Developmental Expression of Pop1/Bves
Department of Biological Sciences, University of Delaware, Newark, Delaware
Correspondence to: Melinda K. Duncan, Dept. of Biological Sciences, University of Delaware, Newark, DE 19716. E-mail: duncanm{at}udel.edu
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Summary |
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Key Words: cardiomyocyte Pop1 Bves plasma membrane blastema myotubes
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Introduction |
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We have recently reported the development of monoclonal antibodies (MAbs) raised against the C-terminal 91358 amino acids of chicken Pop1/Bves (DiAngelo et al. 2001). Here these antibodies are used in Western blotting, confocal microscopy, and immunofluorescence to clarify the discrepancy between the published mRNA and protein localizations.
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Materials and Methods |
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SDS-PAGE and Western Blotting Analysis
All studies using animals conform to guidelines set by the University of Delaware Institutional Animal Care and Use Committee and the National Research Council. Tissue was obtained by microdissecting heart and skeletal muscle from staged embryonic chickens. Similarly, heart, skeletal muscle, brain, and liver tissues were obtained from 5-day post-hatch chicken. Protein extracts were made by homogenizing these tissues using either standard extraction buffer (1 x PBS, 1% Igepal CA-630 (Sigma; St Louis, MO), 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml PMSF, 45 µg/ml aprotinin, and 1 mM sodium orthovanadate) or harsh extraction buffer (0.05 M Tris, pH 7.0, 8.0 M urea, 1% SDS, 0.01% PMSF, and 1% ß-mercaptoethanol). Forty micrograms of the protein was diluted 1:1 with reducing sample buffer (0.125 M Tris-HCl, 0.1% SDS, 4% SDS, 20% glycerol, 32.4 mM DTT, or 0.5 µl ß-mercaptoethanol, pH 6.8) and electrophoresed on a 10% SDS-polyacrylamide gel. Proteins from the gel were transferred to a nitrocellulose membrane (Invitrogen; Carlsbad, CA) and blocked for 1 hr at room temperature (RT) in TBSTween 20 (0.2 M Tris, 0.0025 M Na2HPO4, 0.137 M NaCl, 0.1% Tween-20, pH 7.4), plus 5% wt/v non-fat dry milk. The membrane was then incubated with shaking overnight in TBSTween 20 milk solution with primary antibody (monoclonal mouse anti-chicken, 1:50) at 4C. The incubated membrane was washed three times with TBSTween 20 and then incubated for 1 hr at RT in a 1:2000 dilution of a rabbit anti-mouse horseradish peroxidase (HRP) enzyme conjugate (Cell Signaling Technology; Beverly, MA) in the TBSTween-20milk solution. The membrane was again washed three times with TBSTween-20 and then immersed in the chemiluminescence substrate solution (Cell Signaling Technology) for 1 min and exposed to X-ray film for various amounts of time.
Deglycosylation Analysis
Five-day post-hatch chicken heart protein extract was obtained as above. The deglycosylation analysis was performed according to the manufacturer's protocol (Sigma) with a few modifications as described by FarachCarson et al. (1989). Fifty µg of extracted heart protein was diluted to 45 µl with 50 mM sodium phosphate (pH 7.5) and 50 µg of RNase B was similarly prepared to serve as a positive control. Five µl of denaturing solution (0.2% SDS with 100 mM ß-mercaptoethanol) was added and the solution was incubated at 100C for 10 min to denature the glycoprotein, followed by a 5-min incubation on ice. To this, 5 µl of 0.6% BIGCHAP (Sigma) solution was added. Six µl of the PNGase F enzyme (Sigma) was added to the heart and RNase B experimental tubes and an equal amount of water was added to the heart and RNase B control tubes. Solutions were incubated at 37C for 3 hr. The reaction was stopped by heating the tubes at 100C for 5 min. An aliquot of 25 µl was used for SDS-PAGE and Western blotting analysis.
