Transgenic Bax gene efficiently induces lethality in mouse early embryos

Abstract

Apoptosis is an essential physiological process involved in embryonic development, immune responses, and tissue homeostasis. Despite many studies on pro-apoptotic genes, few reports have directly compared the lethality-inducing potential between them under comparable conditions. In this study, we evaluated the lethality-inducing potential of three representative pro-apoptotic genes, Bax, Casp3, and Casp9, in mouse early embryos under defined conditions using the doxycycline (Dox)-inducible tetracycline-regulated gene expression system in combination with the PiggyBac transposon system. All genes were transcriptionally induced by Dox, and Bax showed the strongest lethal effect, followed by Casp9, while Casp3 did not show any effects. Notably, Bax expression severely impaired blastocyst formation and led to the intense accumulation of the DNA damage marker γH2AX, along with a pronounced increase in the apoptotic cells. These findings suggest that introducing upstream apoptotic regulators leads to the more efficient and widespread activation of the apoptotic cascade. Overall, this study is expected to contribute to a deeper understanding of apoptotic mechanisms and future advancements in regenerative medicine, reproductive engineering, and cancer research.

Apoptosis is a fundamental and essential physiological process in multicellular organisms, playing critical roles in embryonic development, immune responses, and the maintenance of tissue homeostasis. It is a highly regulated mechanism that eliminates unnecessary, aged, or damaged cells without inducing an inflammatory response [1,2,3,4].

Apoptosis is triggered by two major signaling pathways, the extrinsic and intrinsic pathways, both of which ultimately lead to the activation of executioner caspases, such as caspase-3 and caspase-7, resulting in cell death [1,2,3,4,5,6]. The extrinsic pathway is initiated when extracellular death signals, such as Fas ligand or TRAIL, bind to death receptors on the cell membrane, leading to the activation of caspase-8 via FADD and subsequently resulting in the activation of executioner caspases [7, 8]. In contrast, the intrinsic pathway is activated in response to intracellular stress signals, such as DNA damage, oxidative stress, or loss of growth factor [9]. In this pathway, B-cell lymphoma-2 (Bcl-2) family members such as Bax and Bak induce mitochondrial outer membrane permeabilization (MOMP), leading to the release of cytochrome c into the cytosol [10]. Released cytochrome c binds to apoptotic protease activating factor 1 (Apaf-1), which subsequently activates caspase-9. Activated caspase-9 then triggers the activation of executioner caspases [9].

These recent advances in our understanding of the molecular mechanisms of apoptosis have led to increased efforts to intentionally “utilize” this system. Bax, caspase-3, and caspase-9 are central effector molecules that play crucial roles in apoptotic pathways; therefore, they have been frequently investigated in basic and applied studies [11,12,13,14,15,16,17,18,19,20]. Specifically, the overexpression of these genes to induce selective apoptosis in target cells has been widely explored. For example, the overexpression of Bax or caspase-3 has been shown to effectively induce tumor cell death, positioning this approach as a promising strategy for cancer therapy [21, 22]. In addition, caspase-9 overexpression has been demonstrated to induce apoptosis in primary cultures of anterior pituitary cells and HeLa tumor cells, suggesting its potential utility as a model toward understanding the pathological mechanisms of neurodegenerative or malignant diseases and toward developing new therapeutic strategies [23].

Thus, the strategic use of apoptotic pathways holds promising applications in the medical fields including cancer therapy and neurodegenerative diseases. Consequently, the ability to effectively induce apoptosis has become a key focus. In this context, identifying which pro-apoptotic genes can most efficiently trigger cell death is important for expanding its medical and related applications as well as for advancing our fundamental understanding of apoptotic mechanisms. However, few studies have directly compared the apoptotic potency of these genes under comparable experimental conditions.

Therefore, we evaluated the lethality-inducing potential of three representative pro-apoptotic genes: Bax, Casp3, and Casp9. To achieve this, we employed the doxycycline (Dox)-inducible tetracycline-regulated gene expression system (Tet-On system), which enables precise control of gene expression in mammalian cells [24, 25], together with the PiggyBac transposon system, a highly efficient and easy to use method for gene integration in mammalian cells [26,27,28,29]. By introducing either Bax, Casp3, or Casp9 into mouse zygotes using these two tools, and precisely controlling the expression of each pro-apoptotic gene in a Dox-dependent manner, we performed a comparative evaluation of their ability to induce cell lethality under comparable conditions.

This study, using mammalian zygotes as a model, provides a novel framework for comparing the lethality-inducing potential of pro-apoptotic genes under comparable conditions, thereby offering new insights into their apoptosis-inducing potential during the early developmental stages of mammalian embryos. In conventional models, such as cancer cells, the genetic background is often heterogeneous due to their differentiated state, and the genome is unstable, leading to cell cycle abnormalities. Additionally, the anti-apoptotic activity of cancer cells [30] limits our ability to compare and evaluate the effects of pro-apoptotic genes. Embryonic stem (ES) cells are also useful for reproducible apoptotic assays under controlled conditions [31]; however, they lack the developmental context of multicellular embryos and cannot fully reflect the spatial and temporal regulation of gene expression that occurs during normal embryogenesis. In contrast, zygotes possess pluripotency and undergo synchronous and well-ordered developmental processes, making them a suitable model for accurately evaluating the physiological effects of the introduced genes. By deepening our fundamental understanding of pro-apoptotic gene function, this research not only contributes to basic biology but also lays the groundwork for future applications such as reproductive engineering. Ultimately, these findings may support the development of foundational technologies for the medical fields including cancer therapy.

Materials and Methods

In vitro fertilization (IVF)

Female ICR mice (8–12 weeks old; Japan SLC, Shizuoka, Japan) were superovulated by intraperitoneal injection of 7.5 IU equine chorionic gonadotropin (eCG; ASKA Animal Health, Tokyo, Japan), followed by 7.5 IU human chorionic gonadotropin (hCG; ASKA Animal Health) 46–48 h later. At 14–16 h after hCG injection, the mice were euthanized by cervical dislocation, and cumulus oocyte complexes (COCs) were collected and placed in human tubal fluid (HTF) medium supplemented with 4 mg/ml bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) [32]. Male ICR mice (13–16 weeks old; Japan SLC) were euthanized by cervical dislocation, and spermatozoa were collected and cultured in HTF medium for at least 1 h for capacitation. The sperm suspension was then added to the fertilization droplets containing COCs at a final concentration of 1.0 × 10⁶ cells/ml, and the gametes were co-incubated for 2–3 h at 37°C in a humidified atmosphere containing 5% CO₂. After fertilization, the embryos were washed three times in KSOM supplemented with amino acids [33] and 1 mg/ml BSA to remove cumulus cells and excess sperm. Morphologically normal zygotes were used for subsequent microinjection procedures.

Plasmid construction and in vitro transcription (IVT) of hyPBase mRNA

The coding sequence (CDS) of Bax was amplified from total cDNA extracted from mouse ovaries by nested PCR using KOD-Plus polymerase (Toyobo, Osaka, Japan). Outer primers targeting the flanking regions and nested primers specific to the target region were designed to perform a two-step amplification. The amplified product was digested with NotI (New England Biolabs, Ipswich, MA, USA) and XbaI (New England Biolabs) and cloned into a pBluescript II SK (−) plasmid (Agilent Technologies, Santa Clara, CA, USA), which had been inserted with a FLAG tag (DYKDDDDK) and a linker sequence downstream of the T7 promoter (pBS-Bax). Ligation was performed using a Ligation High Kit (Toyobo). From this point onward, DNA fragments were assembled to generate each construct by using Gibson Assembly Master Mix (New England Biolabs). The pTet-One vector from the Tet-One Inducible Expression System (Takara Bio, Kusatsu, Japan) and Bax from pBS-Bax were amplified by PCR using KOD-Plus Neo polymerase (Toyobo), and assembled into pTet-One-Bax. A pAcGFP-membrane (mem) vector (Clontech, Mountain View, CA, USA) and an IRES fragment were used to construct IRES-mem-AcGFP. The pTet-One-Bax and IRES-mem-AcGFP fragments were amplified with KOD-Plus Neo, and IRES-mem-AcGFP was fused downstream of the Tet-On 3G (reverse tetracycline-controlled transactivator, rtTA) gene to generate pTet-One-Bax-IRES-mem-AcGFP. The inverted terminal repeat-flanked backbone, excluding the CAG-TagRFP sequence from pPB-CAG-TagRFP [34], and the genetic cassette of pTet-One-Bax-IRES-mem-AcGFP were each PCR amplified with KOD-Plus Neo and assembled to generate pPB-Tet-One-Bax-IRES-mem-AcGFP. The CDS of Casp3 and Casp9 were PCR amplified from total cDNA derived from mouse brain using KOD-Plus Neo and cloned into pBluescript II SK (−). Subsequently, the pPB-Tet-One-Bax-IRES-mem-AcGFP sequence, excluding the Bax region, and Casp3 or Casp9 sequence were each PCR amplified with KOD-Plus Neo and fused to construct pPB-Tet-One-Casp3-IRES-mem-AcGFP and pPB-Tet-One-Casp9-IRES-mem-AcGFP, respectively. To generate pPB-Tet-One-(no gene)-IRES-mem-AcGFP, the pPB-Tet-One-Casp3-IRES-mem-AcGFP sequence, excluding the Casp3 region, was amplified using a KOD-Plus-Mutagenesis Kit (Toyobo) and self-ligated. Capped and polyadenylated hyPBase mRNA was synthesized in vitro using an mMESSAGE mMACHINE T7 Ultra Kit (Thermo Fisher Scientific, Waltham, MA, USA) from pCAG-hyPBase [29], and purified with an RNeasy Mini Kit (Qiagen, Hilden, Germany). The resulting mRNA was resuspended in nuclease-free water. The primer sequences used for plasmid construction and IVT are listed in Supplementary Table 1.

Plasmid microinjection, Dox treatment, and embryo culture

Approximately 3–5 pl of a mixture containing 30 ng/µl hyPBase mRNA and 30 ng/µl of either pPB-Tet-One-(no gene)-IRES-mem-AcGFP, pPB-Tet-One-Bax-IRES-mem-AcGFP, pPB-Tet-One-Casp3-IRES-mem-AcGFP, or pPB-Tet-One-Casp9-IRES-mem-AcGFP was microinjected into the cytoplasm of each zygote at 3–6 h post-insemination (hpi) [27]. After microinjection, morphologically normal embryos with two pronuclei were collected from each group and divided for culture in KSOM supplemented with (Dox+) or without (Dox−) 100 ng/ml Dox [35] from 6 to 96 hpi. To assess the effect of Dox on embryonic development, uninjected embryos were also cultured under the same conditions. All embryos were incubated at 37°C in a humidified atmosphere containing 5% CO₂.

RNA extraction and reverse transcription-quantitative PCR (RT-qPCR)

By using a QuantAccuracy™ RT-RamDA™ cDNA Synthesis Kit (Toyobo), total RNA was extracted from 5 embryos in each experimental group and reverse transcribed into cDNA. For single-embryo analysis, the same kit was used to extract total RNA and synthesize cDNA from individual embryos. Quantitative PCR was carried out using the synthesized cDNA as a template with gene-specific primers and KOD SYBR qPCR Mix (Toyobo). Amplification and quantification of gene transcripts were conducted following a previously established protocol [36]. H2afz was used as an internal control. Relative expression levels were determined using the comparative Ct method (2^−ΔΔCt) [37]. For the correlation analysis between transgene-derived GFP fluorescence intensity and the expression levels of the introduced pro-apoptotic genes, gene expression level was calculated as the ratio of each target gene to the internal control gene H2afz, based on ΔCt values. The primer sequences used for the RT-qPCR are listed in Supplementary Table1.

Apoptosis detection by γH2AX immunofluorescence

To evaluate apoptosis, the embryos were fixed with 4% paraformaldehyde (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) in phosphate-buffered saline (PBS) for 20 min at 28°C after removal of the zona pellucida with acid Tyrode’s solution (pH 2.5). Subsequently, the embryos were permeabilized in 0.5% Triton X-100 (Sigma-Aldrich) in PBS for 40 min at 28°C. After permeabilization, the embryos were blocked in PBS containing 1.5% BSA, 0.2% sodium azide (FUJIFILM Wako Pure Chemical Corporation), and 0.02% Tween 20 (Nacalai Tesque, Kyoto, Japan) (blocking buffer) for 1 h at 28°C, and then incubated overnight at 4°C with a mouse anti-H2A.X (Ser139) antibody (1:200 dilution; 613401; BioLegend, San Diego, CA, USA). The embryos were washed in blocking buffer and incubated for 1 h at 28°C with an Alexa Fluor 647 donkey anti-mouse IgG (H+L) secondary antibody (1:500 dilution; A31571; Thermo Fisher Scientific). After washing, the embryos were stained with 10 μg/ml Hoechst 33342 (Sigma-Aldrich) in blocking buffer for 20 min at 28°C. Finally, the embryos were mounted on glass slides and observed using a fluorescence microscope (IX73; Olympus, Tokyo, Japan). The fluorescence intensities of γH2AX in embryos was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). First, the embryo region in the image was selected, and the mean fluorescence intensity (Mean Gray Value) within this region was measured. Simultaneously, the area (Area) of the selected region was measured, and the integrated density (Integrated Density) was calculated by multiplying the mean intensity by the area.

