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Keywords:

  • metamorphosis;
  • programmed cell death;
  • thyroid hormone;
  • apoptosis;
  • Xenopus laevis;
  • dominant-negative thyroid hormone receptor

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The tadpole tail, which is twice as long as the body, is induced to resorb completely by thyroid hormone within several days during the anuran metamorphosis. To investigate the underlying mechanism, we undertook two approaches. First, we examined the effect of dominant-negative thyroid hormone receptor (DNTR) on muscle cell death in vitro. The overexpression of DNTR suppressed the death of a tail-derived myoblastic cell line induced by thyroid hormone. Second, tadpole tails were injected with a reporter gene and the DNTR expression construct, and the reporter gene expression in muscle cells was followed during the spontaneous metamorphosis. DNTR overexpression inhibited a decrease of the reporter gene expression that began at stage 57 in the control tadpoles but only delayed massive muscle cell death at stage 63 when tails shrink very rapidly. Some remained even a few weeks after the metamorphosis, although most DNTR-overexpressing cells died by the end of the metamorphosis. These results led us to propose that thyroid hormone induces the suicide of muscle cells (the cell-autonomous death) in the tail between stage 57 and 62 and that both the murder and suicide mechanisms execute muscle cell death in stage 62–64 to remove muscle promptly and completely. Developmental Dynamics 227:246–255, 2003. © 2003 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Amphibian metamorphosis is represented by drastic morphologic and physiological changes to adapt an aquatic tadpole for a terrestrial life. In anurans, almost every organ undergoes modification, including limb development and the loss of the tail and gills. Little is known regarding the molecular mechanism of thyroid hormone (TH) -induced cell death in the regressing tail during the metamorphosis. De novo protein synthesis is necessary for TH-induced tail resorption (Tata, 1966), suggesting that the complex of TH and thyroid hormone receptor (TR) regulates some genes responsible for cell death in the regressing tail. Based on the observation that TH induces the increase of the collagenase activity in explants of tadpole tailfin and gills and the concomitant decrease of their sizes, it has been proposed that TH-induced collagenase production is involved in the remodeling of collagen in these organs (Davis et al., 1975; Derby et al., 1979). Many genes that are up- and down-regulated by TH in the regressing tail have been isolated by PCR-based subtractive hybridization (Wang and Brown, 1993) and characterized by in situ hybridization (Berry et al., 1998a, b). Extracellular matrix (ECM) -degrading enzymes such as stromelysin-3 and collagenase-3 are highly expressed in the subepidermal fibroblasts encircling the entire muscle flank but not in tail muscle. These enzymes are also up-regulated in the myotendinous junctions to which the muscle fibers are attached. These observations lead to the idea that the increase of secreted matrix metalloproteinases (MMPs) induced by TH results in the degeneration of the myotendinous junctions, which detaches muscle cells from ECM and causes their death (a murder model; Berry et al., 1998b). This mechanism is supported by the phenomenon “anoikis,” for which apoptosis is induced by disruption of the interactions between normal epithelial cells and ECM (Meredith et al., 1993; Frisch and Francis, 1994; Ishizuya-Oka et al., 2000).

On the other hand, TH induces apoptosis of a myoblastic cell line derived from a tadpole tail of Xenopus laevis (Yaoita and Nakajima, 1997), suggesting that tail muscle cells die autonomously (a suicide model). However, it is possible that this is an in vitro artifact and does not reflect the physiological death in matrix-interacting cells in vivo. Furthermore, it does not exclude a possibility that TH-treated myoblastic cells kill each other by secreting soluble factors.

DNTR binds to TH response elements but not TH and prevents transcription of TH-responsive genes by the wild-type TR in mammalian cells (Sap et al., 1989; Sakurai et al., 1990; Hao et al., 1994) and Xenopus tadpoles in vivo (Ulisse et al., 1996). To explore the molecular mechanism of cell death in the regressing tail, DNTR expression constructs were introduced into both a tail-derived myoblastic cell line and tail muscle cells in living tadpoles, and the viability of transfected cells was examined in a condition in which programmed cell death (PCD) is induced. Our results suggest that TH induces the commitment of suicide before the regression of tails and promotes the murder and suicide of muscle cells during rapid shortening of tails in stage 62–64.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

