Apoptosis of larval cells during amphibian metamorphosis

MICROSCOPY RESEARCH AND TECHNIQUE 34:228-235 (1996)

Apoptosis of Larval Cells During Amphibian Metamorphosis ATSUKO ISHIZUYA-OKA Department of Anatomy, Dokkyo University School of Medicine, Mibu, Tochigi 321 -02, Japan

KEY WORDS Cell death, Amphibia, Ultrastructure, Thyroid hormone, Cell-cell interaction, Macrophage

ABSTRACT Programmed cell death occurs in a variety of organs during amphibian metamorphosis and is usually identified by electron microscopy as apoptosis or its modifications. Because of the massive cell death that occurs during a short period, amphibian organs serve as an ideal model system for the study of mechanisms underlying programmed cell death. In this article, a series of morphological changes in apoptosis from their nuclear changes to removal by phagocytic macrophages is reviewed, mainly in the small intestine of metamorphosing Xenopus Zaevis tadpoles. It is well known that cell death during amphibian metamorphosis is under the control of thyroid hormone (TH), and changes in gene expression induced by TH have been recently analyzed in a few Xenopus organs. On the other hand, there is a growing body of evidence that cell death is regulated by various kinds of local factors. For example, roles of interactions with other tissue cells and/or participation of immunocompetent cells in cell death have been experimentally shown. Therefore, to clarify the mechanisms of this complicated process, it is important at present that TH-induced changes in gene expression of each cell type comprising the organ are chronologically examined by combining morphological and molecular biological techniques. o 1996 Wiley-Liss, Inc.

INTRODUCTION In general, programmed cell death undergoes mor-

phological changes that are distinctly different from those of necrosis (i.e., passive cell death widely observed in pathological processes). In higher verte- brates, programmed cell death usually occurs by apoptosis, a process characterized by the condensation of nuclear chromatin in tight apposition to the nuclear envelope, followed by fragmentation of the nuclear and the whole cell body into membrane-bounded cell globules (i.e., apoptotic bodies) (Kerr et al., 1972, 1987; Wyllie et al., 1980). The morphological changes in nu- clei have been shown in most systems to be associated with endogenous endonuclease activity which causes internucleosomal DNA fragmentation (Wyllie, 1980; Wyllie et al., 19841, although DNA fragmentation is not a universal feature of apoptosis (Cohen et al., 1992; Oberhammer et al., 1993) and the molecular mechanisms of apoptosis remain obscure. By using DNA fragmentation as a hallmark of this process, a method was recently developed for visualizing individual cells undergoing programmed cell death on histological sec- tions (TUNEL method [Gavrieli et al., 19921). Thus, it is now easy to distinguish cells undergoing programmed cell death in situ by both light and electron microscopy.

During amphibian metamorphosis, the body of the tadpole undergoes remodeling from the larval to the adult form suitable for terrestrial life. In this remodeling process, cell death occurs widely in larval-specific organs such as the tail, the pronephrons, and the gill and also in other organs that exist in both tadpoles and frogs (Atkinson and Just, 1975; Fox, 1971; Lockshin, 1981; Usuku and Gross, 1965). Thus, cell death is one of the most essential components of amphibian remod-

eling, and has been thought of as being “programmed” in the genetic sense (Yoshizato, 1989, 1992). Because cell death occurs in large quantity of tissues during a short developmental period, amphibian metamorphosis serves as an ideal model system for the study of programmed cell death. However, we still have only fragmentary information on cell death during amphibian metamorphosis. Although there have been numerous studies published on the degeneration of amphibian tadpoles, they have often confused the death process a t the cellular level with that occurring at the tissue or organic level. Because every tissue or organ consists of various types of cells possessing different developmental fates, it is important to study the death of individual cells in situ. Such a study is now possible by using modern morphological techniques described above. From this point of view, I intend to reconsider in this review previous data on cell death during amphibian metamorphosis and summarize the information that has been obtained to date at the cellular level.

MORPHOLOGICAL PROCESSES OF CELL DEATH DURING METAMORPHOSIS Larval Cell Death Occurs by Apoptosis

The dramatic degeneration of larval tissue that occurs during amphibian metamorphosis has attracted many biologists. However, the majority of their reports have been mainly focused on histolytic changes such as development of lysosomes (Fox, 1975; Fox et al., 1972; Hourdry and Dauca, 1977; Moulton et al., 1968; Weber,

Received July 15, 199% accepted in revised form October 17, 1994. Address reprint requests to Atauko Ishizuya-Oka, Department of Anatomy,

Dokkyo University School of Medicine, Mibu, Tochigi 321-02, Japan.