Chicken Cardiomyocyte Primary Culture
Chicken cardiomyocyte primary cultures were produced as described by Eschenhagen et al. (1997) with a few modifications. Fertilized eggs from single-comb white leghorn chickens (College of Agriculture and Natural Resources, University of Delaware) were incubated for 11 days (temperature 99.5F, humidity 8587%, and automatic egg rotation). Six E11 chicken embryonic hearts were harvested and washed twice in calcium- and magnesium-free Hank's balanced saline solution (HBSS) (Invitrogen). Hearts were then collected in Dulbecco's minimal essential medium (DMEM) (Invitrogen) containing 10% fetal bovine serum (FBS) and 100 µg/ml gentamicin (Invitrogen) and minced to 1-mm pieces. These pieces were washed once with 0.25% trypsin-1 mM EDTA (Invitrogen) in HBSS and then digested in fresh trypsinEDTA for 15 min at 50 rpm shaker speed at 37C. The supernatant was discarded and the remaining tissue was washed with serum-free DMEM. Tissue was then subjected to digestion with 0.1% collagenase A (Boehringer Mannheim; Indianapolis, IN) in serum-free DMEM for 23 min at 50 rpm shaker speed and 37C. This supernatant was discarded and the pellet was digested further with three additional cycles of collagenase for 15 min each until the pellet was almost completely digested. The supernatant was collected after each cycle in a Petri dish (Falcon Plastics; Oxnard, CA) containing the growth medium (DMEM supplemented with 10% FBS and 100 µg/ml gentamicin) in a CO2 incubator (5% CO2, 37C and humidity). After the third cycle, cells were incubated for an additional 30 min (preplating) in a CO2 incubator. The cell suspension was centrifuged and the resulting supernatant was discarded. The pellet was resuspended in 5 ml of growth medium and centrifuged. Once again the resulting pellet was resuspended in 2 ml of growth medium and cells were plated on 1% gelatin-coated two-chambered slides (Nalge Nunc; Naperville, IL) containing 2.5 ml of the growth medium in each chamber and incubated in a CO2 incubator for 34 days.
Immunohistochemistry
A more detailed description of this procedure has been previously described (Reed et al. 2001). In brief, heart and skeletal muscle from E3 to E21 chicken embryos and 5-day post-hatch chickens were embedded, sectioned at 16 µm, mounted on Colorfrost/Plus glass slides (Fisher Scientific; Pittsburgh, PA). The slides were fixed with pre-chilled 1:1 acetone:methanol for 10 min. After the slides had dried, they were blocked in 1% bovine serum albumin (BSA) in 1 x PBS for 1 hr at RT. Tissue sections were incubated with 100 µl of the primary antibody (1:50 dilution in 1% BSAPBS) at RT for 1 hr. Slides were washed twice with 1 x PBS and then incubated for 1 hr at RT with a 1:50 dilution of Alexa Fluor 568 goat anti-mouse (Molecular Probes; Eugene, OR) secondary antibody as well as a 1:1000 dilution of the nucleic acid stain SYTO13 (Molecular Probes) in 1% BSA1 x PBS. Slides were again washed twice in 1 x PBS and then mounted. The resulting fluorescence of these slides was detected using a Zeiss LSM 510 Confocal Microscope (Zeiss; Gottingen, Germany) configured with an argon/krypton laser (488-nm and 568-nm excitation lines) and helium/neon laser (633-nm excitation line). Images were scanned at various magnifications.
Immunocytochemistry
Growth medium was removed from the cell culture and cells were fixed for 10 min with freshly made 4% formaldehyde in 1 x PBS. The fixative was rinsed twice with 1 x PBS. The cell membranes were permeabilized with 0.2% Triton X-100 in 1 x PBS for 5 min, followed by two rinses with 1 x PBS. The following steps were completed with gentle shaking of the slide chambers. Cells were blocked for 1 hr in 3% BSA in 1 x PBS and incubated overnight with 1000 µl of the primary antibody (1:50 dilution) in 3% BSA in 1 x PBS at 4C. The cells were washed three times with 3% BSA in 1 x PBS and incubated for 1 hr at RT with 1:300 dilution of Alexa Fluor 568 goat anti-mouse (Molecular Probes) as well as a 1:1000 dilution of a nucleic acid stain TO-PRO 3 (Molecular Probes) in 3% BSA in 1 x PBS. The cells were then washed three times with 3% BSA in 1 x PBS, rinsed once with 1 x PBS, and mounted. These immunolabeled cells were then observed with a Zeiss LSM 510 confocal microscope.
