BACKGROUND
as humans and animals age, growth slows and eventually stops.
organs grow until they reach mature size; however, little is known about how growth cessation is regulated and coordinated in the body.
a set of growth regulating genes, which include ezh <dig>  gpc <dig>  mdk, mest, mycn, peg <dig>  and plagl <dig>  has been identified that is dependent upon growth and whose expression declines with age in organs
<cit> .
in general, when expression of these genes decreases, proliferation decreases leading to a reduction in growth.
mutations of these genes also result in reduced viability, growth abnormalities, and diseases such as rhabdomyosarcoma, a skeletal muscle cancer, and simpson-golabi-behmel syndrome
<cit> .
though well characterized in organs, these genes have not been well examined in growing muscle.
it is expected that expression of these genes would differ between muscle, heart, and liver because of the differences in how these tissues grow.
postnatally, muscle grows by mainly hypertrophy, an increase in cell size, and not hyperplasia, an increase in cell number
<cit> .
in mice, muscle fiber number becomes fixed at approximately d  <dig> postnatal
<cit> .
hypertrophy of muscle fibers is accompanied by satellite cell activation.
these cells fuse with existing muscle fibers to support muscle growth
<cit> .
without the activation of satellite cells, postnatal muscle growth and regeneration is severely inhibited.
in muscle, it is known that the expression of ezh <dig>  mdk, mest, peg <dig>  and plagl <dig> is increased during regeneration
<cit> .
in contrast, mycn and gpc <dig> have not been characterized in muscle.
mycn, however, increases proliferation of neural progenitor cells while gpc <dig> is upregulated in the early and middle stages of liver regeneration
<cit> .
because  <dig> of  <dig> of these genes are known to be upregulated with muscle regeneration, it is possible that they are involved in muscle growth by increasing activation, proliferation, or differentiation of satellite cells.
with age, the expression of all of these genes is expected to decline in both organs and muscle because the growth regulating functions of these genes would be less necessary once growth has ceased.
the objectives of this study were to 1) determine if these  <dig> genes were downregulated during postnatal growth of skeletal muscle, 2) determine if expression differed between male and female mice during this time period and 3) determine if expression was altered by the absence of myostatin.
myostatin null mice experience increased muscle growth due to both increased hyperplasia and hypertrophy
<cit>  and exhibit increased satellite cell activity
<cit> .
furthermore, myostatin expression is decreased with increasing age in rats
<cit> , similar to the expression patterns of genes in this study.
therefore, use of myostatin null mice as a model for altered growth to determine the influence of these genes is warranted.
in general, we confirm that this set of genes is downregulated with age in skeletal muscle of mice with minimal differences between males and females.
expression of ezh <dig> was increased at d  <dig> and expression of mdk was increased at d  <dig> in myostatin null mice compared with wild type, but no other genotype effects were present.
these results suggest that this set of genes may globally regulate the cessation of growth in skeletal muscles as well as organs, but likely do not contribute to differences in muscularity of myostatin null mice.
methods
all procedures were approved by the university of illinois institutional animal care and use committee prior to beginning the experiment.
animal procedures and sample collection
mice from an internal c57bl/6 j colony were allowed ad libitum access to pelleted rodent chow  and tap water.
room temperature was maintained at 22°c with a 12 hour light/dark cycle.
male and female mice heterozygous for the myostatin null mutation were bred in monogamous pairs and litters were weaned at 21 days of age.
weaned mice were housed individually and genotyped with pcr based genotyping
<cit> .
at  <dig> ,  <dig>  and  <dig> and 70 days of age,  <dig> mice for each genotype by sex combination were euthanized by co <dig> asphyxiation and decapitation.
only  <dig> male wild type mice were collected at d  <dig>  pregnant dams were also euthanized by co <dig> asphyxiation and decapitation 14 days after breeding  and  <dig> fetuses collected to represent each genotype by sex combination.
at d - <dig>  the entire mouse body was collected.
at d  <dig>  muscles from both hindlimbs were combined.
at all other time points, biceps femoris muscles were weighed and collected.
tissues were stored at -80°c until analysis.
rna extraction and cdna synthesis
total rna was extracted using tri-reagent  and bcp phase separation reagent  according to the manufacturers’ protocols.
rna purity and concentration were measured using a nanodrop 2000c spectrophotometer .
for cdna synthesis, rna was diluted to 500 ng/μl using rnase-free water and 1 μg of rna was reversed transcribed using the qscript cdna super mix .
quantitative real-time pcr
validation experiments were conducted according to the applied biosystems quantitative pcr manual  to determine the appropriate reference gene  and cdna dilution.
eighteen μl of a 1: <dig> dilution of cdna, 2 μl of primer/probe, and 20 μl of perfecta® qpcr fastmix reaction mixes  were mixed together and plated in 18 μl duplicates into a 96-well plate per reaction.
expression of ezh <dig>  gpc <dig>  mdk, mest, mycn, peg <dig>  and plagl <dig>  was determined by quantitative real-time pcr using a steponeplus real-time pcr system thermal cycler .
the thermal cycler performed an initial denaturation at 95°c for 20 seconds followed by 40 cycles of 1 second at 95°c and 20 seconds at 60°c.
expression was normalized in two separate ways.
first, to compare the expression of genes with increasing age, expression was normalized to rn18s  and compared to the average of 70-day-old male wild type expression .
to better visualize the influence of sex and genotype on expression, ct values were normalized to rn18s  and then compared within the fetal and d  <dig> time points to the average male wild type average of each time point .
for  <dig>   <dig> and  <dig> d data, ct values were normalized to rn18s  and then compared to the average male wild type at  <dig> d of age .
values were expressed as a fold change  for statistical analysis.
statistical analysis
data were analyzed using the proc mixed procedure in sas version  <dig>  .
the model included the fixed effects of age , sex, and all 2- and 3-way interactions.
data are presented as least squares means ± 95% confidence intervals.
RESULTS
body and muscle weights
body and muscle weights during postnatal growth were compared between sexes and genotypes to illustrate differences in growth patterns.
at day  <dig> and  <dig>  body weights were similar among all genotype and sex combinations.
mice became heavier with age, and at d  <dig>  myostatin null mice were heavier than wild type mice within sex while males were heavier than females within genotype .
bicep femoris weights were similar among all treatment groups at d  <dig> and  <dig>  but myostatin null muscles were heavier than wild type at d  <dig> .
these results are similar to previous reports of body and muscle weights of myostatin null mice
<cit> .
expression of growth regulating genes in muscle during growth
to determine the expression of these genes during growth, the expression of all genes in wild type male mice was analyzed in comparison to d  <dig> expression .
all genes were affected  by age with an overall decline of expression with increasing age.
for all genes except mycn, expression was not different between d - <dig> and d 0; mycn expression was reduced at d  <dig> compared with d - <dig>  in all genes, expression was reduced  at d  <dig>  d  <dig> and d  <dig> compared with d - <dig> and d  <dig>  expression of ezh <dig>  gpc <dig>  mdk, and plagl <dig> did not decline further after d  <dig>  however, the expression of mest, and peg <dig> was reduced  at d  <dig> and  <dig> compared with d  <dig>  with expression at the later time points being similar.
for mycn, expression was reduced  at d  <dig> compared with d  <dig> with expression at d  <dig> being intermediate and similar to either time point.
therefore, the results of previous experiments detailing the expression of these genes in various organs
<cit>  are confirmed in skeletal muscle.
expression of these genes declined with age in wild type male mice.
expression differences between sexes and genotypes
expression of all  <dig> genes was unaffected by genotype or sex at d - <dig> .
similarly, at birth, expression of ezh <dig>  mdk, mycn, and plagl <dig> were unaffected by sex or genotype .
furthermore, expression of all genes was unaffected by genotype.
expression of gpc <dig>  mest and peg <dig> were increased in female mice compared with male mice.
there were no interactions  of sex and genotype for expression at d - <dig> or birth.
avalues are least squares means of fold changes.
fold change was compared to the average wild type male expression.
bgen = genotype, int = interaction of sex and genotype.
avalues are least squares means of fold changes.
fold change was compared to the average wild type male expression.
bgen = genotype, int = interaction of sex and genotype.
expression data from d  <dig>   <dig> and  <dig> were compared with the average wild type male expression at d  <dig> to calculate fold changes.
these data represent a consistent muscle group as opposed to the previous data that combine biceps femoris with whole hind limb or fetus .
expression of mdk and plagl <dig> did not decline with age from d  <dig> to  <dig>  expression of ezh <dig>  gpc <dig>  and peg <dig> were all increased  at d  <dig> compared with d  <dig> and  <dig>  which were similar to each other.
expression of mest was increased  at d  <dig> compared with d  <dig> and at d  <dig> compared with d  <dig>  expression of mycn actually increased with age as expression was increased  at d  <dig> and  <dig> compared with d  <dig>
expression of all genes was unaffected by sex.
furthermore, with the exception of mdk, expression was also unaffected by genotype and interactions between genotype with age or sex with age.
expression of mdk was increased  at d  <dig> in myostatin null mice compared with wild type, but was similar between genotypes at d  <dig> or  <dig> .
expression of mdk was also increased  at d  <dig> in female mice compared with males, but was similar between sexes at d  <dig> and  <dig> .
expression of ezh <dig> at d  <dig> was also increased  in wild type mice compared with myostatin null.
therefore, despite differences in muscle weights at d  <dig> between wild type and myostatin null mice, only the expression of two genes differed at any time point investigated and in both cases, gene expression was increased in wild type, not the more heavily muscled myostatin null mice.
therefore, these data suggest that increases in satellite cell activity and hypertrophy noted in myostatin null mice is not likely due to increased activity in these growth promoting genes.
nevertheless, we have confirmed that this suite of genes is downregulated with age in skeletal muscle similar to other organs.
ezh <dig>  mdk, mest, peg <dig>  and plagl <dig> are known to be involved in muscle regeneration and all of the genes are overly expressed in rhabdomyosarcoma, a skeletal muscle cancer
<cit> .
it is possible that these genes aid in postnatal muscle growth by activation, proliferation, or differentiation of satellite cells.
ezh <dig> has an established role in satellite cell activation
<cit> ; it can be speculated that mdk, mest, peg <dig>  and plagl <dig> might have similar functions because they are also involved in muscle regeneration
<cit> .
mycn has not been well characterized in muscle, but it promotes the proliferation of neural progenitor cells and inhibits neural differentiation and, therefore, might exhibit similar functions in muscle with regard to satellite cells
<cit> .
given these functions, it is expected that expression of these genes would decrease with age as there is less activation, proliferation, and differentiation of satellite cells and, thus, less need for the growth regulatory functions of these genes as growth slows.
in contrast, gpc <dig> is thought be a negative regulator of growth such that increased expression results in reduced proliferation
<cit> .
gpc <dig> functions in muscle have not been identified; however, gpc <dig> is involved in liver regeneration such that expression peaks during the early and middle stages of the process
<cit> .
this leads to the conclusion that gpc <dig> expression might increase early in postnatal development to negatively regulate growth and proliferation, so overgrowth of organs and muscle does not occur, and decrease as growth slows because proliferation has decreased leading to less need for its regulatory functions.
CONCLUSIONS
in summary, this study examined the expression decline of  <dig> growth regulating genes over time in the muscle of wild type and heavily muscled myostatin null mice.
overall, expression was increased in all genes at the fetal time point or birth compared with  <dig> d of age or later.
despite differences in muscle weights between wild type and myostatin null mice, there were not widespread expression differences between genotypes, nor were there differences between sexes.
therefore, differences observed in muscle weights, especially between those of myostatin null and wild type mice, do not seem to result from differential regulation of the growth-regulating genes examined in this study.
it should be noted, however, that the expression of these genes may be different in the satellite or stem cell populations of skeletal muscle compared with the skeletal muscle as a whole.
future work to differentiate the role of these genes in satellite or stem cell proliferation, differentiation and fusion with existing muscle fibers may be warranted.
competing interests
the authors declare they have no competing interests.
authors’ contributions
jcj carried out the preliminary studies, established the methods, and drafted the manuscript.
kak carried out the study and performed the statistical analysis.
acd conceived the study, and participated in its design and coordination and helped to draft the manuscript.
all authors read and approved the final manuscript.

 as humans and animals age, growth slows and eventually stops. organs grow until they reach mature size; however, little is known about how growth cessation is regulated and coordinated in the body. a set of growth regulating genes, which include ezh gpc mdk, mest, mycn, peg and plagl has been identified that is dependent upon growth and whose expression declines with age in organs. in general, when expression of these genes decreases, proliferation decreases leading to a reduction in growth. mutations of these genes also result in reduced viability, growth abnormalities, and diseases such as rhabdomyosarcoma, a skeletal muscle cancer, and simpson-golabi-behmel syndrome. though well characterized in organs, these genes have not been well examined in growing muscle. it is expected that expression of these genes would differ between muscle, heart, and liver because of the differences in how these tissues grow. postnatally, muscle grows by mainly hypertrophy, an increase in cell size, and not hyperplasia, an increase in cell number. in mice, muscle fiber number becomes fixed at approximately d postnatal. hypertrophy of muscle fibers is accompanied by satellite cell activation. these cells fuse with existing muscle fibers to support muscle growth. without the activation of satellite cells, postnatal muscle growth and regeneration is severely inhibited. in muscle, it is known that the expression of ezh mdk, mest, peg and plagl is increased during regeneration. in contrast, mycn and gpc have not been characterized in muscle. mycn, however, increases proliferation of neural progenitor cells while gpc is upregulated in the early and middle stages of liver regeneration. because of of these genes are known to be upregulated with muscle regeneration, it is possible that they are involved in muscle growth by increasing activation, proliferation, or differentiation of satellite cells. with age, the expression of all of these genes is expected to decline in both organs and muscle because the growth regulating functions of these genes would be less necessary once growth has ceased. the objectives of this study were to 1) determine if these genes were downregulated during postnatal growth of skeletal muscle, 2) determine if expression differed between male and female mice during this time period and 3) determine if expression was altered by the absence of myostatin. myostatin null mice experience increased muscle growth due to both increased hyperplasia and hypertrophy and exhibit increased satellite cell activity. furthermore, myostatin expression is decreased with increasing age in rats, similar to the expression patterns of genes in this study. therefore, use of myostatin null mice as a model for altered growth to determine the influence of these genes is warranted. in general, we confirm that this set of genes is downregulated with age in skeletal muscle of mice with minimal differences between males and females. expression of ezh was increased at d and expression of mdk was increased at d in myostatin null mice compared with wild type, but no other genotype effects were present. these results suggest that this set of genes may globally regulate the cessation of growth in skeletal muscles as well as organs, but likely do not contribute to differences in muscularity of myostatin null mice. methods all procedures were approved by the university of illinois institutional animal care and use committee prior to beginning the experiment. animal procedures and sample collection mice from an internal c57bl/6 j colony were allowed ad libitum access to pelleted rodent chow and tap water. room temperature was maintained at 22°c with a 12 hour light/dark cycle. male and female mice heterozygous for the myostatin null mutation were bred in monogamous pairs and litters were weaned at 21 days of age. weaned mice were housed individually and genotyped with pcr based genotyping. at, and and 70 days of age, mice for each genotype by sex combination were euthanized by co asphyxiation and decapitation. only male wild type mice were collected at d pregnant dams were also euthanized by co asphyxiation and decapitation 14 days after breeding and fetuses collected to represent each genotype by sex combination. at d - the entire mouse body was collected. at d muscles from both hindlimbs were combined. at all other time points, biceps femoris muscles were weighed and collected. tissues were stored at -80°c until analysis. rna extraction and cdna synthesis total rna was extracted using tri-reagent and bcp phase separation reagent according to the manufacturers’ protocols. rna purity and concentration were measured using a nanodrop 2000c spectrophotometer. for cdna synthesis, rna was diluted to 500 ng/μl using rnase-free water and 1 μg of rna was reversed transcribed using the qscript cdna super mix. quantitative real-time pcr validation experiments were conducted according to the applied biosystems quantitative pcr manual to determine the appropriate reference gene and cdna dilution. eighteen μl of a 1: dilution of cdna, 2 μl of primer/probe, and 20 μl of perfecta® qpcr fastmix reaction mixes were mixed together and plated in 18 μl duplicates into a 96-well plate per reaction. expression of ezh gpc mdk, mest, mycn, peg and plagl was determined by quantitative real-time pcr using a steponeplus real-time pcr system thermal cycler. the thermal cycler performed an initial denaturation at 95°c for 20 seconds followed by 40 cycles of 1 second at 95°c and 20 seconds at 60°c. expression was normalized in two separate ways. first, to compare the expression of genes with increasing age, expression was normalized to rn18s and compared to the average of 70-day-old male wild type expression. to better visualize the influence of sex and genotype on expression, ct values were normalized to rn18s and then compared within the fetal and d time points to the average male wild type average of each time point. for and d data, ct values were normalized to rn18s and then compared to the average male wild type at d of age. values were expressed as a fold change for statistical analysis. statistical analysis data were analyzed using the proc mixed procedure in sas version. the model included the fixed effects of age, sex, and all 2- and 3-way interactions. data are presented as least squares means ± 95% confidence intervals.  body and muscle weights body and muscle weights during postnatal growth were compared between sexes and genotypes to illustrate differences in growth patterns. at day and body weights were similar among all genotype and sex combinations. mice became heavier with age, and at d myostatin null mice were heavier than wild type mice within sex while males were heavier than females within genotype. bicep femoris weights were similar among all treatment groups at d and but myostatin null muscles were heavier than wild type at d. these results are similar to previous reports of body and muscle weights of myostatin null mice. expression of growth regulating genes in muscle during growth to determine the expression of these genes during growth, the expression of all genes in wild type male mice was analyzed in comparison to d expression. all genes were affected by age with an overall decline of expression with increasing age. for all genes except mycn, expression was not different between d - and d 0; mycn expression was reduced at d compared with d - in all genes, expression was reduced at d d and d compared with d - and d expression of ezh gpc mdk, and plagl did not decline further after d however, the expression of mest, and peg was reduced at d and compared with d with expression at the later time points being similar. for mycn, expression was reduced at d compared with d with expression at d being intermediate and similar to either time point. therefore, the results of previous experiments detailing the expression of these genes in various organs are confirmed in skeletal muscle. expression of these genes declined with age in wild type male mice. expression differences between sexes and genotypes expression of all genes was unaffected by genotype or sex at d -. similarly, at birth, expression of ezh mdk, mycn, and plagl were unaffected by sex or genotype. furthermore, expression of all genes was unaffected by genotype. expression of gpc mest and peg were increased in female mice compared with male mice. there were no interactions of sex and genotype for expression at d - or birth. avalues are least squares means of fold changes. fold change was compared to the average wild type male expression. bgen = genotype, int = interaction of sex and genotype. avalues are least squares means of fold changes. fold change was compared to the average wild type male expression. bgen = genotype, int = interaction of sex and genotype. expression data from d and were compared with the average wild type male expression at d to calculate fold changes. these data represent a consistent muscle group as opposed to the previous data that combine biceps femoris with whole hind limb or fetus. expression of mdk and plagl did not decline with age from d to expression of ezh gpc and peg were all increased at d compared with d and which were similar to each other. expression of mest was increased at d compared with d and at d compared with d expression of mycn actually increased with age as expression was increased at d and compared with d expression of all genes was unaffected by sex. furthermore, with the exception of mdk, expression was also unaffected by genotype and interactions between genotype with age or sex with age. expression of mdk was increased at d in myostatin null mice compared with wild type, but was similar between genotypes at d or. expression of mdk was also increased at d in female mice compared with males, but was similar between sexes at d and. expression of ezh at d was also increased in wild type mice compared with myostatin null. therefore, despite differences in muscle weights at d between wild type and myostatin null mice, only the expression of two genes differed at any time point investigated and in both cases, gene expression was increased in wild type, not the more heavily muscled myostatin null mice. therefore, these data suggest that increases in satellite cell activity and hypertrophy noted in myostatin null mice is not likely due to increased activity in these growth promoting genes. nevertheless, we have confirmed that this suite of genes is downregulated with age in skeletal muscle similar to other organs. ezh mdk, mest, peg and plagl are known to be involved in muscle regeneration and all of the genes are overly expressed in rhabdomyosarcoma, a skeletal muscle cancer. it is possible that these genes aid in postnatal muscle growth by activation, proliferation, or differentiation of satellite cells. ezh has an established role in satellite cell activation; it can be speculated that mdk, mest, peg and plagl might have similar functions because they are also involved in muscle regeneration. mycn has not been well characterized in muscle, but it promotes the proliferation of neural progenitor cells and inhibits neural differentiation and, therefore, might exhibit similar functions in