DNTR Overexpression Suppresses TH-Induced Death of Cultured Myoblastic Cells

To interrupt the TH signaling that induces cell death, the DNTR expression construct was cotransfected with pEGFP-N2 as a reporter gene to a myoblastic cell line, XLT-15-9 derived from a tadpole tail (Nakajima et al., 2000). The TH-signaling pathway should be hindered only in the DNTR transfected cells but not in the nontransfected surrounding cells, because TR is a nuclear protein. According to a murder model, all cells should be killed by soluble factors that are synthesized by the TH-treated nontransfected cells. In a suicide model, a cell for which the TH signaling is interrupted by the transfection of DNTR gene should survive, whereas the nontransfected cells die (Fig. 1). The vector-transfected cells showed approximately 50% cell death after 3 days of T3 (3,3′,5-triiodo-L-thyronine) treatment, whereas cells overexpressing DNTR were resistant to T3 (Fig. 2A). Nevertheless, the same gelatinolytic activity was observed by zymography in both culture media of the transfected TH-treated cells (Fig. 2B), indicating that these culture media contained a similar amount of TH-induced soluble factors. The nontransfected cells, which are the majority of cells in the culture (around 95%), are expected to release TH-induced soluble factors into the culture medium, which could impact the 5% of the cells that were DNTR-transfected. As expected, gelatinolytic activity (as measured by zymography) was increased by T3 treatment, but the activity did not differ between DNTR and empty vector transfected cultures (Fig. 2B). If cell death occurred by the murder mechanism, a soluble death-inducing factor such as stromelysin-3 and collagenase-3 should kill even DNTR-overexpressing cells. However, our findings show that these cells are not killed by T3-induced secreted proteins, thus supporting a cell-autonomous mechanism for tadpole tail muscle cell death (i.e., suicide).

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Figure 1. Comparison of the murder model and the suicide model. Circles drawn by a dotted line mean dead cells. Blue, dominant-negative thyroid hormone receptor (DNTR) -transfected cells; yellow, nontransfected cells; green arrows, secretion of death factor; red arrows, murder by death factor.

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Figure 2. Overexpression of dominant-negative thyroid hormone receptor (DNTR) inhibits thyroid hormone (TH) -induced death of XLT-15-9 cells. A green fluorescent protein (GFP) reporter gene was cotransfected with a vector or DNTR expression construct into XLT-15-9 cells, cultured for 3 days, and incubated in the absence (closed columns) or presence (open columns) of 10 nM T3 for additional 3 days. A: The percentages of apoptotic round cells were determined in GFP-positive cells. Data are the means (n = 6 to 9). The result represents eight independent experiments. B: The gelatinolytic activity in culture media of the transfected cells (A) was analyzed by zymography (Kinoshita et al., 1998). The result represents three independent experiments.

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Exogenous Gene Transfer Into Tail Muscle Cells In Vivo

One of the main differences between the cultured myoblastic cell line and muscle cells in the living tadpole is the existence of ECM in vivo that surrounds cells. Thus, although the above data indicate that TH is capable of inducing cell-autonomous death in vitro, it remains to be determined whether this is the in vivo mechanism. To examine whether muscle cell death is induced by suicide or murder in vivo, we have used the direct injection of exogenous genes into tail muscle cells of Xenopus laevis (de Luze et al., 1993). Tadpoles were injected with a β-galactosidase expression construct and killed 2 days later for the analysis. Every β-galactosidase-expressing cell was long rectangular and arranged along the anterior–posterior axis within each myomere (Fig. 3A). The immunohistochemical analysis using anti-tropomyosin antibody revealed that β-galactosidase was expressed specifically in muscle cells (Fig. 3B–D).