0 1996 WILEY-LISS, INC.

APOPTOSIS DURING AMPHIBIAN METAMORPHOSIS 229

1964), not on the nuclear changes which are character- istic of apoptosis. Kerr et al. (1974) first described apoptosis of amphibian cells in the metamorphosing tail epidermis and muscle by electron microscopy. Thereaf- ter, unequivocal apoptosis has also been observed by electron microscopy in other organs. Although the number of reports is limited, it should be noted that apoptosis was observed not only in the larval-specific organs but also in organs that exist after metamorphosis, such as the brain (Decker, 1976; Wahnschde et al., 1987) and intestine (Ishizuya-Oka and Shimozawa, 1992a). This suggests that apoptosis occurs widely in the animal body undergoing remodeling. Apoptosis during amphibian metamorphosis has also been his- tochemically observed by the TUNEL method for detection of DNA fragmentation in both the tail (Oofusa et al., 1993) and small intestine (our unpublished data).

Although the morphology of cell death is not exactly the same among different cell types (Kerr et al., 1974), cell death observed during amphibian metamorphosis shares common characteristics of apoptosis as follows: condensation and margination of chromatin against the nuclear envelope (Fig. 11, followed by formation of membrane-bounded cell globules (apoptotic bodies) which contain intact cell membranes and organelles, and a fragmented nucleus (Fig. 2). Apoptotic bodies are then phagocytosed by neighboring healthy cells, mainly by macrophages (Fig. 3) which dramatically increase in number concomitantly with larval cell death (Fig. 4).

Macrophages Function in the Rapid Removal of Apoptotic Bodies

Undoubtedly, macrophages play an important role in the rapid removal of dead cells from remodeling tissue. However, we have only limited information on their origin, differentiation, and function at the molecular level. In this section, I will describe the phagocytic process of apoptotic cells by macrophages, especially in the small intestine which has been recently studied in our laboratory (Ishizuya-Oka and Shimozawa, 1992a, 1994).

Increase in Macrophage Numbers During Am- phibian Metamorpbsis. There still remains a his- torical controversy as to the origin of macrophages which increase rapidly in number within the degenerating larval tissue. One explanation has been that mes- enchymal cells transform into macrophages (Fox and Whitear, 1990; Watanabe et al., 1985; Weber, 1964), whereas the other stipulates that macrophages differentiate from monocytes in blood capillaries (Lehman, 1953). Recently, it has been shown in intestinal fragments isolated from Xenopus tadpoles and cultured or- ganotypically that macrophages rapidly increase in number and phagocytose apoptotic bodies of larval cells as in spontaneous metamorphosis. These results indicate that the macrophages were derived from cells that already existed in the larval intestinal fragment instead of originating from other parts of the body through the blood supply. However, the question still remains whether macrophages transform from other types of intestinal cells or simply differentiate from

preexisting immature macrophages. Considering our previous observations that a small number of immature macrophage-like cells exist in the larval small intestine before metamorphosis and that, during metamorphosis, macrophage-like cells begin to proliferate actively in the connective tissue concomitantly with cell death of the larval epithelium, the latter case seems to account for their increase in number at least partially. In addition, during this period, mitotic pro- files of macrophage-like cells are localized in the connective tissue (Fig. 5) but not in the degenerating epithelium where a large number of mature macrophages exist (Ishizuya-Oka and Shimozawa, 1992a). There- fore, immature macrophages probably increase in number by cell proliferation in the connective tissue and then migrate into the epithelium of the small intestine. Migration of macrophages from the dermis into the epidermis was also observed in the regressing tail (Kinoshita et al., 1985).