Apoptosis detection by TUNEL

To further evaluate apoptosis in embryos, we performed the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay. Briefly, embryos were fixed in 4% paraformaldehyde (FUJIFILM Wako Pure Chemical Corporation) in phosphate-buffered saline (PBS) for 20 min at 28°C and permeabilized in PBS containing 0.5% Triton X-100 (Sigma-Aldrich) for 20 min at 28°C. After washing with PBS containing 5 mg/ml of polyvinylpyrrolidone (PVP), the embryos were incubated for 1 h at 37°C in 50 µl droplets of TUNEL reaction mixture (In Situ Cell Death Detection Kit; Roche, Basel, Switzerland). For negative controls, the TdT enzyme was omitted from the reaction mixture, and positive controls were generated by treating embryos with 100 U/ml DNase I (Nippon Gene, Tokyo, Japan) for 30 min at 37°C prior to the TUNEL reaction. After the TUNEL incubation, the embryos were stained with 10 µg/ml Hoechst 33342 (Sigma-Aldrich) in PBS with 5 mg/ml PVP for 20 min at 28°C. Finally, the embryos were mounted on glass slides and observed using a fluorescence microscope (IX73; Olympus). For each embryo, the total number of nuclei and the number of TUNEL-positive nuclei were manually counted using ImageJ software (National Institutes of Health), and the apoptotic index was calculated as the proportion of TUNEL-positive cells relative to the total cell number. Cells were considered apoptotic when they exhibited TUNEL-positive signals together with characteristic apoptotic nuclear morphology, as described previously [38].

Statistical analysis

Data for RT-qPCR, GFP fluorescence, γH2AX signals, apoptotic index, and Bax/Bcl2 expression ratio were analyzed using Student’s t-test or the Mann–Whitney U test where the data were normally or non-normally distributed, respectively. The normal distribution was assessed using the Shapiro-Wilk test. The correlation between GFP fluorescence intensity and expression levels of transgenes (Bax, Casp3, Casp9) in the Dox‑treated group was assessed using Pearson’s correlation coefficient. Developmental rates were analyzed using a chi-square test. The experimental groups were as follows: (1) non-injected (Non-injected) embryos; (2) gene-lacking construct-injected (Control) embryos; (3) Bax-injected (Bax) embryos; (4) Casp3-injected (Casp3) embryos; and (5) Casp9-injected (Casp9) embryos. For each group, statistical comparisons were made between the Dox− and Dox+ treatments, and P-values < 0.05 were considered statistically significant. This experimental design was chosen to evaluate clearly the impact of Dox-induced gene expression on development, while distinguishing it from the effects of Dox treatment itself and the effects of injection.

Ethical approval for the use of animals

All animal experiments were conducted with approval from the Animal Research Committee of Kyoto University (approval nos. R3–17, R4–17, R5–17, R6–17, and R7–17) and in strict compliance with the committee's ethical guidelines.

Results

Overexpression of pro-apoptotic genes in preimplantation embryos

To induce the overexpression of pro-apoptotic genes, plasmids carrying each gene (Bax, Casp3, or Casp9) were co-injected with hyPBase mRNA into the cytoplasm of mouse zygotes at 3–6 hpi, and the embryos were cultured in KSOM supplemented with (Dox+) or without (Dox−) 100 ng/ml Dox until 96 hpi (Fig. 1B). The expression of each pro-apoptotic transgene was analyzed at the blastocyst stage. The expression of all transgenes was significantly increased in embryos in Dox+ compared with those in Dox− (Fig. 2A). These results confirm that the Tet-On system introduced via the PiggyBac transposon system functioned properly, enabling the Dox-inducible overexpression of pro-apoptotic transgenes.

Fig. 1.

Overview of the experimental procedure. (A) Schematic diagram of a PiggyBac-based all-in-one vector system designed for Dox-inducible gene expression. The construct contains a gene of interest under the control of the TRE3GS promoter, which is activated upon Dox-inducible binding of the Tet-On 3G (rtTA) protein. The Tet-On 3G gene is constitutively expressed under the PGK promoter. The IRES-mem-AcGFP cassette allows for bicistronic expression. PB5′TR and PB3′TR are the terminal repeats required for PiggyBac transposase. (B) Schematic diagram of the experimental design. The embryos were microinjected with hyPBase mRNA and each plasmid, cultured in the presence or absence of Dox, and analyzed for gene expression, developmental rate, and γH2AX signal.

Fig. 2.

Verification of gene introduction and expression levels. The experimental groups were as follows: (1) non-injected (Non-injected) embryos; (2) gene-lacking construct-injected (Control) embryos; (3) Bax-injected (Bax) embryos; (4) Casp3-injected (Casp3) embryos; and (5) Casp9-injected (Casp9) embryos. These experimental groups are indicated at the top of each panel. (A) RT-qPCR of each pro-apoptotic gene (Bax, Casp3, and Casp9), rtTA, Cdx2, and Gata6 in each group was performed using 5 embryos at the blastocyst (96 hpi) stage. Gene expression levels were normalized to H2afz as an internal control. Data are expressed as the mean ± standard error of the mean (n = 5). n.s., not significant, ^★P < 0.05, ^★★★★P < 0.0001, Student’s t-test or the Mann–Whitney U test for normally or non-normally distributed data, respectively. (B) GFP expression in mouse preimplantation embryos in each experimental group at 96 hpi. Scale bar, 100 µm. (C) Box-and-whisker plots depicting the distribution of GFP fluorescence intensity in individual embryos, with the mean indicated by a × symbol. Control (Dox−), n = 11; Control (Dox+), n = 11; Bax (Dox−), n = 13; Bax (Dox+), n = 13; Casp3 (Dox−), n = 14; Casp3 (Dox+), n = 14; Casp9 (Dox−), n = 15; Casp9 (Dox+), n = 15. ^★P < 0.05, ^★★P < 0.01, ^★★★★P < 0.0001, Mann–Whitney U test. (D) Scatter plot showing the correlation between GFP fluorescence intensity and expression levels of transgenes (Bax, Casp3, Casp9) in the Dox‑treated group. Each point corresponds to an individual embryo. Bax, n = 23; Casp3, n = 23; Casp9, n = 24. R and P value were calculated via Pearson’s correlation coefficient analysis.

Simultaneously, we attempted to verify the presence of the genomic insertion by monitoring GFP fluorescence, which was placed downstream of the Tet-On 3G (rtTA) gene regardless of Dox treatment (Fig. 1A). Because rtTA gene expression is considered to be constitutive regardless of Dox treatment [24, 25], comparable levels of GFP fluorescence were expected between the Dox− and Dox+ groups. However, surprisingly, GFP fluorescence intensity was significantly higher in Dox+ embryos than in Dox− embryos in all groups: Control, Bax, Casp3, and Casp9 (Fig. 2B, C). To confirm this unexpected observation, we quantified rtTA gene expression in the above groups. Consequently, rtTA gene expression levels were significantly elevated in Dox+ embryos in all groups. This result deviates from previously established findings [24, 25]. On the other hand, the expression of Cdx2 and Gata6, trophectoderm marker genes, remained unchanged, suggesting that the increased expression of rtTA was not due to abnormal global increases in gene expression, but rather was gene-specific and dependent on Dox treatment (Fig. 2A). Variability in GFP fluorescence in individual embryos observed in the Dox-treated groups (Fig. 2B, C) may reflect differences in transgene expression among embryos possibly due to copy number variation [27]. To evaluate the relationship between transgene copy number and their expression levels, GFP intensity for each embryo was used as a proxy for transgene copy number, because accurate quantification is challenging due to residual plasmid DNA remaining after microinjection. As a result, tending (Bax) or significant (Casp3 and Casp9) positive correlations were observed and some embryos showed sporadically higher GFP signals in Casp3 and Casp9 groups (Fig. 2C, D). However, most individual embryos’ GFP signals were concentrated around 70–90 (arbitrary units) in all groups (Fig. 2D).

Bax overexpression effectively induces apoptosis in mouse embryos

The effects of apoptosis-inducing genes on embryonic development were monitored in each group up to 96 hpi. Among the tested genes, the Dox-induced overexpression of Bax led to a significant reduction in the proportion of embryos reaching the blastocyst stage, indicating the strongest inhibitory effect on development among these three genes. The Dox-induced overexpression of Casp9 inhibited embryonic development to a certain degree, although its inhibitory effect was less pronounced than that of Bax. Embryos overexpressing Bax or Casp9 developed normally until the morula stage but showed lethality around the blastocyst stage under Dox treatment. In contrast, the Dox-induced overexpression of Casp3 had no effect on the developmental rate, showing similar outcomes to the Control embryos with no apparent developmental inhibition (Fig. 3, Table 1). Furthermore, in the Non-injected embryos, the developmental rate was similar between the Dox− and Dox+ conditions, indicating that Dox at a concentration of 100 ng/ml does not adversely affect embryonic development.

Fig. 3.

Embryonic development and apoptosis induction at the blastocyst stage. Developmental morphology of the embryos at the start of culture (6 hpi), at the morula stage (72 hpi), and at the blastocyst stage (96 hpi) in Dox− or Dox+. Scale bar, 100 μm.

Table 1. Developmental rate in the absence (−) or presence (+) of Dox for each treatment group.

To evaluate the cellular responses induced by the expression of these pro-apoptotic genes, we assessed DNA damage in blastocyst stage embryos using γH2AX immunostaining as a marker for apoptosis. Dox-induced Bax overexpression resulted in a robust increase in the γH2AX signal, reflecting widespread DNA damage. A moderate increase was observed in the Casp9 group. In contrast, the Casp3 group did not exhibit any significant differences in γH2AX signal intensity between the Dox− and Dox+ conditions, similar to the Non-injected and Control embryos (Fig. 4A, B).

Fig. 4.

Detection of apoptosis by immunofluorescence. (A) DNA damage in mouse preimplantation embryos at the blastocyst stage (96 hpi) detected by γH2AX immunostaining. The nuclei were counterstained using Hoechst 33342. Scale bar, 100 μm. (B) Box-and-whisker plots depicting the distribution of γH2AX immunofluorescence intensity in individual embryos, with the mean indicated by a × symbol. Non-injected (Dox−), n = 8; Non-injected (Dox+), n = 8; Control (Dox−), n = 10; Control (Dox+), n = 10; Bax (Dox−), n = 10; Bax (Dox+), n = 10; Casp3 (Dox−), n = 9; Casp3 (Dox+), n = 9; Casp9 (Dox−), n = 9; Casp9 (Dox+), n = 9. n.s., not significant, ^★P < 0.05, ^★★★P < 0.001, Student’s t-test or the Mann–Whitney U test for normally or non-normally distributed data, respectively.

Additionally, to confirm apoptosis using a functional apoptotic assay, TUNEL staining was performed for the Bax and Casp9 groups, in which significant increases in γH2AX signals had been observed. The analysis revealed that Dox-induced Bax overexpression caused a pronounced increase in the proportion of TUNEL-positive cells, whereas Casp9 overexpression did not show a significant increase, confirming the differential apoptotic effects of these genes (Fig. 5A, B).

Fig. 5.

Detection of apoptosis by TUNEL assay. (A) Apoptotic cells in mouse preimplantation embryos at the blastocyst stage (96 hpi) detected by TUNEL assay. The nuclei were counterstained using Hoechst 33342. Scale bar, 100 μm. (B) Apoptotic index (the proportion of TUNEL-positive cells relative to the total cell number) at the blastocyst stage. Data are expressed as the mean ± standard error of the mean. Bax (Dox−), n = 9; Bax (Dox+), n = 12; Casp9 (Dox−), n = 10; Casp9 (Dox+), n = 12. n.s., not significant, ^★★P < 0.01, Student’s t-test or the Mann–Whitney U test for normally or non-normally distributed data, respectively.

To further investigate the mechanism of apoptosis induction in the Bax group, which exhibited the most pronounced lethality, we measured the expression levels of Bax and Bcl2 in individual embryos by quantitative RT-PCR. Primers capable of detecting Total-Bax transcripts present in the embryos were used (primer information is provided in the Supplementary Table 1). As a result, the Bax/Bcl2 ratio was significantly elevated in Dox-treated embryos compared with the control group (Supplementary Fig. 1).

