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).
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).
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).
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
| ||Vectorb||Luciferaseb||bc1-XLb||DNTRb||DNTR + vectorc||DNTR + TRc|
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).
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.