BACKGROUND
duchenne muscular dystrophy  is caused by mutations in the gene encoding dystrophin, a subsarcolemmal protein functioning within the dystrophin-associated glycoprotein complex  <cit> .
this complex connects the intracellular cytoskeleton to the extracellular matrix.
the dgc is concentrated at the z-lines of the sarcomere and confers the transmission of force across the muscle fibre <cit> .
disruption of this link results in membrane instability, which eventually leads to sarcolemmal ruptures <cit> .
influx of extracellular calcium alters molecular processes like muscle contraction and activates proteolytic activity.
affected muscle fibres become necrotic or apoptotic, and release mitogenic chemoattractants, which initiate inflammatory processes  <cit> .
cycles of degeneration and regeneration eventually lead to irreversible muscle wasting and replacement by fibrotic and adipose tissue.
muscle has the potential to regenerate by activation of undifferentiated myogenic precursor cells , which are normally quiescent and situated between the basal membrane and the myofibers <cit> .
upon activation, satellite cells proliferate and divide asymmetrically, with the daughter cells having divergent cell fates <cit> .
only one of the daughter cells differentiates, progresses towards the myoblast-stadium, and subsequently fuses with other myoblasts or with damaged muscle fibres to induce muscle fibre repair.
the other daughter cell remains in a proliferating state or returns to quiescence <cit> .
genetic mutations responsible for dmd are also present in satellite cells.
hence, the ability to restore normal muscle function remains obstructed.
a small number of muscle fibres are able to produce functional dystrophin, mostly due to secondary mutations in myogenic precursor cells which restore the reading frame <cit> .
however, these so-called revertant fibres are in a too small minority to alleviate the pathology of the dystrophin-deficiency.
exhaustion of the satellite cell pool due to degeneration and regeneration cycles is thought to critically contribute to the disease <cit> .
the mdx mouse model for dmd has a spontaneous mutation in exon  <dig> of the dmd gene, introducing a premature stopcodon <cit> .
the pathology of the mdx mouse is characterized by histologically well-defined stages with similarity to the human pathology.
neonatal muscle tissue appears to be unaffected.
necrotic or apoptotic processes in combination with inflammation emerge at approximately  <dig> weeks of age <cit> .
regeneration processes are initiated around the age of  <dig> weeks and continue while alternating with ongoing degeneration until  <dig> weeks of age  <cit> .
contrary to the lethal human pathology, the mdx mouse somehow recovers from the progressive muscle wasting, and does not show the accumulation of connective and adipose tissue <cit> .
however, mdx mice do show a decline in their regeneration capacity at advanced age , while necrotic processes persist <cit> .
since the degeneration processes are similar to those seen in human pathology, the regenerational differences may hold one of the clues of restoration of proper muscle function.
although previous studies have studied gene expression levels in the mdx mouse  <cit> , regeneration processes were not studied in full detail.
we studied the regeneration process through genome-wide monitoring of gene expression levels <cit>  in healthy control and mdx mice at  <dig> time points from  <dig> to  <dig> weeks of age, while putting emphasis on time points where regenerative activity is maximal , a period which was not analysed in detail in a previous time course study <cit> .
according to the temporal gene expression profiles, we determined which pathways are active during regeneration with respect to normal muscle aging.
the majority of identified genes presented in this study have not been described before and provide a substantial addition to the elucidation of the temporal phasing of degeneration and regeneration.
by careful annotation based on existing literature, new light is shed on the pathology and subsequent recovery in the mdx mouse.
furthermore, we compared gene expression profiles to those of human dmd patients and found only modest overlap in regeneration-associated genes.
this confirms that regeneration is no longer an active process at the age at which the patients were profiled .
RESULTS
global comparison of mdx and control mice
gene expression levels were determined in hindlimb muscle tissue from mdx and control mice at  <dig> time points, ranging from  <dig> to  <dig> weeks.
differential gene expression levels were calculated per time point by subtraction of the average normalized intensities of control samples from those of the mdx samples to correct for normal aging processes.
the effects of the normal aging processes on gene expression are relatively minor, and are discussed below.
statistical significance was calculated per time point by performing a student's t-test.
differential gene expression was considered significant when p-values were lower than  <dig>  after applying a bonferroni correction for multiple testing .
out of  <dig>  temporal gene expression profiles  <dig>  were selected, which satisfied the significance criterion at one or more time points .
the number of differentially expressed genes per time point changes considerably during the time course, an effect also shown in a previous study by porter et al.
<cit>  .
the number of differentially expressed genes peaks at the age of 8– <dig> weeks, coinciding with the period of maximal muscle regeneration.
interestingly, also at the first two time points , where the histology of the mdx muscle is not different from that of control mice, a large number of genes was differentially expressed, indicating differences in muscle development in dystrophin-deficient animals.
the majority of these genes  also show statistically significant differences in expression at later time points.
this overlap can be explained by the assumption that the repertoire of gene products used for muscle growth and development also functions in muscle regeneration.
in this report we will describe the expression changes of two main categories in more detail: genes coding for proteins within the costamer and the dystrophin-associated glycoprotein complex , and genes involved in regeneration.
the dystrophin-glycoprotein complex
the effect of dystrophin-deficiency on expression levels of dystrophin-glycoprotein complex -related genes, or genes with associated functional relevance within the costamer has not been reported in previous gene expression profiling studies  <cit> , with the exception of the study of porter and co-workers <cit> .
in the porter study, a downregulation of dystrophin was reported, but no changes in gene expression of other components of the dgc.
a selection of  <dig> genes was made based on an overview by ervasti et al.
