Fgfr4-/- mice show abnormal muscle regeneration—
To investigate the role of Fgfr4 in muscle regeneration, we induced staged degeneration/regeneration in gastrocnemii muscles in Fgfr4
-/- mice and controls using CTX, as we have previously described (17
). Six muscles at each time point of 0, 3, 4, 7, and 14 days post-injection were analyzed by H+E for both Fgfr4
null and normal controls, with three muscles in each group selected for more detailed study. The Fgfr4
null mice have an insertion of the NEO gene in exon 6, and have been reported to lack mRNA and protein (21
). RT-PCR was done at day 3 of regeneration in both wild-type and Fgfr4
null muscles, with primer sets to amplify both exons 5-7, and 5-8. Wild-type muscle showed the expected RT-PCR products, while the Fgfr4
null muscles showed an absence of these products (data not shown). This confirmed both the loss-of-function mutation of the Fgfr4
strains used, and the expression of the Fgfr4
gene at the 3 day time point, as we have previously reported (17
H+E histopathology showed similar staining patterns in both Fgfr4-/- and controls at days 0, 3, and 4 (data not shown). However, by day 7 there were clear distinctions between Fgfr4 null and wild type mice by immunostaining with embryonic myosin heavy chain antibodies (). Wild-type muscle showed maturing myofibers of relatively homogeneous size, central nuclei, and moderate staining with embryonic myosin heavy chain. Fgfr4 null mice showed considerable variability in myofiber size, with many small and intensely staining myofibers, suggesting a lag in the timing of regeneration. There were also very small fibers that stained intensely positive for embryonic myosin heavy chain, yet appeared to lack internal nuclei ().
Fig. 1. Fgfr4-/- mouse muscle shows impaired regeneration with embryonic myosin heavy chain staining. Shown is immunostaining of day 7 regenerating muscle frozen sections from normal (A, B, C) and Fgfr4-/- (D, E, F) mice. Positive regenerating muscle fibers in (more ...)
By 14 days, wild-type muscle showed characteristic large, closely packed regenerated myofibers with central nuclei (). Fgfr4 null mice showed a highly variable histopathology, with some areas showing regenerated myofibers of variable size, with larger nuclei (). Other areas showed extensive calcifications by the von Kossa technique (), and others adipose tissue (). Calcifications and adipocytes were not seen in any area of the wild-type regenerated mice. Inflammation, calcification and fat infiltration were quantified. Inflammation was characterized by staining of muscle sections with phycoerythrin-conjugated antibodies against CD16/CD32, which are expressed on a variety of inflammatory cells including natural killer cells, monocytes, macrophages, dendritic cells, granulocytes, mast cells, B lymphocytes, immature thymocytes, and some activated mature T lymphocytes. Fgfr4 null mice showed 2-3 times more CD16/CD32+ cells relative to the total muscle area than wild type mice (). Fgfr4 null muscle showed large numbers of calcifications (three representative fields of three Fgfr4 null mouse muscles), with an average of 60 calcified foci per 10-X field (). Fgfr4 null mice also showed 8-30% of the muscle area replaced by adipocytes, while no adipocytes were seen in wild-type muscle ()
Fig. 2 . Fgfr4-/- mouse muscle shows impaired regeneration at late stage. Shown is H&E staining of day 14 regenerating muscle frozen sections from normal (A) and Fgfr4-/- (B, D), and von Kossa staining of calcifications in Fgfr4-/- mice (C). Wild-type (more ...)
The calcifications were particularly striking in size and number. Chronic degeneration/regeneration in muscle can lead to foci of calcification, presumably due to poor clearance of macrophages. However, the large number and size of the calcifications suggested that these may be bone-like. To test this, we analyzed the mineral composition of the muscle using Fourier Transform infrared microspectroscopy (31
). We compared the infrared spectra from normal murine muscle (), Fgfr4
null muscle 14 days after injection with cardiotoxin (), and normal murine bone (). The mineral peaks at ~900-1200 cm-1 in the injected Fgfr4
-/- muscle are comparable to normal bone, indicating that hydroxyapatite is present. The increased intensity of the mineral relative to the amide I peak in the injected specimen () in contrast to the bone demonstrates that the calcifications are not truly bone-like because the ratio of mineral to collagen differs (either less collagen or more mineral), but have similar components.
Fig. 3. Fourier Transform infrared microspectroscopy of injected Fgfr4-/- muscle indicates the mineral deposit is hydroxyapatite. A, spectrum from a control, uninjected mouse muscle compared with panel B, the spectrum from a Fgfr4-/- muscle containing calcifications (more ...)
Taken together, this data shows that Fgfr4 deficient muscle regenerates abnormally, with poorly timed regeneration after day 4 (myotube formation).