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Results and Discussion |
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Expression of Pop1/Bves in Multiple Tissues
Multiple tissue Western blots were performed on proteins extracted using the standard extraction conditions (RIPA buffer) from 5-day post-hatch chicken heart, brain, liver, and skeletal muscle using the MAb 3F11-D9-E8 (DiAngelo et al. 2001) to detect the expression of endogenous Pop1/Bves (Figure 1A)
. A predominant band of approximately 58 kD was found in the heart. Although this band is significantly larger than the 41-kD protein predicted from the nucleotide sequence of Pop1, its size is consistent with that of myc-tagged Pop1 produced in CHO cells (Andree et al. 2000
) and the molecular weight of the Bves protein detected in embryonic heart extracts using polyclonal peptide antibodies (Reese et al. 1999
). Previously, in vitro translation experiments demonstrated that the addition of canine microsomal membranes altered the molecular weight of Pop1/Bves protein from 41 kD to 58 kD, suggesting that this protein is glycosylated in vivo. In skeletal muscle, the predominant Pop1/Bves protein detected has a molecular weight of approximately 70 kD, and a band of similar molecular weight was also found in the heart protein extract. This suggests that either Pop1/Bves is differentially glycosylated in skeletal muscle and/or that the Pop1/Bves mRNA is differentially spliced in skeletal muscle compared to the heart. In addition, a very weak band of approximately 70 kD was detected in brain extracts. However, we did not detect any specific expression by immunostaining analysis of 5-day post-hatch chicken brain (data not shown).
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Developmental Study of Pop1/Bves Expression in Embryonic Chicken Heart
To further analyze the expression pattern of Pop1/Bves during development, various stages of embryonic chicken hearts were obtained for immunoblotting and immunostaining. Western blotting of embryonic heart protein extracts showed an increase in detectable protein expression during development until E11, after which it appears to plateau (Figure 2A)
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Immunohistochemical analysis of developing chicken heart showed a uniform plasma membrane distribution of Pop1/Bves expression in the myocardium throughout development. This pattern of localization agrees well with the distribution of Pop1 mRNA by in situ hybridization and gene activity by LacZi knock-in reported previously (Andree et al. 2000,2002
) (Figures 3A3F)
. Notably, Pop1/Bves protein was not detected in either the epicardium (except in E6 heart) or the coronary vascular smooth muscle, contradicting previous reports using peptide antibodies (Reese et al. 1999
). The reason for this discrepancy is unclear, but the correlation between these immunohistochemistry results and gene activity suggests that the present data accurately reflect the true distribution of Pop1/Bves protein in vivo.
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The presence of Pop1/Bves at regions of cellcell contact is consistent with previous assertions that this protein may play a role in cellcell adhesion (Wada et al. 2001). When cellcell attachment is made, the Pop1/Bves molecules on the plasma membrane may immediately engage in the adhesion process. Moreover, via an unknown signaling mechanism, they may enhance the mobility of other Pop1/Bves proteins to the plasma membrane from vesicles distributed throughout the cytoplasm. As suggested by Wada et al. (2001)
, these proteins may act in a manner similar to the recently identified "adhesion zipper" molecules (Vasioukhin et al. 2000
; Wada et al. 2001
) and aid in cellcell attachment.
Developmental Study of Pop1/Bves Expression in Skeletal Muscle
Western blotting of embryonic skeletal muscle proteins extracted using standard extraction conditions (RIPA buffer) detected no expression of Pop1/Bves (data not shown), although moderate amounts of protein were detected at post-hatch day 5. This result was surprising because appreciable amounts of Pop1/Bves protein were detected in skeletal muscle from late embryonic chickens by immunohistochemistry (Figure 5). Thus, skeletal muscle tissues were treated in harsh protein extraction solution containing high concentrations of urea and detergent because many membrane proteins are attached to the cytoskeleton and do not solubilize easily. Western blotting of proteins extracted in this manner showed a gradual increase in Pop1/Bves expression throughout development (Figure 4)
, although levels of detectable protein in embryonic skeletal muscle were significantly lower than embryonic heart and post-hatch day 5 skeletal muscle obtained using standard extraction conditions (RIPA buffer).