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Figure 3. Muscle cells remain in the regressing dominant-negative thyroid hormone receptor (DNTR) -injected tails during the metamorphosis. A–D: The injected reporter gene is expressed only in the muscle cells. A total of 100 ng of a β-galactosidase reporter gene was injected into tails of stage 56 tadpoles. Tadpoles were killed 2 days later. A: The lateral view of the tail after the β-galactosidase staining. B–D: The serial cross-sections of the tadpole tail. Muscle cells are identified by staining with anti-tropomyosin antibody TM311 (C,D). Muscle cells with β-galactosidase activity in D are surrounded by dotted lines in C. E: The overexpression of β-galactosidase did not interfere with PCD in a tail. One microgram of a β-galactosidase reporter gene was injected to the ninth myomere in the tail of a stage 57 tadpole without an electric square pulse. The tadpole was raised to stage 62 and killed. The 1 and 2 indicate healthy muscle cells with and without β-galactosidase activity, respectively; the 3 and 4 indicate apoptotic muscle cells with and without this activity, respectively. The result is representative of three independent experiments. F: A stage 58 tadpole just after DNA injection. An arrow indicates an injection site. G: A DNA-injected tadpole (stage 64) just before killing. H–M: β-Galactosidase activity was visualized. H,I: A total of 100 ng of a β-galactosidase reporter gene was injected into tails of stage 59–60 tadpoles. Tadpoles were raised to stage 61 (H) or 64 (I) and killed. The transverse sections were cut at 15 μm. The arrowheads point to the spinal cord. The small arrows in H point to muscle cells with β-galactosidase activity. Notice the size difference between H and I. J–M: Stage 58–61 tadpoles were injected with 10 ng of the β-galactosidase reporter gene and 50 ng of an expression construct encoding Xenopus Bcl-XL (J,K) or DNTR (L,M), raised to stage 59 (J) or 64 (K–M), and killed. The tails were amputated, transverse sections were serially cut at 15 μm, and every sixth section was examined. M: A higher magnification view of L. Muscle cells were identified by visual inspection of the cross-striated structure composed of myofibrils with a Zeiss Axioskop differential interference microscope. NC, notochord; SC, spinal cord. Scale bars = 0.1 mm in A–E, H–M.

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A stage 57 tadpole was injected with a β-galactosidase gene and killed at stage 62. β-Galactosidase-expressing cells also underwent apoptosis as nontransfected cells did (Fig. 3E), suggesting that the overexpression of β-galactosidase does not have any effect on PCD in the regressing tail.

To show the morphology of a PCD-inhibited muscle cell in the regressing tail, a cDNA encoding Xenopus laevis Bcl-XL was cloned and its expression construct was coinjected with a β-galactosidase reporter gene into tails of stage 58–61 tadpoles (Fig. 3F). Bcl-XL is known to repress apoptosis (Boise et al., 1993). These tadpoles were killed at stage 59 (Fig. 3J) or 64 (Fig. 3G,K). Round muscle cells with β-galactosidase activity remained at stage 64 when almost all muscle cells disappear in a tail (Fig. 3I), indicating that the overexpression of Bcl-XL repressed PCD of muscle in vivo.

Individual muscle cells are surrounded by the basement membrane, which is composed mainly of type IV collagen, heparan sulfate proteoglycan, laminin, and entactin. On the other hand, ECM-degrading enzymes such as stromelysin-3 and collagenase-3 are expressed in tail at the climax of the metamorphosis (stage 59–63; Wang and Brown, 1993). To examine the developmental change of ECM during tail regression, tails were analyzed by immunohistochemical analysis (Fig. 4). Type IV collagen was observed not only in the basement membrane within the skin and between muscle cells but also around the spinal cord and in the notochord lamella. As the tail regressed after stage 62, type IV collagen seemed to assemble and fill in the space of dead muscle cells and occupied the area between the skin and spinal cord at stage 64. Tail shortening might be caused by the extinction of muscles and shrinking of basement membrane as well as by contractility of the dorsal and ventral cords after the collapse of the notochord (Elinson et al., 1999). Surviving muscle cells in Bcl-XL-coinjected tails might float in ECM containing type IV collagen (Fig. 3K).

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Figure 4. The developmental change of the distribution of type IV collagen. Tails around the fifth myomere from the base were sectioned. These sections were subjected to the immunohistochemistry using anti-type IV collagen antibody. The arrowheads point to the spinal cord. NC, notochord. Scale bar = 0.1 mm.