of members of the costameric protein network <cit>  .
according to the statistical selection criteria,  <dig> genes were upregulated  and  <dig> were downregulated  in the mdx mouse.
although a decrease in dystrophin expression was found in our study, the stringent statistical criteria were not met.
upregulated dgc related genes
we find that the retina-specific isoform of dystrophin  is expressed in skeletal muscle of mdx mice, whereas dp <dig> cannot be detected in hindlimb muscle of control mice.
expression of dp <dig> was detected by an oligonucleotide probe within the unique first exon of this transcript.
the promoter of the dp <dig> isoform resides in intron  <dig>  downstream of the mdx mutation .
transgenic mdx mice, which overexpress dp <dig> via an alpha-actin promoter, show a restoration of a stable association between costameric actin and the sarcolemma, a re-assembly of the dgc, and an overall alleviation of the pathology <cit> .
increased transcription initiation of dp <dig> might therefore be a natural adaptation for the lack of the muscle specific isoform of dystrophin.
however, in contrast to the artificially raised expression by the alpha-actin promoter, the expression of dp <dig> in the mdx mouse through the original promoter does not seem to be strong enough to compensate for loss of the full-length muscle specific isoform.
we found a continuous upregulation of alpha-dystrobrevin  gene expression with maximum differential expression at  <dig> weeks in mdx mice.
dystrobrevin is a phosphotyrosine-containing protein localized at both the sarcolemma and the postsynaptic side of the neuromuscular junction , where it binds to either dystrophin or utrophin  <cit> .
dtna has been described to function as a signalling mediator within the dgc <cit> .
transcription of dtna is activated when myoblasts differentiate into multinucleated myotubes <cit> .
newey et al.
reported that dtna-protein levels are significantly reduced in the mdx mouse at the sarcolemma, whereas the protein level was unchanged at the nmj.
this would be consistent with a stabilizing action of dtna upon binding to dystrophin or utrophin, since dystrophin is not present at the sarcolemma, whereas utrophin is expressed at the nmj.
they proposed a model, where localized translation of dtna transcripts contributes to synapse formation <cit> .
upregulation of dtna in the mdx mouse might indicate an attempt to compensate for the increased turnover of the protein, in order to stabilize the post-synaptic side of neuromuscular junctions of affected muscle fibres, and retain neuronal connection.
our results show a continuous upregulation of lim domain protein  <dig> .
studies in ldb <dig> knock-out mice demonstrated that ablation of ldb <dig> eradicates the structural integrity of the z-line in contracting striated muscle and causes a severe form of congenital myopathy <cit> .
upregulation of ldb <dig> indicates the necessity for stabilization of the z-line in mdx mice, compensating the undermining effect of dystrophin-deficiency.
downregulated dgc related genes
it can be seen that gene expression levels of several core-proteins of the dgc, e.g.
the transmembrane proteins dystroglycan , sarcospan , and two members of the sarcoglycan-complex , are lower in mdx mice, over the whole time course.
lower expression levels were also detected in other members of the sarcoglycan complex  and in dystrophin , but these were not statistically significant.
the decrease in expression of dgc-related genes was most prominent during regeneration .
interestingly, dgc related gene expression levels restore to pre-regeneration levels subsequent to the regeneration period, but remain lower than normal  level.
similarly, protein levels of core-proteins of the dgc have been shown to be severely reduced in dystrophin-deficient mdx mice <cit> .
it is suggested that the secondary displacement of dgc core-proteins is due to a decrease in protein synthesis and/or assembly, or due to an increase in protein degradation.
similarly, in sarcoglycan-deficiencies the absence of a single subunit causes the loss or strong reduction of the entire sarcoglycan protein complex  <cit> .
since our study reveals a downregulation of mrna levels of the dgc core-proteins, we conclude that alterations in transcriptional activity also contribute to the decrease in protein levels.
as transcription of members of the dgc is likely to be co-ordinately regulated <cit> , downregulation of these members as seen in mdx mice can occur via inhibition or downregulation of shared transcriptional activators.
regeneration
in the mdx mouse, regeneration of affected muscle tissue is most prominent at the age of 6– <dig> weeks, after which a stabilized condition is reached.
to identify pathways active in regeneration, we studied five categories of differentially expressed genes covering major functional characteristics of regenerative tissues .
this selection of  <dig> genes was typed for temporal effects during regeneration, and the pathways to which they belong.
since signal transduction pathways are still poorly annotated in current genomic databases, these pathways were constructed from the literature.
in our study, a pathway is only considered activated or repressed, when multiple members show differential gene expression.
temporal effects during regeneration
differential gene expression profiles, based on the ratio between mdx and control mice, were scaled to the first time point.
differential expression profiles can therefore be compared independent of the ratio level, which enables the detection of temporal effects.
using k-means clustering , differential gene expression profiles were classified according to their temporal similarity.
the unscaled temporal effects of mdx and control gene expression profiles are shown per cluster for the up- and downregulated genes separately .
genes, which show an upregulation in gene expression during the regenerative phase, are present in clusters  <dig> , cluster  <dig> , and cluster  <dig> .
the temporal effect is determined by the gene expression profile of the mdx mouse, since gene expression is continuously low without temporal changes in the control mouse.
downregulation of gene expression in the mdx mouse during regeneration is primarily seen in cluster  <dig> .
during normal aging, which can be seen in the control mouse, gene expression increases until the age of  <dig> weeks, followed by a slow decrease.
during the regenerative phase in the mdx mouse, however, the expression of these genes is downregulated markedly.
notch-delta pathway
gene expression levels of a number of genes functioning in the notch-delta pathway are upregulated , whereas others  are downregulated in the mdx mouse at  <dig> weeks of age .
the gene expression of dll <dig> and numb are switched on in the mdx mouse, while no gene expression can be detected in the control mouse .
the differential expression of the upregulated genes is mostly increased during the regeneration period  .
for several genes in the notch-delta pathway, quantitative rt-pcr experiments were performed to confirm the temporal expression profiles found on the microarray.
in accordance with the microarray results, quantitative rt-pcr experiments demonstrated higher expression of notch <dig>  numb and myogenin in mdx than in control mice at all ages .
previous work by conboy et al.