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Immunohistochemical studies of embryonic chicken skeletal muscle suggest that, unlike the early onset of Pop1/Bves expression in the heart, significant protein expression does not occur until E7 in the skeletal muscle of the leg. This corresponds to the separation of the dorsal and ventral muscle blastema seen at E6 into the anatomically distinguishable muscles seen at E7 (Wortham 1948). Although Pop1/Bves gene activity is detected in the somitic myotome of Pop1/Bves LacZi knock-in mice (Andree et al. 2002
), the present data suggest that little protein is produced from this expression. Furthermore, during the early stages of chick muscle development (E7E11), Pop1/Bves is not clearly localized to the sarcolemma. Instead, it is seen in a punctate distribution throughout the cell (Figure 5)
, suggesting that it can not participate in cell adhesion functions (Wada et al. 2001
) at this stage. This observation may explain the apparently normal skeletal muscle phenotype in homozygous Pop1/Bves LacZi knock-in mice (Andree et al. 2002
).
By Western blotting, Pop1/Bves protein levels are significantly upregulated in skeletal muscle at about E13 (Figure 4). Furthermore, this protein is now clearly detectable in the sarcolemma by immunolocalization (Figure 5). In chicken, this correlates with the onset of satellite cell proliferation at E12 and the generation of secondary myotubes from these cells shortly thereafter (Cossu and Molinaro 1987). Notably, in LacZi knock-in mice, myotube development into myofibers is abnormally slow during skeletal muscle regeneration after chemical injury (Andree et al. 2002
). Because satellite cells contribute to muscle regeneration as well as secondary myotube formation, this supports the idea that Pop1/Bves participates in the transition from satellite cell to myofibril, perhaps through its function as a cell adhesion molecule.
Between E13 and E17, Pop1/Bves persists at high levels in the sarcolemma of myotubes and remains in the sarcolemma as these myotubes develop into myofibers between E17 and E19 (Figure 5) (Tokuyasu et al. 1985). After hatching, strong Pop1/Bves localization is still observed in the sarcolemma of mature myofibers, although it may have different molecular interactions with the cytoskeleton because Pop1/Bves is easily extractable from post-hatch but not pre-hatch skeletal muscles. This is in contrast to the loss of LacZi staining from the nuclei of mature skeletal muscle of Pop1/Bves knock-in mice. This suggests that Pop1/Bves protein is stable in myofibrils once produced, unlike LacZi, which is known to be rapidly degraded in adult skeletal muscle after its translation (Newlands et al. 1998
).
Our findings support the idea that Pop1/Bves is localized to the cell membrane of cardiac and skeletal muscles and may be involved in cell adhesion. Although further work will be needed to understand the regulation of this expression pattern, it is notable that a genomic fragment near the Pop1/Bves gene was found to bind the transcription factor Pax3 by cyclic amplification and selection of targets (CASTing). CASTing is performed to conduct a genome-wide search to identify cis-regulatory elements and putative target genes of a particular transcription factor. In this method, DNA fragments bound to a transcription factor are separated from unbound genomic DNA by gel eletrophoresis and amplified by PCR. The isolated genomic DNA fragments containing the binding site are evaluated by BLAST analysis against GenBank to identify putative target genes. Because Pop1/Bves mRNA levels were reduced in Pax3-null embryos, which die in utero due to multiple defects including abnormalities in the heart and skeletal muscle, it appears likely that Pop1/Bves is a true Pax3 target gene. (Barber et al. 2002).
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Acknowledgments |
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We are grateful to Dr William Cain for his help with the generation of the monoclonal antibody against chicken Pop1/Bves, Mr Bob Nardone for maintaining the bioreactor used for its production, Dr Gary Laverty for advice on the chicken cardiomyocyte cultures, Dr Kirk Czymmek for his help with confocal imaging, and Dr Deni Galileo for the supply of fertilized chicken eggs.
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Footnotes |
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Literature Cited |
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