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DNTR Overexpression Delays Muscle Cell Death in the Regressing Tail During the Spontaneous Metamorphosis

To inhibit the TH signaling in tail muscle cells, tadpoles were coinjected with the β-galactosidase gene and the DNTR expression construct just before the beginning of the tail regression (stage 58–61; Fig. 3F) and killed at stage 64 (Fig. 3G). Muscle cells with β-galactosidase activity remained in ECM containing type IV collagen in tails that had almost resorbed (Fig. 3L,M), whereas β-galactosidase-expressing cells were not observed in tails of 12 tadpoles and 19 tadpoles that were coinjected with a vector and the luciferase expression construct as negative controls, respectively (Table 1). When a β-galactosidase reporter gene was coinjected with both DNTR and TR expression constructs, no muscle cells with β-galactosidase activity were found at stage 64. The result demonstrated that the PCD inhibition by means of DNTR overexpression is mediated specifically by blocking the TH signaling, because an injection of this amount of DNTR expression construct inhibited death of muscle cells with β-galactosidase activity. These data indicated that the suicide mechanism by TH plays an important role in muscle cell death during stage 58–64.

Table 1. Inhibition of Muscle Cell Death in Stage 58–64 by the Overexpression of DNTRa
 VectorbLuciferasebbc1-XLbDNTRbDNTR + vectorcDNTR + TRc
  • a

    Data represent the number of tadpole tails with (Surviving) or without (Dead) β-galactosidase–expressing muscle cells. When blue muscle cells were observed in the right (injected) side of a tail and any healthy cells did not remain in the left (noninjected) side, the tail was recognized as one containing the surviving transfected cells (Surviving). However, even if one muscle cell existed in the left side, this tail was neglected. When no blue muscle cell was left in the right side of a tail, it was considered as one that lost transfected cells (Dead).

  • b

    Tails of stage 58–61 tadpoles were injected with 10 ng of a β-galactosidase reporter gene and 50 ng of a vector, luciferase, Bc1-XL, or DNTR expression construct.

  • c

    50 ng of a β-galactosidase reporter gene and 10 ng of DNTR expression construct were coinjected with 50 ng of a vector or TRα expression construct into tails of stage 58–61 tadpoles.

Surviving0061040
Dead12198126

To confirm this idea, we have developed the GFP-reporter gene assay instead of the β-galactosidase assay to follow the process of PCD in tails of living tadpoles. Tails of stage 57–58 tadpoles were injected with the GFP expression construct and subject to the time-lapse analysis under a fluorescent microscope. GFP-positive cells shrank in vector-coinjected tails, and their fluorescence decreased dramatically at stage 63 when a tail resorbs rapidly (Fig. 5A,C). The prompt decrease of fluorescence intensity at stage 63–64 reflects muscle cell death and digestion after phagocytosis, because a developmental loss of cellular fluorescence occurred in an “all or nothing” manner (Fig. 6A), and the ratio of apoptotic area to total muscle area becomes maximum at these stages (Nishikawa and Hayashi, 1995).

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Figure 5. The overexpression of dominant-negative thyroid hormone receptor (DNTR) delays programmed cell death during the climax of metamorphosis. A: A total of 50 ng of a green fluorescent protein (GFP) reporter gene was coinjected with 50 ng or 300 ng of an expression construct encoding no protein or DNTR into tails of stage 57–58 tadpoles. The numbers between the panels indicate the number of days required for the development. White lines trace contours of regressing tails other than fins. Note that every muscle cell changed its shape at stage 63. B: A GFP reporter gene and DNTR expression vector were injected into alternate tail myomeres of stage 57 tadpole. Note that GFP-negative myomeres between GFP-positive ones have shrunk at stage 63. C: The quantification of the GFP signal was carried out by calculating a ratio of a fluorescent intensity to the maximum value of each tadpole during the climax of metamorphosis. Data are the mean ± SE (n = 4 to 18). D: A ratio of the number of tails with GFP-positive muscle cells to the total number of injected tails is shown (n = 4 to 24). E–P: Stage 57 tadpoles were injected with 50 ng of a GFP reporter gene and the DNTR expression construct (E–J and N–P) or a vector (K–M) and raised to stage 66 (E–J), 62 (K–M), or 63 (N–P). The transverse sections of GFP-positive regions were serially cut at 15 μm. Muscle cells were stained (red) with TM228 (F), TM311 (I), and anti-active caspase-3 antibody (L,O). The arrowheads point to the spinal cord. h, hind limb; r, rectum. Similar results were obtained in two (E–J), two (K–M), and five (N–P) independent experiments. Scale bars = 1 mm in A,B, 0.1 mm in E–P.