Recognition and Phagocytosia of Apoptotic Cells by Macrophages. In the intestinal epithelium during amphibian metamorphosis, larval cells undergoing apoptosis coexist with basally disposed undifferentiated cells that will later form the intestinal absorptive epithelium during adulthood (adult cells) (Fig. 6a). In- terestingly, macrophages are distributed only among the larval cells but not among the adult cells (Fig. 6b). This suggests that macrophages can recognize larval cells before phagocytosing them. Although the precise mechanisms are still unknown, our previous histo- chemical studies indicated that binding patterns of wheat germ agglutinin (WGA) and soybean agglutinin (SBA) are greatly altered in epithelial cells during cell death (Ishizuya-Oka and Shimozawa, 1990). In mammals, macrophages are known to possess lectin-like molecules on their cell surface and also recognize cells undergoing apoptosis (Duvall et al., 1985; Savill et al., 1993). Therefore, one may speculate that macrophages recognize modified carbohydrates on the apoptotic cell surface via their surface lectin molecules.

The ultimate fate of macrophages after phagocytosis of apoptotic bodies is almost unknown, as is their origin. Exceptionally, in the case of the small intestine, they have been observed to become enlarged by en- gulfed phagosomes and be finally extruded into the gut lumen (Fig. 7). Even in the lumen they retain an intact cell membrane and organelles and contain the apoptotic bodies (Fig. 8).

REGULATION OF LARVAL CELL DEATH Endocrine Controls

It is widely accepted that amphibian metamorphic changes including programmed cell death are initiated and controlled by thyroid hormone (TH) (Yoshizato, 1989,1992). In fact, THdependent degeneration of tissues has been repeatedly observed in several organ culture systems (Derby et al., 1979; Gona, 1969; Pouyet et al., 1983; Tata, 1966). In addition, in the small intestine, it has been shown that cell death induced by TH in vitro occurs by apoptosis as in spontaneous metamorphosis (Ishizuya-Oka and Shimozawa, 1992a); numerous apoptotic bodies are formed within the epithelium in the presence of TH (Fig. 9) but not in its

230 A. ISHIZUYA-OKA

Fig. 1. An apoptotic cell in the larval intestinal epithelium ofXe- MPUS Zaevis during metamorphosis. Its nucleus (N) possesses condensed chromatin close to the nuclear envelope (arrow). Its cell mem-

Fig. 3. A macrophage (arrowheads) including an apoptotic body (AB) within the larval intestinal epithelium (LE) during Xenopus metamorphosis. L, lumen; N, nucleus. Bar = 1 km.

brane and organelles are intact. Bar = 1 pm. Fig. 2. Apoptotic bodies in the intestinal connective tissue during

Xenopus metamorphosis. One cell body contains a nuclear fragment

Fig. 4. Cross-section of the small intestine during Xenopus metamorphosis. Many macrophages are observed (arrows) in both the epithelium (LE) and the connective tissue (CT). L, lumen. Bar = 10 wm.

(arrow). Bar = 1 bm.

absence of TH (Fig. 10). Furthermore, in the tail epidermal cells and fibroblasts, it has been shown by cell culture techniques that TH directly shortens the life span of the larval cells in vitro (Nishikawa and Yoshizato, 1985, 1986; Nishikawa et al., 1989).

At present, the precise molecular mechanisms of TH action on programmed cell death during amphibian metamorphosis are unknown. However, there is no doubt that the first step of TH action is its binding to the thyroid hormone receptors (TRs) of target cells. Re- cently, the amphibian TRs genes whose protein products probably mediate the effects of TH during amphibian metamorphosis have been cloned and characterized in Xenopus laeuis tadpoles (Shi and Brown, 1992; Yao- ita and Brown, 1990; Yaoita et al., 1990). Moreover, several TH response genes have been isolated in various organs including the tail (Wang and Brown, 1993)

Fig. 5. Mitosis of a macrophage-like round cell possessing small vacuoles (arrows) in the intestinal connective tissue during Xenopus metamorphosis. ECM, extracellular matrix. Bar = 2 pm.

Fig. 6. Coexistence of the larval intestinal epithelium (LE) with the primordia of adult epithelium (AE) during Xenopus metamorphosis. a: Primordia of the adult epithelium growing into the connective tissue ( 0 . Arrowheads indicate the boundaries between the larval and adult epithelia. CM, circular muscle; L, lumen; LM, longitudinal muscle. Bar = 20 pm. b Numerous macrophages (arrows) within the larval epithelium but not within the undifferentiated adult epithelium. CT, connective tissue; L, lumen. Bar = 2 pm.

Fig. 7. Large macrophages (arrows) extruding from the larval intestinal epithelium (LE) into the lumen (L) during Xenopus metamorphosis. Bar = 2 pm.

