in vertebrates , the process of skeletal muscle development occurs in subsequent steps that involve distinct populations of myogenic progenitors , the myoblasts , which arise from the dermomyotomal domain of somitic mesoderm ( christ and ordahl , 1995 ) .
the process of myogenic differentiation is initiated in mesodermal cells by a family of basic - helix - loop - helix ( bhlh ) transcription factors , named muscle regulatory factors ( mrfs ) , that are able to activate transcription of muscle - specific markers such as the myosin heavy chain ( myhc ) isoforms ( pinney et  al . , 1988 , cao et  al . , 2010 ) .
embryonic myoblasts sustain a first wave of myogenesis between embryonic day 10 ( e10 ) and e12 in the mouse and give rise to primary myofibers that establish the primitive shape of muscle and express high levels of the slow myhc isoform ( myhc - i , encoded by the myh7 gene ) and of the embryonic myhc isoform ( myhc - emb , encoded by myh3 ) ( schiaffino et  al . , 1986 , stockdale , 1992 ) .
a second wave of muscle differentiation takes place between e15 and e18 , driven by fetal myoblasts that form secondary fibers , characterized by low levels of myhc - i and high levels of neonatal myhc ( myhc - neo , encoded by myh8 ) ( eusebi et  al . , 1986 , lyons et  al . , 1990 , daou et  al . ,
eventually , primary fibers conserve the slow - twitch phenotype typical of embryonic muscle , while secondary fibers lose the expression of several embryonic - specific markers such as myhc - i and acquire expression of fast - twitch markers ( ferrari et  al . , 1997 ,
embryonic and fetal myoblasts , once isolated from the embryo , are committed to a specific fiber type , suggesting the involvement of intrinsic factors rather than nerve activity in the establishment of fiber phenotype ( page et  al . , 1992 ) .
these observations suggest that the proper transition of skeletal muscle from the embryonic to the fetal / post - natal phenotype requires a switch in the transcriptional status of differentiating myoblasts . in our previous study
, we have shown that the transcription factor nfix , a member of the nuclear factor i ( nfi ) family , has a key role in the establishment of fetal muscle phenotype and in the downregulation of slow myhc both in fetal and adult muscles ( messina et  al . , 2010 , rossi et  al . , 2016 ) .
we have shown that nfix is strongly expressed in fetal myoblasts and indirectly represses myhc - i expression via the transcription factor nfatc4 , a positive regulator of myhc - i in skeletal muscle ( calabria et  al .
we have also reported that the zebrafish ( danio rerio ) nfix ortholog nfixa has an evolutionarily conserved role in the transition from slow - twitch to fast - twitch myogenesis ( pistocchi et  al . , 2013 ) .
in the past few years , it has been shown that sox6 , a member of the sry - related hmg box ( sox ) factor family , which is highly conserved in vertebrates , plays a critical role in fetal fiber specification through direct repression of myhc - i by binding to the 5-upstream region in two different binding sites .
the first is located 200  bp from the transcription start site ( tss ) in the proximal promoter and is sufficient for sox6-dependent myhc - i repression in fetal myotubes ( hagiwara et  al . , 2007 , an et  al . , 2011 ) , and the second is located 2,900  bp from the tss in a distal muscle enhancer that is required for full promoter activity ( giger et  al . ,
sox6-null mouse muscle displays increased levels of myhc - i and a general switch toward a slower phenotype ( hagiwara et  al .
, studies in zebrafish have shown that sox6 is restricted to fast - twitch fibers during embryonic muscle development and that ectopic sox6 expression in adaxial cells ( the slow muscle progenitors in zebrafish ) leads to the silencing of slow - twitch genes ( such as the slow myhc isoform smyhc1 and the transcription factor prox1a ) ( von hofsten et  al . , 2008 , wang et  al . , 2011 ,
, we observed that sox6 , at variance with nfix , is expressed at comparable levels in embryonic and fetal myoblasts , despite its role of inhibitor of slow twitching program .
intriguingly , we demonstrated that sox6 has opposite roles in regulating myhc - i expression between embryonic and fetal myogenesis in mouse .
specifically , during embryonic myogenesis , sox6 indirectly promotes myhc - i expression via transcriptional activation of mef2c . as a consequence ,
sox6 deficiency in embryonic muscle leads to a strong downregulation of myhc - i . on the contrary , during fetal myogenesis
, sox6 cooperates with nfix to repress myhc - i in a complex in which nfix is necessary for the proper binding of sox6 to the myhc - i promoter in fetal myotubes .