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Figure 6. The overexpression of dominant-negative thyroid hormone receptor (DNTR) inhibits programmed cell death (PCD) from stage 57 to stage 62. A total of 50 ng of a green fluorescent protein (GFP) reporter gene and an expression construct encoding no protein or DNTR were injected into tails of stage 52–56. A: PCD of muscle in a vector-coinjected tail has begun at stage 57. A muscle cell that is indicated by a white arrowhead in early stage 57 tadpole has disappeared at late stage 57. The numbers between the panels show the number of days required for development. B: The GFP signal was quantified by calculating a ratio of a fluorescent intensity to the value of stage 57 in each tadpole. Data are the mean ± SE (n = 13 to 17). C: Model for the mechanism of muscle cell death in the tail. At stage 63, the notochord (blue) is degenerated and muscle cells (yellow) and ECM (red) change the shape and distribution, respectively. See text for details. Scale bar = 1 mm in A.

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Because all muscle cells rounded at the same time (Fig. 5B), this morphologic change might result from a loss of the tension by the tail shortening. Another possibility is that TH induces the expression of MMPs, which leads to the degradation of ECM between muscle cells. Although type IV collagen still remained at stage 64 (Fig. 4), we cannot exclude a possibility that the collagen was digested, but only its antigenicity was maintained.

Stage 57–58 tadpoles were injected with the DNTR expression construct and GFP gene and analyzed. DNTR expression retarded PCD by a few days, but approximately 70% of GFP fluorescence disappeared from stage 62 to stage 64 (Fig. 5A–C). The former supports the suicide model, but the latter favors the hypothesis that cell death is promoted by changes of extracellular environment such as a loss of the cell's tension and the degradation of ECM (the murder model).

Approximately 10% of GFP fluorescence remained at the end of metamorphosis (Fig. 5C). Furthermore, GFP-positive cells were found even 3 weeks after it in all DNTR-injected tails and then faded away, whereas control GFP-expressing muscle cells were almost extinguished at stage 64 and completely at stage 65 (Fig. 5D). These observations support the suicide model. One explanation for the survival of muscle cells by the DNTR overexpression is that the GFP-positive cells are slow peripheral muscles or cords that persist until the very end of tail resorption and express a slow muscle-specific tropomyosin (Elinson et al., 1999). If these TH-resistant cells express DNTR, they are expected to become unresponsive to TH. However, it appears to be inconsistent with the observation that most GFP-positive cells after the metamorphosis still expressed a muscle-specific protein, tropomyosin, but not a slow muscle-specific tropomyosin (Elinson et al., 1999; Fig 5E–J). The second possibility is that GFP-positive surviving cells expressed more DNTR. It seems unlikely, because the injection with 50 ng and 300 ng of the DNTR expression construct resulted in the similar rate of survival cells (Fig. 5A,C). The third possibility is that all DNTR-expressing cells survive and that a contraction of the cell volume is responsible for the fluorescence reduction. The apparent size of muscle cells did not change significantly in cross-sections of stage 64 tails (Fig. 3J–L), although tail muscle cells rounded. Moreover, at least 50 GFP-positive cells were observed at stage 62 in a DNTR-injected tail, but less than 10 cells were detected at stage 66 in photographs of both the whole tail and the cross-sections (data not shown). Once muscle cells survive the climax of the metamorphosis by chance, they could remain for a long period, because endogenous TH drops to 10% of its peak level after the metamorphosis (Leloup and Buscaglia, 1977).

A DNTR-injected tail was examined at stage 63 by the immunohistochemistry using anti-active caspase-3 antibody (Fig. 5N–P). The result demonstrated that caspase-3 was activated in some GFP-positive cells. This observation shows that apoptosis is responsible for the fluorescence reduction in DNTR-injected tails at stage 63–64 as well as that in vector-injected tails at stage 62–63 (Fig. 5K–M).

DNTR Overexpression Inhibits Muscle Cell Death Before the Tail Regression

To examine when the muscle cell death starts, the GFP expression construct was injected into younger tadpole tails (stage 52–56) with the expression vector (Fig. 6A,B). A decrease of the fluorescence was observed without any morphologic changes of the remaining cells after stage 57 when the endogenous T3 becomes scarcely detectable in plasma (Leloup and Buscaglia, 1977) and type II iodothyronine deiodinase (activating enzyme of thyroxine) is not expressed in tail (Huang et al., 2001). Some GFP-positive cells disappeared between early and late stage 57. This observation indicates that muscle cell death in a tail begins at stage 57 and that a drastic morphologic change is not required for PCD.