muscle with regard to satellite cells. given these functions, it is expected that expression of these genes would decrease with age as there is less activation, proliferation, and differentiation of satellite cells and, thus, less need for the growth regulatory functions of these genes as growth slows. in contrast, gpc is thought be a negative regulator of growth such that increased expression results in reduced proliferation. gpc functions in muscle have not been identified; however, gpc is involved in liver regeneration such that expression peaks during the early and middle stages of the process. this leads to the conclusion that gpc expression might increase early in postnatal development to negatively regulate growth and proliferation, so overgrowth of organs and muscle does not occur, and decrease as growth slows because proliferation has decreased leading to less need for its regulatory functions.  in summary, this study examined the expression decline of growth regulating genes over time in the muscle of wild type and heavily muscled myostatin null mice. overall, expression was increased in all genes at the fetal time point or birth compared with d of age or later. despite differences in muscle weights between wild type and myostatin null mice, there were not widespread expression differences between genotypes, nor were there differences between sexes. therefore, differences observed in muscle weights, especially between those of myostatin null and wild type mice, do not seem to result from differential regulation of the growth-regulating genes examined in this study. it should be noted, however, that the expression of these genes may be different in the satellite or stem cell populations of skeletal muscle compared with the skeletal muscle as a whole. future work to differentiate the role of these genes in satellite or stem cell proliferation, differentiation and fusion with existing muscle fibers may be warranted. competing interests the authors declare they have no competing interests. authors’ contributions jcj carried out the preliminary studies, established the methods, and drafted the manuscript. kak carried out the study and performed the statistical analysis. acd conceived the study, and participated in its design and coordination and helped to draft the manuscript. all authors read and approved the final manuscript.
genes that decline in expression with age and are thought to coordinate growth cessation have been identified in various organs, but their expression in skeletal muscle is unknown. gene expression was determined by quantitative real-time pcr. therefore, our objective was to determine expression of these genes in skeletal muscle from birth to maturity. we hypothesized that expression of these genes would decline with age in skeletal muscle but differ between sexes and between wild type and myostatin null mice. female and male wild type and myostatin null mice were sacrificed by carbon dioxide asphyxiation followed by decapitation at d - and days of age. expression of ezh was increased at d and expression of mdk was increased at d in myostatin null mice compared with wild type, but no other genotype effects were present. in general, expression of these growth-regulating genes was reduced at d compared with day and d - expression of gpc mest, and peg was further reduced at d and compared with d however the expression of mycn increased from d to d and myostatin null mice, as expected, were heavier with increased biceps femoris weight at d however, with respect to sex and genotype, there were few differences in expression. overall, these data suggest the downregulation of these growth-regulating genes with age might play a role in the coordinated cessation of muscle growth similar to organ growth but likely have a limited role in the differences between sexes or genotypes.
 genes that decline in expression with age and are thought to coordinate growth cessation have been identified in various organs, but their expression in skeletal muscle is unknown. therefore, our objective was to determine expression of these genes in skeletal muscle from birth to maturity. we hypothesized that expression of these genes would decline with age in skeletal muscle but differ between sexes and between wild type and myostatin null mice.  female and male wild type and myostatin null mice were sacrificed by carbon dioxide asphyxiation followed by decapitation at d - and days of age. whole bodies at d - all muscles from both hind limbs at d and bicep femoris muscle from d and were collected. gene expression was determined by quantitative real-time pcr. in general, expression of these growth-regulating genes was reduced at d compared with day and d - expression of gpc mest, and peg was further reduced at d and compared with d however the expression of mycn increased from d to d and myostatin null mice, as expected, were heavier with increased biceps femoris weight at d however, with respect to sex and genotype, there were few differences in expression. expression of ezh was increased at d and expression of mdk was increased at d in myostatin null mice compared with wild type, but no other genotype effects were present. expression of mdk was increased in females compared to males at d but no other sex effects were present.  overall, these data suggest the downregulation of these growth-regulating genes with age might play a role in the coordinated cessation of muscle growth similar to organ growth but likely have a limited role in the differences between sexes or genotypes. ezh2gpc3mdkmestmycnpeg3plagl1muscle growthmyostatin 