BACKGROUND
texel sheep, a typical "double muscle" breed due to a gdf <dig> mutation  <cit> , are now commercially produced throughout the world, with no adverse effects detected by objective assessments of meat quality  <cit> .
however, evidence for an association between g+6723g > a and decreased intramuscular fat and reduced eating quality has been observed  <cit> .
compared with texel sheep, indigenous chinese ujumqin sheep, with no gdf <dig> mutation  <cit> , are less muscular and have a higher fat content, but they are superior in terms of perceived meat quality.
therefore, these two sheep breeds provide a good natural model for studying muscle and fat development, as well as for identifying myostatin genes.
prenatal skeletal muscle development is an important determinant of both muscularity and meat quality  <cit> .
in large precocial species such as sheep  <cit>  and cattle  <cit> , the maximum myofiber complement of a muscle is achieved prior to birth.
more than three waves of myogenic cells appear in sheep, and most myofibers form during the second half of gestation  <cit> .
however, whether the myofibers increase with a pulsed-wave mode at certain developmental stages or whether they augment evenly across developmental stages in fetal sheep remains unclear.
myostatin, a member of the transforming growth factor-β  family, is predominantly expressed and secreted by skeletal muscle and functions as a negative regulator of muscle growth.
mutations in the myostatin gene lead to a hypertrophic phenotype in mice, sheep, cattle, dog, and human  <cit> .
the effect of myostatin on gene expression in prenatal muscles at the genome-wide level was recently explored in fetal cattle  <cit> , but no studies have been conducted dynamically at multiple fetal stages comparing two pure breeds with extreme phenotypes.
a recent mice study demonstrated that myoblasts from embryonic and fetal stages not only had different fusion abilities, proliferation, differentiation and responses to tgf-β, phorbol ester 12-o-tetradecanoylphorbol-13-acetate, and bone morphogenetic protein- <dig> in vitro, but they also differed in gene expression profiles  <cit> , indicating that complicated and obvious changes in physiology and biochemistry occur during the prenatal stage in vivo.
therefore, investigating the subtle changes of the effect of a myostatin mutation on skeletal muscle development at multiple fetal stages using our experimental model is necessary.
here, we examined gene expression and myofiber development in texel and ujumqin sheep at the transcriptome and histological levels.
our findings contribute to understanding the dynamic effects of myostatin on the biology of prenatal skeletal muscle development in large mammals.
the results also provide clues into human myopathy and obesity during prenatal stages.
we also identified putative candidate genes that are valuable for meat-quality traits in farm animals.
RESULTS
histological characteristics of ovine fetal skeletal muscle development
we examined the number and diameter of myofibers at each developmental stage in texel and ujumqin sheep via histological analysis  .
significant difference was observed in myofiber diameter between texel and ujumqin sheep at each development stage, except at  <dig> d. however, the myofiber diameter patterns of the two breeds were similar throughout the five developmental stages.
unexpectedly, we found that the myofiber hyperplasia profiles differed between texel and ujumqin fetuses .
the proliferation of myofibers in texel sheep was fastest at  <dig> and  <dig> d, particularly at  <dig> d, suggesting that this period is vital for muscle development in fetal texel sheep.
myofiber hyperplasia reached a peak at  <dig> d in ujumqin sheep, when the number of myofibers exceeded that of texel sheep.
notably, with development, the number of myofibers in texel fetuses recovered to that of ujumqin fetuses at  <dig> d, although myofiber hypertrophy in texel sheep was greater than that in ujumqin sheep at this stage.
therefore, a new wave of myogenesis most probably occurs at  <dig> d in texel sheep, given that the number of myofibers increased in ujumqin sheep during this period.
additionally, many small-diameter fibers occurred in the muscle at  <dig> d in both breeds.
the small fibers could be due either to intrafascicular terminations  <cit>  or have been generated by the fusion of myoblasts during the period investigated.
therefore, to explore the myogenic potential in the two breeds of sheep, we identified pax7-positive cells in skeletal muscle at  <dig> d using immnofluorescence staining  and found that the ratio of the number of pax7-positive cells  to the fibers on each muscle slide was significantly higher in the texel fetuses than in the ujumqin fetuses .
in combination with figure  <dig>  this observation suggests that a period of 100- <dig> d is another new proliferative stage for myofiber hyperplasia in texel sheep.
higher numbers of pax7-positive cells were correlated with a higher muscle mass in texel sheep during both the prenatal and postnatal stages.
microarray experiment
a mean transcript sequence identity of 97% was found between ovine and bovine orthologs, which probably highlights the utility of the microarray technique with ovine samples  <cit> .
however, some no-reporting probe sets were found, partly because of the poor performance of the probe sets with samples from a related species.
the best way to resolve this type of problem is to use a species-specific microarray.
in the present study, we applied the first specialized and standardized transcriptome-wide sheep oligo dna microarray .
due to improvements in species specificity, the average detectable rate of all probe sets reached  <dig> % , which enabled us to capture the subtle changes in gene expression.
a pool of differentially expressed  probes from the two groups was used for a systematic hierarchical clustering to gain insight into the transcriptome-wide similarities among all  <dig> individuals investigated .
we found that individuals at the same developmental stage were clustered together regardless of their genetic background, indicating that differences derived during development were larger than those from the genetic backgrounds between the breeds.
gene expression in skeletal muscle at various developmental stages between breeds
according to the primary functions of different genes including muscle, lipid, and the immune system, we visualized the data on ma and bar plots .
combined with figures  <dig> and  <dig>  figure  <dig> also suggests the phenotypic profile in muscle and the profile of a given category of de genes at different developmental stages.
the biological functions and canonical pathways were explored at various developmental stages in texel and ujumqin sheep .
the partial de genes in the two breeds of sheep at various developmental stages are listed in tables  <dig> and  <dig> .
<dig>  the p-value was calculated using a right-tailed fisher's exact test.
<dig>  the number of genes enriched in corresponding functions.
<dig>  the ratio was calculated as the number of genes from our data set that overlap with the canonical pathway in question divided by the total number of genes represented in that canonical pathway.
<dig>  italicized terms such as "cancer" and "cardiovascular disease" represent a biased annotation or are not related to the aim of the study.
ipa version:  <dig>
content version:  <dig>
reference set: ingenuity knowledge base
relationships to include: direct and indirect
includes endogenous chemicals
filter summary: consider all molecules and/or relationships
symbols and analytical settings are the same as in table  <dig>
symbols and analytical settings are the same as in table  <dig>
symbols and analytical settings are the same as in table  <dig>
symbols and analytical settings are the same as in table  <dig>
differentially expressed probes were detected from five t-test analyses using genespring  <dig>  .
<dig>  original probe name in microarray analysis.
<dig>  genbank accession number of expressed sequence tags provided by the manufacturer according to the specific probe name.
<dig>  the highly homologous human sequences acquired using blastn search in ncbi .
differentially expressed probes were detected from five t-test analyses using genespring  <dig>  .
<dig>  original probe name in the microarray analysis.
<dig>  the genbank accession number of expressed sequence tags provided by the manufacturer according to the specific probe name.
<dig>  the highly homologous human sequences acquired by a blastn search in ncbi .
t <dig> vs. u70
the rapid increase in total muscle fiber number in the ovine fetus begins at  <dig> d  <cit> .
we found that the number of de genes was highest at this stage compared with other stages:  <dig> probe sets, including  <dig> known genes and  <dig> expressed sequence tags  were differentially expressed in the t <dig> and u <dig> samples.
compared with u <dig>   <dig> genes were upregulated and  <dig> genes were downregulated in t <dig>  a gene ontology analysis by david revealed that these upregulated genes encoded cell activation, negative regulation of apoptosis, positive regulation of peptidyl-tyrosine phosphorylation, and vesicle-mediated transport.
genes that were downregulated encoded hexose catabolism, monosaccharide catabolism, postembryonic development, alcohol catabolism, and lipid biosynthesis.
to ascertain whether some molecular events or cascades were associated with the process of skeletal muscle development, we identified four canonical pathways by david and the ipa platforms, which are involved in myogenesis and muscle metabolism, including the glycolysis/gluconeogenesis pathway, n-glycan biosynthesis, calcium signaling, and chemokine  receptor  <dig>  signaling).
we also revealed through ipa software that genes associated with the skeletal and muscular system development and function, as well as lipid metabolism, were top biological functions .
we further analyzed some transcripts and found that eukaryotic translation elongation factor  <dig> alpha  <dig>  was highly expressed in t <dig>  in contrast, the expression levels of signal transducer and activator of transcription  <dig> , aldolase a , and the leptin receptor  were upregulated in u <dig>
t <dig> vs. u85
in total,  <dig> probe sets were differentially expressed in t <dig> and u <dig>  including  <dig> annotated genes and  <dig> ests.
in contrast to u <dig>   <dig> genes were upregulated and  <dig> were downregulated in t <dig>  of these genes, immune response, cellular carbohydrate catabolism, and fatty acid metabolism were significantly overrepresented in the upregulated genes, whereas cell migration regulation was significantly overrepresented in the downregulated genes.
for all de genes between u <dig> and t <dig>  we identified four canonical pathways that were overrepresented between the two breeds: glycolysis/gluconeogenesis, fructose and mannose metabolism, the pentose phosphate pathway, and vascular endothelial growth factor  signaling.
moreover, hematological system development and function and lipid metabolism were still significantly represented as top biofunctions.
in particular, we found a cluster of genes enriched in the biofunction of skeletal and muscular disorders, suggesting a difference in skeletal muscle development between texel and ujumqin fetal sheep .
among the de genes, eukaryotic translation elongation factor  <dig> alpha  <dig> , fructose- <dig> -bisphosphatase  <dig> , actinin alpha  <dig>  proteoglycan  <dig> , myosin heavy chain  <dig>  were more highly expressed in texel than in ujumqin sheep.
however, the stathmin-like  <dig>  and phosphoinositide-3-kinase, regulatory subunit  <dig> alpha  were more highly expressed in u <dig>
t <dig> vs. u100
at this fetal developmental stage, the difference in muscle development, particularly the rate of myofiber proliferation, was most significant between the breeds.
ninety probe sets, including  <dig> annotated genes and  <dig> ests, were differentially expressed in t <dig> and u <dig>  among these genes,  <dig> were upregulated and  <dig> were downregulated in t <dig>  the upregulated genes were involved in dna binding, rna binding, and ribonucleoproteins, whereas those that were downregulated were mostly related to steroid biosynthesis, lipid metabolism, and skeletal muscle fiber development.
for all de genes at  <dig> d, hematological system development and function remained the most overrepresented physiological system, and lipid metabolism was also more active at this stage.
inositol metabolism, steroid biosynthesis, eukaryotic initiation factor  <dig>  signaling, and vegf signaling were overrepresented between t <dig> and u <dig> .
within the de genes, thyroid stimulating hormone receptor  and paraoxonase  <dig>  were more highly expressed in ujumqin sheep than in texel sheep, whereas ovar-dqb <dig> , pyruvate dehydrogenase kinase, isozyme  <dig> , and thioredoxin reductase  were upregulated in t <dig>
t <dig> vs. u120
the discrepancy in fetal muscle development was more significant between texel and ujumqin sheep at  <dig> d. in total,  <dig> probe sets, including  <dig> annotated genes and  <dig> ests, were differentially expressed in t <dig> and u <dig>  of these genes,  <dig> were upregulated in t <dig> and  <dig> were upregulated in u <dig>  the upregulated genes were specific to the immune response, leukocyte activation, and the i-kappab kinase/nf-kappab cascade, whereas the downregulated genes were significantly associated with the response to ethanol, the enzyme-linked receptor protein signaling pathway, and histone h2a acetylation.
hematological system development and function, immune cell trafficking, and nervous system development and function were most overrepresented in physiological systems development and function.
furthermore, lipid metabolism was also the most overrepresented molecular and cellular function.
several canonical pathways were associated with immune and lipid metabolism disorders, such as nuclear factor -kb signaling and atherosclerosis signaling .
among de genes between t <dig> and u <dig>  ovar-dqb <dig> , guanylate-binding protein 2; interferon-inducible, guanylate-binding protein 4; and ccaat/enhancer-binding protein  beta  were upregulated in t <dig>  both stmn <dig> and transformation/transcription domain-associated protein  were upregulated in u <dig>
t <dig> vs. u135
seventy-two probe sets, including  <dig> annotated genes and  <dig> ests, were differentially expressed in t <dig> and u <dig>  of these genes,  <dig> were upregulated in t <dig> and another  <dig> were more highly expressed in u <dig>  the immune response process was overrepresented in the upregulated genes, and mitosis was represented significantly in the downregulated genes.
for the pool of de genes, embryonic development and tissue development, hematological system development and function, cell-mediated immune response, and nervous system development and function were listed at the top of physiological system development and function.
cellular functions such as the cell cycle, cell death, and cell-to-cell signaling and interaction were more active.
notably, a pathway involved in glycerolipid metabolism was identified between t <dig> and u <dig> , two members of which  were upregulated in u <dig>
in terms of physiological system development and functions, differences in the immune and hematological systems were most significant between texlel and ujumqin sheep during muscle development.
then, differences in nervous system development and function became more prominent at later gestational stages.
the most obvious differences in molecular and cellular functions were lipid metabolism and cell communication between the two breeds.
several overrepresented canonical pathways related to muscle and adipose development , which were particularly noteworthy, underpinned the differences in skeletal muscle and adipose tissue between the two breeds at the fetal stage.
quantitative pcr validation of microarray gene expression
to confirm the differential expression of the genes in the microarray analysis, we selected  <dig> genes to validate by quantitative pcr .
among these, eight genes  encode myofibrillar proteins and two  are transcription factors that regulate the genes associated with myogenesis.
pik3r <dig> is involved in fat metabolism regulation, whereas trrap, txnrd <dig>  and insig <dig> participate in the regulation of gene expression and cell proliferation.
additionally, we also detected mstn expression during skeletal muscle development.
as in previous studies  <cit> , rplp <dig> was chosen as an ideal reference gene to normalize the data.
the ratios of  <dig> de genes at particular developmental stages in the two sheep breeds in the microarray analysis agreed with the qpcr results .
<dig>  the data in the rows are the ratio of the 2ΔΔct values for the qpcr in a texel sheep sample relative to that in the ujumqin sheep sample at the same developmental stage.
the microarray row provides gene expression ratios in texel sheep samples divided by those in ujumqin sheep samples at the same developmental stage.
*differentially expressed genes in the microarray analysis with a statistically significant difference at p <  <dig> .
discussion
characteristics of prenatal muscle fiber development in sheep
wilson et al.
and maier et al.
showed that at least three waves of myogenesis occurred until d  <dig> of gestation in sheep  <cit> .
we demonstrated that the myofiber numbers increase sharply with a pulsed-wave mode at a particular developmental stage in sheep.
this is the first time that the mode of fetal myofiber proliferation was revealed in a large mammal during the second half of gestation.
in addition to the longissimus dorsi muscle, similar patterns of myofiber proliferation were found in the semitendenosus, gluteus medius, gastrocnemius, and triceps brachii muscles in texel and ujumqin fetal sheep .
these findings indicate that the specific developmental stage during which myogenic surges of myofiber hyperplasia appeared is pivotal for fetal muscle development as well as postnatal muscularity potential in sheep.
unlike the studies of wilson et al.
and maier et al., who identified different generations of myogenic cells using electron microscopy  <cit> , we counted the muscle fibers under a light microscope and found many small-diameter fibers until  <dig> d in both breeds.
we considered that at least part of the small fibers must have been generated by the fusion of myoblasts  during the study period.
pax <dig> plays an important role conferring myogenic potential to these progenitor cells  <cit> , and pax <dig> is an ideal cell marker of myogenic progenitor cells such as myoblasts and satellite cells  <cit> .
our findings indicate a higher potential for postnatal muscle growth that entails more pax7-positive cells in the fetal stage.
gene expression in the two breeds at various developmental stages
of the five prenatal stages, the numbers of de genes in the two sheep breeds were highest at  <dig> and  <dig> d, indicating that rapid myofiber proliferation included numerous genes during the myogenic process.
however, two surges of myofiber proliferation appeared at  <dig> and  <dig> d in texel fetuses, probably because the expression of more genes associated with myoblast proliferation and differentiation at  <dig> d prepared for the myofiber hyperplasia that followed at  <dig> d, and more genes related to the immune system were differentially expressed between t <dig> and u <dig> , which led to larger total counts of de genes.
the difference between the two sheep breeds in the timing of myogenesis in terms of transcriptomic levels was similar to that found between pig breeds  <cit> .
cagnazzo et al.
showed that myogenesis-related gene expression is greater in early duroc  embryos than in early pietrain  embryos at 14- <dig> d of gestation, whereas the opposite was found in late embryos   <cit> .
the present findings suggest that highly muscled breeds have a longer myogenic process during prenatal stages and that myogenesis is more intense in late-stage fetuses, which was validated by pax7-cell staining in longissimus muscle cross sections .
however, whether this myogenic process is the same in sheep as in pigs at earlier embryonic stages remains to be investigated.
we found that the immune and hematological systems' development and function were most overrepresented of all physiological systems and functions, accompanied by muscle system development, which was consistent with a microarray developmental analysis in cattle fetuses  <cit> .
recent discoveries have revealed complex interactions between skeletal muscle and the immune system that regulate muscle regeneration and myogenesis, and many immune molecules, such as tumor necrosis factor-alpha , nf-kb, interleukin - <dig>  il- <dig>  il- <dig>  and leukemia inhibitory factor are involved in muscle cell proliferation and differentiation  <cit> .
in most cases, whether perturbation in the same signaling pathways related to myogenesis occurs will determine normal myogenic development or muscle disorders.
therefore, the pathways associated with immune and muscle disorders in this work are valuable for human myopathy at the prenatal stage.
the hematological system is also implicated in myogenesis  <cit> , which probably reflects the systemic requirement for muscle function during fetal development.
nervous system development was overrepresented in the last two development stages in the two sheep breeds.
neurons are indispensable for maintaining normal muscle physiological function and also parallel the skeletal muscle in auxology.
comprehensive and close interactions occur between muscles and the nervous system during development  <cit> .
muscle development is regulated by the central nervous system in drosophila and pigs  <cit> , and muscle fiber type is dependent on the pattern of innervation of a muscle established due to differential projection patterns between fast and slow motoneurons  <cit> .
animals with different muscle phenotypes undergo diverse innervation patterns during fetal development.
double-muscled cattle have an additional 13-26% increased branching in terminal axons compared to that in normal cattle caused by a real increase in the number of myofibers  <cit> .
we identified several valuable canonical pathways directly associated with muscle development and function, myogenesis, myoblast proliferation, and the cell cycle in muscle at various developmental stages, such as calcium  <cit> , cxcr <dig>  <cit> , and vegf signaling  <cit> .
furthermore, other pathways involved in adipose and muscle metabolism, such as inositol metabolism, steroid biosynthesis, eif <dig> signaling, glycerolipid metabolism, the glycolysis/gluconeogenesis pathway, and n-glycan biosynthesis, are significantly affected during development.
steelman et al.
suggested that wnt signaling is a potential downstream target of myostatin for postnatal skeletal muscle growth and hypertrophy in mice  <cit> .
however, we focused on the transcriptome during prenatal stages rather than postnatal stages and found that various canonical pathways are predominant in skeletal muscle development at different stages due to a myostatin mutation in sheep.
de genes involved in muscle and adipose development
stat <dig>  a member of the stat family and a cooperator in the janus kinase pathway, plays a dual role in the regulation of myoblast proliferation and differentiation, but is dependent on interactions with various cofactors  <cit> .
we found that stat <dig> expression was higher in u <dig> than t <dig>  which suggests that stat <dig> is most probably associated with the differentiation rather than proliferation of myoblasts.
the go analysis of de genes between t <dig> and u <dig> confirmed our presumption, and muscle fiber hyperplasia was more intense in t <dig>  as well.
aldoa is responsible for significant activation during the differentiation of chicken primary myoblasts and plays an important role in muscle gene transcription  <cit> .
in our study, a higher aldoa expression level in u <dig> indicated that more myoblasts exited the cell cycle and entered differentiation in u <dig> than those in t <dig>  the leptin receptor , a protein secreted from adipocytes, is responsible for fat mass regulation via leptin in the hypothalamus  <cit> .
lepr expression was higher in u <dig> than in t <dig>  whether the higher lepr expression at prenatal stages is associated with high postnatal adipose deposition remains to be investigated.
both hyperplasia and hypertrophy continued in skeletal muscle at  <dig> d. fbp <dig>  which encodes a gluconeogenesis regulatory enzyme that catalyzes the hydrolysis of fructose  <dig> -bisphosphate to fructose 6-phosphate and inorganic phosphate, was expressed higher in t <dig> than in u <dig>  a previous study suggested that fbpase participates in some nuclear processes during the development and regeneration of skeletal muscle  <cit> .
additionally, prg <dig> expression was also higher in t <dig>  previous studies have shown that prg <dig> plays a role in some early aspects of skeletal myogenesis  <cit> .
the expression levels of pik3r <dig>  tshr, and pon <dig> were higher in u <dig> and u <dig> than that in t <dig> or t <dig>  previous studies have demonstrated that these genes are involved in fat metabolism and adipocyte development  <cit> .
pdk <dig>  a gene that was upregulated in t <dig>  is a key regulatory enzyme involved in switching the energy source from glucose to fatty acids in response to physiological conditions.
the pyruvate dehydrogenase complex occupies a central and strategic position in muscle intermediary metabolism and is primarily regulated by phosphorylation/dephosphorylation  <cit> .
in addition, pdk <dig> is significantly associated with intramuscular fat and muscle water content  <cit> , indicating that pdk <dig> is involved in meat quality.
txnrd <dig> is a variant of thioredoxin reductase, an important selenoprotein that maintains cellular redox balance and regulates several redox-dependent processes during apoptosis, cell proliferation, and differentiation  <cit> .
lef <dig> is a transcription factor involved in the regulation of myogenesis.
loss of lef1-mediated repression results in an increased number of cells expressing pax- <dig> and pax- <dig>  suggesting that wnt signaling via lef <dig> acts to regulate the number of premyogenic cells in somites  <cit> .
in our study, lef <dig> expression was higher in u <dig> than that in t <dig>  which is consistent with the rapid proliferation of myofibers in u <dig>  although transcription factor activity is predominantly manifested at the protein level, the difference in lef <dig> expression level indicated a potential difference of lef <dig> between breeds.
ovar-dqb <dig>  was expressed much higher in t <dig> and t <dig>  particularly in t <dig> , compared with u  <dig> and u <dig>  results from karpati et al.
indicated that the class i molecule may be involved in the fusion of myogenic cells during muscle regeneration  <cit> .
honda and rostami demonstrated that the expression of class i antigens on muscle cells is not only immunologically modulated but also developmentally regulated, and that these antigens may play a role in cell recognition and interaction during the myogenic fusion process.
the presence of the antigens, however, was transitory, and they disappeared as myoblasts fused and differentiated into multinucleate myotubes  <cit> .
cebpb, an important transcription factor, acts as a indispensable regulator of adipocyte differentiation during adipogenesis  <cit> .
we found that cebpb expression was higher in skeletal muscle in t <dig> than in u <dig>  indicating a key difference in adipogenesis between texel and ujumqin sheep.
however, which breeds contain more adipose mass than the other at this developmental stage remains to be investigated.
trrap, which was downregulated in t <dig>  in contrast to that in u <dig>  is vital for embryonic survival and control of the mitotic checkpoint  <cit> .
deletion of trrap leads to a reduced level of beta-catenin ubiquitination, a lower degradation rate, and accumulation of the beta-catenin protein, whereas trrap knockdown results in abnormal retention of beta-catenin at chromatin and concomitant hyperactivation of the canonical wnt pathway  <cit> .
however, the canonical wnt pathway is downregulated in the absence of myostatin through beta-catenin, whereas the wnt/calcium pathway is upregulated  <cit> , suggesting that trrap may negatively regulate skeletal muscle development between the canonical wnt and the wnt/calcium pathways through beta-catenin at  <dig> d. further efforts are warranted to test the effect of trrap on skeletal muscle development.
other than the genes discussed above, we found that mstn expression was lower in texel than in ujumqin sheep through the five development stages .
this result indicated that mstn downregulation contributed to the hyperplasia and hypertrophy of myofibers in texel sheep through the second half of gestation.
moreover, the genes discussed above are potential myostatin targets for further investigation.
CONCLUSIONS
we demonstrated that fetal myofiber number increased sharply in a pulsed-wave mode at a particular developmental stage and was not augmented evenly across developmental stages in large mammal.
the two surges in myofiber hyperplasia occurred at  <dig> and  <dig> d in texel sheep, whereas a single surge appeared at  <dig> d in ujumqin sheep during the second half of gestation.
a myostatin mutation changed the gene expression profile in prenatal skeletal muscle, particularly disrupting some pivotal signaling pathways governing muscle development and function at some developmental stages, which explains much of the variation in myofiber phenotypes between texel and ujumqin sheep.
further studies on the crucial de genes and signaling pathways involved would be helpful for revealing the mystery of muscle development in mammals.

 texel sheep, a typical "double muscle" breed due to a gdf mutation, are now commercially produced throughout the world, with no adverse effects detected by objective assessments of meat quality. however, evidence for an association between g+6723g > a and decreased intramuscular fat and reduced eating quality has been observed. compared with texel sheep, indigenous chinese ujumqin sheep, with no gdf mutation, are less muscular and have a higher fat content, but they are superior in terms of perceived meat quality. therefore, these two sheep breeds provide a good natural model for studying muscle and fat development, as well as for identifying myostatin genes. prenatal skeletal muscle development is an important determinant of both muscularity and meat quality. in large precocial species such as sheep and cattle, the maximum myofiber complement of a muscle is achieved prior to birth. more than three waves of myogenic cells appear in sheep, and most myofibers form during the second half of gestation. however, whether the myofibers increase with a pulsed-wave mode at certain developmental stages or whether they augment evenly across developmental stages in fetal sheep remains unclear. myostatin, a member of the transforming growth factor-β family, is predominantly expressed and secreted by skeletal muscle and functions as a negative regulator of muscle growth. mutations in the myostatin gene lead to a hypertrophic phenotype in mice, sheep, cattle, dog, and human. the effect of myostatin on gene expression in prenatal muscles at the genome-wide level was recently explored in fetal cattle, but no studies have been conducted dynamically at multiple fetal stages comparing two pure breeds with extreme phenotypes. a recent mice study demonstrated that myoblasts from embryonic and fetal stages not only had different fusion abilities, proliferation, differentiation and responses to tgf-β, phorbol ester 12-o-tetradecanoylphorbol-13-acetate, and bone morphogenetic protein- in vitro, but they also differed in gene expression profiles, indicating that complicated and obvious changes in physiology and biochemistry occur during the prenatal stage in vivo. therefore, investigating the subtle changes of the effect of a myostatin mutation on skeletal muscle development at multiple fetal stages using our experimental model is necessary. here, we examined gene expression and myofiber development in texel and ujumqin sheep at the transcriptome and histological levels. our findings contribute to understanding the dynamic effects of myostatin on the biology of prenatal skeletal muscle development in large mammals. the results also provide clues into human myopathy and obesity during prenatal stages. we also identified putative candidate genes that are valuable for meat-quality traits in farm animals.  histological characteristics of ovine fetal skeletal muscle development we examined the number and diameter of myofibers at each developmental stage in texel and ujumqin sheep via histological analysis. significant difference was observed in myofiber diameter between texel and ujumqin sheep at each development stage, except at d. however, the myofiber diameter patterns of the two breeds were similar throughout the five developmental stages. unexpectedly, we found that the myofiber hyperplasia profiles differed between texel and ujumqin fetuses. the proliferation of myofibers in texel sheep was fastest at and d, particularly at d, suggesting that this period is vital for muscle development in fetal texel sheep. myofiber hyperplasia reached a peak at d in ujumqin sheep, when the number of myofibers exceeded that of texel sheep. notably, with development, the number of myofibers in texel fetuses recovered to that of ujumqin fetuses at d, although myofiber hypertrophy in texel sheep was greater than that in ujumqin sheep at this stage. therefore, a new wave of myogenesis most probably occurs at d in texel sheep, given that the number of myofibers increased in ujumqin sheep during this period. additionally, many small-diameter fibers occurred in the muscle at d in both breeds. the small fibers could be due either to intrafascicular terminations or have been generated by the fusion of myoblasts during the period investigated. therefore, to explore the myogenic potential in the two breeds of sheep, we identified pax7-positive cells in skeletal muscle at d using immnofluorescence staining and found that the ratio of the number of pax7-positive cells to the fibers on each muscle slide was significantly higher in the texel fetuses than in the ujumqin fetuses. in combination with figure this observation suggests that a period of 100- d is another new proliferative stage for myofiber hyperplasia in texel sheep. higher numbers of pax7-positive cells were correlated with a higher muscle mass in texel sheep during both the prenatal and postnatal stages. microarray experiment a mean transcript sequence identity of 97% was found between ovine and bovine orthologs, which probably highlights the utility of the microarray technique with ovine samples. however, some no-reporting probe sets were found, partly because of the poor performance of the probe sets with samples from a related species. the best way to resolve this type of problem is to use a species-specific microarray. in the present study, we applied the first specialized and standardized transcriptome-wide sheep oligo dna microarray. due to improvements in species specificity, the average detectable rate of all probe sets reached %, which enabled us to capture the subtle changes in gene expression. a pool of differentially expressed probes from the two groups was used for a systematic hierarchical clustering to gain insight into the transcriptome-wide similarities among all individuals investigated. we found that individuals at the same developmental stage were clustered together regardless of their genetic background, indicating that differences derived during development were larger than those from the genetic backgrounds between the breeds. gene expression in skeletal muscle at various developmental stages between breeds according to the primary functions of different genes including muscle, lipid, and the immune system, we visualized the data on ma and bar plots. combined with figures and figure also suggests the phenotypic profile in muscle and the profile of a given category of de genes at different developmental stages. the biological functions and canonical pathways were explored at various developmental stages in texel and ujumqin sheep. the partial de genes in the two breeds of sheep at various developmental stages are listed in tables and. the p-value was calculated using a right-tailed fisher's exact test. the number of genes enriched in corresponding functions. the ratio was calculated as the number of genes from our data set that overlap with the canonical pathway in question divided by the total number of genes represented in that canonical pathway. italicized terms such as "cancer" and "cardiovascular disease" represent a biased annotation or are not related to the aim of the study. ipa version: content version: reference set: ingenuity knowledge base relationships to include: direct and indirect includes endogenous chemicals filter summary: consider all molecules and/or relationships symbols and analytical settings are the same as in table symbols and analytical settings are the same as in table symbols and analytical settings are the same as in table symbols and analytical settings are the same as in table differentially expressed probes were detected from five t-test analyses using genespring. original probe name in microarray analysis. genbank accession number of expressed sequence tags provided by the manufacturer according to the specific probe name. the highly homologous human sequences acquired using blastn search in ncbi. differentially expressed probes were detected from five t-test analyses using genespring. original probe name in the microarray analysis. the genbank accession number of expressed sequence tags provided by the manufacturer according to the specific probe name. the highly homologous human sequences acquired by a blastn search in ncbi. t vs. u70 the rapid increase in total muscle fiber number in the ovine fetus begins at d. we found that the number of de genes was highest at this stage compared with other stages: probe sets, including known genes and expressed sequence tags were differentially expressed in the t and u samples. compared with u genes were upregulated and genes were downregulated in t a gene ontology analysis by david revealed that these upregulated genes encoded cell activation, negative regulation of apoptosis, positive regulation of peptidyl-tyrosine phosphorylation, and vesicle-mediated transport. genes that were downregulated encoded hexose catabolism, monosaccharide catabolism, postembryonic development, alcohol catabolism, and lipid biosynthesis. to ascertain whether some molecular events or cascades were associated with the process of skeletal muscle development, we identified four canonical pathways by david and the ipa platforms, which are involved in myogenesis and muscle metabolism, including the glycolysis/gluconeogenesis pathway, n-glycan biosynthesis, calcium signaling, and chemokine receptor signaling). we also revealed through ipa software that genes associated with the skeletal and muscular system development and function, as well as lipid metabolism, were top biological functions. we further analyzed some transcripts and found that eukaryotic translation elongation factor alpha was highly expressed in t in contrast, the expression levels of signal transducer and activator of transcription, aldolase a, and the leptin receptor were upregulated in u t vs. u85 in total, probe sets were differentially expressed in t and u including annotated genes and ests. in contrast to u genes were upregulated and were downregulated in t of these genes, immune response, cellular carbohydrate catabolism, and fatty acid metabolism were significantly overrepresented in the upregulated genes, whereas cell migration regulation was significantly overrepresented in the downregulated genes. for all de genes between u and t we identified four canonical pathways that were overrepresented between the two breeds: glycolysis/gluconeogenesis, fructose and mannose metabolism, the pentose phosphate pathway, and vascular endothelial growth factor signaling. moreover, hematological system development and function and lipid metabolism were still significantly represented as top biofunctions. in particular, we found a cluster of genes enriched in the biofunction of skeletal and muscular disorders, suggesting a difference in skeletal muscle development between texel and ujumqin fetal sheep. among the de genes, eukaryotic translation elongation factor alpha, fructose- -bisphosphatase, actinin alpha proteoglycan, myosin heavy chain were more highly expressed in texel than in ujumqin sheep. however, the stathmin-like and phosphoinositide-3-kinase, regulatory subunit alpha were more highly expressed in u t vs. u100 at this fetal developmental stage, the difference in muscle development, particularly the rate of myofiber proliferation, was most significant between the breeds. ninety probe sets, including annotated genes and ests, were differentially expressed in t and u among these genes, were upregulated and were downregulated in t the upregulated genes were involved in dna binding, rna binding, and ribonucleoproteins, whereas those that were downregulated were mostly related to steroid biosynthesis, lipid metabolism, and skeletal muscle fiber development. for all de genes at d, hematological system development and function remained the most overrepresented physiological system, and lipid metabolism was also more active at this stage. inositol metabolism, steroid biosynthesis, eukaryotic initiation factor signaling, and vegf signaling were overrepresented between t and u. within the de genes, thyroid stimulating hormone receptor and paraoxonase were more highly expressed in ujumqin sheep than in texel sheep, whereas ovar-dqb, pyruvate dehydrogenase kinase, isozyme, and thioredoxin reductase were upregulated in t t vs. u120 the discrepancy in fetal muscle development was more significant between texel and ujumqin sheep at d. in total, probe sets, including annotated genes and ests, were differentially expressed in t and u of these genes, were upregulated in t and were upregulated in u the upregulated genes were specific to the immune response, leukocyte activation, and the i-kappab kinase/nf-kappab cascade, whereas the downregulated genes were significantly associated with the response to ethanol, the enzyme-linked receptor protein signaling pathway, and histone h2a acetylation. hematological system development and function, immune cell trafficking, and nervous system development and function were most overrepresented in physiological systems development and function. furthermore, lipid metabolism was also the most overrepresented molecular and cellular function. several canonical pathways were associated with immune and lipid metabolism disorders, such as nuclear factor -kb signaling and atherosclerosis signaling. among de genes between t and u ovar-dqb, guanylate-binding protein 2; interferon-inducible, guanylate-binding protein 4; and ccaat/enhancer-binding protein beta were upregulated in t both stmn and transformation/transcription domain-associated protein were upregulated in u t vs. u135 seventy-two probe sets, including annotated genes and ests, were differentially expressed in t and u of these genes, were upregulated in t and another were more highly expressed in u the immune response process was overrepresented in the upregulated genes, and mitosis was represented significantly in the downregulated genes. for the pool of de genes, embryonic development and tissue development, hematological system development and function, cell-mediated immune response, and nervous system development and function were listed at the top of physiological system development and function. cellular functions such as the cell cycle, cell death, and cell-to-cell signaling and interaction were more active. notably, a pathway involved in glycerolipid metabolism was identified between t and u, two members of which were upregulated in u in terms of physiological system development and functions, differences in the immune and hematological systems were most significant between texlel and ujumqin sheep during muscle development. then, differences in nervous system development and function became more prominent at later gestational stages. the most obvious differences in molecular and cellular functions were lipid metabolism and cell communication between the two breeds. several overrepresented canonical pathways related to muscle and adipose development, which were particularly noteworthy, underpinned the differences in skeletal muscle and adipose tissue between the two breeds at the fetal stage. quantitative pcr validation of microarray gene expression to confirm the differential expression of the genes in the microarray analysis, we selected genes to validate by quantitative pcr. among these, eight genes encode myofibrillar proteins and two are transcription factors that regulate the genes associated with myogenesis. pik3r is involved in fat metabolism regulation, whereas trrap, txnrd and insig participate in the regulation of gene expression and cell proliferation. additionally, we also detected mstn expression during skeletal muscle development. as in previous studies, rplp was chosen as an ideal reference gene to normalize the data. the ratios of de genes at particular developmental stages in the two sheep breeds in the microarray analysis agreed with the qpcr results. the data in the rows are the ratio of the 2ΔΔct values for the qpcr in a texel sheep sample relative to that in the ujumqin sheep sample at the same developmental stage. the microarray row provides gene expression ratios in texel sheep samples divided by those in ujumqin sheep samples at the same developmental stage. *differentially expressed genes in the microarray analysis with a statistically significant difference at p <. discussion characteristics of prenatal muscle fiber development in sheep wilson et al. and maier et al. showed that at least three waves of myogenesis occurred until d of gestation in sheep. we demonstrated that the myofiber numbers increase sharply with a pulsed-wave mode at a particular developmental stage in sheep. this is the first time that the mode of fetal myofiber proliferation was revealed in a large mammal during the second half of gestation. in addition to the longissimus dorsi muscle, similar patterns of myofiber proliferation were found in the semitendenosus, gluteus medius, gastrocnemius, and triceps brachii muscles in texel and ujumqin fetal sheep. these findings indicate that the specific developmental stage during which myogenic surges of myofiber hyperplasia appeared is pivotal for fetal muscle development as well as postnatal muscularity potential in sheep. unlike the studies of wilson et al. and maier et al., who identified different generations of myogenic cells using electron microscopy, we counted the muscle fibers under a light microscope and found many small-diameter fibers until d in both breeds. we considered that at least part of the small fibers must have been generated by the fusion of myoblasts during the study period. pax plays an important role conferring myogenic potential to these progenitor cells, and pax is an ideal cell marker of myogenic progenitor cells such as myoblasts and satellite cells. our findings indicate a higher potential for postnatal muscle growth that entails more pax7-positive cells in the fetal stage. gene expression in the two breeds at various developmental stages of the five prenatal stages, the numbers of de genes in the two sheep breeds were highest at and d, indicating that rapid myofiber proliferation included numerous genes during the myogenic process. however, two surges of myofiber proliferation appeared at and d in texel fetuses, probably because the expression of more genes associated with myoblast proliferation and differentiation at d prepared for the myofiber hyperplasia that followed at d, and more genes related to the immune system were differentially expressed between t and u, which led to larger total counts of de genes. the difference between the two sheep breeds in the timing of myogenesis in terms of transcriptomic levels was similar to that found between pig breeds. cagnazzo et al. showed that myogenesis-related gene expression is greater in early duroc embryos than in early pietrain embryos at 14- d of gestation, whereas the opposite was found in late embryos. the present findings suggest that highly muscled breeds have a longer myogenic process during prenatal stages and that myogenesis is more intense in late-stage fetuses, which was validated by pax7-cell staining in longissimus muscle cross sections. however, whether this myogenic process is the same in sheep as in pigs at earlier embryonic stages remains to be investigated. we found that the immune and hematological systems' development and function were most overrepresented of all physiological systems and functions, accompanied by muscle system development, which was consistent with a microarray developmental analysis in cattle fetuses. recent discoveries have revealed complex interactions between skeletal muscle and the immune system that regulate muscle regeneration and myogenesis, and many immune molecules, such as tumor necrosis factor-alpha, nf-kb, interleukin - il- il- and leukemia inhibitory factor are involved in muscle cell proliferation and differentiation. in most cases, whether perturbation in the same signaling pathways related to myogenesis occurs will determine normal myogenic development or muscle disorders. therefore, the pathways associated with immune and muscle disorders in this work are valuable for human myopathy at the prenatal stage. the hematological system is also implicated in myogenesis, which probably reflects the systemic requirement for muscle function during fetal development. nervous system development was overrepresented in the last two development stages in the two sheep breeds. neurons are indispensable for maintaining normal muscle physiological function and also parallel the skeletal muscle in auxology. comprehensive and close interactions occur between muscles and the nervous system during development. muscle development is regulated by the central nervous system in drosophila and pigs, and muscle fiber type is dependent on the pattern of innervation of a muscle established due to differential projection patterns between fast and slow motoneurons. animals with different muscle phenotypes undergo diverse innervation patterns during fetal development. double-muscled cattle have an additional 13-26% increased branching in terminal axons compared to that in normal cattle caused by a real increase in the number of myofibers. we identified several valuable canonical pathways directly associated with muscle development and function, myogenesis, myoblast proliferation, and the cell cycle in muscle at various developmental stages, such as calcium, cxcr, and vegf signaling. furthermore, other pathways involved in adipose and muscle metabolism, such as inositol metabolism, steroid biosynthesis, eif signaling, glycerolipid metabolism, the glycolysis/gluconeogenesis pathway, and n-glycan biosynthesis, are significantly affected during development. steelman et al. suggested that wnt signaling is a potential downstream target of myostatin for postnatal skeletal muscle growth and hypertrophy in mice. however, we focused on the transcriptome during prenatal stages rather than postnatal stages and found that various canonical pathways are predominant in skeletal muscle development at different stages due to a myostatin mutation in sheep. de genes involved in muscle and adipose development stat a member of the stat family and a cooperator in the janus kinase pathway, plays a dual role in the regulation of myoblast proliferation and differentiation, but is dependent on interactions with various cofactors. we found that stat expression was higher in u than t which suggests that stat is most probably associated with the differentiation rather than proliferation of myoblasts. the go analysis of de genes between t and u confirmed our presumption, and muscle fiber hyperplasia was more intense in t as well. aldoa is responsible for significant activation during the differentiation of chicken primary myoblasts and plays an important role in muscle gene transcription. in our study, a higher aldoa expression level in u indicated that more myoblasts exited the cell cycle and entered differentiation in u than those in t the leptin receptor, a protein secreted from adipocytes, is responsible for fat mass regulation via leptin in the hypothalamus. lepr expression was higher in u than in t whether the higher lepr expression at prenatal stages is associated with high postnatal adipose deposition remains to be investigated. both hyperplasia and hypertrophy continued in skeletal muscle at d. fbp which encodes a gluconeogenesis regulatory enzyme that catalyzes the hydrolysis of fructose -bisphosphate to fructose 6-phosphate and inorganic phosphate, was expressed higher in t than in u a previous study suggested that fbpase participates in some nuclear processes during the development and regeneration of skeletal muscle. additionally, prg expression was also higher in t previous studies have shown that prg plays a role in some early aspects of skeletal myogenesis. the expression levels of pik3r tshr, and pon were higher in u and u than that in t or t previous studies have demonstrated that these genes are involved in fat metabolism and adipocyte development. pdk a gene that was upregulated in t is a key regulatory enzyme involved in switching the energy source from glucose to fatty acids in response to physiological conditions. the pyruvate dehydrogenase complex occupies a central and strategic position in muscle intermediary metabolism and is primarily regulated by phosphorylation/dephosphorylation. in addition, pdk is significantly associated with intramuscular fat and muscle water content, indicating that pdk is involved in meat quality. txnrd is a variant of thioredoxin reductase, an important selenoprotein that maintains cellular redox balance and regulates several redox-dependent processes during apoptosis, cell proliferation, and differentiation. lef is a transcription factor involved in the regulation of myogenesis. loss of lef1-mediated repression results in an increased number of cells expressing pax- and pax- suggesting that wnt signaling via lef acts to regulate the number of premyogenic cells in somites. in our study, lef expression was higher in u than that in t which is consistent with the rapid proliferation of myofibers in u although transcription factor activity is predominantly manifested at the protein level, the difference in lef expression level indicated a potential difference of lef between breeds. ovar-dqb was expressed much higher in t and t particularly in t, compared with u and u results from karpati et al. indicated that the class i molecule may be involved in the fusion of myogenic cells during muscle regeneration. honda and rostami demonstrated that the expression of class i antigens on muscle cells is not only immunologically modulated but also developmentally regulated, and that these antigens may play a role in cell recognition and interaction during the myogenic fusion process. the presence of the antigens, however, was transitory, and they disappeared as myoblasts fused and differentiated into multinucleate myotubes. cebpb, an important transcription factor, acts as a indispensable regulator of adipocyte differentiation during adipogenesis. we found that cebpb expression was higher in skeletal muscle in t than in u indicating a key difference in adipogenesis between texel and ujumqin sheep. however, which breeds contain more adipose mass than the other at this developmental stage remains to be investigated. trrap, which was downregulated in t in contrast to that in u is vital for embryonic survival and control of the mitotic checkpoint. deletion of trrap leads to a reduced level of beta-catenin ubiquitination, a lower degradation rate, and accumulation of the beta-catenin protein, whereas trrap knockdown results in abnormal retention of beta-catenin at chromatin and concomitant hyperactivation of the canonical wnt pathway. however, the canonical wnt pathway is downregulated in the absence of myostatin through beta-catenin, whereas the wnt/calcium pathway is upregulated, suggesting that trrap may negatively regulate skeletal muscle development between the canonical wnt and the wnt/calcium pathways through beta-catenin at d. further efforts are warranted to test the effect of trrap on skeletal muscle development. other than the genes discussed above, we found that mstn expression was lower in texel than in ujumqin sheep through the five development stages. this result indicated that mstn downregulation contributed to the hyperplasia and hypertrophy of myofibers in texel sheep through the second half of gestation. moreover, the genes discussed above are potential myostatin targets for further investigation.  we demonstrated that fetal myofiber number increased sharply in a pulsed-wave mode at a particular developmental stage and was not augmented evenly across developmental stages in large mammal. the two surges in myofiber hyperplasia occurred at and d in texel sheep, whereas a single surge appeared at d in ujumqin sheep during the second half of gestation. a myostatin mutation changed the gene expression profile in prenatal skeletal muscle, particularly disrupting some pivotal signaling pathways governing muscle development and function at some developmental stages, which explains much of the variation in myofiber phenotypes between texel and ujumqin sheep. further studies on the crucial de genes and signaling pathways involved would be helpful for revealing the mystery of muscle development in mammals. 
using the first specialized transcriptome-wide sheep oligo dna microarray and histological methods, we investigated the gene expression profile and histological characteristics of developing fetal ovine longissimus muscle in texel sheep, as a myostatin model of natural mutation, and ujumqin sheep. the surges in myofiber hyperplasia occurred at and d in texel sheep, whereas a unique proliferative surge appeared at d in ujumqin sheep. analysis of the microarray demonstrated that immune and hematological systems' development and function, lipid metabolism, and cell communication were the biological functions that were most differentially expressed between texel and ujumqin sheep during muscle development. the myostatin mutation changed the gene expression pattern in skeletal muscle at a transcriptome-wide level, resulting in variation in myofiber phenotype between texel and ujumqin sheep during the second half of gestation. whether myofibers increase with a pulsed-wave mode at particular developmental stages or whether they augment evenly across developmental stages in large mammals is unclear. our findings provide a novel and dynamic description of the effect of myostatin on skeletal muscle development, which contributes to understanding the biology of muscle development in large mammals. moreover, we identified some differentially expressed genes between the two breeds that could be potential myostatin targets for further investigation. myofiber number increased sharply with a pulsed-wave mode at certain developmental stages but was not augmented evenly across developmental stages in fetal sheep. fetal skeletal muscles were sampled at and d of gestation. proliferation of myofibers proceeded in a pulsed-wave mode at particular fetal stages in the sheep. additionally, the molecular mechanisms of myostatin in myofiber hyperplasia at the fetal stage in sheep remain unknown. pathways associated with myogenesis and the proliferation of myoblasts, such as calcium signaling, chemokine receptor signaling, and vascular endothelial growth factor signaling, were affected significantly at specific fetal stages, which underpinned fetal myofiber hyperplasia and postnatal muscle hypertrophy.
 whether myofibers increase with a pulsed-wave mode at particular developmental stages or whether they augment evenly across developmental stages in large mammals is unclear. additionally, the molecular mechanisms of myostatin in myofiber hyperplasia at the fetal stage in sheep remain unknown. using the first specialized transcriptome-wide sheep oligo dna microarray and histological methods, we investigated the gene expression profile and histological characteristics of developing fetal ovine longissimus muscle in texel sheep, as a myostatin model of natural mutation, and ujumqin sheep. fetal skeletal muscles were sampled at and d of gestation.  myofiber number increased sharply with a pulsed-wave mode at certain developmental stages but was not augmented evenly across developmental stages in fetal sheep. the surges in myofiber hyperplasia occurred at and d in texel sheep, whereas a unique proliferative surge appeared at d in ujumqin sheep. analysis of the microarray demonstrated that immune and hematological systems' development and function, lipid metabolism, and cell communication were the biological functions that were most differentially expressed between texel and ujumqin sheep during muscle development. pathways associated with myogenesis and the proliferation of myoblasts, such as calcium signaling, chemokine receptor signaling, and vascular endothelial growth factor signaling, were affected significantly at specific fetal stages, which underpinned fetal myofiber hyperplasia and postnatal muscle hypertrophy. moreover, we identified some differentially expressed genes between the two breeds that could be potential myostatin targets for further investigation.  proliferation of myofibers proceeded in a pulsed-wave mode at particular fetal stages in the sheep. the myostatin mutation changed the gene expression pattern in skeletal muscle at a transcriptome-wide level, resulting in variation in myofiber phenotype between texel and ujumqin sheep during the second half of gestation. our findings provide a novel and dynamic description of the effect of myostatin on skeletal muscle development, which contributes to understanding the biology of muscle development in large mammals. 