The strong inhibition of the fluorescence reduction from stage 57 to stage 62 was observed in the DNTR-coinjected tails (Fig. 6A,B), showing that the overexpression of DNTR repressed PCD. This result corroborated that PCD of muscle is dependent on the cell-autonomous TH signaling and executed by the suicide mechanism before the tail regression.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Tail Muscle Cells Start PCD at Stage 57 in Response to TH

The rapid growth and differentiation of hindlimb are observed in prometamorphosis (stage 55–57), but only when pharynx resorption is almost complete (stage 62) does the tail length decrease (Dodd and Dodd, 1976). The first ultrastructural changes in muscle of the regressing tail are detectable 3 days (stage 59–60) after the onset of metamorphosis, before any measurable reduction in tail length occurs (Weber, 1964). Apoptotic bodies first appeared in early climax (stage 59) when the T3 level is quite high (Nishikawa and Hayashi, 1995). However, our result showed muscle cell death at stage 57 when T3 is detectable at first (0.89 nM; Leloup and Buscaglia, 1977). Although the thyroxine concentration is 3.7 nM at this stage, T3 could not be generated from thyroxine in tail, because the tail has almost no mRNA of type II iodothyronine deiodinase (St. Germain et al., 1994; Huang et al., 2001). Therefore, tail muscle cells dying at this stage should be very sensitive to T3. This idea is supported by our previous report that a tail-derived myoblastic cell line can respond to 0.3 nM of T3 (Yaoita and Nakajima, 1997).

Treatment at late stage 57 or 58 with the goitrogen methimazole (an inhibitor of TH synthesis) produces feeding frogs that retain their muscle-less tail (Elinson et al., 1999). A tailed frog created by overexpressing type III iodothyronine deiodinase (inactivating enzyme of TH) also has a similar tail (Huang et al., 1999). Consistent with our results, these reports suggest that fast muscle cells, the majority of muscle, in a tail are as sensitive as most of the other organs and that the rest of a tail are resistant to T3. Both the methimazole treatment and the overexpression of type III iodothyronine deiodinase might decrease the intracellular concentration of T3 so that a tail, except fast muscle, can remain even after the end of the metamorphosis.

Tail Muscle Cells Die in a DNTR-Sensitive Manner Before the Tail Regression

Muscle cell death in tails of stage 57–62 tadpoles was almost inhibited by the overexpression of DNTR, which blocks the TH signaling. This result indicates that PCD occurs cell autonomously by TH, which is supported by the observation that the overexpression of DNTR repressed the TH-induced death of a tail-derived myoblastic cell line (XLT-15-9). Because any gross morphologic changes were not observed before a loss of a cellular fluorescence, an individual muscle cell might die without rounding, or with rounding for the very short period. In the process of dissolution of muscle cells, the occurrence of longitudinal clefts between myofibrils is followed by fragmentation of the cytoplasm into several apoptotic bodies (oval fragmented muscle fibers; Fig. 3E; Kerr et al., 1974). Many apoptotic bodies were observed in the long rectangular area like a muscle cell but not in the roundish area at stage 61 (data not shown). Muscle cell death might be executed by the suicide mechanism without rounding before stage 62.

A recent communication (Das et al., 2002) reports that fast tail muscle has cell-autonomous death induced by TH. It is shown that fast tail muscle is protected from TH-induced death in transgenic tadpoles that express DNTR exclusively in muscle cells. This observation is consistent with the TH-induced apoptosis of a tail-derived myoblastic cell line (Yaoita and Nakajima, 1997). Because every tail muscle cell expresses DNTR in transgenic tadpoles, it is not clear in a physiological condition whether muscle cells die in a cell-autonomous manner or they kill each other by a death-inducing factor. Our assay is able to discriminate these possibilities, because transfected cells are the minority, and, in the murder model, killed by a death-inducing factor that the majority of nontransfected cells secrete (Fig. 1).