<cit>  indicates the role of the notch-delta signalling pathway in the regulation of proliferation versus differentiation of asymmetrically dividing satellite cells by notch or numb, respectively.
according to delfini et al.
<cit> , notch is expressed in immature myoblasts, while delta  expressing cells are more advanced in myogenesis .
notch activation is thought to inhibit transcription factors containing a basic helix loop helix domain   <cit> , via the induction of hairy and enhancer of split  <dig>  <cit> , thereby inhibiting myogenic differentiation.
numb-expressing cells are able to undergo myogenic differentiation, because the notch-delta pathway is inhibited .
based on our results, the notch-delta signalling pathway, notably the expression of notch or numb, is responsible for the determination towards proliferation or differentiation of activated satellite cells in the mdx mouse.
since gene expression profiling detects proliferation and differentiation processes simultaneously, satellite cell activation and commitment are ongoing, parallel processes.
bmp pathway
various members of a bmp-associated pathway  are differentially expressed in the mdx mouse .
the gene expression of bmp <dig>  bmpr1a, inhbc, inhbe, and idb <dig> is switched on in the mdx mouse, while expression cannot be detected in the control mouse.
bone morphogenetic protein  <dig>  is a member of the transforming growth factor-β  family.
bmp <dig> induces transcription of inhibitors of dna binding proteins  via binding to bone morphogenetic protein receptor type i , and the downstream transportation of smad-complex  to the nucleus <cit> .
idb-proteins function as positive regulators of cell growth by binding to retinoblastoma  <dig> .
this leads to the activation of the e2f transcription factor, which plays a role in cell-cycle regulation.
furthermore, idb-proteins inhibit myogenic differentiation through binding to mrfs <cit> .
the activation of idb <dig> in the pre-regeneration period is indicative of an inhibition of the myogenic differentiation.
this inhibition seems to be alleviated during the regeneration period by a decrease in differential expression of idb <dig>  although most of the differentially expressed genes in the bmp pathway are continuously upregulated, the expression of a number of genes peaks during regeneration .
the inhibin proteins , likely to be antagonists of bone morphogenetic proteins <cit> , are also upregulated.
altogether, this points to a positively and negatively controlled regulation of the bmp <dig> pathway.
our data suggests that the bmp <dig> pathway has an important function in the balancing of proliferation and differentiation of myoblasts, necessary for effective upscaling of muscle-mass.
neuregulin pathway
in our study we found that several members of the epidermal growth factor-like  neuregulin pathway are differentially expressed .
the signalling cascade is activated by the binding of neuregulin <dig>  to the extracellular domain of the upregulated protein tyrosine kinase v-erb-a erythroblastic leukemia viral oncogene homolog  <dig>  <cit> .
both nrg <dig> and erbb <dig> are expressed in the mdx mouse and cannot be detected in the control mouse.
the interaction between nrg <dig> and erbb <dig> activates epidermal growth factor-like signal transduction via binding of the adaptor protein growth factor receptor bound protein  <dig> , which peaks at the initiation of regeneration .
grb <dig> can activate mitogen activated kinase kinase  <dig>  <cit> , whose differential gene expression is increased at  <dig>  weeks as well as during regeneration.
the activation of the map kinase pathways eventually leads to transcriptional induction  through members of activating protein complex  <dig> , like jund <dig> and jun.
depending on the protein complexes formed, specific transcription activation will lead to different biological processes ranging from proliferation to differentiation.
furthermore, we find that other grb-interacting proteins like vav  <dig> oncogene  <cit> , and p21-activated kinase  <dig>  <cit>  are switched on and upregulated, respectively.
the differential gene expression of vav <dig> increases during the initiation of regeneration, and might play a role in the clustering of integrins for cell adhesion <cit> .
pak <dig> differential gene expression is increased during regeneration.
downstream genes activated by pak <dig> regulate cytoskeletal dynamics, proliferation and cell survival signalling <cit> .
furthermore, our results show that the erk pathway is downregulated in the mdx mouse as well as the p <dig> pathway.
comparisons with other studies
in contrast to previously published studies of temporal gene expression profiling in the mdx mouse  <cit> , we primarily focus on regeneration.
the majority of differentially expressed genes in regeneration  in our study have not been reported as differentially expressed in the mdx mouse in other studies.
apart from important differences in gene coverage , we explain the limited overlap by differences in cut-off levels: as we applied very stringent statistical tests, we could avoid setting a cut-off level for the fold change, thereby picking up genes with small but consistent fold changes, which can be biologically very relevant, especially in the case of transcription factors.
this may also explain the large difference between the study of porter et al.
and our study in the number of genes found differentially expressed at the early timepoints , where mainly subtle expression changes are expected.
goetsch et al.
reported results from a gene expression profiling studies during muscle regeneration induced by cardiotoxin injection in wildtype mice <cit> .
the authors concluded that muscle regeneration is a complex process that requires the coordinated modulation of the inflammatory response, myogenic precursor cells, growth factors, and the extracellular matrix for complete regeneration of muscle architecture.
a similar study of cardiotoxin-induced muscle regeneration, recently published by zhao and hoffman, reported that embryonic positional cues  were not induced, whereas expression of factors involved in satellite cell proliferation and differentiation  was recapitulated <cit> .
our study, which also asserts satellite cell activation, proliferation, and differentiation, shows differences in muscle regeneration between mdx and wildtype mice.
bmp and egf-like signalling pathways are activated during regeneration in the mdx mouse, as well as upregulation of members of the notch-delta pathway.
in contrast to the upregulation of pax <dig> in wildtype mice, we have found upregulation of pax <dig>
altogether, these findings suggest that dystrophin-deficiency might lead to enhanced regeneration processes in hindlimb muscles over and above those found in wildtype mice.
it is likely that the regeneration pathways identified in our study are also active in the mdx diaphragm, given that the expression of their downstream targets, the muscle regulatory factors myf- <dig>  myod, and myogenin, are even more elevated in mdx diaphragm than in hindlimb <cit> .
as demonstrated in another recent study <cit> , the regeneration capacity per se is high and does not explain why the muscle wasting in the mdx diaphragm is more severe than in the hindlimb.
other factors such as higher workload and different involvement of the immune system are likely to contribute.
to discern active processes between the lethal human and regenerative murine dystrophin-deficiency, the selected murine gene expression profiles at  <dig> weeks of age were compared to those of dmd patients <cit> .
out of  <dig> regeneration-associated transcripts,  <dig> genes could be detected that are differentially expressed in both the human and murine muscular dystrophy .
seven of these overlapping genes showed an opposite differential expression between human dmd and mdx, of which platelet derived growth factor beta  and paired box  <dig>  are discussed below.
gene expression of pdgfb is upregulated in the mdx mouse , where it is downregulated  in dmd patients.
in the mdx mouse, pdgfb shows an increase in gene expression during regeneration .
pdgfb was immunolocalized in infiltrating macrophages, regenerating muscle fibres, and myofibre nuclei of affected dystrophic muscle tissue <cit> .
the mitogen pdgfb stimulates myoblast proliferation, while inhibiting myoblast differentiation <cit> .
it seems to have a similar role during regeneration.
paired box  <dig>  gene expression is activated in the mdx mouse relative to the control.
its gene expression increases during regeneration, peaking at  <dig> weeks of age, while hpax <dig> is downregulated in dmd patients .
pax <dig> is capable of activating the expression of the muscle regulatory factors myod <dig>  myf <dig> or myogenin, and thereby activating the myogenic program <cit> .
the limited amount of overlapping genes between mdx mice and human dmd patients, as well as a number of genes showing opposite expression , suggests that processes active in regenerating mouse muscle are not active in human patients at the time gene expression was profiled .
this corresponds with clinical findings that patients older than  <dig> years have surpassed active regeneration processes <cit> .
the discovery of genes showing opposite regulation may partly explain the differences in regeneration efficiency and lethal manifestation of the pathology between dystrophin-deficient human and murine muscles.
CONCLUSIONS
mdx mice lack a functional dgc at the sarcolemma.
as a consequence, gene expression of most dgc members is downregulated.
mdx mice suffer from massive muscle fibre necrosis starting at the age of  <dig> weeks.
regenerative processes, starting approximately at the age of  <dig> weeks, largely restore muscle tissue architecture, although muscle fibres remain centrally nucleated.
recovered muscles of mdx mice have slightly diminished strength and higher fatigability.
by analysing temporal expression profiles in mdx and control mice, we have identified genes and pathways involved in regeneration.
the expression of these genes peaks between the ages of 6– <dig> weeks.
based on the observation that several of these genes are not expressed in control muscle and based on gene ontology classification and further literature annotation, we suggest a role for these genes in activation, proliferation, or differentiation of satellite cells and myoblasts.
we propose the following model .
muscular dystrophy leads to muscle fibre necrosis, which attracts inflammatory cells, and release of trophic factors.
these factors activate quiescent satellite cells, which as a consequence start to proliferate and differentiate.
this divergent cell-fate is controlled by the notch-delta pathway.
activated satellite cells differentiate to myoblasts, which proliferate and differentiate as well.
the balance between these cell-fates may be regulated by the level of numb and the activation of bmp <dig> and nrg <dig> signalling pathways.
differentiation of myoblasts eventually leads to fusion with affected muscle fibres or to the formation of new muscle fibres.
the genes and pathways active in regeneration are reminiscent of embryonic myogenesis.
we hypothesize that the newly formed or repaired muscle fibres are similar to those in the pre-necrotic phase, but are more able to adapt to dystrophin-deficiency through remodelling of muscle structure and fibre composition.
since many regeneration-related genes remain higher expressed in mdx than in control muscle, it seems that regeneration processes are active throughout the life span of the animal.
regenerative processes appear to be most effective when mice reach adulthood, and normal growth processes cease.
regeneration and muscle development are both dependent on the availability of satellite cells, and these processes will therefore compete for the satellite cell availability when activated simultaneously.
in the human situation, regeneration processes seem to be exhausted before growth is finished.
together with the accumulation of fibrotic and adipose tissue, exhaustion is thought to be the reason of the lethal manifestation of the disease in human patients.
prolongation of the regenerative capacity by activating the described pathways and/or replenishment with a pool of 'regeneration-primed' cells may therefore provide an attractive strategy in the treatment of muscular dystrophy.

 duchenne muscular dystrophy is caused by mutations in the gene encoding dystrophin, a subsarcolemmal protein functioning within the dystrophin-associated glycoprotein complex. this complex connects the intracellular cytoskeleton to the extracellular matrix. the dgc is concentrated at the z-lines of the sarcomere and confers the transmission of force across the muscle fibre. disruption of this link results in membrane instability, which eventually leads to sarcolemmal ruptures. influx of extracellular calcium alters molecular processes like muscle contraction and activates proteolytic activity. affected muscle fibres become necrotic or apoptotic, and release mitogenic chemoattractants, which initiate inflammatory processes. cycles of degeneration and regeneration eventually lead to irreversible muscle wasting and replacement by fibrotic and adipose tissue. muscle has the potential to regenerate by activation of undifferentiated myogenic precursor cells, which are normally quiescent and situated between the basal membrane and the myofibers. upon activation, satellite cells proliferate and divide asymmetrically, with the daughter cells having divergent cell fates. only one of the daughter cells differentiates, progresses towards the myoblast-stadium, and subsequently fuses with other myoblasts or with damaged muscle fibres to induce muscle fibre repair. the other daughter cell remains in a proliferating state or returns to quiescence. genetic mutations responsible for dmd are also present in satellite cells. hence, the ability to restore normal muscle function remains obstructed. a small number of muscle fibres are able to produce functional dystrophin, mostly due to secondary mutations in myogenic precursor cells which restore the reading frame. however, these so-called revertant fibres are in a too small minority to alleviate the pathology of the dystrophin-deficiency. exhaustion of the satellite cell pool due to degeneration and regeneration cycles is thought to critically contribute to the disease. the mdx mouse model for dmd has a spontaneous mutation in exon of the dmd gene, introducing a premature stopcodon. the pathology of the mdx mouse is characterized by histologically well-defined stages with similarity to the human pathology. neonatal muscle tissue appears to be unaffected. necrotic or apoptotic processes in combination with inflammation emerge at approximately weeks of age. regeneration processes are initiated around the age of weeks and continue while alternating with ongoing degeneration until weeks of age. contrary to the lethal human pathology, the mdx mouse somehow recovers from the progressive muscle wasting, and does not show the accumulation of connective and adipose tissue. however, mdx mice do show a decline in their regeneration capacity at advanced age, while necrotic processes persist. since the degeneration processes are similar to those seen in human pathology, the regenerational differences may hold one of the clues of restoration of proper muscle function. although previous studies have studied gene expression levels in the mdx mouse, regeneration processes were not studied in full detail. we studied the regeneration process through genome-wide monitoring of gene expression levels in healthy control and mdx mice at time points from to weeks of age, while putting emphasis on time points where regenerative activity is maximal, a period which was not analysed in detail in a previous time course study. according to the temporal gene expression profiles, we determined which pathways are active during regeneration with respect to normal muscle aging. the majority of identified genes presented in this study have not been described before and provide a substantial addition to the elucidation of the temporal phasing of degeneration and regeneration. by careful annotation based on existing literature, new light is shed on the pathology and subsequent recovery in the mdx mouse. furthermore, we compared gene expression profiles to those of human dmd patients and found only modest overlap in regeneration-associated genes. this confirms that regeneration is no longer an active process at the age at which the patients were profiled.  global comparison of mdx and control mice gene expression levels were determined in hindlimb muscle tissue from mdx and control mice at time points, ranging from to weeks. differential gene expression levels were calculated per time point by subtraction of the average normalized intensities of control samples from those of the mdx samples to correct for normal aging processes. the effects of the normal aging processes on gene expression are relatively minor, and are discussed below. statistical significance was calculated per time point by performing a student's t-test. differential gene expression was considered significant when p-values were lower than after applying a bonferroni correction for multiple testing. out of temporal gene expression profiles were selected, which satisfied the significance criterion at one or more time points. the number of differentially expressed genes per time point changes considerably during the time course, an effect also shown in a previous study by porter et al.. the number of differentially expressed genes peaks at the age of 8– weeks, coinciding with the period of maximal muscle regeneration. interestingly, also at the first two time points, where the histology of the mdx muscle is not different from that of control mice, a large number of genes was differentially expressed, indicating differences in muscle development in dystrophin-deficient animals. the majority of these genes also show statistically significant differences in expression at later time points. this overlap can be explained by the assumption that the repertoire of gene products used for muscle growth and development also functions in muscle regeneration. in this report we will describe the expression changes of two main categories in more detail: genes coding for proteins within the costamer and the dystrophin-associated glycoprotein complex, and genes involved in regeneration. the dystrophin-glycoprotein complex the effect of dystrophin-deficiency on expression levels of dystrophin-glycoprotein complex -related genes, or genes with associated functional relevance within the costamer has not been reported in previous gene expression profiling studies, with the exception of the study of porter and co-workers. in the porter study, a downregulation of dystrophin was reported, but no changes in gene expression of other components of the dgc. a selection of genes was made based on an overview by ervasti et al. of members of the costameric protein network. according to the statistical selection criteria, genes were upregulated and were downregulated in the mdx mouse. although a decrease in dystrophin expression was found in our study, the stringent statistical criteria were not met. upregulated dgc related genes we find that the retina-specific isoform of dystrophin is expressed in skeletal muscle of mdx mice, whereas dp cannot be detected in hindlimb muscle of control mice. expression of dp was detected by an oligonucleotide probe within the unique first exon of this transcript. the promoter of the dp isoform resides in intron downstream of the mdx mutation. transgenic mdx mice, which overexpress dp via an alpha-actin promoter, show a restoration of a stable association between costameric actin and the sarcolemma, a re-assembly of the dgc, and an overall alleviation of the pathology. increased transcription initiation of dp might therefore be a natural adaptation for the lack of the muscle specific isoform of dystrophin. however, in contrast to the artificially raised expression by the alpha-actin promoter, the expression of dp in the mdx mouse through the original promoter does not seem to be strong enough to compensate for loss of the full-length muscle specific isoform. we found a continuous upregulation of alpha-dystrobrevin gene expression with maximum differential expression at weeks in mdx mice. dystrobrevin is a phosphotyrosine-containing protein localized at both the sarcolemma and the postsynaptic side of the neuromuscular junction, where it binds to either dystrophin or utrophin. dtna has been described to function as a signalling mediator within the dgc. transcription of dtna is activated when myoblasts differentiate into multinucleated myotubes. newey et al. reported that dtna-protein levels are significantly reduced in the mdx mouse at the sarcolemma, whereas the protein level was unchanged at the nmj. this would be consistent with a stabilizing action of dtna upon binding to dystrophin or utrophin, since dystrophin is not present at the sarcolemma, whereas utrophin is expressed at the nmj. they proposed a model, where localized translation of dtna transcripts contributes to synapse formation. upregulation of dtna in the mdx mouse might indicate an attempt to compensate for the increased turnover of the protein, in order to stabilize the post-synaptic side of neuromuscular junctions of affected muscle fibres, and retain neuronal connection. our results show a continuous upregulation of lim domain protein. studies in ldb knock-out mice demonstrated that ablation of ldb eradicates the structural integrity of the z-line in contracting striated muscle and causes a severe form of congenital myopathy. upregulation of ldb indicates the necessity for stabilization of the z-line in mdx mice, compensating the undermining effect of dystrophin-deficiency. downregulated dgc related genes it can be seen that gene expression levels of several core-proteins of the dgc, e.g. the transmembrane proteins dystroglycan, sarcospan, and two members of the sarcoglycan-complex, are lower in mdx mice, over the whole time course. lower expression levels were also detected in other members of the sarcoglycan complex and in dystrophin, but these were not statistically significant. the decrease in expression of dgc-related genes was most prominent during regeneration. interestingly, dgc related gene expression levels restore to pre-regeneration levels subsequent to the regeneration period, but remain lower than normal level. similarly, protein levels of core-proteins of the dgc have been shown to be severely reduced in dystrophin-deficient mdx mice. it is suggested that the secondary displacement of dgc core-proteins is due to a decrease in protein synthesis and/or assembly, or due to an increase in protein degradation. similarly, in sarcoglycan-deficiencies the absence of a single subunit causes the loss or strong reduction of the entire sarcoglycan protein complex. since our study reveals a downregulation of mrna levels of the dgc core-proteins, we conclude that alterations in transcriptional activity also contribute to the decrease in protein levels. as transcription of members of the dgc is likely to be co-ordinately regulated, downregulation of these members as seen in mdx mice can occur via inhibition or downregulation of shared transcriptional activators. regeneration in the mdx mouse, regeneration of affected muscle tissue is most prominent at the age of 6– weeks, after which a stabilized condition is reached. to identify pathways active in regeneration, we studied five categories of differentially expressed genes covering major functional characteristics of regenerative tissues. this selection of genes was typed for temporal effects during regeneration, and the pathways to which they belong. since signal transduction pathways are still poorly annotated in current genomic databases, these pathways were constructed from the literature. in our study, a pathway is only considered activated or repressed, when multiple members show differential gene expression. temporal effects during regeneration differential gene expression profiles, based on the ratio between mdx and control mice, were scaled to the first time point. differential expression profiles can therefore be compared independent of the ratio level, which enables the detection of temporal effects. using k-means clustering, differential gene expression profiles were classified according to their temporal similarity. the unscaled temporal effects of mdx and control gene expression profiles are shown per cluster for the up- and downregulated genes separately. genes, which show an upregulation in gene expression during the regenerative phase, are present in clusters, cluster, and cluster. the temporal effect is determined by the gene expression profile of the mdx mouse, since gene expression is continuously low without temporal changes in the control mouse. downregulation of gene expression in the mdx mouse during regeneration is primarily seen in cluster. during normal aging, which can be seen in the control mouse, gene expression increases until the age of weeks, followed by a slow decrease. during the regenerative phase in the mdx mouse, however, the expression of these genes is downregulated markedly. notch-delta pathway gene expression levels of a number of genes functioning in the notch-delta pathway are upregulated, whereas others are downregulated in the mdx mouse at weeks of age. the gene expression of dll and numb are switched on in the mdx mouse, while no gene expression can be detected in the control mouse. the differential expression of the upregulated genes is mostly increased during the regeneration period. for several genes in the notch-delta pathway, quantitative rt-pcr experiments were performed to confirm the temporal expression profiles found on the microarray. in accordance with the microarray results, quantitative rt-pcr experiments demonstrated higher expression of notch numb and myogenin in mdx than in control mice at all ages. previous work by conboy et al. indicates the role of the notch-delta signalling pathway in the regulation of proliferation versus differentiation of asymmetrically dividing satellite cells by notch or numb, respectively. according to delfini et al., notch is expressed in immature myoblasts, while delta expressing cells are more advanced in myogenesis. notch activation is thought to inhibit transcription factors containing a basic helix loop helix domain, via the induction of hairy and enhancer of split, thereby inhibiting myogenic differentiation. numb-expressing cells are able to undergo myogenic differentiation, because the notch-delta pathway is inhibited. based on our results, the notch-delta signalling pathway, notably the expression of notch or numb, is responsible for the determination towards proliferation or differentiation of activated satellite cells in the mdx mouse. since gene expression profiling detects proliferation and differentiation processes simultaneously, satellite cell activation and commitment are ongoing, parallel processes. bmp pathway various members of a bmp-associated pathway are differentially expressed in the mdx mouse. the gene expression of bmp bmpr1a, inhbc, inhbe, and idb is switched on in the mdx mouse, while expression cannot be detected in the control mouse. bone morphogenetic protein is a member of the transforming growth factor-β family. bmp induces transcription of inhibitors of dna binding proteins via binding to bone morphogenetic protein receptor type i, and the downstream transportation of smad-complex to the nucleus. idb-proteins function as positive regulators of cell growth by binding to retinoblastoma. this leads to the activation of the e2f transcription factor, which plays a role in cell-cycle regulation. furthermore, idb-proteins inhibit myogenic differentiation through binding to mrfs. the activation of idb in the pre-regeneration period is indicative of an inhibition of the myogenic differentiation. this inhibition seems to be alleviated during the regeneration period by a decrease in differential expression of idb although most of the differentially expressed genes in the bmp pathway are continuously upregulated, the expression of a number of genes peaks during regeneration. the inhibin proteins, likely to be antagonists of bone morphogenetic proteins, are also upregulated. altogether, this points to a positively and negatively controlled regulation of the bmp pathway. our data suggests that the bmp pathway has an important function in the balancing of proliferation and differentiation of myoblasts, necessary for effective upscaling of muscle-mass. neuregulin pathway in our study we found that several members of the epidermal growth factor-like neuregulin pathway are differentially expressed. the signalling cascade is activated by the binding of neuregulin to the extracellular domain of the upregulated protein tyrosine kinase v-erb-a erythroblastic leukemia viral oncogene homolog. both nrg and erbb are expressed in the mdx mouse and cannot be detected in the control mouse. the interaction between nrg and erbb activates epidermal growth factor-like signal transduction via binding of the adaptor protein growth factor receptor bound protein, which peaks at the initiation of regeneration. grb can activate mitogen activated kinase kinase, whose differential gene expression is increased at weeks as well as during regeneration. the activation of the map kinase pathways eventually leads to transcriptional induction through members of activating protein complex, like jund and jun. depending on the protein complexes formed, specific transcription activation will lead to different biological processes ranging from proliferation to differentiation. furthermore, we find that other grb-interacting proteins like vav oncogene, and p21-activated kinase are switched on and upregulated, respectively. the differential gene expression of vav increases during the initiation of regeneration, and might play a role in the clustering of integrins for cell adhesion. pak differential gene expression is increased during regeneration. downstream genes activated by pak regulate cytoskeletal dynamics, proliferation and cell survival signalling. furthermore, our results show that the erk pathway is downregulated in the mdx mouse as well as the p pathway. comparisons with other studies in contrast to previously published studies of temporal gene expression profiling in the mdx mouse, we primarily focus on regeneration. the majority of differentially expressed genes in regeneration in our study have not been reported as differentially expressed in the mdx mouse in other studies. apart from important differences in gene coverage, we explain the limited overlap by differences in cut-off levels: as we applied very stringent statistical tests, we could avoid setting a cut-off level for the fold change, thereby picking up genes with small but consistent fold changes, which can be biologically very relevant, especially in the case of transcription factors. this may also explain the large difference between the study of porter et al. and our study in the number of genes found differentially expressed at the early timepoints, where mainly subtle expression changes are expected. goetsch et al. reported results from a gene expression profiling studies during muscle regeneration induced by cardiotoxin injection in wildtype mice. the authors concluded that muscle regeneration is a complex process that requires the coordinated modulation of the inflammatory response, myogenic precursor cells, growth factors, and the extracellular matrix for complete regeneration of muscle architecture. a similar study of cardiotoxin-induced muscle regeneration, recently published by zhao and hoffman, reported that embryonic positional cues were not induced, whereas expression of factors involved in satellite cell proliferation and differentiation was recapitulated. our study, which also asserts satellite cell activation, proliferation, and differentiation, shows differences in muscle regeneration between mdx and wildtype mice. bmp and egf-like signalling pathways are activated during regeneration in the mdx mouse, as well as upregulation of members of the notch-delta pathway. in contrast to the upregulation of pax in wildtype mice, we have found upregulation of pax altogether, these findings suggest that dystrophin-deficiency might lead to enhanced regeneration processes in hindlimb muscles over and above those found in wildtype mice. it is likely that the regeneration pathways identified in our study are also active in the mdx diaphragm, given that the expression of their downstream targets, the muscle regulatory factors myf- myod, and myogenin, are even more elevated in mdx diaphragm than in hindlimb. as demonstrated in another recent study, the regeneration capacity per se is high and does not explain why the muscle wasting in the mdx diaphragm is more severe than in the hindlimb. other factors such as higher workload and different involvement of the immune system are likely to contribute. to discern active processes between the lethal human and regenerative murine dystrophin-deficiency, the selected murine gene expression profiles at weeks of age were compared to those of dmd patients. out of regeneration-associated transcripts, genes could be detected that are differentially expressed in both the human and murine muscular dystrophy. seven of these overlapping genes showed an opposite differential expression between human dmd and mdx, of which platelet derived growth factor beta and paired box are discussed below. gene expression of pdgfb is upregulated in the mdx mouse, where it is downregulated in dmd patients. in the mdx mouse, pdgfb shows an increase in gene expression during regeneration. pdgfb was immunolocalized in infiltrating macrophages, regenerating muscle fibres, and myofibre nuclei of affected dystrophic muscle tissue. the mitogen pdgfb stimulates myoblast proliferation, while inhibiting myoblast differentiation. it seems to have a similar role during regeneration. paired box gene expression is activated in the mdx mouse relative to the control. its gene expression increases during regeneration, peaking at weeks of age, while hpax is downregulated in dmd patients. pax is capable of activating the expression of the muscle regulatory factors myod myf or myogenin, and thereby activating the myogenic program. the limited amount of overlapping genes between mdx mice and human dmd patients, as well as a number of genes showing opposite expression, suggests that processes active in regenerating mouse muscle are not active in human patients at the time gene expression was profiled. this corresponds with clinical findings that patients older than years have surpassed active regeneration processes. the discovery of genes showing opposite regulation may partly explain the differences in regeneration efficiency and lethal manifestation of the pathology between dystrophin-deficient human and murine muscles.  mdx mice lack a functional dgc at the sarcolemma. as a consequence, gene expression of most dgc members is downregulated. mdx mice suffer from massive muscle fibre necrosis starting at the age of weeks. regenerative processes, starting approximately at the age of weeks, largely restore muscle tissue architecture, although muscle fibres remain centrally nucleated. recovered muscles of mdx mice have slightly diminished strength and higher fatigability. by analysing temporal expression profiles in mdx and control mice, we have identified genes and pathways involved in regeneration. the expression of these genes peaks between the ages of 6– weeks. based on the observation that several of these genes are not expressed in control muscle and based on gene ontology classification and further literature annotation, we suggest a role for these genes in activation, proliferation, or differentiation of satellite cells and myoblasts. we propose the following model. muscular dystrophy leads to muscle fibre necrosis, which attracts inflammatory cells, and release of trophic factors. these factors activate quiescent satellite cells, which as a consequence start to proliferate and differentiate. this divergent cell-fate is controlled by the notch-delta pathway. activated satellite cells differentiate to myoblasts, which proliferate and differentiate as well. the balance between these cell-fates may be regulated by the level of numb and the activation of bmp and nrg signalling pathways. differentiation of myoblasts eventually leads to fusion with affected muscle fibres or to the formation of new muscle fibres. the genes and pathways active in regeneration are reminiscent of embryonic myogenesis. we hypothesize that the newly formed or repaired muscle fibres are similar to those in the pre-necrotic phase, but are more able to adapt to dystrophin-deficiency through remodelling of muscle structure and fibre composition. since many regeneration-related genes remain higher expressed in mdx than in control muscle, it seems that regeneration processes are active throughout the life span of the animal. regenerative processes appear to be most effective when mice reach adulthood, and normal growth processes cease. regeneration and muscle development are both dependent on the availability of satellite cells, and these processes will therefore compete for the satellite cell availability when activated simultaneously. in the human situation, regeneration processes seem to be exhausted before growth is finished. together with the accumulation of fibrotic and adipose tissue, exhaustion is thought to be the reason of the lethal manifestation of the disease in human patients. prolongation of the regenerative capacity by activating the described pathways and/or replenishment with a pool of'regeneration-primed' cells may therefore provide an attractive strategy in the treatment of muscular dystrophy. 
in contrast, dystrophin-deficient mdx mice recover due to effective regeneration of affected muscle tissue. to characterize the molecular processes associated with regeneration, we compared gene expression levels in hindlimb muscle tissue of mdx and control mice at timepoints, ranging from 1– weeks of age. the following pathways are activated, as shown by upregulation of multiple members per signalling pathway: the notch-delta pathway that plays a role in the activation of satellite cells, and the bmp and neuregulin signalling pathways that may regulate proliferation and differentiation of satellite cells. we found that genes coding for components of the dystrophin-associated glycoprotein complex are generally downregulated in the mdx mouse. out of genes, were differentially expressed between mdx and control muscle at at least one timepoint. based on the observed expression profiles, we describe a model for muscle regeneration in mdx mice, which may provide new leads for development of dmd therapies based on the improvement of muscle regeneration efficacy. duchenne muscular dystrophy, caused by mutations in the dystrophin gene, is lethal. in dmd patients, only few of the identified regeneration-associated genes were found activated, indicating less efficient regeneration processes in humans.
 duchenne muscular dystrophy, caused by mutations in the dystrophin gene, is lethal. in contrast, dystrophin-deficient mdx mice recover due to effective regeneration of affected muscle tissue. to characterize the molecular processes associated with regeneration, we compared gene expression levels in hindlimb muscle tissue of mdx and control mice at timepoints, ranging from 1– weeks of age.  out of genes, were differentially expressed between mdx and control muscle at at least one timepoint. we found that genes coding for components of the dystrophin-associated glycoprotein complex are generally downregulated in the mdx mouse. based on functional characteristics such as membrane localization, signal transduction, and transcriptional activation, differentially expressed genes with possible functions in regeneration were analyzed in more detail. the majority of these genes peak at the age of weeks, where the regeneration activity is maximal. the following pathways are activated, as shown by upregulation of multiple members per signalling pathway: the notch-delta pathway that plays a role in the activation of satellite cells, and the bmp and neuregulin signalling pathways that may regulate proliferation and differentiation of satellite cells. in dmd patients, only few of the identified regeneration-associated genes were found activated, indicating less efficient regeneration processes in humans.  based on the observed expression profiles, we describe a model for muscle regeneration in mdx mice, which may provide new leads for development of dmd therapies based on the improvement of muscle regeneration efficacy. 