Activators of Fgfr4: Identification of Tead as a candidate—
To determine transcriptional activators for Fgfr4
relevant to myogenesis, we obtained mouse Fgfr4
(NM_008011) promoter sequence from Genome Browser (http://genome.ucsc.edu/cgi-bin/hgGateway
), and queried the promoter region (600 bp) and first intron of the Fgfr4
genomic sequence for potential transcriptional factor binding sites using Transcription Element Search System (TESS, http://www.cbil.upenn.edu/tess/
). We identified an M-CAT motif (CATTCCT) 49 bp from the transcription start site of mouse Fgfr4
. This M-CAT motif is conserved in human FGFR4
. The M-CAT motif has been found in promoters of muscle specific genes, and has been shown to be bound by TEA domain proteins (Tead) proteins. To determine which Tead
isoform (gene) was a possible regulator of Fgfr4
, we queried our previously reported 27-time point muscle regeneration expression profiling series for all Tead transcripts (17
). The transcripts corresponding to Tead1
(TEF-1) and Tead3
(TEF-5) were not detectable at any time points by Affymetrix GeneChips (absent calls). There are two different probe sets for Tead4
(TEF-3). One showed expression at all time points without differential regulation and the other showed upregulation at day 3.5. Tead2
(TEF-4) showed a characteristic expression pattern similar to MyoD
, with ~10-fold upregulation at peak time point (day 3) (). Upregulation of Tead2
at this critical time point in muscle regeneration was confirmed by both quantitative multiplex fluorescent (QMF) RT-PCR using LI-COR DNA Analyzer and real time RT-PCR using ABI Prism 7900HT Sequence Detection System (). The fold change difference observed by QMF RT-PCR and real time RT-PCR is probably caused by the different signal quantitation methods each system employed. Because Tead2
expression is commensurate with Fgfr4
, whereas Tead4
is half day later, we focused on Tead2
in this study.
Fig. 4. Expression of Tead2 in muscle regeneration. A, shown is the temporal expression of Tead2, Fgfr4 and MyoD over the entire 27 time point series. Tead2 was transiently upregulated at day 3 post-injection, corresponding to a stage of proliferating myoblast (more ...)
To further study Tead2 expression, we produced affinity purified polyclonal antibodies to a 15 amino acid sequence unique to Tead2 N-terminal region before the TEA DNA-binding domain. Immunoblotting showed a band of the predicted molecular weight (45 kD), as well as additional bands (). Immunoprecipitation of muscle cell nuclear extracts with the same Tead2 antibodies pulled down only the expected 45 kD protein, suggesting that the additional bands seen by immunoblot were non-specific (data not shown). Tead2 was not detectable in normal non-regenerating muscle, but was induced by degeneration/regeneration by day 3, consistent with the mRNA data (microarray and RT-PCR).
Tead2 antibodies were then used for immunolocalization of the protein in regenerating muscle (). Double immunostaining with antibodies to Tead2 and a basal membrane protein Laminin a2 showed Tead2 protein to be localized to regenerating myofiber nuclei, with some cells showing staining patterns suggestive of concentration of Tead2 at the nuclear envelope. Tead2 was not detectable in non-injected control muscle nuclei.
Fig. 5. Tead2 is expressed in regenerating muscle fibers. Shown are normal murine muscle (A, B, C, D), and regenerating muscle at 7 days (E, F, G, H). Cryosections were immunostained with Tead2 (A and E), Laminin a2 (B and F), or stained with H&E (D and (more ...)
Tead2 activates the Fgfr4 promoter—The data presented above was consistent with Tead2 as a candidate for regulating Fgfr4. To test this hypothesis, a 226 bp Fgfr4 genomic region containing the M-CAT motif was subcloned into two different reporter constructs. One of these constructs harbored the viral SV40 promoter directing expression of the luciferase gene (SV40P-luc), the second contained the luciferase gene only (Fgfr4-SV40P-luc and Fgfr4-luc reporters) (). These Fgfr4 constructs were transiently transfected into mouse C2C12 skeletal muscle cells with or without the co-transfection of Tead2 expression constructs. C2C12 cells were grown in growth medium (GM) for 24 hours after transfection. C2C12 cell differentiation was then induced by switching to differentiation medium (DM) and continuing culture for 2 days. Cells were harvested and luciferase activity measured. In myoblasts and myotubes transfected with Fgfr4-SV40P-luc, luciferase activity significantly increased in the presence of Tead2 constructs compared to that in the absence of Tead2 constructs (p<0.0048 and 0.0009, respectively; data not shown). In myotubes transfected with Fgfr4-luc, luciferase activity showed a 3-fold increase in the presence of Tead2 constructs relative to that in the absence of Tead2 constructs (p<0.0002) (). Luciferase activity did not show significant difference between myoblasts transfected with Fgfr4-luc alone and myoblasts transfected with Fgfr4-luc and Tead2 ().
Fig. 6. Fgfr4 promoter contains Tead2 sensitive positive regulatory elements. A, shown is a schematic of the constructs containing the 226 bp of Fgfr4 promoter and the luciferase reporter. B, shown is the luciferase activity in proliferating myoblast C2C12 cells (more ...)
These results indicate that Tead2 can enhance the transcriptional activation of the SV40 promoter linked to the Fgfr4 genomic region in both myoblasts and myotubes. Tead2 alone is sufficient to activate transcription of the Fgfr4-luc construct in myotubes, but not in myoblasts. This suggests that co-activators of Tead2 are present in differentiated myotubes, but not myoblasts.
To test whether the Fgfr4-luc construct responds to Tead2 in a dose-dependent manner, the Fgfr4-luc construct was co-transfected with 0, 50ng, 100ng, 150ng and 200ng of the Tead2 construct respectively. Luciferase activity showed a slight increase with increasing amount of Tead2 from 50ng to 200ng, although the dose-response was not dramatic (data not shown).
Transcriptional activation of Fgfr4 is dependent on the presence of the M-CAT motif—We further investigated whether the Fgfr4 promoter region was necessary for this activation. Fgfr4-luc constructs or constructs containing only luciferase (luc) were transfected into C2C12 cells in the presence of Tead2 constructs. Transfected cells were then allowed to differentiate for 48 hours. Cells transfected with the Fgfr4-luc construct showed a significant upregulation of luciferase activity compared to cells transfected with the luc construct (). These results suggest that the Fgfr4 genomic region is necessary for the transcriptional activation in response to Tead2.
Finally, we tested whether regulation of Fgfr4 promoter region by Tead2 was dependent on the M-CAT motif. We interrupted the integrity of the Fgfr4 M-CAT motif by introducing six single point mutations () and co-transfected the resulting mutated Fgfr4-luc construct in C2C12 cells with a Tead2 expression vector. While the Fgfr4-luc wild-type construct could be efficiently transactivated by Tead2, the Fgfr4-M-CAT mutant-luc did not respond to Tead2 indicating that Tead2 activates the Fgfr4 promoter through the M-CAT site (). These results suggest that Tead2 is a direct regulator of the Fgfr4 promoter.
Co-activators of Tead2 are specific to differentiated myogenic cells—Our finding of activation of Fgfr4-luc by co-transfected Tead2 in myotubes, but not in myoblasts suggested that Tead2 co-activators may be specific to differentiated myogenic cells. To test this, we transfected Fgfr4-luc constructs into non-myogenic 10T1/2 cells with or without co-transfection of Tead2 constructs, with increasing amounts of the myogenic differentiation factor, MyoD. Co-transfection of Fgfr4 promoter reporter with Tead2 and MyoD expression constructs did not result in significant reporter expression in growth medium (GM) (). When cells were switched into differentiation medium (DM) for 48 hours, Tead2 was still unable to activate Fgfr4-luc, suggesting the co-activator of Tead2 is not present in 10T1/2 cells (). However, co-transfection of MyoD with Tead2 constructs activated Fgfr4-luc in a dose dependent manner ().
Fig. 7. Tead2 activation of the Fgfr4 promoter requires co-factors in differentiated myogenic cells. A, shown is transfection into non-myogenic 10T1/2 cells of Fgfr4-luc reporter construct, with or without co-transfection of Tead2 expression construct, and with (more ...)
This data suggested that either MyoD could activate Fgfr4-luc directly, or co-activators of Tead2 were induced by MyoD during 10T1/2 cell differentiation. To distinguish the two possibilities, we transfected 10T1/2 cells with MyoD and Fgfr4-luc without co-transfection of Tead2 constructs. MyoD alone activated Fgfr4-luc to a similar extent compared to transfection of the same amount of MyoD plus Tead2 constructs (). This indicates that MyoD activated Fgfr4-luc constructs directly rather than through the induction of Tead2 co-activators. This is not surprising because the 226bp Fgfr4 promoter region contains an E-box 6bp before the M-CAT motif (). Although the M-CAT motif is conserved between mouse and human FGFR4, the E-box is not conserved in human. This suggests that the E-box may play a less important role in Fgfr4 regulation across species boundaries.
Expression of Vestigial like 2 in muscle regeneration—
Vestigial like 2 (Vgl2) is a co-activator of Tead transcription factors (32
). Vgl2 appears to be the only known Tead interacting protein expressed specifically in both embryonic and adult muscles, although a potential role of Vgl2 has not been investigated in muscle regeneration. The U74Av2 GeneChips do not contain a probe set for Vgl2
. The probe set for Vgl2
on MOE 430 2.0 array showed that Vgl2
is present in day 0 normal muscle, down-regulated at early muscle regeneration, and then expression increased towards normal level. We further examined the expression of Vgl2
in muscle regeneration using real time RT-PCR. Vgl2
mRNA was detected in uninjected control muscle. Following CTX injection, the expression of Vgl2
was down-regulated with the onset of necrosis (day 2). Although the level of Vgl2
at day 3 was still lower than that of uninjected muscle, its expression increased when compared to day 2 and gradually regained commensurate with satellite cell proliferation and differentiation (day 3 and after; ). This observation suggests Vgl2 is a relevant co-activator for Tead2 mediated activation of transcription in muscle regeneration.
Fig. 8. Expression of Vgl2 is regulated during muscle regeneration. Shown is expression of Vgl2 detected by real time RT-PCR at selected muscle regeneration time points (0, 2d, 3d, 3.5d, 5d, 16d). Two independent muscle samples were studied at each of the time (more ...) MyoD directly binds to the first intron of Tead2—
The transient upregulation of Tead2
at day 3 is the characteristic expression pattern of MyoD
and some MyoD downstream targets (26
). We therefore investigated whether the Tead2
gene is a potential direct target of MyoD transcriptional activation. MyoD binds to a consensus sequence (CANNTG) called the E-box (34
). By searching Tead2
genomic sequence, we identified multiple E-boxes within 1 kb of the upstream promoter sequence and first intron. However, it has been shown that MyoD does not bind to all E-boxes equally. The affinity of MyoD to E-boxes is affected by the bases in the middle of E-boxes, as well as in the flanking region (25
). By carefully examining the sequences of these E-boxes, we found two closely located (15 bp interval) E-boxes in the first intron region have high similarity to an expanded 9 bp consensus high affinity MyoD binding site. We have previously reported this expanded 9 bp consensus site on both the muscle creatine kinase (MCK
) promoter and Slug
promoter where we had previously shown functionality by both gel shift and chromatin immunoprecipitation assays (25
; ). The two Tead2
intron E-boxes are conserved between mouse and human ().
Fig. 9. MyoD directly binds to the first intron of Tead2 gene. A, shown are E-boxes and flanking sequences in the mouse Tead2 first intron, Slug promoter and muscle creatine kinase (MCK) promoter. Tead2 E-boxes (Tead2 E1 and E2) show high similarity to Slug and (more ...)
To test whether MyoD was able to bind to the two Tead2
E-boxes, an in vitro
gel shift assay was performed using myoblast nuclear extracts and synthesized oligonucleotides containing each E-box. A band shift similar to that of the positive control E-box from MCK
promoter was seen with both Tead2
E-boxes (). The DNA/protein complexes were competed by excess unlabeled wild-type MCK
probes, but not excess unlabeled mutant MCK
probes. We further tested whether MyoD binds to the Tead2
first intron region by chromatin immunoprecipitation assays in both myoblasts and myotubes. In myoblasts, Tead2
showed a 3.6-fold enrichment in MyoD precipitates compared to IgG precipitates. In myotubes, Tead2
was 7.4-fold enriched in MyoD precipitates compared to IgG precipitates (). This suggests that Tead2
is a direct downstream target of MyoD, and that MyoD binding to Tead2 intron increases with myoblast differentiation. As a positive control, we used Rapsn
promoter; this is one of the genes we previously reported as a strong MyoD target (26
). The Rapsn
promoter showed very high affinit y to MyoD in both myoblasts (~220 fold enrichment) and myotubes (~1300 fold enrichment). Gapdh
was used as a negative control.
MyoD activates the first intron of Tead2—To investigate whether MyoD can bind and activate the Tead2 gene via sequences in the first intron, a 213bp Tead2 intron region containing the two E-boxes were subcloned into pGL3-Promoter luciferase reporter vector contain ing a luciferase gene driven by a SV40 promoter (). The constructs were transiently transfected into 10T1/2 cells without MyoD or with co-transfection of increasing amount of MyoD constructs. Cells were switched from growth medium to differentiation medium 24 hours after transfection, and allowed to differentiate for 48 hours before being harvested. Luciferase activity increased when MyoD constructs were added from a lower amount to a higher amount (). When the two E-boxes were mutated (), activation the constructs by MyoD was greatly reduced (). These results suggest that the Tead2 first intron region can serve as an enhancer and be activated by MyoD.
Fig. 10. MyoD activates Tead2 intron region through the E-boxes. A, shown is a schematic of the constructs containing the 213 bp of Tead2 first intron region, a SV40 promoter and the luciferase reporter. B, shown is the dose response of Tead2-SV40P-luc constructs (more ...)