Muscle Cell Death Occurs Irrespective of the Overexpression of DNTR at Stage 63–64

DNTR-overexpressing cells in tail could survive at stage 61–62 when endogenous TH reaches a peak (Leloup and Buscaglia, 1977), but approximately 80% of them disappeared until the end of metamorphosis. The overexpression of DNTR delayed the death of tail muscle cells, which indicates the contribution of a suicide type of muscle cell death. The suicide mechanism during tail regression is also supported by the observation that some of GFP-positive muscle cells survived in all DNTR-coinjected tails a few weeks after the metamorphosis, whereas GFP-positive cells were extinguished at stage 65 in all vector-coinjected tails (Fig. 5A–D). This observation means that a loss of tension by tail regression and the degeneration of ECM due to the increasing expression of MMPs is clearly insufficient for the death of all tail muscle cells.

However, the overexpression of DNTR could not protect most tail muscle cells from PCD at stage 63–64. Furthermore, there was no difference between tails injected with 50 ng and 300 ng of the DNTR expression construct, suggesting that even the saturated expression of DNTR cannot inhibit PCD. Some extracellular environmental changes during the climax of the metamorphosis might force muscle cells to die. Stromelysin-3 and collagenase-3 are highly expressed in subepidermal fibroblast encircling the entire muscle flank (Berry et al., 1998b), and the functional inhibition of the stromelysin-3 by antibody blocks TH-induced ECM remodeling and apoptosis of the larval epithelium in the intestine (Ishizuya-Oka et al., 2000). Gelatinase A is also up-regulated in subepidermal fibroblasts of tail at stage 63–64 (Jung et al., 2002) and can cleave type IV collagen, one of the main components of the basement membrane surrounding each muscle cell. High-level expression of secreted ECM-degrading enzymes in the subepidermal fibroblasts could lead to widespread ECM loss within the muscle flank, which induces PCD of muscle (Berry et al., 1998b). The molecular mechanism of murder remains to be elucidated. Our data indicate that both suicide and murder mechanisms mediate tail muscle cell death at stage 63–64.

Model for the Mechanism of Muscle Cell Death in the Tail During the Metamorphosis

In conclusion, we propose a model to explain the molecular mechanism of tail muscle cell death during spontaneous metamorphosis (Fig. 6C). Tail muscle cells start apoptosis at stage 57 in response to a scarcely detectable level of T3 by the suicide mechanism (cell autonomously) without any morphologic changes. This type is the DNTR-sensitive cell death. When many muscle cells are absorbed after death and a notochord is degenerated, a tail regresses rapidly in stage 62–64 by contraction of cords and shrinkage of ECM, and all muscle cells become round and die simultaneously by both the murder and suicide mechanisms. This process is only partially inhibited by the overexpression of DNTR (the DNTR-resistant cell death). What is a suicide gene and how the murder is executed remain to be elucidated.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Expression Constructs

The Xenopus DNTRα cDNA was generated by PCR by using pBluescript-TRαA (gifts of Drs. A. Kanamori and D.D. Brown; Yaoita et al., 1990) by deleting 42 base pairs (14 amino acids) from the C-terminus (Ulisse et al., 1996). The DNTRα cDNA was cloned into the expression vector, pcDNA1/Amp (Invitrogen).

Transient Transfection Assay

XLT-15-9 (a subline of XLT-15-11; Nakajima et al., 2000) was used and maintained as described previously (Yaoita and Nakajima, 1997). XLT-15-9 cells were transfected with 0.1 μg of the green fluorescent protein (GFP) expression vector pEGFP-N2 (Clontech) as a reporter gene and 0.1 μg of pcDNA1/Amp or the DNTR gene expression constructs using 3 μl of FuGENE-6 transfection reagent (Roche Molecular Biochemicals) according to the instructions of the manufacturer. Apoptotic round cells were identified by visual inspection of GFP-positive cells (Nakajima et al., 2000).