Discussion

This study was conducted to compare the lethality-inducing potential of three representative pro-apoptotic genes—Bax, Casp3, and Casp9—in mouse early embryos under comparable conditions, by combining the Tet-On system with transgenesis using the PiggyBac transposon system.

The variability in GFP fluorescence among Dox-treated embryos likely reflects mosaic integration and/or differences in transgene copy number, consistent with a previous report [27]. Indeed, there was a tendency or significant correlations such that the higher the GFP fluorescence, the higher the expression levels of the transgenes, but most GFP fluorescence levels were concentrated within a narrow range (70–90 in Fig. 2D). These results further indicate comparable transgene introduction efficiency, validating these embryos as a suitable model for comparing gene-specific effects on early development.

As a result, all three genes (Bax, Casp3, and Casp9) exhibited Dox-dependent upregulation at the transcriptional level; however, their effects on embryonic development and apoptosis were gene-dependent. Among the three genes tested, the induction of Bax expression most strongly inhibited embryonic development and markedly increased γH2AX signals and TUNEL-positive apoptotic cells, suggesting robust DNA damage and the induction of apoptosis. This is further supported by the significant increase in the Bax/Bcl2 ratio in Dox-treated embryos, as higher Bax/Bcl2 ratios reflect ongoing apoptosis and indicate stronger activation of the apoptotic pathway. In addition, although sporadically high GFP fluorescence was observed in the Casp3 and Casp9 groups, embryonic lethality was not as pronounced as in the Bax group. This result also suggests the highest pro-apoptotic effects of Bax among the genes tested. This may reflect the fact that Bax is an upstream molecule that directly initiates apoptosis by inducing MOMP [10], a key triggering event in the apoptotic pathway, consistent with a previous finding that Bax alone can trigger apoptosis in mammalian cells [39]. In contrast, caspase-3 and caspase-9 function downstream of Bax and require upstream activators for their activation. This may be a reason for their weaker inhibitory effects on embryonic development upon induction. In the case of Casp3, no impact was shown. The induction of Casp9 expression resulted in a certain degree of developmental inhibition, possibly because it acts upstream of caspase-3 and can activate not only caspase-3 but also caspase-7 and, indirectly, caspase-6 [40, 41], resulting in a stronger effect than that of Casp3 alone.

Interestingly, in all of the experimental groups in which the Tet-On system was introduced, the addition of Dox resulted in increased rtTA mRNA levels and GFP fluorescence. This was an unexpected result, given that rtTA is typically driven by a constitutive promoter and its expression is Dox-independent [24, 25]. The improved Tet-On system used in this study is an all-in-one vector [42, 43]. Therefore, feedback or crosstalk between its components may be involved, although the details remain unclear. Further analysis of the molecular mechanism is needed.

In this study, we used closed-colony ICR mice to perform the assays within a certain degree of genetic diversity; however, this methodology is also considered applicable to inbred mouse strains. A limitation of this study is that gene expression was assessed only at the mRNA level, without direct evaluation of protein translation or the activation states of caspases. The potential variation in copy number of introduced genes due to random integration can also be considered a limitation in this study. In addition, we treated the embryos with Dox continuously from 6 to 96 hpi and observed the Dox-induced transgene expression at the end of the treatment. Therefore, we did not identify when these expressions were induced. However, a previous report has shown that Dox-induced gene expression can be detected as early as 15 min after Dox treatment in mouse embryos [35], suggesting that induction may have occurred soon after the treatment in our system as well.

In summary, this study demonstrated that Bax exhibits the strongest lethality-inducing potential in mouse early embryos, followed by Casp9, while Casp3 did not show any lethal effects. These results suggest that the further upstream an introduced molecule is, the wider range of the apoptotic cascade it can efficiently activate. In contrast, Casp9 and Casp3 are highly dependent on the activation conditions, indicating the need for optimization for their effective use. Additionally, an unexpected behavior was observed in the Tet-On system, highlighting the need for further investigation. Overall, these findings enhance our understanding of apoptotic mechanisms and provide a foundation for future applications in regenerative medicine, reproductive engineering, and cancer research.

Conflict of interests

The authors declare no conflicts of interest.