finally , we show that nfixa and sox6 together regulate smyhc in zebrafish embryos , revealing an evolutionarily conserved mechanism that is required for the acquisition of normal muscle phenotype .
sox6 has been intensively studied as an inhibitor of slow muscle phenotype during the fetal period .
however , we observed that sox6 is also expressed during embryonic myogenesis , which is mainly characterized by the expression of typical slow genes such as the slow myhc isoform myhc - i .
myf5 embryos and fetuses were collected at e12.5 or e16.5 , and gfp - positive myoblasts were isolated via fluorescence - activated cell sorting ( facs ) as previously described ( messina et  al . , 2010 ) . using quantitative real - time pcr and western blot
, we found that sox6 levels do not significantly change , whereas nfix , as known , is drastically upregulated in fetal progenitors ( figures 1a1c ) .
importantly , these data show that sox6 and nfix proteins are co - expressed only during fetal myogenesis .
we also performed extensive immunofluorescence analysis on frozen mouse embryo sections from e10.5 to e18.5 in order to follow sox6 protein expression throughout development .
notably , sox6 is first expressed between e11.5 and e12.5 in primary fibers that express high levels of myhc - i ( figures s1a  s1h ) , whereas at e17.5 , the localization of sox6 is almost completely associated with secondary fibers that are negative for myhc - i ( figures s1i  s1l ) , as previously described ( an et  al . , 2011 ,
we therefore decided to investigate the possible function of sox6 during embryonic myogenesis , a function not apparently linked to repression of myhc - i . to this aim , we performed immunofluorescence analysis on e12.5 muscle sections from homozygous mice carrying the sox6 allele ( hence referred to as sox6-null mice ) ( smits et  al . , 2001 ) .
surprisingly , the staining for myhc - i is strongly decreased in sox6 null in comparison to wild - type ( wt ) muscle ( figures 1d and 1e ) .
importantly , no differences in total myhc content were assessed in embryonic muscle groups of sox6-null embryos ( figures 1 g and 1h ) , implying that the decrease in myhc - i expression is not due to delayed or aberrant muscle differentiation .
we also performed western blot and quantitative real - time pcr on embryonic muscle lysates to confirm the immunofluorescence data , and our results showed a decrease of myhc - i protein in sox6-null samples , without major changes in total myhc content ( figures 1j and 1k ) .
interestingly , the phenotype of sox6-null embryonic muscle is reminiscent of the tg : mlc1f - nfix2 gain - of - function embryo ( figures 1f and 1i ) , in which the nfix2 splice variant is ectopically expressed in muscle cells from e11.5 , leading to a fetal - like muscle phenotype ( kelly et  al . , 1997 , messina et  al . , 2010 )
importantly , nfix expression in embryonic skeletal muscle is not altered in the absence of sox6 at both the protein and mrna levels ( figures s2a  s2d ) . taken together ,
these results show that sox6 is expressed at equal levels in skeletal muscle during embryonic and fetal myogenesis and that deletion of sox6 during the embryonic period unexpectedly leads to downregulation of myhc - i . in order to define a possible mechanism
by which sox6 regulates the transcription of myhc - i during embryonic myogenesis , we performed chromatin immunoprecipitation ( chip ) for sox6 in differentiated embryonic myoblasts .
we found that sox6 does not significantly bind either to the proximal or to the distal regulative regions upstream of myhc - i ( figure  2a ) , thus suggesting that sox6 is not able to directly regulate myhc - i transcription in embryonic muscle . to confirm this hypothesis , we performed luciferase assays on wt and sox6-null embryonic differentiated myoblasts with vectors containing the 3,500-bp myhc - i full 5-upstream region ( myhc - i 3500 ) , the 408-bp myhc - i proximal promoter sequence ( myhc - i 408 ) , or the mutated forms of the  distal and proximal canonical sox6 binding sites , myhc - i 3500  m and myhc - i 408  m ( figure  s3a ) ( hagiwara et  al . , 2007 , an et  al . , 2011 ) . as expected , in the absence of sox6 , we found a significant reduction of firefly luciferase activity in all the conditions with the only notable exception of the 408 wt construct ( figure  2b ) , suggesting that sox6 is promoting myhc - i expression in embryonic myocytes without direct binding to its canonical binding sites .
in order to identify a possible indirect mechanism by which sox6 enhances myhc - i expression in embryonic muscle , we focused on the transcription factor mef2c , a known positive regulator of the slow phenotype ( wu et  al . , 2000
, potthoff et  al . , 2007 , anderson et  al . , 2015 ) .