Zymography

Gelatinolytic activity was detected as described elsewhere (Kinoshita et al., 1998). A total of 10 μl of culture medium was incubated with 5 μl of 10 mM phosphate buffer, pH 7.0, containing 6% sodium dodecyl sulfate, 0.02% bromphenol blue, and 30% glycerol at 37°C for 30 min. Samples were loaded in a 10% polyacrylamide gel impregnated with 1.6 mg/ml gelatin. After electrophoresis, the gel was washed twice in 2.5% Triton X-100 and incubated in 50 mM Tris-HCl, pH 8.0, containing 5 mM CaCl2 at 37°C for 2 days. Bands containing gelatinases appear as clear bands in a dark blue background after staining with Coomassie brilliant blue R-250 (Polysciences, Inc.).

Cloning of Xenopusbcl-XL Gene

A Xenopus bcl-XL cDNA fragment was amplified by PCR using degenerate primers. Its full-length cDNA was obtained by screening a cDNA library of the stage 49 tadpole. The predicted amino acid sequence of Xenopus Bcl-XL (DDBJ accession no. AB055494) contained eight different amino acid residues compared with that of Xenopus xR11 (Cruz Reyes and Tata, 1995).

Microinjection of DNA Into Muscle Cells of the Tadpole Tail

The β-galactosidase, luciferase, Xenopus bcl-XL, DNTR, and TRα genes were cloned into the expression vector pcDNA1/Amp containing 5′ and 3′ untranslated regions of a Xenopus β-globin gene (Liman et al., 1992). Microinjection of DNA was performed as described previously (de Luze et al., 1993) with the following modifications. A 0.5-μl aliquot containing expression constructs was injected into the fifth and sixth myomeres from the base of a tail, and the electric square pulses were applied to the injected site for the loading period of 200 msec/pulse at 9 V/2 mm three (stage 54–56) or five times (stage 57–61) in a few seconds (Muramatsu et al., 1997). Administration of the electric pulse enhanced the expression of the injected gene (data not shown). The cotransfection efficiency was determined by coinjection of pEGFP-N2 and pDsRed1-N1 (Clontech) expression vectors, which express green and red fluorescent proteins, respectively. The inspection of the whole tails and the cross-sections revealed that every muscle cell with a red fluorescent protein also had green fluorescence. But red fluorescence was not observed in approximately 5% of cells that express a scarcely detectable level of green fluorescent protein, which might be caused by low intensity of red fluorescence (data not shown). Developmental stages followed the criteria of Nieuwkoop and Faber (Nieuwkoop and Faber, 1956).

Immunohistochemistry

The primary antibodies were a mouse monoclonal anti-tropomyosin antibody TM228 (Sigma) diluted 1/1,000, a mouse monoclonal anti-tropomyosin antibody TM311 (Sigma) diluted 1/400–3,000, and a rabbit monoclonal antibody against the active form of caspase-3 (BD Pharmingen) diluted 1/200. TM311 stains all muscle fibers (Nishikawa and Hayashi, 1994), whereas TM228 recognizes slow muscle (Elinson et al., 1999). The secondary antibodies were anti-mouse and anti-rabbit IgG goat polyclonal antibodies conjugated with Alexa Fluor 568 (Molecular Probes) that were diluted 1/320 and 1/200, respectively, and anti-mouse IgG goat polyclonal antibody conjugated with peroxidase (Cappel) diluted 1/100.

Immunohistochemistry for type IV collagen was performed as described previously (Utoh et al., 2000) with the following modifications. The rabbit polyclonal anti-mouse type IV collagen antibody (LSL Co., Ltd.) and Cy3-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, Inc.) were diluted in 1/1,000 and 1/400, respectively.

Quantification of the Fluorescent Intensity by Expressed GFP in Tadpole Tail

Tadpoles were anesthetized, and the GFP expression pattern was photographed under a fluorescent dissecting microscope (MZ FLIII, Leica) equipped with a color chilled 3CCD camera C5810 (Hamamatsu Photonics). The fluorescent signal of the photographs over the threshold was extracted by a routine of Adobe Photoshop 6.0.1 (Adobe Systems). The fluorescent intensity was quantified as the sum of brightness of extracted pixels using a routine of matrix calculator 2.3.1 (http://www.akita-noken.go.jp/provide/mc/index_mcF.html). The samples with lower intensity than the threshold were eliminated.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank Dr. A. Kanamori (Nagoya University, Japan) and Dr. D.D. Brown (Carnegie Institution, Baltimore) for the Xenopus TRα cDNA, and Dr. K. Yoshizato (Hiroshima University, Japan) for critical comments.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES