BACKGROUND
genetic background and development prior to birth are known to influence the composition of bovine muscle tissue  <cit> .
prenatal muscle development is therefore a promising source of gene discovery for the molecular events that determine adult muscle phenotype.
prenatal muscle development in mammals begins with the determination of myogenic cells  in the dermamyotome and their migration to their sites of differentiation  <cit> .
myoblasts then fuse to form multinucleated muscle cells, myotubes.
in cattle, primary myotubes appear prior to  <dig> days of gestation  <cit> .
primary skeletal muscle fibers subsequently differentiate from this generation of myotubes  <cit> .
secondary muscle fibers are derived from a separate population of myotubes arising around day  <dig>  <cit> .
the number of muscle fibers is roughly fixed at two thirds of bovine fetal development and muscle fibers differentiate before birth, making cattle a relatively mature species at birth compared to other mammals.
during the final third of gestation in cattle, fetal isoforms of the muscle contractile proteins disappear and are replaced by adult isoforms.
also over the final third of gestation, metabolic differentiation takes place, with an increase in glycolytic and oxidative pathway enzymes  <cit> .
histologically, a steady decrease in the extracellular matrix contribution to muscle mass can be observed over fetal development in the bovine  <cit> .
large mammals such as pigs and cattle offer an advantageous model for the study of myogenesis in vivo, as individual muscles can be identified and reliably dissected from early stages.
in addition, livestock breeds represent useful genetic models for muscle hypertrophy and other muscle traits, as these have been subject to intensive genetic selection due to their impact on meat quality.
for example, array studies of pig fetal development have characterized the coordinate genome-wide expression changes during a developmental time course  <cit>  and also exploited the contrasting muscle phenotypes of the pietrain and duroc pig breeds to compare prenatal events that may underlie the adult muscle phenotype  <cit> .
similarly, sudre et al.
<cit>  studied expression profiles of bovine fetal muscles using a human skeletal muscle macroarray.
a number of human macroarray elements detected significant differential expression signals between fetal developmental stages.
their study highlighted the crucial developmental changes that occur during the final trimester of gestation, as most of the differentially expressed genes in this study were identified in the comparison between  <dig> and  <dig> d of gestation.
a human microarray was employed in a study of fetal muscle from fetuses carrying myostatin loss-of-function mutations  <cit> .
this work described breed-related differences in the expression of genes relevant to several skeletal muscle compartments .
in the present study, we used a bovine cdna microarray derived from adult muscle and adipose tissue libraries to characterize genome-wide transcriptional changes that accompany muscle differentiation in cattle and to determine the impact of two contrasting genotypes on prenatal muscle development.
we carried out a transcriptional profiling study of longissimus muscle from fetuses of both wagyu × hereford and piedmontese × hereford crosses, sampled at d  <dig>   <dig> and  <dig>  as well as newborn calves.
the double-muscled mutations of gdf <dig> carried by beef breeds such as belgian blue or piedmontese cattle  <cit>  have a large impact on muscle yield, and animals of the japanese black  breed of cattle are genetically predisposed to accumulate intramuscular fat  <cit> .
the postnatal phenotypes of offspring from hereford dams sired by these breeds have been well-characterised and are highly divergent for muscle growth and intramuscular fat content  <cit> .
the sampling time points chosen in this study represent primary myogenesis , secondary myogenesis , as well as beginning  and final stages  of functional differentiation of muscle fibers.
RESULTS
the entire set of expression data was deposited on gene expression omnibus database  <cit>  and can be accessed using accession numbers gsm <dig> and gsm <dig>  in this microarray profiling experiment of developing bovine longissimus muscle, we detected  <dig> individual genes  that met the criteria for differential gene expression  either across developmental time or between the two sire breeds.
temporal changes in gene expression
tables  <dig> and  <dig> list expression values for differentially expressed  genes for which reliable annotations could be obtained using the basic local alignment search tool .
these genes were differentially expressed across developmental time, regardless of breed.
twenty genes showed decreasing expression over time and  <dig> genes showed increasing gene expression over time.
all genes in tables  <dig> and  <dig> showed expression changes of more than 2-fold over the time course examined.
gene ontology annotation of the de genes using ease  <cit>  showed that genes involved with the biological processes, morphogenesis and cellular growth were overrepresented in the genes that show decreasing expression over time .
when annotations with respect to cellular component were examined, 69% of the genes with decreasing expression could be classified as belonging to "extracellular matrix" .
collagen genes alone represent 68% of the de genes in table  <dig>  on the other hand, in the list of genes with increasing expression over time, genes with biological process annotations belonging to muscle cell contraction and metabolism are overrepresented .
the main observation from the examination of the developmental time course is therefore that transcripts associated with extracellular matrix synthesis are increasingly less represented as a proportion of total tissue rna as muscle development proceeds, while at the same time transcripts of muscle fiber structural, contractile and metabolism genes are gaining in prevalence when maturation of muscle fibres takes place in preparation for postnatal life.
in most instances, the most marked increase or decrease in gene expression can be observed between d  <dig> of fetal development and birth .
1* indicates a statistically significant decrease in gene expression at p <  <dig>  level; ** indicates a statistically significant decrease in gene expression at p <  <dig>  level.
2sequence annotation was carried out using approved gene symbols  via the ibiss <dig> database  <cit> .
blast  <cit>  scores >  <dig> and e-values <  <dig> e- <dig> were accepted for annotation purposes.
3in cases where more than  <dig> microarray element was used to calculate the expression data, the accession number of  <dig> representative element is shown.
4number of microarray elements representing the same gene that were used to calculate the expression data.
5to obtain the ratios shown, averaged absolute microarray signal intensity values for d  <dig>   <dig> and birth were divided by the signal intensities measured at d  <dig>
1* indicates a statistically significant increase in gene expression at p <  <dig>  level; ** indicates a statistically significant increase in gene expression at p <  <dig>  level.
