Live in vivo imaging of Egr-1 promoter activity during neonatal development, liver regeneration and wound healing
- Philipp Dussmann†1,
- Judith I Pagel†1,
- Sabina Vogel2,
- Terese Magnusson3,
- Rene Zimmermann4,
- Ernst Wagner3,
- Wolfgang Schaper2,
- Manfred Ogris3Email author and
- Elisabeth Deindl1Email author
© Dussmann et al; licensee BioMed Central Ltd. 2011
Received: 10 December 2010
Accepted: 20 May 2011
Published: 20 May 2011
The zinc finger transcription factor Egr-1 (Early growth response 1) is central to several growth factors and represents an important activator of target genes not only involved in physiological processes like embryogenesis and neonatal development, but also in a variety of pathophysiological processes, for example atherosclerosis or cancer. Current options to investigate its transcription and activation in vivo are end-point measurements that do not provide insights into dynamic changes in the living organism.
We developed a transgenic mouse (Egr-1-luc) in which the luciferase reporter gene is under the control of the murine Egr-1 promoter providing a versatile tool to study the time course of Egr-1 activation in vivo. In neonatal mice, bioluminescence imaging revealed a high Egr-1 promoter activity reaching basal levels three weeks after birth with activity at snout, ears and paws. Using a model of partial hepatectomy we could show that Egr-1 promoter activity and Egr-1 mRNA levels were increased in the regenerating liver. In a model of wound healing, we demonstrated that Egr-1 promoter activity was upregulated at the site of injury.
Taken together, we have developed a transgenic mouse model that allows real time in vivo imaging of the Egr-1 promoter activity. The ability to monitor and quantify Egr-1 activity in the living organism may facilitate a better understanding of Egr-1 function in vivo.
The transcription factor Egr-1 belongs to the Egr family (Egr-1 to -4) of zinc finger proteins [1, 2]. The growth factor inducible gene was discovered after stimulation of neuronal cells with nerve growth factor (NGF) and therefore initially referred to as NGF inducible A (NGFI-A) . Fibroblast growth factor 1 (FGF-1), platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and general serum proteins are also capable of activating Egr-1 (for a recent review see ). Egr-1 is an important activator of target genes such as angiopoetin 1  or cell division cycle 20 gene (cdc20) , which in turn are key players in cell proliferation, migration and differentiation [5, 6]. Furthermore, Egr-1 itself has been shown to promote haematopoietic cell differentiation towards the macrophage lineage [7, 8]. Being in the crossfire of different growth signals makes Egr-1 an interesting candidate to be studied during embryogenesis and neonatal development . In addition, Egr-1 has been associated with atherosclerosis , diabetes , wound healing  and tumor growth . Although Egr-1 knockout mice are viable, liver regeneration after hepatectomy is decreased due to impaired progression of mitosis . Hence, Egr-1 relates to various physiological and pathological processes. However, most of the gathered data on Egr-1 gene activation have been evaluated within in vitro studies and could not be confirmed when being re-evaluated in in vivo models . For this reason, it is inevitable to study activation patterns in the living organism over time. In knockout mice, however, compensation of Egr-1 loss of function by other Egr family members cannot be excluded (Pagel et al, manuscript submitted). Since dynamic changes over time cannot be examined by end-point measurements, studying Egr-1 activity within the living organism could help in gaining new information on in vivo Egr-1 gene activation.
The firefly luciferase has been applied as a bioluminescent reporter in living mice using a photon imaging system for studying gene induction noninvasively . We have established a transgenic mouse model using the murine Egr-1 promoter to control the expression of the luciferase reporter and utilized noninvasive bioluminescence imaging (BLI) to study the dynamics of Egr-1 gene activity in the same animal over time. This model was applied to analyze Egr-1 promoter driven luciferase expression during the development of neonatal mice between the ages of 7 to 21 days after birth, where we observed a continuous decrease in Egr-1 promoter activity over time within the examined areas (snout and paw). The activation pattern of Egr-1 during wound healing and tissue regeneration was followed in a model for wound healing of ear tissue and in liver regeneration after partial liver hepatectomy.
Results & Discussion
To analyze exemplarily whether primary cells from Egr-1-luc mice might also be suitable for in vitro investigations, we isolated vascular smooth muscle cells (SMC) from adult Egr-1-luc mice, cultured them and measured luciferase activity in cell lysates. On average, 4,000 RLU were measured per well (12-well plate, luc-negative cells give a background value of <300 RLU/well). This Egr-1 promoter activity in proliferating in vitro cultures of SMC is in line with Egr-1 activities described in the literature for SMC . As the major aim of this work was to monitor Egr-1 activity in vivo, we did not further pursue in vitro cultures.
In vivomonitoring of Egr-1 promoter activity during postnatal development and embryogenesis
Egr-1 activation in regenerating liver
Egr-1 activation in wound healing
In summary, the present study followed the Egr-1 activation pattern over time in the transgenic Egr-1-luc animal model and showed the spatial expression patterns and their time dependent changes in vivo. This transgenic mouse provides a convenient model for studying Egr-1 expression during neonatal development and wound healing at areas were the fur of mice with C57Bl/6 background does not interfere with BLI imaging. Monitoring Egr-1 activity within internal organs, such as in the liver regeneration model presented, was only possible by endpoint measurements with animals having an opened body cavity. To further improve its usability for BLI, cross-breeding into hairless mice will improve its sensitivity. Moreover, it will then offer a useful tool for monitoring effects of pharmaceutical drugs over time in vivo.
Transgenic mice (Egr-1-luc)
The vector containing the murine Egr-1 promoter was a generous gift from Martin Braddock (Glaxo Wellcome, United Kingdom). From this vector, the Egr-1 promoter  compassing the sequence from -930 to +237 base pairs relative to the Egr-1 promoter transcriptional start site [34, 35] was isolated by SalI restriction. 5'ends were filled-in with DNA polymerase I (Klenow enzyme) and cloned into the pUHC13-2 vector by blunt end ligation thereby replacing the CMV promoter. The pUHC13-2 vector, which was a generous gift from H. Bujard (ZMBH, Germany), is a derivate of pUHD10-1  and was originally developed by U. Baron in the laboratory of H. Bujard. In short, the reporter plasmid pUHC13-2 containing the promoter-enhancer sequence of the CMV promoter followed by a polylinker and the luciferase gene of Photinus pyralis (firefly) fused to the SV40 small-t intron and poly(A) signal was digested with HindIII and XhoI to excise the CMV promoter. The 5'ends were filled in with Klenow enzyme and ends were dephosphorylated with alkaline phosphatase. After cloning the Egr-1 promoter into pUHC13-2 vector, the Egr-1 promoter - luciferase reporter gene - SV40 small-t intron fragment was isolated by AseI and PvuI digestion. Finally the transgene was purified using a QIAquick Gel Extraction Kit (Qiagen). All constructs obtained were reviewed and verified by sequencing.
Establishing Egr-1-luc transgenic mouse lines
Egr-1-luc transgenic mice were established by micro-injecting 2 pl of the transgene (5 ng/μl) into male pronuclei (identified by size) of murine zygotes and transferred into pseudopregnant females (strain C57BL/6). The presence of the transgene was confirmed by means of PCR using a specific primer combination spanning the region between the reporter gene luciferase and the SV40 small-t intron (forward primer: 5'- GAG ATC GTG GAT TAC GTC GC - 3'; reverse primer: 5'- TGC TCC CAT TCA TCA GTT CC -3').
In vivo imaging of luciferase activity
Animals were housed in individually vented cages with a 12 h day/night cycle and chow and water provided ad libitum. All animal procedures were approved and controlled by the local ethics committee and carried out according to the guidelines with the German law for protection of animal life.
In vivo imaging was performed using the IVIS Lumina Imaging System (Caliper Life Sciences GmbH) as recently described . For the developmental studies, Egr-1-luc transgenic mice were anesthetized by i.p. injection of xylazin/ketamin (0.375 ml/0.635 ml in PBS, respectively); for liver regeneration and wound healing studies animals were anaesthetized with 2.5% isofluorane in oxygen. Ten minutes after i.p. injection of 300 mg/kg luciferin (Promega, Hilden, Germany) the bioluminescence signal was collected for one to three minutes. Reflected light pictures were taken during illumination with four white LED. Image acquisition and processing was carried out using Living Image 2.60.1 - IGOR Pro 4.09 Software.
For immunhistochemical detection of luciferase and Egr-1 in liver, the tissue was fixed in 4% paraformaldehyde (PFA) over night at 4°C and subsequently embedded in paraffin. Embryos (n = 6, littermates) were collected at day 14 of development for detection of luciferase and Egr-1, fixed in 4% PFA for three days and placed in a solution of Na4EDTA (ethylenediaminetetraacetic acid tetrasodium salt), 200 g/L, pH 7.1 (adjusted using 20% w/v citric acid) for decalcification before being embedded. Four μm sections were mounted on Super Frost® Plus slides (Thermo Scientific Gerhard Menzel). Antigen retrieval for luciferase staining was achieved with Pronase E (Sigma-Aldrich) diluted in 0.5 M Tris buffer (0.1% w/v) for 20 min at room temperature; Egr-1 antigen retrieval was performed in a steamer with sodium citrate buffer (10 mM sodium citrate, pH 6.0) for 20 min. Endogenous peroxidase activity was quenched by treatment with 1% H2O2 for 30 min. Slides were incubated over night at 4°C with an anti-luciferase goat polyclonal horseradish peroxidase (HRP) conjugated antibody (Abcam, 1:50 in Tris-buffered saline (TBS)/0.3% BSA (TBS-B)) and an Egr-1 rabbit monoclonal antibody (clone: 15F7, # 4153, Cell Signaling, dilution 1:50 in TBS-B), respectively. For Egr-1 staining, a biotinylated secondary anti-rabbit antibody was prepared using a rabbit ABC kit (VECTASTAIN® Elite ABC system, Vector). Immunoreactivity was visualized with the chromogen 3-amino-9-ethyl-carbazole (AEC) (AEC single solution, Invitrogen) for 20 min.