to verify the interaction between mef2c and the myhc - i promoter in embryonic myoblasts , we performed a chip assay , which showed direct binding of mef2c on the proximal myhc - i promoter ( figure  s3b ) .
interestingly , mef2c mrna is downregulated in e12.5 sox6-null muscle , at variance with the closely related mef2a ( figure  2c ) . by chip on embryonic differentiated myoblasts
, we found that sox6 directly binds to a region located in the mef2c promoter ( figure  2d ) , indicating that sox6 is a direct activator of mef2c during embryonic myogenesis . in order to validate a possible mef2c - mediated mechanism of myhc - i regulation , we transfected wt and sox6-null unpurified embryonic myoblasts with a vector overexpressing mef2c .
mef2c overexpression in sox6-deficient cells ( figure  s3c ) leads to an increase of myhc - i expression when normalized on the levels of myhc - emb to account for the total number of myogenic cells ( figure  2e ) . to validate these data
, we also transduced purified embryonic myoblasts with a lentiviral vector expressing small hairpin rna ( shrna ) against sox6 to achieve in  vitro sox6 knockdown ( figure  s3d ) .
we then transfected cells with mef2c - overexpression vector ( figure  s3d ) and assessed the levels of myhc - i mrna by quantitative real - time pcr ( figure  2f ) .
strikingly , overexpression of mef2c was able to partially rescue myhc - i expression in shsox6 cells ( up to 30% of the levels of scramble - transduced cells ) .
these results show that during the embryonic period , sox6 acts indirectly as a positive regulator of myhc - i via a mef2c - dependent mechanism , in sharp contrast with its well - known and characterized function during fetal myogenesis ( an et  al .
, 2011 , quiat et  al . , 2011 ; see below ) . since sox6 has opposite roles in myhc - i regulation in embryonic and fetal muscle , and since nfix is only expressed during fetal myogenesis ( messina et  al . , 2010 )
, we decided to investigate the possible cooperation between nfix and sox6 during fetal myogenesis .
2011 ) , fetal fiber specification is completely disrupted in the absence of sox6 . indeed , in contrast to wt , sox6-null fetal muscle displays very high levels of myhc - i by immunofluorescence , quantitative real - time pcr , and western blot ( figures s4a  s4d ) .
we also looked at nfix on sections from the sox6-null mice and found that despite the dramatic increase in myhc - i expression , nfix is correctly expressed in the nuclei of muscle fibers in the absence of sox6 ( figures 3a3d ) , and western blot analysis did not reveal differences in nfix protein content ( figure  s4c ) .
these data suggest that nfix is normally expressed in fetal muscles lacking sox6 but unable to properly repress myhc - i .
to verify whether sox6 is required for nfix function , we performed chip for nfix in wt fetal myotubes , which revealed binding of nfix to myhc - i promoter in the same region of the proximal sox6 binding site ( 200  bp ) ( figure  3e ) .
interestingly , we observed that in sox6-null fetal myotubes , the binding of nfix to the myhc - i promoter is significantly reduced .
importantly , nfix binding to nfatc4 promoter is not impaired in the absence of sox6 ( figure  3e ) , suggesting that nfix is still able to bind to other transcriptional targets in the absence of sox6 .
we further validated the repressive role of nfix on the myhc - i proximal promoter by performing a luciferase assay with myhc - i 408 and myhc - i 408  m vectors in wt and nfix - null fetal myotubes ( figure  3f ) .
our results showed increased luciferase expression in the absence of nfix with the vector carrying the wt sequence , suggesting that indeed nfix represses myhc - i by acting on the  proximal promoter regulative region , in spite of the absence of any nfix consensus sequences ( data not shown ) .
importantly , nfix - dependent negative regulation of myhc - i is lost when the proximal sox6 binding sequence is mutated , demonstrating that sox6 is required for nfix binding to the proximal myhc - i promoter ( figure  3f ) .
overall , these results suggest a possible crosstalk between nfix and sox6 in regulating myhc - i expression at the proximal promoter region . to better investigate a possible reciprocal interplay between nfix and sox6 , we performed immunofluorescence for sox6 and total myhc on frozen muscle sections from e16.5 wt and nfix - null mice ( campbell et  al . , 2008 ) .
we found that sox6 is expressed in both wt and nfix - null muscles ( figures s4e and s4f ) , implying that nfix is dispensable for normal sox6 expression in fetal muscle .
interestingly , immunofluorescence for sox6 and myhc - i on sections from the same mice revealed that in the absence of nfix , there is a marked increase in the number of sox6-positive fibers that co - express myhc - i , in contrast to wt muscles ( figures 4a4d ) .
we further validated these data by western blot and quantitative real - time pcr on differentiated fetal myotubes and observed that nfix - null myotubes express higher levels of myhc - i than wt cells ( figures 4e and 4f ) .
these data show that in the absence of nfix , the repressive activity of sox6 on myhc - i is partially impaired , even if the normal expression pattern of sox6 is maintained .
the results obtained in both sox6 and nfix - null fetuses led us  to hypothesize a functional cooperation between nfix and sox6 in myhc - i regulation .
therefore , we tested the ability of nfix and sox6 to bind to each other in a multi - protein complex . to this aim , we used fetal myotubes transiently transfected with an nfix2 hemagglutinin ( ha ) vector ( see experimental procedures ) and performed a co - immunoprecipitation ( coip ) assay from nuclear extracts for ha and sox6 .
coip revealed the presence of a complex containing both sox6 and nfix2-ha , as shown in figure  5a .
therefore , we wondered whether , as observed for nfix fetuses in absence of sox6 , nfix might be required for the  binding of sox6 to the myhc - i promoter as well .
hence , we performed chip for sox6 in wt and nfix - null fetal myotubes .
our results showed a decrease in sox6 binding to the two different sites in the myhc - i 5-upstream region ( figure  5b ) .
notably , the binding with the proximal promoter was completely lost in the absence of nfix , whereas the binding with the distal enhancer was reduced by 50% .
interestingly , chip for sox6 on the mef2c promoter reveals that in wt fetal myotubes , sox6 is not able to bind to the mef2c promoter , at variance with what happens during the embryonic stage . on the contrary
, sox6 binding on the mef2c promoter still occurs in nfix - null fetal myotubes .
these data indicate that nfix is required for the proper binding of sox6 to the myhc - i promoter during fetal myogenesis . finally , we  performed chip for sox6 on wt and tg : mlc1f - nfix2 embryonic myoblasts ( figure  5c ) .
strikingly , nfix overexpression leads to a switch in the binding properties of sox6 ; indeed , we found that sox6 is bound to the myhc - i promoter , but not to the mef2c promoter , in tg : mlc1f - nfix2 embryonic myoblasts .
these data demonstrate that nfix is necessary and sufficient for the binding of sox6 to the myhc - i proximal promoter and therefore for sox6 repressive activity on myhc - i .
it was previously demonstrated that both sox6 and nfixa have an evolutionarily conserved role in the repression of slow - twitch genes in zebrafish ( von hofsten et  al . , 2008 ,
we thus wondered whether a functional interplay between nfixa and sox6 is conserved in zebrafish myogenesis . as a preliminary analysis , we performed quantitative real - time pcr on trunk and tail regions isolated from zebrafish embryos at 1 , 2 , and 3  days post - fertilization ( dpf ) and found that the sox6 transcript is expressed at high levels at 1 dpf and is steadily downregulated up to 3 dpf ( figure  s5a ) , whereas the nfixa transcript peaks at 2 dpf ( figure  s5b ) , as  shown previously ( pistocchi et  al . , 2013 ) . additionally , we performed immunofluorescence for sox6 and total myhc ( mf20 antibody ) or the slow myhc isoform smyhc ( f59 antibody ) on 2 dpf embryos and found that sox6 protein is specifically expressed in the nuclei of fast muscle fibers that are negative for smyhc ( figures 6a and 6b ) , whereas the outer superficial fibers positive for smyhc are mostly negative for sox6 ( data not shown ) .
interestingly , we observed that a minority of slow superficial fibers show cytoplasmic staining for sox6 ( figures 6c6j ) , suggesting that sox6 subcellular localization might be spatially regulated in the different muscle domains . in order to elucidate the role of sox6 in slow - twitch genes regulation , we performed morpholino ( mo)-mediated knockdown of sox6 ( von hofsten et  al . , 2008 ) . by quantitative real - time pcr analysis ( figures s5c and s5d ) , we found that the expression of smyhc1 is markedly increased in sox6 morphants at 2 dpf , whereas the fast - twitch gene mylpfa ( fast myosin light chain isoform ) is expressed at equal levels .
moreover , the nfixa transcript is drastically upregulated in sox6 morphants , suggesting that sox6 might negatively regulate nfixa ( figure  s5e ) .
we conclude that sox6 is a critical repressor of the slow - twitch phenotype in zebrafish and that nfixa is not able to compensate for sox6 knockdown .
to verify a possible cooperation between nfixa and sox6 , we performed co - injections of morpholinos against sox6 and nfixa at lower doses  with respect to those previously described ( von hofsten et  al . , 2008 , pistocchi et  al . , 2013 ) in order to minimize their effect when injected separately ( see experimental procedures ) .
co - injection of these doses of mos resulted in synergistic defects in motility in touch - response assays ( figure  s6 ) .
control embryos and the vast majority of sox6 or nfixa morphants readily swam away when touch - stimulated . on the contrary ,
66% of double partial morphants were either shivering or bending their tails before eventually moving away but within a shorter distance ( figures s6a and s6c ; movie s1 ) .
the synergistic effect is more evident when lowering the doses ( see figures s6b and s6d ) .
strikingly , quantitative real - time pcr results show that smyhc1 is significantly upregulated only in the double morphants at 48 hpf , whereas the level of myl1 , an early marker for differentiating fast muscle cells ( burguire et  al . , 2011 ) , does not change ( figure  6k ) .
moreover , we validated the increased expression of smyhc ( f59 antibody ) in sox6/nfixa double morphants by western blot ( figure  6l ) . we thus conclude that functional cooperation of sox6 and nfix is required for proper skeletal muscle development and that this cooperation is evolutionarily conserved in mouse and zebrafish .
adult skeletal muscle is composed of two major fiber types presenting a wide range of physiological and biochemical differences .
slow - twitch type i fibers use oxidative metabolism and express the slow myhc isoform myhc - i ; in contrast , fast - twitch type ii fibers present glycolytic or mixed metabolism and express three fast myhc isoforms ( myhc - iia , iix / d , and iib ) ( peter et  al . , 1972 , schiaffino et  al . , 1988 , chakkalakal et  al . , 2012 ) .
the phenotype of post - natal muscle fibers is strictly regulated by extrinsic signals such as muscle activity and hormones ( butler - browne et  al . , 1982 , gambke et  al . , 1983 ,
additionally , different factors controlling adult muscle plasticity have been identified , including the nfatc ( calabria et  al . , 2009 ) and mef2 ( wu et  al . , 2000 , potthoff et  al .
transcription factor families and pgc-1 ( li et  al . , 2002 ) . on the contrary ,
the molecular mechanisms by which muscle fiber diversity is achieved during pre - natal development are still poorly understood .
it was shown that the intrinsic transcriptional properties of embryonic and fetal myogenic progenitors are important to set the fiber type in the absence of nerve activity ( cho et  al . , 1993 , cusella - de angelis et  al . , 1994 ) . moreover , in recent years , several transcription factors contributing to developmental muscle fiber specification have been identified , including sox6 ( hagiwara et  al . , 2007 , an et  al . , 2011 ) , nfix ( messina et  al . , 2010 ) , six1/six4 ( richard et  al . , 2011 ) , and nfatc2 ( daou et  al . , 2013 ) . however , the network of  transcription factors controlling fiber specification during embryogenesis is still far from being fully characterized , and until now , functional interactions among the different regulators were completely unknown . in this work , we provided evidence for functional interplay between nfix and sox6 in controlling expression of the slow myhc isoform during mouse pre - natal muscle development .
in contrast to nfix , which is a specific marker of fetal myogenesis ( messina et  al . , 2010 , mourikis et  al . ,
2012 ) , sox6 is expressed in both embryonic and fetal purified myoblasts at the mrna and protein levels .
consistently , we found that sox6 protein is expressed in skeletal muscle in  vivo starting between e11.5 and e12.5 .
this was unexpected , since sox6 is known to be a repressor of myhc - i , which along with myhc - emb is expressed in all embryonic fibers ( hagiwara et  al .
it is known that sox6 transcript is absent in mouse primary myotome between e9.5 and e10.75 ( vincent et  al . , 2012 ) , suggesting that sox6 is quickly activated in embryonic myoblasts at the beginning of primary myogenesis .
unexpectedly , sox6 deficiency during primary myogenesis leads to a transient faster muscle phenotype with low levels of myhc - i .
this is  followed in sox6-null fetuses by dramatic upregulation of myhc - i at the transcription level , consistently with previous characterizations of the sox6-null phenotype ( hagiwara et  al .
our results demonstrate that sox6 plays opposite roles in myhc - i expression during development .
importantly , in embryonic myogenic cells , sox6 does not  bind to the two canonical myhc - i binding sites , which are both dispensable for sox6-dependent embryonic regulation of myhc - i , at least in our culturing conditions .
we found that sox6 directly binds to the promoter of mef2c and activates its expression in primary myofibers .
mef2c is part of a transcription factor family that is constitutively expressed in muscle cells since early embryogenesis ( edmondson et  al . , 1994 ) and plays an important role in the specification and maintenance of type i fibers ( chin et  al . , 1998 , wu et  al . , 2000 , anderson et  al
importantly , conditional deletion of mef2c in skeletal muscle leads to a drastic reduction in slow - twitch fibers ( potthoff et  al . , 2007 ) .
our rescue experiment , although partial , clearly shows that one of the sox6-dependent effects on myhc - i transcription in embryonic myofibers is mediated by mef2c , which is directly targeted by sox6 in embryonic , but not fetal , muscle .
this function led us to hypothesize that the binding ability of sox6 is differentially regulated in discrete myogenic progenitor populations or at different times during development according to different co - factors , which contribute to the high versatility of sox6 functions .
it is known that soxd factors , lacking trans - acting functional domains , have a critical requirement for co - factors in order to regulate transcription of target genes ( reviewed in  hagiwara , 2011 ) .
therefore , it is likely that the reversal in sox6 function is due to different factors that are progressively recruited and activated during muscle development .
indeed , we found that during fetal myogenesis , nfix acts as a fundamental co - factor of sox6 and is able to form a complex with sox6 , which is no longer able to bind to the mef2c promoter but can bind to myhc - i regulative regions , in particular the proximal promoter that was shown to be critical for sox6-dependent fetal repression of myhc - i ( hagiwara et  al . , 2007 ) .
in our previous study , we showed that nfix negatively regulates myhc - i by repressing nfatc4 , a positive regulator of myhc - i
interestingly , we have now found that during fetal myogenesis , nfix is also present at the myhc - i proximal promoter along with sox6 , suggesting that a physical association between these two proteins may be required for proper myhc - i downregulation . since nfix presents both a trans - repression domain and a  trans - activation domain , we hypothesized that formation of a complex with sox6 might provide the basis for the transcriptional repression at the myhc - i locus ( figure  7 ) .
indeed , nfix and sox6 were found to be part of the same complex in fetal myotubes and are both present at the myhc - i proximal promoter .
moreover , our study on nfix - null and sox6-null fetuses clearly shows that sox6 and nfix are independently expressed during secondary myogenesis and that neither sox6 nor nfix is able to correctly downregulate myhc - i expression when the other one is not present .
a scheme of the scenario in which sox6 and nfix behave and cooperate during pre - natal muscle development is shown in figure  7 . in this work ,
we have also provided evidence for a conserved transcriptional cooperation of sox6 and nfixa in zebrafish .
we have shown that sox6 is crucial for the regulation of smyhc1 , in line with the findings of jackson et  al .
it was shown that sox6 expression is silenced in zebrafish slow fibers by the combined effects of prdm1a and mir-499 ( von hofsten et  al . , 2008 ,
we found that a minority of smyhc - positive cells display cytoplasmic staining for sox6 , suggesting that sox6 might also be regulated through subcellular localization in different fiber types .
notably , our double partial knockdown performed with suboptimal doses of sox6-mo ( von hofsten et  al
. , 2008 ) and nfixa - mo ( pistocchi et  al . , 2013 ) caused a severe impairment in the touch - evoked escape response , suggesting that sox6 and nfixa cooperate in proper muscle function .
molecularly , our data suggest that the two transcription factors can act together to repress smyhc1 expression in zebrafish embryos , whereas the expression of a typical fast - twitch gene , such as myl1 ( burguire et  al . , 2011 ) ,
, we have presented a complex model of regulation of embryonic to fetal myhc - i regulation that involves a functional interplay between nfix and sox6 that is conserved in mammals and teleosts .
the following murine lines were used : myf5 ( kassar - duchossoy et  al . , 2004 ) , nfix null ( campbell et  al . , 2008 ) , sox6 ( smits et  al . , 2001 ) , and tg : mlc1f - nfix2 ( messina et  al . , 2010 ) .
for each of these lines , the genotyping strategy has been described in the references .
mice were kept in pathogen - free and controlled conditions , and all procedures conformed to italian law ( d. lgs n 2014/26 , implementation of the 2010/63/ue ) and were approved by the animal welfare body of the university of milan and the italian minister of health ( 1212/2015pr ) .
the following line was used : ab ( from carole wilson , ucl , london , uk ) . mouse or zebrafish embryos were fixed in 4% paraformaldehyde , extensively washed in pbs , and incubated overnight in pbs containing 15% sucrose .
samples were then frozen in nitrogen - chilled isopentane and kept at 80c until use .
0.2% triton x-100 in pbs for 30  min at room temperature and then incubated for 1  hr in blocking solution ( 10% goat serum in pbs ) and overnight with the primary antibody or with mock pbs .
after incubation for 45  min with the fluorescent - conjugated secondary antibody ( jackson immunoresearch laboratories ) , sections were washed in pbs 0.2% triton x-100 and mounted , and fluorescent immunolabeling was recorded with a dm6000 leica microscope .
the following primary antibodies were used : rabbit anti - sox6 ( abcam , 1:300 ) , rabbit anti - nfix ( novus biological , 1:200 ) , mouse anti - myhc - i ( sigma , 1:200 ) , bad5 ( monoclonal , 1:2 ) , mf20 ( monoclonal , 1:2 ) , and f59 ( monoclonal , 1:10 ) .
nuclei were stained with hoechst ( 1:1,000 ) . dissected myf5 embryonic or fetal muscles were digested by 0.15  mg / ml collagenase ( sigma ) , 1.5  mg / ml dispase ( gibco ) , and 0.1  mg / ml dnase i ( sigma ) for 30  min at 37c in agitation as described in biressi et  al .
after centrifugation and filtration , cells were collected in dmem , 20% fetal bovine serum ( fbs ) , 2  mm edta , and 20  mm hepes . for cell
gfp - positive cells were collected for mrna and protein extraction . for the preparation of unpurified fetal myoblasts after digestion of tissue , cells were pre - plated for 30  min on a plastic dish to lose fibroblasts , which normally adhere to plastic .
unpurified cells were kept in incubation at  37c in 20% hs ( 20% horse serum in dmem ) and 24  hr later allowed to differentiate in dm ( 2% horse serum in dmem ) for 48  hr .
preparation of viral particles were performed by co - transfecting plko.1-shsox6 vectors ( thermo fisher scientific ) or non - targeting shrna vectors ( 30  g ) , together with the packaging plasmids pmdlg / p ( 16.25  g ) , pcmv - vsvg ( 9  g ) , and prsv - rev ( 6.25  g ) , in hek293 t cells . transfection was performed using the calcium phosphate transfection method .
viral  particles were collected 40  hr after transfection and subjected to ultracentrifugation at 20,000  rpm for 2  hr at 20c ( beckman coulter , optima l-100 xp ) .
the concentrated viral particles were re - suspended in pbs and stored in aliquots at 80c until further use .
these preparations were used to transduce embryonic myoblasts at an moi of 10 . the day after transduction
, embryonic myoblasts were transfected with mef2c ( pcdnai / a - mef2c ) or the empty vector as a negative control using lipofectamine ltx ( invitrogen ) .
cultured cells were washed twice in ice - cold pbs and then lysed ( 30  min in ice ) with ripa buffer ( 10  mm tris - hcl [ ph 8.0 ] , 1  mm edta , 1% triton - x , 0.1% sodium deoxycholate , 0.1% sds , and 150  mm nacl in deionized water ) plus protease inhibitors ( 1  mm pmsf ) . for zebrafish ,
only trunk and tail regions were used , cutting out the head , and protein was extracted in laemmli buffer ( 3  l per embryo ) .
protein was harvested after centrifugation , quantified by  absorbance reading at 750  nm , and stored at 80c .
30  g protein was resolved on 8%12% polyacrylamide gels or on miniprotean tdx gels ( bio - rad ) after denaturation at 95c for 5  min with sds - page loading sample buffer 2x ( 100  mm tris [ ph 6.8 ] , 4% sds , 0.2% bromophenol blue , 20% glycerol , and 10  mm dithiothreitol ) . for western blot analysis , proteins were transferred onto nitrocellulose with the iblot dry blotting system ( invitrogen ) . following transfer ,
membranes were blocked in 5% milk in tris - buffered saline ( tbs)-t ( tbs plus 0.02% tween20 ) for 1  hr at room temperature .
the primary antibodies used were rabbit anti - sox6 ( abcam ; 1:1,000 ) , mouse anti - myhc - i ( sigma ; 1:5,000 ) , mouse anti--tubulin ( santa cruz biotechnology ; 1:5,000 ) , rabbit anti - nfix ( novus biologicals ; 1:500 ) , mouse anti - ha ( covance ; 1:500 ) , mouse anti - vinculin ( sigma ; 1:1,000 ) , and mouse anti - smyhc ( f59 , hybridoma bank ) anti - mouse sarcomeric myhc ( mf20 , hybridoma bank ) .
horseradish peroxidase ( hrp)-conjugated antibodies ( bio - rad ) were used as secondary antibodies , and the signal was revealed with the chemidoc mp system ( bio - rad ) . for transfection of fetal myoblasts , wt or sox6-null cells were plated on 90-mm dishes and allowed to reach 80% confluence in proliferating conditions .