2sequence annotation was carried out using approved gene symbols  via the ibiss <dig> database  <cit> .
blast  <cit>  scores >  <dig> and e-values <  <dig> e- <dig> were accepted for annotation purposes.
3in cases where more than  <dig> microarray element was used to calculate the expression data, the accession number of  <dig> representative element is shown.
4number of microarray elements representing the same gene that were used to calculate the expression data.
5to obtain the ratios shown, averaged absolute microarray signal intensity values for d  <dig>   <dig> and birth were divided by the signal intensities measured at d  <dig>
two genes, fstl <dig> and igfbp <dig> , which are implicated in the regulation of cell growth and differentiation, are significantly downregulated in developing bovine muscle.
these genes were chosen for quantitative reverse transcription-pcr  validation .
twelve microarray probes that reported increasing expression levels during development all matched to a single consensus sequence  in the ibiss <dig> database  <cit> .
using the netblast function in ibiss <dig>  a match with a non-coding  rna transcript orthologous with the recently described neat <dig> transcript was identified  <cit>  .
gene expression differences correlated with sire breed
a subset of gene expression differences according to sire breed is summarized in table  <dig>  in total,  <dig> genes were differentially expressed according to the breed of the fetus' sire in at least one time point .
the temporal changes in gene expression profiles were greater than the expression differences that were correlated to sire breed.
only  <dig> of these de genes  show a fold-change of more than  <dig> .
aldoa was around two-fold less expressed in the longissimus muscle of wagyu-sired fetuses at d <dig>  while fabp <dig> showed more than two-fold less expression in the longissimus muscle of piedmontese-sired calves at birth.
fabp <dig> expression is also highlighted in table  <dig>  as this gene showed less gene expression in the longissimus muscle of piedmontese-sired fetuses samples at d  <dig>  but at no other time point.
matr <dig> showed less gene expression in the longissimus muscle of day  <dig> wagyu-sired fetuses.
1* indicates a statistically significant gene expression difference at p <  <dig>  level; ** indicates a statistically significant gene expression difference at p <  <dig>  level.
2sequence annotation was carried out using approved gene symbols  via the ibiss <dig> database  <cit> .
blast  <cit>  scores >  <dig> and e-values <  <dig> e- <dig> were accepted for annotation purposes.
3in cases where more than  <dig> microarray element was used to calculate the expression data, the accession number of  <dig> representative element is shown.
4number of microarray elements representing the same gene that were used to calculate the expression data.
5to obtain the ratios shown, averaged absolute microarray signal intensity values for p  fetuses were divided by the signal intensities measured for w  fetuses at d  <dig>  d  <dig>  d  <dig> and birth.
a number of genes showed smaller, yet still significant differences in gene expression between breeds in at least one time point .
table  <dig> provides a summary for some of these classes of genes, for example the collagen genes, which were more highly represented in the transcriptome of late-stage wagyu fetuses.
all differentially expressed ribosomal protein genes and cytochrome c oxidase genes were detected as significantly up-regulated in the muscle of piedmontese crossbred calves at birth .
ribosomal protein and cytochrome c oxidase genes were not detected as differentially expressed over developmental time .
qrt-pcr validation of microarray gene expression patterns
we conducted qrt-pcr studies on the amplified rna used in the microarray study.
assays for fabp <dig>  fabp <dig>  fstl <dig>  igfbp <dig>  and matr <dig> were developed to confirm temporal and sire-breed specific gene expression differences.
in order to confirm that breed-specific differences caused by the piedmontese myostatin  mutation could be detected by gene expression measurements, and because gdf <dig> was not represented on the microarray, gdf <dig> expression was studied using qrt-pcr.
goodness of fit, as measured by the r <dig>  resulted in the anova model explaining  <dig> %,  <dig> %,  <dig> %,  <dig> %,  <dig> % and  <dig> % of the total variation in the expression of gdf <dig>  fabp <dig>  fabp <dig>  igfbp <dig>  fstl <dig>  and matr <dig>  respectively .
temporal gene expression patterns detected by microarray were confirmed for igfbp <dig> and fstl <dig> .
the breed-specific differences in fabp <dig> and fabp <dig> expression documented by microarray were accurately confirmed by qrt-pcr .
fabp <dig> expression was higher in the longissimus muscle of wagyu-sired fetuses at d <dig> and birth.
fabp <dig> expression was higher in the longissimus muscle of wagyu-sired calves at birth, but no significant differences were detected at any other time point.
gdf <dig> expression was significantly higher in the longissimus muscle of piedmontese-sired fetuses at d <dig>  d <dig> and birth .
no difference could be detected at d <dig>
1to obtain fold ratios, mean normalized  qrt-pcr values at  <dig> d gestation,  <dig> d gestation and birth were divided by the values at  <dig> d gestation.
** significance at p <  <dig> .
1to obtain fold ratios, mean normalized  qrt-pcr values from p  samples were divided by those from w  samples at  <dig> d gestation,  <dig> d gestation,  <dig> d gestation and birth.
* significance at p <  <dig> ; ** significance at p <  <dig> .
using the matr <dig> qrt-pcr assay, we were unable to confirm the > <dig> fold higher gene expression detected by microarray in d <dig> muscle rna samples from wagyu-sired fetuses .
in order to clarify what may have caused this discrepancy, qrt-pcr assays for matr <dig> were performed on the amplified rna samples that had been used for the microarray analysis.
this analysis showed that the rna samples amplified from the d  <dig> longissimus muscle of piedmontese-sired fetuses contained higher amounts of matr <dig> rna than the non-amplified rna sample .
specifically, this elevated expression was due to one individual rna sample .
qrt-pcr testing of matr <dig> expression therefore confirmed that the microarray analysis, which was carried out with amplified rna, had accurately identified the elevated matr <dig> expression levels in the d  <dig> sample.
the results did, however uncover a discrepancy between amplified and total rna for at least one particular individual and one gene.
discussion
this study was designed to map out gene expression changes that accompany four discrete phases of muscle development: the first wave of myogenesis , the second generation myoblast formation  and the beginning  and end  stages of contractile and metabolic differentiation of muscle fibers.
as two different breeds of sire  were used to generate the fetuses in this study, we also attempted to detect gene expression differences that may be associated with the genetic background of the fetus.
the fact that hereford crossbreeds rather than purebred fetuses were used in this comparison is a limitation imposed by resource constraints.
however, the postnatal phenotypes of offspring from hereford dams sired by these breeds have been studied in a separate investigation and were found to be highly divergent for muscle growth and intramuscular fat content  <cit> .
at d  <dig>  the longissimus muscle was not identifiable as a distinct anatomical entity.
an area of tissue corresponding to the future position of the longissimus muscle was therefore collected at this time point.
at the d  <dig> time point it was more problematic to accurately dissect the overlying skin tissue away from the muscle.
reliable and consistent sampling of the longissimus muscle, using position along the vertebral column as a positional marker, was achieved from d  <dig> onwards.
due to limiting sample amounts, particularly at the d  <dig> time point, all rna samples were subjected to a linear amplification procedure before labelling for microarray hybridization.
the gene expression analysis of longissimus muscle rna samples provided evidence that the linear amplification procedure may have introduced bias into the representation of mrna sequences in the d  <dig> microarray sample from one individual.
while numerous studies have shown that linear amplification will conserve transcript abundance to an acceptable level, the potential to introduce significant bias to gene expression studies does exist  <cit> .
for the above reasons, the discussion of differential gene expression will focus on the time points d  <dig>  d  <dig> and birth.
temporal changes in gene expression
this microarray study of fetal bovine muscle development detected a large number of differentially expressed genes across developmental time.
the expression pattern of muscle fiber structural and metabolism genes observed in this study is correlated to the timing of contractile differentiation of muscle fibers during fetal development.
the overall trend of decreasing representation of extracellular matrix gene expression in fetal bovine muscle over time is in accordance with histological observations that show the proportion of extracellular matrix material on a muscle section decreasing from 70% to 27% over the time course studied here  <cit> .
the increasing representation of muscle fiber-specific gene expression over time may therefore be a reflection of the proliferation and differentiation of muscle fibers within a connective tissue "scaffold" established early during development  <cit> .
an earlier transcriptome study of bovine muscles during ontogenesis  <cit>  did not identify connective tissue gene expression as a significant contributor to differential gene expression across developmental time.
this may be a consequence of the skeletal muscle focused macroarray that was used in their study.
the bovine fat/muscle microarray used in this study was constructed from muscle and subcutaneous fat cdna libraries  <cit> .
the differentially expressed microarray elements that showed decreasing gene expression over time, mainly had their origin in the subcutaneous fat cdna library.
the observation that the most marked gene expression changes occur between d  <dig> and birth is in accordance with other studies that have shown the dramatic adaptations of skeletal muscle in preparation for birth  <cit> .
as the postnatal muscle samples studied here were collected within  <dig> hours after birth, our study may, in addition, detect gene expression differences that reflect birth trauma.
we were unable to detect transcriptional changes that reflect the replacement of fetal with adult isoforms of contractile protein transcripts during the final trimester of pregnancy, as we are using a microarray based on adult skeletal muscle gene expression  <cit> .
the vast majority of temporal changes in gene expression in fetal longissimus muscle comprise structural and metabolic components of extracellular matrix and muscle fibers.
two exceptions are fstl1and igfbp <dig>  which have been implicated in muscle cell growth and differentiation.
the expression patterns for these two genes were confirmed by qrt-pcr and their reduced expression over the developmental time course is in accord with postulated roles in the inhibition of muscle cell differentiation  <cit> .
both genes have been reported as early targets of myod expression in a mouse model of myod-induced differentiation  <cit> .
in this model of myogenesis, fst <dig> was down-regulated, whereas igfbp <dig> was strongly up-regulated.
this discrepancy with our results may indicate species-specific differences in igfbp <dig> action in bovine muscle development, be due to the fact that our results are derived from an in vivo, not an in vitro model of myogenesis, or hint at the proposed dual role of this gene in the regulation of muscle cell development  <cit> .
our results lend further support to the notion that fstl <dig>  while its precise function is still unknown, has a crucial role in muscle development.
the neat1orthologue, a ncrna species, is found at increasing levels during development of cattle muscle.
neat <dig> was one of only three large abundant ncrnas found enriched in the nucleus and highly conserved across mammalian species  <cit> .
this, together with its association with sc <dig> splicing domains, suggests a fundamental function in mrna metabolism for the neat <dig> transcript.
the increasing representation of this ncrna species in developing cattle muscle may be a reflection of the increasing number of nuclei in developing myofibres.
myonuclei accumulate in muscle cross-sections at an increasing rate up to about  <dig> days post conception  <cit> .
the  <dig>  fold higher representation of the neat <dig> orthologue transcript in the muscle of newborn cattle, compared with the muscle at d <dig>  may therefore indicate a correspondingly higher proportion of nuclei per cell volume sampled.
the observations of ansay  <cit>  on the dna content of developing cattle muscle lend support to this view.
individual gene expression differences correlated with sire breed
while the microarray analysis detected a large number of genes that were significantly differentially expressed in at least one of the time points studied, only  <dig> of those genes showed a more than two-fold difference between the two sire breeds.
we therefore examined the expression of myostatin  itself by qrt-pcr.
gdf <dig> rna was expressed at significantly higher levels in the muscle of piedmontese-sired fetuses at d  <dig> and d  <dig>  in accordance with the findings of forbes et al.
<cit>  that the non-functional gdf <dig> molecule of the piedmontese breed failed to repress the gdf <dig> promoter and therefore led to higher gdf <dig> transcript levels in the fetal muscle.
our observations therefore confirm that the gdf <dig> mutation did affect gene regulation in the fetal longissimus muscle as predicted.
one of the genes showing more than two-fold elevated expression in the muscle of newborn wagyu-sired calves, fabp <dig>  may indicate a higher level of adipogenic differentiation in the longissimus muscle of these animals, even at this early stage of their development.
fabp <dig> showed a similar pattern of breed-specific differences, and the differential expression of both genes was confirmed by qrt-pcr .
fatty acid binding proteins are expressed at very high levels by adipocytes and are themselves, by interaction with peroxisome proliferator-activated receptors  involved in mediating the effects of fatty acids on gene regulation  <cit> .
genetic variation in the bovine fabp <dig> gene has been found to be associated with intramuscular fat content and subcutaneous fat depth  <cit> .
the possibility that fabp <dig> and fabp <dig> may be involved in defining the early onset of intramuscular fat deposition in the wagyu breed remains to be explored.
on the other hand, this finding may be an indication of relatively lower expression of fabp <dig> and fabp <dig> in piedmontese-sired fetuses and therefore a consequence of the biochemical changes initiated by the myostatin mutation.
in support of this latter possibility, a recent study of bovine fetuses carrying a myostatin mutation found a gene marker of adipocyte differentiation down-regulated in semitendinosus muscle at d  <dig> of gestation  <cit> .
global gene expression differences correlated with sire breed
the microarray used in these experiments was based on adult muscle gene expression and is therefore less likely to detect expression differences in developmental regulatory genes involved with determining the double-muscled phenotype.
however, the comprehensive view of developing muscle biochemistry that these experiments describes, points to subtle differences in timing of gene expression, as well as differences in the translational capacity of the piedmontese muscle and an altered biochemical profile as the main consequences of the gdf <dig> mutation.
our study provides evidence for gene expression timing differences by the large number of ribosomal genes, which tend to be expressed at a relatively lower level in the longissimus muscle of piedmontese-sired fetuses at the earlier time points and are then relatively more highly expressed at birth.
this confirms the findings by steelman et al.
<cit> , who found genes involved in translation overrepresented among the up-regulated genes in 5-week-old myostatin null mice.
a study of d 30- d  <dig> bovine embryos had identified a number of ribosomal protein genes as differentially expressed in embryos carrying the gdf <dig> mutation  <cit> .
cassar-malek et al.
<cit> , in a gene expression study in bovine semitendinosus muscle from d  <dig> fetuses carrying myostatin mutations also described higher levels of expression of ribosomal protein genes at this time point.
support for the view that timing differences characterize the development of gdf <dig> mutants comes from the work of deveaux et al <cit> , which showed evidence for a delay in contractile differentiation of muscle from belgian blue fetuses, and cagnazzo et al.
<cit> , who showed that the fetuses from a more highly muscled breed of pigs showed a later peak of myogenesis-related gene expression during fetal development.
our study confirms the findings by cassar-malek et al.
<cit>  of decreased collagen gene expression in the muscle of late stage bovine fetuses carrying a myostatin mutation.
this finding lends support to our interpretation of the temporal changes of collagen gene expression .
as the gdf <dig> mutation results in larger numbers of muscle fibers that have larger cross-sectional areas on average, connective tissue fibroblasts  should contribute relatively less to the overall gene expression profile.
gdf <dig> mutants are known to have a more glycolytic adult muscle phenotype than wild type animals  <cit>  and this is clearly reflected in the gene expression comparisons between breeds at this time point.
for example, the piedmontese-sired fetuses show higher expression of genes that reflect fast, glycolytic fiber structural differentiation such as actn <dig>  myh <dig> and myh <dig>  and mybpc <dig>  coupled with higher expression of genes for glycolytic metabolic enzymes such as aldoa and ldha.
these findings confirm the observations of cassar-malek et al <cit>  on bovine muscle and also of steelman et al.
<cit> , who documented a shift away from slow muscle fiber isoforms in myostatin null mice.
at birth, a suite of gene expression changes suggesting an increased atp supply to the muscle of the piedmontese-sired fetuses is observed.
in fact, there is a coordinated up-regulation of genes encoding enzymes that determine mitochondrial activity at multiple steps in cellular respiration.
these include key oxidative enzymes, in particular isoforms of cytochrome c oxidase , the terminal enzyme in the citric acid cycle.
cagnazzo et al <cit>  made a similar observation in their comparison of transcription profiles of developing muscles from two contrasting breeds of pigs.
they found that a highly muscled breed of pigs  showed more active transcription of atp metabolism-related genes during late fetal development.
this surge in atp supply may reflect increased energetic demand in the immediate postnatal period in animals that experience very rapid postnatal growth, such as piedmontese-sired calves or pietrain piglets.
two major atp consuming cellular processes are the maintenance of transmembrane ion gradients and protein turnover  <cit> .
the demand for sarcolemmal ion gradients is unlikely to increase at this time.
in fact, the expression of atpase genes involved in this process  is relatively lower in the muscle of piedmontese-sired calves.
therefore, taken together with the observation that a suite of ribosomal proteins is upregulated in piedmontese-sired calves, we suggest that the extra atp might be supporting more active ribosomal synthetic machinery.
consequently, the gene expression changes captured by this microarray appear to describe the energetic modifications necessary for the rapid muscular growth of newborn gdf <dig> mutants.
CONCLUSIONS
this study describes the molecular events accompanying skeletal muscle differentiation in the bovine, namely a coordinate down-regulation of extracellular matrix-related gene expression at the same time as increasing gene expression levels of structural and metabolic constituents of muscle fibers.
this study also highlights the developmental expression pattern of fstl <dig> and igfbp <dig>  which have previously been implicated in myogenesis regulation, as well as describing the changing representation of a recently-described ncrna  in developing cattle muscle.
a large number of breed-related gene expression differences were detected by contrasting the development of wagyu-sired and piedmontese-sired fetuses.
while no breed-related differences in regulatory pathways were detected in this study, it documents the differential timing and magnitude of gene expression that result in the more glycolytic muscle fiber profile as well as the lower level of intramuscular fat observed in animals carrying myostatin mutations.

 genetic background and development prior to birth are known to influence the composition of bovine muscle tissue. prenatal muscle development is therefore a promising source of gene discovery for the molecular events that determine adult muscle phenotype. prenatal muscle development in mammals begins with the determination of myogenic cells in the dermamyotome and their migration to their sites of differentiation. myoblasts then fuse to form multinucleated muscle cells, myotubes. in cattle, primary myotubes appear prior to days of gestation. primary skeletal muscle fibers subsequently differentiate from this generation of myotubes. secondary muscle fibers are derived from a separate population of myotubes arising around day. the number of muscle fibers is roughly fixed at two thirds of bovine fetal development and muscle fibers differentiate before birth, making cattle a relatively mature species at birth compared to other mammals. during the final third of gestation in cattle, fetal isoforms of the muscle contractile proteins disappear and are replaced by adult isoforms. also over the final third of gestation, metabolic differentiation takes place, with an increase in glycolytic and oxidative pathway enzymes. histologically, a steady decrease in the extracellular matrix contribution to muscle mass can be observed over fetal development in the bovine. large mammals such as pigs and cattle offer an advantageous model for the study of myogenesis in vivo, as individual muscles can be identified and reliably dissected from early stages. in addition, livestock breeds represent useful genetic models for muscle hypertrophy and other muscle traits, as these have been subject to intensive genetic selection due to their impact on meat quality. for example, array studies of pig fetal development have characterized the coordinate genome-wide expression changes during a developmental time course and also exploited the contrasting muscle phenotypes of the pietrain and duroc pig breeds to compare prenatal events that may underlie the adult muscle phenotype. similarly, sudre et al. studied expression profiles of bovine fetal muscles using a human skeletal muscle macroarray. a number of human macroarray elements detected significant differential expression signals between fetal developmental stages. their study highlighted the crucial developmental changes that occur during the final trimester of gestation, as most of the differentially expressed genes in this study were identified in the comparison between and d of gestation. a human microarray was employed in a study of fetal muscle from fetuses carrying myostatin loss-of-function mutations. this work described breed-related differences in the expression of genes relevant to several skeletal muscle compartments. in the present study, we used a bovine cdna microarray derived from adult muscle and adipose tissue libraries to characterize genome-wide transcriptional changes that accompany muscle differentiation in cattle and to determine the impact of two contrasting genotypes on prenatal muscle development. we carried out a transcriptional profiling study of longissimus muscle from fetuses of both wagyu × hereford and piedmontese × hereford crosses, sampled at d and as well as newborn calves. the double-muscled mutations of gdf carried by beef breeds such as belgian blue or piedmontese cattle have a large impact on muscle yield, and animals of the japanese black breed of cattle are genetically predisposed to accumulate intramuscular fat. the postnatal phenotypes of offspring from hereford dams sired by these breeds have been well-characterised and are highly divergent for muscle growth and intramuscular fat content. the sampling time points chosen in this study represent primary myogenesis, secondary myogenesis, as well as beginning and final stages of functional differentiation of muscle fibers.  the entire set of expression data was deposited on gene expression omnibus database and can be accessed using accession numbers gsm and gsm in this microarray profiling experiment of developing bovine longissimus muscle, we detected individual genes that met the criteria for differential gene expression either across developmental time or between the two sire breeds. temporal changes in gene expression tables and list expression values for differentially expressed genes for which reliable annotations could be obtained using the basic local alignment search tool. these genes were differentially expressed across developmental time, regardless of breed. twenty genes showed decreasing expression over time and genes showed increasing gene expression over time. all genes in tables and showed expression changes of more than 2-fold over the time course examined. gene ontology annotation of the de genes using ease showed that genes involved with the biological processes, morphogenesis and cellular growth were overrepresented in the genes that show decreasing expression over time. when annotations with respect to cellular component were examined, 69% of the genes with decreasing expression could be classified as belonging to "extracellular matrix". collagen genes alone represent 68% of the de genes in table on the other hand, in the list of genes with increasing expression over time, genes with biological process annotations belonging to muscle cell contraction and metabolism are overrepresented. the main observation from the examination of the developmental time course is therefore that transcripts associated with extracellular matrix synthesis are increasingly less represented as a proportion of total tissue rna as muscle development proceeds, while at the same time transcripts of muscle fiber structural, contractile and metabolism genes are gaining in prevalence when maturation of muscle fibres takes place in preparation for postnatal life. in most instances, the most marked increase or decrease in gene expression can be observed between d of fetal development and birth. 1* indicates a statistically significant decrease in gene expression at p < level; ** indicates a statistically significant decrease in gene expression at p < level. 2sequence annotation was carried out using approved gene symbols via the ibiss database. blast scores > and e-values < e- were accepted for annotation purposes. 3in cases where more than microarray element was used to calculate the expression data, the accession number of representative element is shown. 4number of microarray elements representing the same gene that were used to calculate the expression data. 5to obtain the ratios shown, averaged absolute microarray signal intensity values for d and birth were divided by the signal intensities measured at d 1* indicates a statistically significant increase in gene expression at p < level; ** indicates a statistically significant increase in gene expression at p < level. 2sequence annotation was carried out using approved gene symbols via the ibiss database. blast scores > and e-values < e- were accepted for annotation purposes. 3in cases where more than microarray element was used to calculate the expression data, the accession number of representative element is shown. 4number of microarray elements representing the same gene that were used to calculate the expression data. 5to obtain the ratios shown, averaged absolute microarray signal intensity values for d and birth were divided by the signal intensities measured at d two genes, fstl and igfbp, which are implicated in the regulation of cell growth and differentiation, are significantly downregulated in developing bovine muscle. these genes were chosen for quantitative reverse transcription-pcr validation. twelve microarray probes that reported increasing expression levels during development all matched to a single consensus sequence in the ibiss database. using the netblast function in ibiss a match with a non-coding rna transcript orthologous with the recently described neat transcript was identified. gene expression differences correlated with sire breed a subset of gene expression differences according to sire breed is summarized in table in total, genes were differentially expressed according to the breed of the fetus' sire in at least one time point. the temporal changes in gene expression profiles were greater than the expression differences that were correlated to sire breed. only of these de genes show a fold-change of more than. aldoa was around two-fold less expressed in the longissimus muscle of wagyu-sired fetuses at d while fabp showed more than two-fold less expression in the longissimus muscle of piedmontese-sired calves at birth. fabp expression is also highlighted in table as this gene showed less gene expression in the longissimus muscle of piedmontese-sired fetuses samples at d but at no other time point. matr showed less gene expression in the longissimus muscle of day wagyu-sired fetuses. 1* indicates a statistically significant gene expression difference at p < level; ** indicates a statistically significant gene expression difference at p < level. 2sequence annotation was carried out using approved gene symbols via the ibiss database. blast scores > and e-values < e- were accepted for annotation purposes. 3in cases where more than microarray element was used to calculate the expression data, the accession number of representative element is shown. 4number of microarray elements representing the same gene that were used to calculate the expression data. 5to obtain the ratios shown, averaged absolute microarray signal intensity values for p fetuses were divided by the signal intensities measured for w fetuses at d d d and birth. a number of genes showed smaller, yet still significant differences in gene expression between breeds in at least one time point. table provides a summary for some of these classes of genes, for example the collagen genes, which were more highly represented in the transcriptome of late-stage wagyu fetuses. all differentially expressed ribosomal protein genes and cytochrome c oxidase genes were detected as significantly up-regulated in the muscle of piedmontese crossbred calves at birth. ribosomal protein and cytochrome c oxidase genes were not detected as differentially expressed over developmental time. qrt-pcr validation of microarray gene expression patterns we conducted qrt-pcr studies on the amplified rna used in the microarray study. assays for fabp fabp fstl igfbp and matr were developed to confirm temporal and sire-breed specific gene expression differences. in order to confirm that breed-specific differences caused by the piedmontese myostatin mutation could be detected by gene expression measurements, and because gdf was not represented on the microarray, gdf expression was studied using qrt-pcr. goodness of fit, as measured by the r resulted in the anova model explaining %, %, %, %, % and % of the total variation in the expression of gdf fabp fabp igfbp fstl and matr respectively. temporal gene expression patterns detected by microarray were confirmed for igfbp and fstl. the breed-specific differences in fabp and fabp expression documented by microarray were accurately confirmed by qrt-pcr. fabp expression was higher in the longissimus muscle of wagyu-sired fetuses at d and birth. fabp expression was higher in the longissimus muscle of wagyu-sired calves at birth, but no significant differences were detected at any other time point. gdf expression was significantly higher in the longissimus muscle of piedmontese-sired fetuses at d d and birth. no difference could be detected at d 1to obtain fold ratios, mean normalized qrt-pcr values at d gestation, d gestation and birth were divided by the values at d gestation. ** significance at p <. 1to obtain fold ratios, mean normalized qrt-pcr values from p samples were divided by those from w samples at d gestation, d gestation, d gestation and birth. * significance at p <; ** significance at p <. using the matr qrt-pcr assay, we were unable to confirm the > fold higher gene expression detected by microarray in d muscle rna samples from wagyu-sired fetuses. in order to clarify what may have caused this discrepancy, qrt-pcr assays for matr were performed on the amplified rna samples that had been used for the microarray analysis. this analysis showed that the rna samples amplified from the d longissimus muscle of piedmontese-sired fetuses contained higher amounts of matr rna than the non-amplified rna sample. specifically, this elevated expression was due to one individual rna sample. qrt-pcr testing of matr expression therefore confirmed that the microarray analysis, which was carried out with amplified rna, had accurately identified the elevated matr expression levels in the d sample. the results did, however uncover a discrepancy between amplified and total rna for at least one particular individual and one gene. discussion this study was designed to map out gene expression changes that accompany four discrete phases of muscle development: the first wave of myogenesis, the second generation myoblast formation and the beginning and end stages of contractile and metabolic differentiation of muscle fibers. as two different breeds of sire were used to generate the fetuses in this study, we also attempted to detect gene expression differences that may be associated with the genetic background of the fetus. the fact that hereford crossbreeds rather than purebred fetuses were used in this comparison is a limitation imposed by resource constraints. however, the postnatal phenotypes of offspring from hereford dams sired by these breeds have been studied in a separate investigation and were found to be highly divergent for muscle growth and intramuscular fat content. at d the longissimus muscle was not identifiable as a distinct anatomical entity. an area of tissue corresponding to the future position of the longissimus muscle was therefore collected at this time point. at the d time point it was more problematic to accurately dissect the overlying skin tissue away from the muscle. reliable and consistent sampling of the longissimus muscle, using position along the vertebral column as a positional marker, was achieved from d onwards. due to limiting sample amounts, particularly at the d time point, all rna samples were subjected to a linear amplification procedure before labelling for microarray hybridization. the gene expression analysis of longissimus muscle rna samples provided evidence that the linear amplification procedure may have introduced bias into the representation of mrna sequences in the d microarray sample from one individual. while numerous studies have shown that linear amplification will conserve transcript abundance to an acceptable level, the potential to introduce significant bias to gene expression studies does exist. for the above reasons, the discussion of differential gene expression will focus on the time points d d and birth. temporal changes in gene expression this microarray study of fetal bovine muscle development detected a large number of differentially expressed genes across developmental time. the expression pattern of muscle fiber structural and metabolism genes observed in this study is correlated to the timing of contractile differentiation of muscle fibers during fetal development. the overall trend of decreasing representation of extracellular matrix gene expression in fetal bovine muscle over time is in accordance with histological observations that show the proportion of extracellular matrix material on a muscle section decreasing from 70% to 27% over the time course studied here. the increasing representation of muscle fiber-specific gene expression over time may therefore be a reflection of the proliferation and differentiation of muscle fibers within a connective tissue "scaffold" established early during development. an earlier transcriptome study of bovine muscles during ontogenesis did not identify connective tissue gene expression as a significant contributor to differential gene expression across developmental time. this may be a consequence of the skeletal muscle focused macroarray that was used in their study. the bovine fat/muscle microarray used in this study was constructed from muscle and subcutaneous fat cdna libraries. the differentially expressed microarray elements that showed decreasing gene expression over time, mainly had their origin in the subcutaneous fat cdna library. the observation that the most marked gene expression changes occur between d and birth is in accordance with other studies that have shown the dramatic adaptations of skeletal muscle in preparation for birth. as the postnatal muscle samples studied here were collected within hours after birth, our study may, in addition, detect gene expression differences that reflect birth trauma. we were unable to detect transcriptional changes that reflect the replacement of fetal with adult isoforms of contractile protein transcripts during the final trimester of pregnancy, as we are using a microarray based on adult skeletal muscle gene expression. the vast majority of temporal changes in gene expression in fetal longissimus muscle comprise structural and metabolic components of extracellular matrix and muscle fibers. two exceptions are fstl1and igfbp which have been implicated in muscle cell growth and differentiation. the expression patterns for these two genes were confirmed by qrt-pcr and their reduced expression over the developmental time course is in accord with postulated roles in the inhibition of muscle cell differentiation. both genes have been reported as early targets of myod expression in a mouse model of myod-induced differentiation. in this model of myogenesis, fst was down-regulated, whereas igfbp was strongly up-regulated. this discrepancy with our results may indicate species-specific differences in igfbp action in bovine muscle development, be due to the fact that our results are derived from an in vivo, not an in vitro model of myogenesis, or hint at the proposed dual role of this gene in the regulation of muscle cell development. our results lend further support to the notion that fstl while its precise function is still unknown, has a crucial role in muscle development. the neat1orthologue, a ncrna species, is found at increasing levels during development of cattle muscle. neat was one of only three large abundant ncrnas found enriched in the nucleus and highly conserved across mammalian species. this, together with its association with sc splicing domains, suggests a fundamental function in mrna metabolism for the neat transcript. the increasing representation of this ncrna species in developing cattle muscle may be a reflection of the increasing number of nuclei in developing myofibres. myonuclei accumulate in muscle cross-sections at an increasing rate up to about days post conception. the fold higher representation of the neat orthologue transcript in the muscle of newborn cattle, compared with the muscle at d may therefore indicate a correspondingly higher proportion of nuclei per cell volume sampled. the observations of ansay on the dna content of developing cattle muscle lend support to this view. individual gene expression differences correlated with sire breed while the microarray analysis detected a large number of genes that were significantly differentially expressed in at least one of the time points studied, only of those genes showed a more than two-fold difference between the two sire breeds. we therefore examined the expression of myostatin itself by qrt-pcr. gdf rna was expressed at significantly higher levels in the muscle of piedmontese-sired fetuses at d and d in accordance with the findings of forbes et al. that the non-functional gdf molecule of the piedmontese breed failed to repress the gdf promoter and therefore led to higher gdf transcript levels in the fetal muscle. our observations therefore confirm that the gdf mutation did affect gene regulation in the fetal longissimus muscle as predicted. one of the genes showing more than two-fold elevated expression in the muscle of newborn wagyu-sired calves, fabp may indicate a higher level of adipogenic differentiation in the longissimus muscle of these animals, even at this early stage of their development. fabp showed a similar pattern of breed-specific differences, and the differential expression of both genes was confirmed by qrt-pcr. fatty acid binding proteins are expressed at very high levels by adipocytes and are themselves, by interaction with peroxisome proliferator-activated receptors involved in mediating the effects of fatty acids on gene regulation. genetic variation in the bovine fabp gene has been found to be associated with intramuscular fat content and subcutaneous fat depth. the possibility that fabp and fabp may be involved in defining the early onset of intramuscular fat deposition in the wagyu breed remains to be explored. on the other hand, this finding may be an indication of relatively lower expression of fabp and fabp in piedmontese-sired fetuses and therefore a consequence of the biochemical changes initiated by the myostatin mutation. in support of this latter possibility, a recent study of bovine fetuses carrying a myostatin mutation found a gene marker of adipocyte differentiation down-regulated in semitendinosus muscle at d of gestation. global gene expression differences correlated with sire breed the microarray used in these experiments was based on adult muscle gene expression and is therefore less likely to detect expression differences in developmental regulatory genes involved with determining the double-muscled phenotype. however, the comprehensive view of developing muscle biochemistry that these experiments describes, points to subtle differences in timing of gene expression, as well as differences in the translational capacity of the piedmontese muscle and an altered biochemical profile as the main consequences of the gdf mutation. our study provides evidence for gene expression timing differences by the large number of ribosomal genes, which tend to be expressed at a relatively lower level in the longissimus muscle of piedmontese-sired fetuses at the earlier time points and are then relatively more highly expressed at birth. this confirms the findings by steelman et al., who found genes involved in translation overrepresented among the up-regulated genes in 5-week-old myostatin null mice. a study of d 30- d bovine embryos had identified a number of ribosomal protein genes as differentially expressed in embryos carrying the gdf mutation. cassar-malek et al., in a gene expression study in bovine semitendinosus muscle from d fetuses carrying myostatin mutations also described higher levels of expression of ribosomal protein genes at this time point. support for the view that timing differences characterize the development of gdf mutants comes from the work of deveaux et al, which showed evidence for a delay in contractile differentiation of muscle from belgian blue fetuses, and cagnazzo et al., who showed that the fetuses from a more highly muscled breed of pigs showed a later peak of myogenesis-related gene expression during fetal development. our study confirms the findings by cassar-malek et al. of decreased collagen gene expression in the muscle of late stage bovine fetuses carrying a myostatin mutation. this finding lends support to our interpretation of the temporal changes of collagen gene expression. as the gdf mutation results in larger numbers of muscle fibers that have larger cross-sectional areas on average, connective tissue fibroblasts should contribute relatively less to the overall gene expression profile. gdf mutants are known to have a more glycolytic adult muscle phenotype than wild type animals and this is clearly reflected in the gene expression comparisons between breeds at this time point. for example, the piedmontese-sired fetuses show higher expression of genes that reflect fast, glycolytic fiber structural differentiation such as actn myh and myh and mybpc coupled with higher expression of genes for glycolytic metabolic enzymes such as aldoa and ldha. these findings confirm the observations of cassar-malek et al on bovine muscle and also of steelman et al., who documented a shift away from slow muscle fiber isoforms in myostatin null mice. at birth, a suite of gene expression changes suggesting an increased atp supply to the muscle of the piedmontese-sired fetuses is observed. in fact, there is a coordinated up-regulation of genes encoding enzymes that determine mitochondrial activity at multiple steps in cellular respiration. these include key oxidative enzymes, in particular isoforms of cytochrome c oxidase, the terminal enzyme in the citric acid cycle. cagnazzo et al made a similar observation in their comparison of transcription profiles of developing muscles from two contrasting breeds of pigs. they found that a highly muscled breed of pigs showed more active transcription of atp metabolism-related genes during late fetal development. this surge in atp supply may reflect increased energetic demand in the immediate postnatal period in animals that experience very rapid postnatal growth, such as piedmontese-sired calves or pietrain piglets. two major atp consuming cellular processes are the maintenance of transmembrane ion gradients and protein turnover. the demand for sarcolemmal ion gradients is unlikely to increase at this time. in fact, the expression of atpase genes involved in this process is relatively lower in the muscle of piedmontese-sired calves. therefore, taken together with the observation that a suite of ribosomal proteins is upregulated in piedmontese-sired calves, we suggest that the extra atp might be supporting more active ribosomal synthetic machinery. consequently, the gene expression changes captured by this microarray appear to describe the energetic modifications necessary for the rapid muscular growth of newborn gdf mutants.  this study describes the molecular events accompanying skeletal muscle differentiation in the bovine, namely a coordinate down-regulation of extracellular matrix-related gene expression at the same time as increasing gene expression levels of structural and metabolic constituents of muscle fibers. this study also highlights the developmental expression pattern of fstl and igfbp which have previously been implicated in myogenesis regulation, as well as describing the changing representation of a recently-described ncrna in developing cattle muscle. a large number of breed-related gene expression differences were detected by contrasting the development of wagyu-sired and piedmontese-sired fetuses. while no breed-related differences in regulatory pathways were detected in this study, it documents the differential timing and magnitude of gene expression that result in the more glycolytic muscle fiber profile as well as the lower level of intramuscular fat observed in animals carrying myostatin mutations. 
all fetuses and newborns were from hereford dams and crossed with either wagyu or piedmontese sires, genotypes that vary markedly in muscle and compositional characteristics later in postnatal life. in order to discover genes that are involved in determining adult muscle phenotypes, we studied the gene expression profile of developing fetal bovine longissimus muscle from animals with two different genetic backgrounds using a bovine cdna microarray. fetal longissimus muscle was sampled at stages of myogenesis and muscle maturation: primary myogenesis, secondary myogenesis, as well as beginning and final stages of functional differentiation of muscle fibers. quantitative reverse transcription-pcr analysis of rna from developing longissimus muscle was able to validate the differential expression patterns observed for a selection of differentially expressed genes, with one exception. the developing longissimus muscle of fetuses carrying the piedmontese mutation shows an emphasis on glycolytic muscle biochemistry and a large-scale up-regulation of the translational machinery at birth. taken together, these findings provide a detailed description of molecular events accompanying skeletal muscle differentiation in the bovine, as well as gene expression differences that may underpin the phenotype differences between the two breeds. fstl and igfbp were two genes implicated in growth and differentiation that showed developmentally regulated expression levels in fetal muscle. in addition, this study has highlighted a non-coding rna, which is abundantly expressed and developmentally regulated in bovine fetal muscle. we also observed differences in gene expression profiles between the two different sire breeds. we obtained expression profiles of three individuals for each time point and genotype to allow comparisons across time and between sire breeds. we detected large-scale changes in temporal gene expression between the four developmental stages in genes coding for extracellular matrix and for muscle fiber structural and metabolic proteins. wagyu-sired calves showed higher expression of fatty acid binding protein rna at birth.
 the muscle fiber number and fiber composition of muscle is largely determined during prenatal development. in order to discover genes that are involved in determining adult muscle phenotypes, we studied the gene expression profile of developing fetal bovine longissimus muscle from animals with two different genetic backgrounds using a bovine cdna microarray. fetal longissimus muscle was sampled at stages of myogenesis and muscle maturation: primary myogenesis, secondary myogenesis, as well as beginning and final stages of functional differentiation of muscle fibers. all fetuses and newborns were from hereford dams and crossed with either wagyu or piedmontese sires, genotypes that vary markedly in muscle and compositional characteristics later in postnatal life.  we obtained expression profiles of three individuals for each time point and genotype to allow comparisons across time and between sire breeds. quantitative reverse transcription-pcr analysis of rna from developing longissimus muscle was able to validate the differential expression patterns observed for a selection of differentially expressed genes, with one exception. we detected large-scale changes in temporal gene expression between the four developmental stages in genes coding for extracellular matrix and for muscle fiber structural and metabolic proteins. fstl and igfbp were two genes implicated in growth and differentiation that showed developmentally regulated expression levels in fetal muscle. an abundantly expressed gene with no functional annotation was found to be developmentally regulated in the same manner as muscle structural proteins. we also observed differences in gene expression profiles between the two different sire breeds. wagyu-sired calves showed higher expression of fatty acid binding protein rna at birth. the developing longissimus muscle of fetuses carrying the piedmontese mutation shows an emphasis on glycolytic muscle biochemistry and a large-scale up-regulation of the translational machinery at birth. we also document evidence for timing differences in differentiation events between the two breeds.  taken together, these findings provide a detailed description of molecular events accompanying skeletal muscle differentiation in the bovine, as well as gene expression differences that may underpin the phenotype differences between the two breeds. in addition, this study has highlighted a non-coding rna, which is abundantly expressed and developmentally regulated in bovine fetal muscle. 