Copyright ©The Histochemical Society, Inc.

Developmental Expression of Pop1/Bves

Trusha K. Vasavada, Justin R. DiAngelo and Melinda K. Duncan

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


    Summary
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 Summary
 Introduction
 Materials and Methods
 Results and Discussion
 Literature Cited
 
Initial studies have suggested that Pop1/Bves protein is exclusively expressed in the smooth muscle walls of the coronary vessels, implying its possible importance in coronary diseases. However, the mRNA and activity of this gene are detected in both skeletal and cardiac muscles, not coronary smooth muscle, and Pop1/Bves knockout mice have defects in skeletal muscle regeneration. Here we used specific monoclonal antibodies (MAbs) raised against chicken Pop1/Bves and demonstrated the presence of this protein in cardiomyocytes through development and its apparent absence in coronary vessels. Immunostaining of cardiomyocytes cultured in vitro confirmed the membrane localization of this protein in cells that participate in cell adhesion, with significant intracellular staining seen in isolated cells. In skeletal muscle, Pop1 protein becomes detectable at embryonic day (E) 7, coincident with the differentiation of morphologically distinct muscle masses from the limb muscle blastema, but the protein is not found at high levels in the cell membrane of myotubes until E11, coincident with the formation of secondary myotubes from satellite cells. These data support the hypothesis that Pop1/Bves is a cell adhesion molecule present in skeletal and cardiac muscle. (J Histochem Cytochem 52:371–377, 2004)

Key Words: cardiomyocyte • Pop1 • Bves • plasma membrane • blastema • myotubes


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results and Discussion
 Literature Cited
 
The Popeye genes, Pop1, Pop2, and Pop3, are members of a recently identified gene family with predominant expression in heart and skeletal muscle. These genes have no significant sequence similarity to any previously described gene family but are highly conserved among vertebrates. Northern blotting has detected high levels of Pop1 mRNA in the heart of chickens, Xenopus, mice, and humans (Andree et al. 2000Go; Hitz et al. 2002Go) as well as adult mouse and human skeletal muscle (Andree et al. 2000Go). In situ hybridization of early chicken, mouse, and Xenopus embryos detected high levels of Pop1 mRNA in the myocardium, with no detectable expression in the epicardial or endocardial layers of the heart (Andree et al. 2000Go). Knock-in of LacZi into the mouse Pop1 gene confirmed the activity of the Pop1 gene in the heart and further demonstrated Pop1 expression in the myotomal component of the somite, in developing skeletal muscle of the limb, and in the satellite cells of adult muscles. Homozygous Pop1 knockout mice are viable and apparently healthy. However, they have impaired skeletal muscle regeneration after muscle damage by cardiotoxin (Andree et al. 2002Go). In contrast to these results at the level of gene expression, Pop1 protein was detected in the proepicardial organ, epicardium, and cardiac vascular smooth muscle of chicken embryos using a polyclonal peptide antibody. This result led to the conclusion that this protein is the earliest known marker of cardiac vascular smooth muscle and it was therefore named blood vessel/epicardial substance (Bves) (Reese et al. 1999Go). Further investigations at the protein level using this and other polyclonal peptide antibodies have suggested that Pop1(Bves) is a novel cell adhesion molecule found at points of cell–cell contact in mature vascular smooth muscle (Wada et al. 2001Go).

We have recently reported the development of monoclonal antibodies (MAbs) raised against the C-terminal 91–358 amino acids of chicken Pop1/Bves (DiAngelo et al. 2001Go). Here these antibodies are used in Western blotting, confocal microscopy, and immunofluorescence to clarify the discrepancy between the published mRNA and protein localizations.


    Materials and Methods
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 Summary
 Introduction
 Materials and Methods
 Results and Discussion
 Literature Cited
 
Production of MAb Against Chicken Pop1/Bves
The MAb against recombinant chicken Pop1/Bves was produced as previously described by DiAngelo et al. (2001)Go. Antibody from the cell line 3F11-D9-E8 was used for Western blotting analysis and immunostaining experiments.

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 TBS–Tween 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 TBS–Tween 20 milk solution with primary antibody (monoclonal mouse anti-chicken, 1:50) at 4C. The incubated membrane was washed three times with TBS–Tween 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 TBS–Tween-20–milk solution. The membrane was again washed three times with TBS–Tween-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 Farach–Carson et al. (1989)Go. 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)Go 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 85–87%, 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 trypsin–EDTA 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 3–4 days.

Immunohistochemistry
A more detailed description of this procedure has been previously described (Reed et al. 2001Go). 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% BSA–PBS) 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% BSA–1 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.


    Results and Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results and Discussion
 Literature Cited
 
Previous immunohistochemical studies using polyclonal antibodies have suggested that Pop1/Bves protein is predominantly found in the coronary vascular smooth muscle (Reese et al. 1999Go), whereas in situ hybridization and data from LacZi knock-in mice have shown the gene to be preferentially expressed in cardiomyocytes and in skeletal muscle (Andree et al. 2000Go). Here we have reexamined the tissue specificity and subcellular localization of Pop1/Bves during development using MAbs against the chicken Pop1/Bves protein (DiAngelo et al. 2001Go).

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. 2001Go) 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. 2000Go) and the molecular weight of the Bves protein detected in embryonic heart extracts using polyclonal peptide antibodies (Reese et al. 1999Go). 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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Figure 1

(A) Western blot of multiple tissues from 5-day post-hatch chicken. Pop1/Bves expression is detected in heart and skeletal muscle protein extracts. Sk, skeletal muscle; Lv, liver; B, brain; H, heart. (B) Western blot of PNGase F-digested 5-day post-hatch chicken heart protein extract. (C) Control (no PNGase F treatment); PNGase, PNGase F-treated.

 
To confirm that the bands detected in the heart protein extract were indeed glycosylated forms of Pop1/Bves, we treated the heart protein extract with Peptide-N-glycosidase F (PNGase F), an enzyme used for deglycosylation analysis of glycoproteins (Figure 1B). Our data show that this treatment resulted in deglycosylation of Pop1/Bves protein. A predominant band of approximately 41 kD was detected in the PNGase F-treated sample in addition to bands of lower and higher molecular weights, suggesting various degrees of deglycosylation or detection of various splice forms (Andree et al. 2000Go). This result is consistent with a recent report demonstrating that tagged forms of Pop1/Bves are glycosylated by similar PNGase F treatments (Knight et al. 2003Go).

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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Figure 2

(A) Developmental Western blot of embryonic chicken heart protein extracts. During development, the amount of protein detected increases until E11 stage, then stabilizes. Arrow indicates the Pop1/Bves band at 58 kD. E5–E15, embryonic day 5–15; P5, 5-day post-hatch. (B) Western blot of E8 embryonic chicken heart extracts in the presence and absence of a reducing agent. Under non-reducing conditions, Pop1/Bves has a molecular weight of 116 kD, which is double the molecular weight of Pop1/Bves under reducing conditions. R, reducing condition; NR, non-reducing condition.

 
Pop1/Bves protein has nine cysteine residues (Andree et al. 2000Go), allowing the formation of four disulfide bridges with one cysteine residue remaining to form a covalent linkage with a free cysteine residue of another protein. To test this hypothesis, Western blotting was performed on protein extracted from E8 chicken hearts in the presence and absence of a reducing agent. Because a band of roughly twice the molecular weight of the expected 58-kD band was detected in the nonreduced sample (Figure 2B), Pop1/Bves may form a disulfide-crosslinked dimer in vivo.

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. 2000Go,2002Go) (Figures 3A–3F) . 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. 1999Go). 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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Figure 3

Confocal images of Pop1/Bves expression in developing chicken heart and cultured cardiomyocytes. (A) E3; (B) E5; (C) E6; (D) E9; (E) E16; (F) P5. Myocardial expression is detected as early as E3 (A). No epicardial expression is detected except at E6 (C). Myocardial expression is detected at all stages analyzed from E3 to P5 (A–F). (E) No protein expression was detected in the coronary vessels. (G,H) Confocal images of E11 chicken cardiomyocyte primary cultures. Arrows point to the plasma membrane distribution of Pop1/Bves. Some expression is also detected in cytoplasmic vesicles (H). mc, myocardium; ep, epicardium; cr, coronary vessel; pm, plasma membrane; v, vesicle; E, embryonic day; P5, 5-day post-hatch. Red, Pop1/Bves; green (A–F), blue (G–H), Nuclear DNA. Bars: A–C = 38.4 µm; D–F = 19.2 µm; G = 12.8 µm; H = 25.6 µm.

 
Next, the apparent plasma membrane distribution of Pop1/Bves was investigated further by immunocytochemical studies of live and fixed chicken cardiomyocyte primary cultures (Figures 3G–3H). In differentiated cardiomyocyte aggregates, Pop1/Bves is predominately found at the plasma membrane in regions of cell–cell contact (Figure 3G). In contrast, in single cells, a significant amount of Pop1/Bves is found in a punctate distribution inside the cell and, in addition, some protein is detected at the plasma membrane (Figure 3H). Although the observed protein distribution could reflect either an integral membrane protein or an intracellular protein localized to the plasma membrane, it has been recently confirmed that Pop1/Bves is an integral membrane protein with an extracellular amino-terminus, three transmembrane domains, and a cytoplasmic carboxyl-terminus using epitope-tagged recombinant Pop1/Bves (Knight et al. 2003Go).

The presence of Pop1/Bves at regions of cell–cell contact is consistent with previous assertions that this protein may play a role in cell–cell adhesion (Wada et al. 2001Go). When cell–cell 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)Go, these proteins may act in a manner similar to the recently identified "adhesion zipper" molecules (Vasioukhin et al. 2000Go; Wada et al. 2001Go) and aid in cell–cell 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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Figure 5

Confocal images of embryonic chicken skeletal muscle. (A) E7; (B) E13; (C) E15; (D) E19; (E) E21; (F) P5. The onset of Pop1/Bves expression occurs around E7. However, localization to the sarcolemma is not detected until E13 and does not become exclusively associated with the sarcolemma until hatching. Red, Pop1/Bves; green, nuclear DNA. Bars = 19.2 µm.

 


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Figure 4

Developmental Western blot of embryonic chicken skeletal muscle protein extracted using harsh conditions. Very low amounts of protein are detected at E11. The amount of detectable protein appears to increase during development, with the highest levels obtained after hatching. Multiple bands were detected in the heart and in the skeletal muscle lanes, possibly indicating detection of isoforms. Arrow indicates 58-kD Pop1/Bves bands. P5 heart, 5-day post-hatch heart; E11–E19, embryonic day 11–19; P5 Sk, 5-day post-hatch skeletal muscle.

 
Furthermore, multiple bands were detected in the skeletal muscle protein extracts, with the major form of Pop1/Bves in skeletal muscle being larger than the major form found in heart (Figure 4). It has been previously shown that the chicken Pop1/Bves gene is alternatively spliced, producing four distinct isoforms; Pop1A, Pop1B, Pop1C, and Pop1D, of predicted molecular weights of 41 kD, 33 kD, 35 kD and 80 kD, respectively (Andree et al. 2000Go). Because Pop1A is posttranslationally modified to run at an apparent molecular weight of 58 kD, it is likely that the multiple bands observed derive from a combination of alternate splicing and posttranslational modification. However, it is unlikely that Pop1D is present at significant levels in skeletal muscle because all of the prominent bands detected are smaller than 80 kD. Therefore, in contrast to heart, Pop1/Bves in embryonic skeletal muscle may be tightly bound to insoluble cellular elements, an association that may result from differences in molecular form.

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 1948Go). Although Pop1/Bves gene activity is detected in the somitic myotome of Pop1/Bves LacZi knock-in mice (Andree et al. 2002Go), the present data suggest that little protein is produced from this expression. Furthermore, during the early stages of chick muscle development (E7–E11), 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. 2001Go) at this stage. This observation may explain the apparently normal skeletal muscle phenotype in homozygous Pop1/Bves LacZi knock-in mice (Andree et al. 2002Go).

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 1987Go). Notably, in LacZi knock-in mice, myotube development into myofibers is abnormally slow during skeletal muscle regeneration after chemical injury (Andree et al. 2002Go). 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. 1985Go). 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. 1998Go).

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. 2002Go).


    Acknowledgments
 
Supported by a grant from the American Heart Association, Delaware–Pennsylvania Affiliate. JRD was an HHMI scholar.

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.


    Footnotes
 
Received for publication May 23, 2003; accepted October 8, 2003


    Literature Cited
 Top
 Summary
 Introduction
 Materials and Methods
 Results and Discussion
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