One-third liver hepatectomy
Hepatectomy was carried out based on a currently published protocol  with slight modifications. In brief, mice were anaesthetized with isoflurane and 50 μl carprofen given i.p. for pain reduction. All surgical steps were carried out as described , except that only the median lobe was resected leaving a small ischemic stump behind. After surgery, mice received daily 50 μl carprofen i.p.. In sham operated animals, only the midline incision was performed and sutured. At indicated time points after surgery, animals were anaesthetized, the liver was exposed after luciferin injection and images of the ventral view of the fully exposed liver were collected.
Ear wound healing model
A punch wound of approximately 1.5 mm in diameter was inflicted with an ear notcher on one ear of Egr-1-luc mice according to the standard procedure of animal labeling. At indicated time points after the wound setting, BLI from the ear region was carried out as described above, only that the ear was immobilized with adhesive tape during imaging. As a control, the untreated ear was measured.
Primary cultures of murine aortic SMC were established as previously described  and cultured on gelatin-coated plates with standard HAM's F12/Waymouth 1:1 medium (Biochrom), 10% FCS (fetal calf serum) and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B). For stimulation, cells between passages three and four were serum starved for two days. Luciferase quantification was performed in cell lysates as described . In brief, an aliquot of cell lysate was quantified in a tube luminometer after injection of substrate solution for 10 sec, background values from wildtype cells were deducted from the measurement; two nanogram recombinant luciferase (Promega, Mannheim, Germany) correspond to 107 relative light units (RLU).
RNA isolation and quantitative Real Time PCR (qRT-PCR)
Total RNA was isolated according to the procedure of Chromzynski and Sacchi  from frozen liver samples isolated 12 h after hepatectomy or sham operation (n = 4). One microgram of DNase treated total RNA was reverse transcribed using random nonamers (Roche) and a 1st Strand cDNA Synthesis Kit for RT-PCR (Roche). qRT-PCR was performed with a Light Cycler 1.5 (Roche) in a reaction volume of 10 μl using a Light Cycler® FastStart DNA MasterPlus SYBR Green I Kit (Roche) and 50 pmol of each primer (Egr-1, forward: 5'- CGA ACA ACC CTA TGA GCA CCT G - 3'; reverse: 5'- CAG AGG AAG ACG ATG AAG CAG C - 3'; luciferase, forward: 5'- CAG ATG CAC ATA TCG AGG TG - 3'; reverse: 5'- CAT ACT GTT GAG CAA TTC ACG - 3'; 18S rRNA, forward: 5'- GGA CAG GAT TGA CAG ATT GAT AG - 3'; reverse: 5'- CTC GTT CGT TAT CGG AAT TAA C - 3'). Three independent qRT-PCR reactions were performed on each template. An initial denaturation step at 95°C for 10 min was followed by 40 cycles of denaturation (95°C, 10 sec), annealing (64°C for Egr-1; 58°C for luciferase, 64°C for 18S rRNA, 5 sec), and extension (72°C, 15 sec). Melt curve analyses were performed to control specific amplification. Results were normalized to the expression levels of the 18S rRNA.
Protein extracts of liver tissue samples were isolated 48 h after hepatectomy as described . Equal amounts of protein were separated on a 4-20% Tris-glycine gel (Serva) and immunoreactive bands were visualized using Super-Signal-Femto-West (Pierce) with a HRP conjugated rabbit polyclonal antibody against firefly luciferase (1:1000, Santa Cruz Biotechnology), a rabbit monoclonal antibody against Egr-1 (1:500, Cell Signaling) or β-actin (1:2000, Sigma), respectively. Luminescence was evaluated using Hamamatsu 1394 ORCA-ERA camera, AequoriaMDSTM Macroscopic Imaging System and Wasabi software (Hamamatsu Photonics, Herrsching, Germany). Protein bands were quantified by densitometry, and results expressed as Luc/ß-actin and Egr-1/ß-actin ratio, respectively. For negative control, the first antibody was omitted. Blots were repeated at least twice.
Statistical analyses were performed using WinStat. p-values <0.05 were regarded as statistical significant and calculated using either the non-parametric U-test (according to Mann-Whitney) or the Wilcoxon test.
Early growth response 1
vascular smooth muscle cells
relative light units
region of interest
We are grateful to Christine Csapo, Mei-Ping Wu, Nanette Rink and Alke Schropp for technical assistance. This study was in part funded by the Nanosystems Initiative Munich (NIM), the DFG research priority programme SPP1230 and the "Förderprogramm für Forschung und Lehre (FöFoLe)" of the medical faculty at the Ludwig-Maximilians-University Munich.
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