas 15 days after amputation. Surprisingly,

FERRETTI

ET AL.

Keratin

Expre-ssion

in

Mesenchymal

FIG. 1. Reactivity of anti-keratin monoclonal antibodies RGE53 and LPlK with 15-day regenerated rescence. Strong reactivity is observed in the majority of blastemal cells, but not. in normal tissue,

6 of the 32 monoclonal antibodies that cross-reacted with the newt stained the blastema without staining normal epidermis or differentiated mesenchymal tissues. Two of these (KG8.1317 (Gigi et aZ., 1982) and A45-B/B3 (Karsten et al., 1985)) react with several type I and II keratins (including keratins 8 and 18) and thus were not studied in detail. The other four monoclonal antibodies are selective for keratins 8 or 18 in human tissues, which constitute a coexpressed pair characteristic of simple epithelial cells (Sun et aZ., 1984; Fuchs et al., 1987). Three of these are monospecific for keratin 18 (RGE53 and CK18.2; Ramaekers et ah, 1984; Broers et al, 1986) or keratin 8 (LE41; Lane et al, 1985; Morgan et aZ., 1987). The fourth (LPlK; Lane et aZ., 1985) recognizes several type II keratins by immunoblotting but is selective in tissue sections for keratin 8 (or keratin 7 in human tissues). No staining of blastemal cells was detected with these antibodies 3 days after amputation, but reactivity in the blastema was observed after 7 days and steadily increased up to 15-22 days, before the onset of differentiation. RGE53 and LPlK reactivities on sections of a 15-day-regeneration blastema are shown in Fig. 1. The majority of the blastemal cells were strongly labeled and so were cells associated with the regenerating nerve (Fig. 2). Reactivity with a subset of cartilage cells was observed at the onset of cartilage condensation (not shown). A variable number of epithelial cells scattered through the wound epithelium were stained by LPlK, while LE41 consistently stained a subset of basal cells in the wound (but not normal) epithelium (Table 1). CK18.2 and LE41 staining was weaker than that observed with RGE53 and LPlK. Subepidermal glands, perineurium, and some blood vessels reacted with these antibodies in both regenerating and normal

Cells

417

blastema were assayed by immunofluowith both antibodies. Scale bar is 200 am.

limbs, but all other differentiated tissues, including normal epidermis, were negative (Figs. 2 and 3). We have previously shown that the blastemal phenotype recognized by the antibody 22/18 is induced in long-term cultures derived from normal limb mesenthyme (Ferretti and Brockes, 1988). Such cultured limb cells appear to be mesenchymal since they grow as well-spread single cells, do not make desmosomes, express vimentin (Ferretti and Brockes, 1987), and differentiate into myotubes (Ferretti and Brockes, 1988). When these cultured cells (Fig. 4) or cells from dissociated blastema (not shown) were reacted with the anti-keratin 8 and 18 antibodies a filamentous staining pattern was observed in all the cells. RGE53, CK18.2 and LPlK gave a very bright staining, while LE41 staining was less intense but consistently present. A mixed intermediate filament phenotype (vimentin/keratin) is thus observed both in tivo and in vitro. Previous findings have demonstrated that the differential expression of the 2208 antigen in developing versus regenerating limbs reflects a difference in the mechanisms controlling these processes, such as nerve dependence (Fekete and Brockes, 1987, Fekete and Brockes, 1988). It was therefore of interest to compare keratin expression in the regenerating blastema with that in the embryonic limb bud. The distinctive regenerative pattern of staining was not seen in the developing limb, further supporting a difference in the cellular composition of the two systems. P. waltz embryos were examined at two different stages of limb development corresponding to an early, undifferentiated bud (stage 33) and a late bud (stage 37) in which a few axons and myoblasts are present (Fekete and Brockes, 1987). At the early stage all monoclonal antibodies reacted with

418

FIG .2. RGE53 staining of a regenerating cells ( possibly Schwann cells) are present. bar is 50 fim.

DEVELOPMENTALBIOLOGY

VOLUME 133.1989

nerve 15 days after forelimb amputation (a) and corresponding Nomarski Perineural cells (arrow), as observed to some extent also in normal nerves,

notochord, nephric tubules, gut, and liver. Some staining was observed also in yolk cells as well as other scattered cells. Immunostaining with monospecific antibodies (RGE53, CK18.2, and LE41), however, showed either no reactivity (Fig. 5) or only rare positive cells within the limb mesenchyme, while nearly all limb cells were very weakly labeled by LPlK; the intensity of the staining was much lower than in other tissues of the embryo and in the regenerating blastema. It was diffi-

image (b). Many positive are strongly labeled. Scale

cult to assess the significance of this reactivity, since this monoclonal antibody is not monospecific and we do not know whether it recognizes other keratins in the embryo. At the later stage, all monoclonal antibodies showed some reactivity with a few cells mainly located in the central part of the bud (Fig. 6). The molecular weight of the newt proteins detected by LPlK/LE41 and RGE53/CK18.2 are consistent with those of human keratins 8 and 18 (Moll et ah, 1982). The

FERRETTI

POLYPEPTIDES DETECTED MONOCLONAL ANTIBODIES CYTOCHEMISTRY W-epithel”

TABLE 1 IN NEWT TISSUES BY IMMUNOBLOTTING

Kerutin

ET AL.

Expresskm in Mesench~mal

BY ANTI-KERATIN AND IYMUNO-

Blasternab

Cultured

cells

mAb

kDa

imm

kDa

imm

kDa

imm

LPlK LE41 RGE53 CK18.2

52-57 52-59 -

(+I (+I’ -

52-57 52-59 43 43

+ + + +

52 52 43 43

+ + + +

I‘ Wound epithelium. ’ Blastema without ’ Subset of cells.

epidermis

and wound

epithelium.

bands recognized by LPlK (Fig. 7) on immunoblots of cytoskeletal fractions of blastema with epidermis, blastema without epidermis, wound epithelium, and normal epidermis had apparent molecular weights of 52 and 57 kDa, while only the 52-kDa band was detected in

Ceils

419

fractions from cultured newt cells (Table 1). A similar pattern of reactivity was observed in the various cytoskeletal fractions with the monospecific monoclonal antibody LE41 (Table 1). LPlK and LE41 both recognized a 52-kDa band in the blastema and in the cultured cells and their immunostaining of cultured cells differed only in intensity. It appears therefore that these monoclonal antibodies recognize two different epitopes on the same polypeptide, with the LE41 epitope being less accessible in the native than in the denatured molecule. CK18.2 stained a band of apparent molecular weight of 43 kDa in all of the cytoskeletal fractions tested (Table 1). Although RGE53 reactivity on immunoblots was very weak, the positive band seemed identical to that detected with CK18.2 (Table l), suggesting that these two monoclonal antibodies also recognize different epitopes on the same molecule. DISCUSSION

There is little doubt that we are observing expression of simple epithelia keratins 8 and 18 since filamentous

FIG. 3. RGE53 reactivity in normal limb tissue (a) and at the junction between the blastema blood vessels (arrow) are stained. (b) The boundary between regenerating and normal tissue whereas the stump tissue is completely negative. Scale bars are 100 pm (a) and 20 nrn (b).

and the stump (b). (a) Subepidermal glands and is evident: Blastemal cells are brightly stained,

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FIG. 4. Reactivity of anti-keratin 18 monoclonal antibodies RGE53 and CK18.2, and anti-keratin 8 monoclonal antibodies LPlK and LE41 with newt limb cells in culture. All the antibodies stain the cultured cells, although LE41 staining is less bright. Note the fibroblastic morphology of the cells. Scale bar is 50 pm.

FERRETTI

FIG. 5. Transverse section through an early (b). Limb buds (arrows) are negative; positive cells are highly autofluorescent. The abundance Irm.

ET AL.

Keratin

Expression

in Mesenchymal

Cells

421

Pleurodeles embryo

stained with RGE53 (a) and counterstained with the DNA dye, Hoxhst 33258 tissues include notochord, nephric tubules, gut, liver, yolk cells, and other scattered cells. Yolk of yolk resulted in artifactual tearing of the tissue during processing (asterisk). Scale bar is 200

structures are seen by multiple specific antibodies, which recognize appropriately sized proteins on immunoblotting. The similar biochemical and immunocytochemical cross-reactivities of the antibodies between human and newt keratins are consistent with the high degree of evolutionary conservation of keratin gene families (Fuchs and Marchuk, 1983; Hoffmann and Franz, 1984; Franz and Franke, 1986). We are currently attempting to clone the newt genes coding for the regeneration-associated proteins recognized by the antikeratin 8 and 18 monoclonal antibodies. These results show for the first time that keratins typical of simple epithelia can be expressed by multipotential mesenchymal cells in an adult animal. On the other hand the distribution of keratins 8 and 18 in the normal tissue of the newt limb is consistent with the expression of these keratins in endothelial cells and in a subset of smooth muscle of Xenop-us Zaevis recently reported by Jahn et al. (1987). Early in embryogenesis homologs of human keratins 8 and 18 are expressed by all cells of Xenopus and mouse embryos (Franz and Franke, 1986; Chisholm and Houliston, 1987; Lehtonen et al., 1988). Subsequently one or more transitions occur between epithelial and mesenchymal phenotypes along certain mesodermal lineages, until organogenesis seems to stabilize cell differentiation (Viebhan et al., 1988); changes in this apparently

stable phenotype can be, in some cases, induced in vitro (Lehtonen et ah, 1985; Semat et ah, 1986; Greenburg and Hay, 1988). Thus the coexpression of keratin and vimentin by blastemal cells following dedifferentiation may reflect a regression to this early embryonic stage where epithelial/mesenchymal transitions occur, since at a cellular level a recapitulation of development may be necessary for tissue respecification during regeneration. An alternative explanation would be that a few multipotential embryonic cells with mixed phenotypes are left in the adult newt to maintain the regenerative potential of the limb and that dedifferentiation of stump tissues constitutes a minor contribution to the blastema. This seems a less likely explanation because of previous studies (Wallace, 1981), and because of the lack of keratin expression 3 days after amputation, when cells start to accumulate under the wound epithelium to form a blastema. The tissue specificity of intermediate filament expression has lead to the increasing use of keratin antibodies in diagnostic histopathology (Broers et al, 1986; Osborn and Weber, 1986; Morgan et al., 1987), where recent reports of anomalous expression of keratins in mesenchymal tumors and normal tissues (Khong et ab, 1986; Ramadan and Goudie, 1986; Gusterson, 1987) have threatened to undermine the usefulness of these probes. However if a mesenchymal cell type analogous to the

FIG. 6. Transverse sections through the limb bud of a stage 37 Pleurodeles corresponding Nomarski images (right). A few bright cells are observed antibody was omitted. Scale bar is 50 Frn. 422

embryo stained with anti-keratin monoclonal in the limb bud. In the control the incubation

antibodies (lt !ft) and with the mon oclonal

FERRETTI ET AL.

Keratin

Expression

in Mesenchymal

Cells

clonal antibodies; J. P. Brockes for the useful support; and B. B. Boycott for his comments

116

discussions and constant on the manuscript.

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FIG. 7. Western blot analysis of soluble protein and cytoskeletal fractions from l&day blastemas and cultured newt cells stained either with India ink (left panel) or the monoclonal antibody LPlK (right panel). Soluble protein fraction (l), cytoskeletal fraction of blastema with epidermis (2), blastema without epidermis (3), wound epithelium (4), normal epidermis (5), newt cells (6). The positions of standard molecular weight proteins are indicated. The molecular weight of the two proteins detected by LPlK are 52 and 5’7 kDa.

blastemal cells can arise in human tissues, it may be the source of “anomalous” expression of keratins. One might predict such a cell type to be multipotent, and possibly to play a role in tissue restructuring. The differential expression of simple epithelia keratins in development and regeneration confirms the existence of differences in cellular contribution to the developing versus the regenerating limb that may reflect a different mechanism of growth control in these two systems (Fekete and Brockes, 1987, 1988). It has been shown that retinoids affect differentiation of both tumor and normal cells (Edwards and McBurney, 1983; Ramaekers et al, 1984; Kim et ab, 1987, Kopan et al, 1987). In addition they represent the first group of compounds that have been unequivocally shown to act as morphogens when applied exogenously (Maden, 1985). In urodeles, retinoids can induce morphogenetic duplications in the regenerating but not the developing limb (Maden, 1985; Scadding and Maden, 1986). They can also regulate the expression of certain keratins including keratins 8 and 18 (Ramaekers et al., 1984; Kim et al, 1987; Kopan et al., 1987). The differential expression of these keratins in development and regeneration is therefore consistent with the different morphogenetic effects induced by retinoic acid. The expression of keratins 8 and 18 in the regeneration blastema strengthens the possibility that retinoids are indeed endogenous morphogens in regeneration and that morphogenesis may be effected in part through changes in intermediate filament expression. We wish Ramaekers,

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U. Karsten, C. Makin, F. for the use of their mono-

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