cells were transfected with pch - nfix2-ha or pch - ha plasmids with lipofectamine ltx ( invitrogen ) overnight at 37c .
cells were kept in dm for 36  hr after transfection , and then nuclear extracts were prepared by collecting 5   10 cells in 400  l ice - cold hypotonic buffer a ( 10  mm hepes [ ph 7.9 ] , 10  mm kcl , 0.1  mm egta , 1  mm dtt , protease inhibitors , 1  mm pmsf , and phosphatase inhibitors ) for 15  min in ice .
then , np40 was added to a final concentration of 0.625% , and cells were centrifuged at 5,000  rpm for 5  min at 4c .
the supernatant was discarded as cytoplasmatic extract , while the pelleted nuclei were resuspended in 100  l immunoprecipitation lysis buffer containing 50  mm tris - hcl ( ph 7.5 ) , 1% np40 , 1  mm edta , 100  mm nacl , and protease and phosphatase inhibitors .
hr with gentle rotation at room temperature with 5  g rabbit anti - sox6 antibody ( abcam ) , rabbit anti - ha antibody ( santa cruz ) , or non - related rabbit immunoglobulin g ( igg ) ( santa cruz ) .
then , the bead - antibody complex was incubated with gentle rotation for 2  hr at 4c with 1.5  mg total protein lysates per condition .
the eluted proteins were denatured with non - reducing sds - page loading sample buffer ( 100  mm tris [ ph 6.8 ] , 4% sds , 0.2% bromophenol blue , and 20% glycerol ) and loaded onto a gel .
chip for sox6 and luciferase assays were performed as previously published ( an et  al . , 2011 ) .
rna from homogenized mouse or zebrafish embryos and isolated cells was  extracted with nucleospin rna kits ( macherey - nagel ) following the manufacturer s instructions . eluted rna was checked on agarose gels , quantified with  a nanodrop spectrophotomer , and stored at 80c .
approximately 0.5  g rna was used with the improm - ii reverse transcriptase kit ( promega ) .
real - time pcr was performed on cdna using sybr green supermix ( bio - rad ) and the cfx connect real time system ( bio - rad ) .
after amplification , relative mrna expression levels were calculated using standard curves from cdna dilutions and normalized on the gapdh expression levels . for quantitative real - time pcr in zebrafish
the antisense mos ( gene tools ) used in this study , sox6-mo1 ( von hofsten et  al . , 2008 ) and nfixa - mo ( pistocchi et  al . , 2013 ) , were previously described .
mos , diluted in danieau buffer ( nasevicius and ekker , 2000 ) , were injected at the one- to two - cell stage .
escalating doses of each mo were tested for phenotypic effects ; as control for unspecific effects , each experiment was performed in parallel with an std - mo ( standard control oligo ) with no target in zebrafish embryos .
we usually injected 0.25 pmol sox6-mo per embryo . for combined knockdown experiments , we injected sox6-mo and nfixa - mo at 0.1 and 0.25 or 0.08 and 0.125 pmol per embryo , respectively .
statistical significance was assessed by unpaired student s t test with prism 5 software . statistical significance with a probability of less than 5% , 1% , or 0.1%
is indicated in each graph with a single , double , or triple asterisk , respectively , followed by the number of independent experiments ( n ) .
conceptualization , g. maroli , v.t . , and g. messina ; methodology , s.c .
; writing  original draft , g. maroli and v.t . ; writing  review & editing , v.t .
, m.b . , and g. messina ; supervision , m.b . and g. messina ; funding acquisition , g. messina .
 summarysox6 belongs to the sox gene family and plays a pivotal role in fiber type differentiation , suppressing transcription of slow - fiber - specific genes during fetal development . here 
 , we show that sox6 plays opposite roles in myhc - i regulation , acting as a positive and negative regulator of myhc - i expression during  embryonic and fetal myogenesis , respectively . during embryonic myogenesis 
 , sox6 positively regulates myhc - i via transcriptional activation of mef2c , whereas during fetal myogenesis , sox6 requires and  cooperates with the transcription factor nfix in repressing myhc - i expression . 
 mechanistically , nfix is necessary for sox6 binding to the myhc - i promoter and thus for sox6 repressive function , revealing a key role for nfix in driving sox6 activity . 
 this feature is evolutionarily conserved , since the orthologs nfixa and sox6 contribute to repression of the slow - twitch phenotype in zebrafish embryos . 
 these data demonstrate functional cooperation between sox6 and nfix in regulating myhc - i expression during prenatal muscle development . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions