NF-Y–dependent cyclin B2 promoter fragment drives transcription in MITO-Luc mice
For the generation of the MITO-Luc reporter mice, we used a transgene harboring the luciferase gene under control of an NF-Y–dependent cyclin B2 promoter fragment as a sensor of NF-Y activity in vivo. To minimize the influence of the surrounding chromatin status at the integration site, we flanked this construct with insulators that were reported to overcome position effects to a large extent (Ciana et al., 2003
; ). As a control, we cloned the same promoter regions with all CCAAT boxes mutated (CCAAT mutated into TTACT). To test the functionality of these constructs, we performed transient transfections in murine C2C12 () and NIH 3T3 cells (unpublished data). These experiments revealed that the mutation of the CCAAT boxes led to a significant reduction of luciferase units, showing the dependency of this promoter construct on NF-Y activity. The transgene was injected in the pronucleus of fertilized C57BL/6xDBA/2 eggs using standard protocols (Hogan et al., 1994
). We obtained three independent founder animals, of which two were fertile. Semi-quantitative PCR analysis with genomic DNA from tail biopsies showed clear differences in the transgene amount in the two lines, #10 and #26 (Supplemental Figure S1). We injected the luciferase substrate d
-luciferin, into anesthetized transgenic mice and visualized luciferase activity with a charge-coupled device (CCD) camera (Signore et al., 2010
). Both male and female mice were imaged in ventral and dorsal positions (). Light emission, although to different extents, was detected in areas corresponding to femur, skull, sternum, vertebral column, and spleen, and it was equal in both genders for common tissues. In males, testes were also luminescent (Figure S2A).
FIGURE 1: NF-Y–dependent luciferase transcription in MITO-Luc mice. (a) Scheme of the transgene used. (b) C2C12 cells transiently transfected with the wild-type (white bar) or mutated (black bar) CCAAT (CCAAT versus TTACT) boxes. Transfection efficiency (more ...)
In vivo imaging is carried out in two dimensions; thus the definition of the organ/tissue contributing to the photon emission is limited; furthermore, tissue penetration can cause some emitted light to be lost. Therefore we carried out ex vivo experiments in which we measured luciferase activity of dissected organs from the killed mice. The measurement of the enzyme activity was done by bioluminescence, exposing the dissected organ to the CCD camera. As seen in vivo, overall light emission was equal in both genders (unpublished data). As shown in , luciferase activity, as measured ex vivo, generally reproduces and extends what is observed in vivo. Indeed, we detected high luciferase activity in the vertebral column, sternum, spleen, testis, femur, and skull as expected, based on in vivo analysis. Several tissues (e.g., lung, brain, heart, aorta, skeletal muscle, liver, and kidney) did not emit light. All the tissues noted were imaged with the same color scale bar. In these experimental conditions, the intestine, stomach, uterus, and ovaries also did not emit light. Interestingly, using a more stringent color scale bar, we also observed luciferase activity along these tissues, suggesting that their lower luminescence in vivo was indeed due to tissue penetration light loss. To demonstrate that these signals were specific, we imaged the spleen and the lung with the same color scale bar as negative and positive tissues, respectively (Figure S2B).
To further verify tissue distribution of luciferase, we measured enzymatic activity in homogenates of 21 different tissues from both male and female 6- to -8-wk-old mice (Figure S3). The in vitro results closely resembled those seen in vivo and ex vivo. Transgenic line #26 showed similar in vivo, ex vivo, and in vitro results but with lower intensities (unpublished data), due to its lower transgene copy number.
To demonstrate the dependence of luciferase expression on NF-Y transcriptional activity, we injected MITO-Luc mice intravenously with an adenovirus expressing a dominant-negative mutant of NF-Y, dn-YA (Ad-dnYA), that impairs the DNA binding of the resulting complex (Mantovani et al., 1994
; Gurtner et al., 2008
). This molecule is an NF-YA protein with a triple amino acid substitution in the DNA-binding domain that impairs its ability to bind DNA (). It is still able to interact with an NF-YB/-YC dimer, but the resulting trimer is inactive in terms of CCAAT recognition (). Ad-dnYA injection resulted in almost complete inhibition of luciferase activity in every body area after 2 d, returning to almost basal level at day 8 ( and S4). These results definitely demonstrate that the luciferase gene is expressed in an NF-Y–dependent manner in every area of the MITO-Luc mice.
FIGURE 2: NF-Y–dependent luciferase transcription in MITO-Luc mice. (a) Scheme of the dominant-negative NF-YA protein (dn-YA). (b) The NF-Y subunits, A, B, and C, form a complex that binds DNA. dn-YA is still able to interact with the NF-YB/-YC dimer, but (more ...)
The cellular distribution of NF-Y and luciferase was analyzed by immunohistochemistry (). Adjacent slices from testis, spleen, and, as a negative control, skeletal muscle, were stained with antibodies against NF-YA or luciferase. Immunoreactivity for both was clearly detected in testis and spleen, while no staining was observed in skeletal muscle. Both testis and spleen contain proliferating cells, while postmitotic cells are the main constituents of skeletal muscles. To detect proliferating cells, we stained the slices with the antibody anti-PCNA, an antigen expressed in proliferating cells and not in quiescent cells. As expected, PCNA is expressed in the proliferating germ cells and spermatogonium inside the seminiferous tubules of the testis, and in several cells in the spleen. Of note, cells expressing the proliferation marker PCNA contain high levels of NF-YA and luciferase, indicating that in live animals, as in cultured cells, the NF-Y complex is active in proliferating, but not in postmitotic, cells. Most notably, the results in and point to the MITO-Luc reporter mice as a powerful tool for visualizing proliferation events in live animals.
Analysis of luciferase activity upon inhibition of proliferation in MITO-Luc mice
To further understand whether the luciferase activity does occur in proliferating cells, we treated MITO-Luc mice with 5-fluorouracil (5-FU), a well-known antiproliferative drug (Hofer et al., 2006
). The main effects of 5-FU are on rapidly proliferating tissues, particularly bone marrow. After 5 d of treatment, we collected bone marrow from MITO-Luc mice femurs and observed that the luciferase activity was strongly inhibited by the drug treatment (). This inhibition correlates well with the inhibition of the S phase of bone marrow cell mediated by the drug treatment, as observed by fluorescence-activated cell sorting (FACS) analysis (). We next observed the effects of 5-FU in a long-term experiment. We monitored the drug effects over time, reimaging the same animal at various time points posttreatment. Representative images are shown at 5, 10, 15, 23, 30, and 75 d posttreatment (). In the first 5 d after treatment, we observed a significant decrease of luminescence over the entire body. Fifteen days later, we observed luminescence in the spleen, which indicated that proliferation was occurring in the tissue at that time, and it is already known that spleen cells proliferate at this time to repopulate the bone marrow. Whole-body luminescence was completely restored at 75 d posttreatment. It has been shown that ionizing irradiation of normal tissues leads to tissue damage through cell cycle arrest and apoptosis (Stone et al., 2003
). Thus MITO-Luc mice were subjected to whole-body cesium γ-irradiation with sublethal doses of 3 and 5 Gy. Baseline images were obtained before treatment, and mice were then reimaged at various time points posttreatment. Representative images are shown at 3, 6, 11, 15, 20, and 35 d posttreatment (). At 3 d posttreatment, luciferase activity was inhibited in the entire body, indicating that both radiation doses induced tissue damage. At 11 and 20 d, in mice treated with 3 and 5 Gy, respectively, we observed an increase of luciferase activity, indicating that tissue repair mediated by proliferation was occurring at those times. In both cases, luminescence declined to the baseline until 35 d posttreatment. Taken together, these data strongly support the idea that luciferase activity maps whole-body proliferation events in MITO-Luc mice.
FIGURE 3: Analysis of luciferase activity upon inhibition of proliferation in MITO-Luc mice. (a) In vitro luciferase activity and (b) FACS analysis of MITO-Luc mouse bone marrow 5 d after treatment with 5-FU antiproliferative drug (150 mg/kg). Error bars represent (more ...)
Luciferase activity is high in proliferating cells derived from MITO-Luc mice
We observed high luciferase activity in MITO-Luc mouse embryos at 19 d postcoitum (dpc; ). Luciferase activity was analyzed in embryonic tissues at 19 dpc and found present in all, although to different extents (). Since cell proliferation is widespread in embryonic tissues, these results support the hypothesis that NF-Y activity in intact animals correlates with this process. We have previously demonstrated that NF-Y exerts its transcriptional activity in primary proliferating myoblasts and fibroblasts but is inactive in terminally differentiated myotubes and quiescent fibroblasts (Gurtner et al., 2008
). We asked whether NF-Y was active in primary proliferating myoblasts from MITO-Luc mice. shows that luciferase activity is high in primary myoblasts obtained from skeletal muscles of newborn MITO-Luc mice, while it is shut down in terminally differentiated myotubes. As a control for proliferation, myoblasts and myotubes were cultured for 24 h in the presence of BrdU, a marker of DNA synthesis, and were then immunostained for BrdU. As expected, 95% of the myoblasts incorporated BrdU, while myotubes did not. As a control, myoblasts and myotubes were immunostained for muscle-specific myosin heavy chain (MHC), a marker of terminal differentiation in skeletal muscle. As expected, the majority of the myotubes expressed MHC, while myoblasts did not. Next, mouse embryonic fibroblasts (MEFs) were isolated from MITO-Luc mice and, as shown in , luciferase activity was high in proliferating MEFs, but was shut down in quiescent MEFs. Moreover, activity was greatly down-regulated in MEFs at 24 h postinfection with Ad-dnYA (Figure S5). These results demonstrate that the luciferase gene is expressed in an NF-Y–dependent manner in proliferating cells isolated from MITO-Luc mice. Moreover, these results also give information about the leakage of the transgene. Indeed, both in myotubes and in quiescent fibroblasts the luciferase activity is very low, suggesting that the expression of the luciferase is not influenced by the surrounding chromatin.
FIGURE 4: Luciferase activity in MITO-Luc mouse embryos and derived cells. (a) BLI of a representative MITO-Luc embryo at 19 dpc. Both positive and negative littermates are shown. The luciferase activity is visualized in pseudocolor scaling. (b) in vitro luciferase (more ...)
Luciferase activity is induced in MITO-Luc mouse tissues upon induction of proliferation
We asked whether it was possible to induce luciferase expression upon induction of proliferation in MITO-Luc mice. Aberrant proliferation represents an early occurring preneoplastic event. We therefore induced formation of skin papillomas by local administration of 7,12-dimethylbenz(a)anthracene and phorbol ester 12-O-tetradecanoylphorbol-13 acetate (DMBA-TPA) to the skin of MITO-Luc mice. DMBA and TPA were put directly onto shaved ventral skin (), with DMBA applied once-weekly and TPA applied twice-weekly. Representative images are shown for 2, 4, 6, and 8 wk after the first DMBA treatment. We detected luminescence at the treated sites directly after the first DMBA application and during the DMBA-TPA treatment period prior to tumor occurrence, which took place between 12 and 14 wk after treatment. Quantification of luminescence detected at different time points is shown in . The same results were achieved by applying the DMBA-TPA protocol to the mouse ear. Representative images are shown at 1, 2, 4, 5, 7, 10, 11, 12, and 14 wk after the first DMBA treatment (), and data are presented graphically in .
FIGURE 5: Luciferase activity in MITO-Luc mouse–induced papillomas. (a) BLI of a representative MITO-Luc mouse after 2, 4, 6, and 8 wk of DMBA-TPA treatments on ventral skin. (b) Quantification of emitted light from MITO-Luc mouse skin papillomas. The sites (more ...)
We next asked whether induction of luciferase expression upon induction of proliferation/regeneration is a general feature of MITO-Luc mouse tissues. We analyzed the light emitted by skeletal muscle during regeneration following acute hind limb ischemia. This rapid repair process is mainly carried out by satellite cells (SCs) that become active and proliferate upon injury (Zaccagnini et al., 2007
). Unilateral hind limb ischemia was induced in MITO-Luc mice by removing the femoral artery. As shown in Figure S6A, light emission decreased in the injured limb at 1 d postischemia, compared with the contralateral limb, which emitted light from the femur and bone marrow (). Of note, at 2, 4, and 7 d postischemia, adductor and gastrocnemius muscles from injured limb emitted light, as demonstrated by in vitro luciferase assays in protein extracts from these tissues (Figure S6B). Part of the light in this and the contralateral limb originated from the femoral bone marrow (Figure S6C).
Finally, we took advantage of a regeneration model after hepatic injury with carbon tetrachloride (CCl4
) administration. During regeneration of mammalian liver, quiescent hepatocytes reenter the cell cycle and divide synchronously once or twice before returning to quiescence (Fausto, 2000
). This process is concomitant with transcriptional induction of several NF-Y target genes regulating cell proliferation, for example, members of the cyclin
families. Using in vivo (), ex vivo (), and in vitro (Figure S7) BLI experiments, we observed that CCl4
-induced liver injury resulted in transient luciferase activation in the liver of MITO-Luc mice, peaking at 4 d after treatment and returning to almost basal level after 6 d. Staining of adjacent slices from livers of CCl4
-treated mice with antibodies against PCNA or luciferase showed significant overlap in the expression of both markers ().
FIGURE 6: Luciferase activity during MITO-Luc mouse tissue regeneration. (a) BLI of a representative MITO-Luc mouse after 1, 4, and 6 d of CCl4-induced liver damage. After fur removal in the ventral region, the images were collected in a ventral close-range mode. (more ...)
Luciferase activity was not present in CCl4-treated MITO-LUC mouse liver pretreated with a cobra toxin, MCTX (), which impairs entry of hepatocytes into S phase. Moreover, Ad-dnYA injection in CCl4-treated mice strongly inhibited luciferase activation in liver, compared with luciferase activation observed in mice injected with an adenovirus carrying a wild-type NF-YA protein (Ad-NFYA), as demonstrated by in vivo () and ex vivo () BLI and in vitro experiments (). As expected, an empty adenovirus, used as a control, showed intermediate luciferase activation (unpublished data). Of note, the Ad-dnYA effect was accompanied by a strong reduction of a well-known NF-Y target gene, cyclin A, during cell cycle progression, demonstrating that the luciferase inhibition in the liver of Ad-dnYA–injected mice is due to inhibition of proliferation (). These results indicate that induction of luminescence strictly correlates with induction of proliferation in MITO-Luc mice and strongly suggest that NF-Y activity is essential for hepatocyte proliferation during liver regeneration in intact animals.
FIGURE 7: NF-Y activity during liver regeneration. (a) BLI of a representative MITO-Luc mouse after CCl4-treatment and intravenous injection of Ad-NFYA or Ad-dnYA. Experiments were conducted in six animals per group. (b) Ex vivo BLI of representative MITO-Luc mouse (more ...)