Fig. 8. A macrophage including apoptotic bodies (AB) in the intestinal lumen (L) during Xenopus metamorphosis. Its cell membrane and nucleus (N) are still intact. Bar = 2 pm.


Figs. 5-8.

232 A. ISHIZUYA-OKA

Fig. 9. The Xenopus small intestine cultured in the presence of TH. Apoptotic bodies possessing nuclear fragments (arrows) exist between and within the larval epithelial cells (LE). CT, connective tissue; L, lumen. Bar = 2 pm.

Fig. 10. The Xenopus small intestine cultured in the absence of TH. Neither apoptotic bodies nor macrophages are observed. CT, connective tissue; L, lumen; LE, larval epithelial cells. Bar = 2 pm.

and the intestine (Shi and Brown, 1993), where massive cell death occurs. If cell death is truly programmed at the genetic level, its process could be explained by changes in expression of TH response genes. In the small intestine, expression of one TH response gene, the intestinal fatty acid-binding protein (IFABP) gene, was recently examined by in situ hybridization techniques (Ishizuya-Oka et al., 1994; Shi and Hayes, 1994). IFABP mRNA was observed exclusively in the absorptive epithelial cells of the intestine throughout the larval period (Fig. 11) but suddenly decreased in expression at the end of prometamorphosis, just before apoptosis was morphologically detected (Fig. 12). This suggests that changes in gene expression which in- clude cell-specific genes such as IFABP precede the morphological phenotype of programmed cell death. Considering a time lag of 2-3 days for the morphological detection of apoptosis following TH addition in vitro (Ishizuya-Oka and Shimozawa, 1992a), many genes seem to be associated with this process. Charac- terization of all genes that are involved in the cell death pathway and clarification of how this cascade may be regulated are now eagerly awaited. Among potential candidate genes, there may be a gene that regulates the choice between cell death and cell proliferation, such as c-myc reported in mammalian programmed cell death (Cohen, 1993; Evan et al., 1992). This is noteworthy because intestinal epithelial cells during amphibian metamorphosis adopt one of two fates, cell death or cell proliferation to form a new adult epithelium.

Effects of hormones other than TH on cell death during amphibian metamorphosis have been also reported;

prolactin inhibits TH-induced degeneration of the tail in vitro (Tata et al., 1991), probably by preventing the TH induction of TRs mRNAs (Baker and Tata, 1992). In contrast, corticoids administered along with TH ac- celerate tail regression in vitro (Kikuyama et al., 1983; Shimizu-Nishikawa and Miller, 1992). An increase of nuclear TH-binding sites by the corticoids has been postulated as a potential mechanism (Kikuyama et al., 1993).

Influences of Local Cell-Cell Interactions Although programmed cell death during amphibian

metamorphosis is commonly triggered and controlled by the endocrine system, there is an accumulating body of evidence which suggests that the cell death pathway may be regulated locally by other types of cells.

Participation of Immunocompetent Cells. Dur- ing amphibian metamorphosis, a dramatic transition of major histocompatibility complex antigens occurs, and lymphocytes also undergo a transition from larval to adult type (Du Pasquier and Flajnik, 1990; Flajnik and Du Pasquir, 1988; Flajnik et al., 1987). Therefore, there are two presumed immunological difficulties: the first is that adult-type cells may be recognized immu- nologically as nonself by larval-type lymphocytes, and the second is that larval-type cells may be recognized as nonself by adult-type lymphocytes. However, as to the former case, tadpoles can overcome this problem by induction of tolerance (Cohen et al., 1985; Nakamura et al., 1987). In contrast, the latter possibility seems likely, because it has been experimentally shown that larval skin grafts are rejected in the adult body (Izutu and Yoshizato, 1993; Yoshizato, 1992). This rejection


Fig. 11. The Xenopus small intestine before metamorphosis, hybridized with an antisense probe derived from the IFABP gene cDNA. Signals are observed exclusively in larval epithelial cells (LE). bb, brush border; CT, connective tissue. Bar = 10 pm.

Fig. 12. The Xenopus small intestine at the end of prometamorphosis, hybridized with an antisense probe derived from the IFABP gene cDNA. Although larval epithelial cells (LE) still retain intact structures such as the brush border (bb), their signals become negative. CT, connective tissue. Bar = 10 pm.

may be of biological significance to prevent larval cells from remaining in the adult body. However, the extent to which adult-type lymphocytes participate in larval cell death during spontaneous metamorphosis remains unknown. Morphologically, there is evidence to suggest that a large number of immunocompetent cells other than macrophages are distributed within tissues destined to degenerate (Marshall and Dixon, 1978; Ki- noshita et al., 1985). Their function during amphibian metamorphosis is worthy of further study. Roles of Tissue Interactions. Several experiments

have demonstrated the importance of tissue interactions in the degeneration of amphibian larval tissues. A good example is the dermis-dependent degeneration of the skin epidermis (Kinoshita et al., 1986,1989). The tail epidermis, which is fated to die during spontaneous metamorphosis, could survive when combined with the dermis of the back region. In contrast, the back epidermis, which is fated to survive in adults, degenerated when combined with the tail dermis. Although degeneration in these studies was not analyzed at the cellular level, these findings strongly suggest that the dermis can somehow affect the death pathway.

By contrast, in epithelium<onnective tissue recom- bination experiments of the small intestine, cell death of the epithelium occurred independently of the region from which the connective tissue was derived (Ishi- zuya8ka and Shimozawa, 199213). Instead, a strong correlation between cell death and ultrastructural changes in the basal lamina was observed (Ishizuya- Oka and Shimozawa, 1987). Interestingly, it has been recently shown that, when the basal lamina is altered during metamorphosis, a TH response gene, the stromelysin-3 gene, is activated in the Xenopus small intestine (Shi and Brown, 1993). In mammals, this gene was found to be one of the metalloproteinase genes whose products degrade/modify the extracellular

matrix (ECM) and is expressed in the connective tissue surrounding tissues undergoing cell death (Basset et al., 1990; Lefebre et al., 1992; Wolf et al., 1993). In similar fashion, within the amphibian intestine, its expression is restricted to the connective tissue of the small intestine (Patterton et al., 1995). Therefore, these authors have speculated that stromelysin-3 may be involved in ECM remodeling including the basal lamina and that changes in the ECM could in turn influence cell death of the epithelium. Further analysis of this epithelial-connective tissue interaction will focus on the precise distribution of cells expressing this gene and attempt to provide a more detailed correlation between stromelysin-3 gene expression and alter- ation of the basal lamina. This work is currently under investigation.

On the other hand, an important role of the epithelium in connective tissue degeneration has been also reported in tail skin cultures in vitro. Dermis deprived of epidermis did not degenerate even in the presence of TH (Niki et al., 1982, 1984). In addition, in cultured intestine in vitro, it has been observed that the epithelium facilitates hormonal effects on proliferation and/or differentiation of macrophages (Ishizuya-Oka and Shimozawa, 1994). In both situations, the exis- tence of chemical substances produced by epithelial cells has been speculated, but the epithelial products have not yet been identified.

As described above, various kinds of cell-cell and cell-ECM interactions are associated with programmed cell death during amphibian metamorphosis. The mechanisms of these interactions remain unsolved at present but may also be controlled by TH. Although the biological significance of these interactions is unclear, they may be important local regulators of larval cell death, thus insuring survival of the whole body throughout metamorphic processes.

234 A. ISHIZUYA-OKA

CONCLUSIONS AND PERSPECTIVES Amphibian metamorphosis gives us an excellent

model system appropriate for the study of programmed cell death, because cell death 1) occurs by apoptosis, 2) occurs in large quantity during a short developmental period, and 3) is easily induced by TH both in vivo and in vitro. Morphological studies have been further strengthened by recent developments of gene technol- ogy in this field.

Previously, many experiments have shown that cell death during amphibian metamorphosis is triggered and regulated by an endocrine system including TH. However, there is a growing body of evidence that the cell death pathway is very complex and locally influ- enced by other types of cells andlor their products. Therefore, to elucidate the mechanism of programmed cell death in this model system, it is important to ex- amine chronological changes in gene expression induced by TH for each cell type comprising the organ, using combined morphological and molecular biological techniques. In the tail and small intestine, some TH response genes have already been isolated and are available for morphological studies. With the use of in situ hybridization, a more precise analysis of programmed cell death is now in progress in these amphibian organs.

ACKNOWLEDGMENTS I thank Dr. A. Shimozawa for his continuous encour-

agement in my work. The work described in this article was partly funded by a grant in aid for scientific research from the Ministry of Education, Science, and Culture of Japan.

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Apoptosis of larval cells during amphibian metamorphosis