BACKGROUND
the musculature of each animal species is composed of a complex array of body wall muscles that enable precise and stereotypic movements.
muscle formation involves fusion of mononucleated myoblasts.
the coupling of muscle differentiation with multinucleation raises the general question of transcriptional -programming of nuclei within a syncytium.
whereas the transcriptional control of generic aspects of myogenesis has been largely decrypted in vertebrates, elucidating the mechanisms that confer each muscle a specific morphology, i.e.
distinctive shape and orientation, number of nuclei, size, skeleton attachment sites and innervation, remains a major challenge in myology research.
because of its relative simplicity, the somatic musculature of the drosophila larva is a classical model to approach generic and morphology aspects of muscle formation.
every abdominal hemisegment displays approximately  <dig> distinct body wall muscles, each corresponding to a single multinucleated fibre with a unique morphology  <cit> .
each muscle develops by fusion of one founder cell  with a given number of fusion competent myoblasts   <cit> .
fc-fcm fusion is driven by the mutually exclusive expression of ig domain proteins, notably dumbfounded /kirre in fcs and sticks and stones  in fcms .
thus, drosophila muscle development involves the mandatory asymmetric fusion of two cell types that display intrinsically different gene expression programmes.
alongside myoblast fusion, each prospective muscle elongates towards epidermal tendon cells to establish stable junctions with the exoskeleton at specific positions, after which myofibres become contractile  <cit> .
this requires assembly of generic core sarcomeric proteins such as myosin heavy chain   <cit> .
the specific morphology of each muscle reflects the expression of a specific combination of identity transcription factors  such as apterous, even-skipped and slouch/s <dig> by its fc  <cit> .
fcs originate from asymmetric, terminal division of progenitor cells  that are selected from equivalence groups of myoblasts called promuscular clusters , while unselected myoblasts become fcms  <cit> .
specification of the fc itf code integrates positional, temporal and homeotic information as well as extensive cross-regulations between different itf genes at the pc and fc stages  <cit> .
by contrast, all fcms express the lameduck  and tramtrack ttk <dig> transcription factors .
yet, fcms are derived from a pmc that transiently expresses specific itfs  <cit> .
whether this transient itf expression has an impact on fcm fate is a long-standing question.
whereas the fc/fcm fusion process has been decrypted in detail, the fate of syncytial fcm nuclei has not received the same attention.
expression studies on the s <dig> and collier  itfs showed that fcm nuclei activate itf gene transcription after fusion, revealing a conversion of syncytial nuclei towards fc-specific identity  <cit> .
the kinetics and selectivity of this conversion, and whether all nuclei in a syncytium behave in the same manner, remain to be established.
one current hypothesis is that the fc itf code controls fibre-specific expression of ‘realisation’ genes responsible for the stereotyped morphology of each muscle  <cit> .
how the activation of realisation genes and that of generic differentiation genes common to all body wall muscles articulate with each other also remains unresolved.
here, we addressed these different questions by studying the dynamics of transcription of three muscle itf genes   <cit> ), one fcm-generic gene , one fc-generic gene , the generic muscle differentiation gene   <cit> , and four putative realisation genes   <cit> , kon-tiki/perdido   <cit> , m-spondin  and paxillin  ), in a group of dorsolateral muscles.
using fish with intronic probes to detect nascent transcripts, we describe the temporal window of transcription of each gene in individual nuclei within different syncytia.
statistical analyses take into account the variations in signal due to the discontinuous character of transcription, which is comprised of a succession of transcriptional bursts, followed by periods of little or no transcription  <cit> .
we could thus compare the transcriptional status of different genes in individual nuclei, within and between syncytia.
our data show that fcms from different pmcs can be incorporated into a given myofibre and are equally competent to be converted to a specific muscle identity.
transcriptional re-programming of fcm nuclei is sequential, with the loss of fcm-specific transcription preceding activation of fc genes.
fibre-specific expression of realisation genes is only activated in nuclei transcribing itfs, and is regulated at both the level of transcription and the period of transcriptional activity.
finally, we show that the muscle generic and identity transcription programmes are regulated independently.
our results provide a novel view of the dynamics of transcriptional reprogramming of post-mitotic nuclei within a syncytium, and a new framework for understanding the transcriptional control of muscle identity, a fundamental process in the animal kingdom.
RESULTS
fcms are naive myoblasts
one key step in generating the stereotyped drosophila skeletal muscle pattern is the specification of muscle pc and fc identity.
pcs and fcms both derive from equivalent groups of cells, called pmcs   <cit> .
all myoblasts in a given cluster express some itfs in response to positional information.
itf expression is maintained in pcs and some fcs, while lost in fcms   <cit> .
hence, a long-standing question in the field is whether transient activation of specific itf in prospective fcms biases their fate?fig.
<dig> tracking fusion competent myoblasts  derived from the col+ promuscular cluster.
a schematic representation of dorso-lateral muscle formation, with embryonic stages indicated.
the da3/do <dig>  ll1/do <dig> and dt1/do <dig> progenitor cells  are selected from a promuscular cluster expressing col ; unselected myoblasts become fcms .
each pc generates two founder cells  that fuse with fcms to form syncytial fibres, which attach to tendon cells to form contractile muscles.
the ll <dig> and dt <dig> fcs express kr and s <dig>  respectively .
col expression is maintained in the da <dig> muscle .
out-group dorsal da <dig>  lateral lt <dig> and ventral va <dig> muscles are circled in blue, other muscles in black.
b–b’ stage  <dig> col
ecrm
-h2brfp; duf-lacz embryo stained for rfp  to identify the col+ promuscular cluster  nuclei, lacz  and lmd  to visualise all fcs and all fcms, respectively.
b external and  internal layers where fcs and fcms are located, respectively; c–f repartition of rfp+ fcm nuclei at stage  <dig>  box plots indicate the numbers of rfp+ relative to mef2+ nuclei in da <dig>  dt <dig>  lt <dig> and va <dig> ; col
ecrm
-h2brfp; col
lcrm
-moegfp , col
ecrm
-h2brfp; s59-mcd8gfp  and col
ecrm
-h2brfp; kr
gmr80h11
-gal <dig> uas-mcd8gfp  embryos stained for rfp  and gfp  to outline the da <dig> , dt <dig> and va <dig> , and lt <dig> and ll <dig>  contours; mef <dig> staining  visualises all myoblast nuclei.
lateral views of embryos, dorsal up, anterior left; two adjacent abdominal hemisegments in b, b’, three in d-f; scale bar: 20 μm.
in c, bar graphs indicate the mean, and error bars the sd
in order to explore this question, we used as a paradigm the col-expressing pmc from which the da3/do <dig>  dt1/do <dig> and ll1/do <dig> pcs are selected, after which only the da <dig> fc and muscle maintain col expression   <cit> .
in order to specifically label col-expressing pmc myoblasts and follow their fate, we expressed h2b-rfp under the control of the pmc col cis-regulatory module , col
ecrm .
lacz expression under the control of cis-regulatory elements of the fc-specific gene duf  and lmd served to visualise all fcs and fcms, respectively, at stage  <dig>  as expected, h2b-rfp staining was detected in nuclei of fcs and fcms .
due to its stability and nuclear retention, h2b-rfp expression allowed tracking of the repartition of myoblasts derived from the col pmc in developing myofibres up to stage  <dig>  it showed that rfp+ nuclei were mainly incorporated in dorsolateral muscles and occasionally found in more dorsal as well as ventral-lateral muscles.
this repartition suggests that fcm repartition follows neighbouring cues.
we precisely quantified the fraction of rfp+ nuclei within da <dig> and dt <dig>  each of the four lateral transverse  muscles, and the ventral va <dig> muscle .
for this quantification, all myoblast nuclei were visualised by mef <dig> staining .
the da <dig> and dt <dig> plus va <dig> contours were identified by col
lcrm-  and s59-driven gfp expression, respectively , and the contours of lts by mcd8-gfp expression under control of a kr muscle crm , which we identified by systematically testing kr-linked gmr lines .
in either da <dig> or dt <dig> dorsolateral muscles, approximately 50% of nuclei were rfp+ .
a similar proportion was observed in ll <dig>  a third dorsolateral muscle .
in contrast, both lateral lts, or ventral va <dig> muscles contained few rfp+ nuclei , supporting the conclusion that fcm recruitment follows local cues.
recruitment of both rfp+ and rfp– fcms into da <dig> and dt <dig> further shows that fcms are not pre-determined to contribute to a given muscle by early itf expression.
to further strengthen this conclusion, we analysed the repartition of h2b-rfp nuclei in col
<dig> mutant embryos expressing col
ecrm
-h2b-rfp, with col
ecrm activity being independent of col. in col
<dig> embryos, the da <dig> muscle is transformed into a more dorsal, da2-like muscle , while dt <dig> morphology is unchanged  <cit>  .
we found that the average number of rfp+ nuclei incorporated into dt <dig> was unchanged .
however, it was lower in the dorsalised da3 > da <dig> muscle than in da <dig> , while remaining higher than in dorsal da <dig> .
these data show that the recruitment of fcms in distinct muscles during the elongation process follows topological rules.fig.
<dig> promuscular col expression does not impact fusion competent myoblast  fate.
stage  <dig> col
ecrm
-h2b-rfp  and col
1
,col
ecrm
-h2b-rfp  embryos stained for rfp  and β3-tub  to visualise the nuclei of col+ pmc myoblasts and all muscles, respectively;  red channel only;  close up of three abdominal hemisegments.
c number of rfp+ nuclei in da <dig>  da <dig> and dt <dig> at stage  <dig> in col
ecrm
-h2brfp and da <dig>  da3 > da <dig> and dt <dig> in col
1
,col
ecrm
-h2brfp embryos.
the da <dig> transformation into a da2-like muscle  in col
<dig> embryos is schematised on the right.
d fish of nascent col transcripts  in stage  <dig> col
ecrm
-h2brfp embryos stained for rfp  and col ; col is transcribed in a fraction of both rfp+ and rfp– da <dig> nuclei.
scale bar: 20 μm
finally, we investigated whether fused fcms were differentially competent to transcribe fc itf, depending upon their pmc origin by analysing col transcription in da <dig> muscles of col
ecrm
-h2b-rfp embryos, which contain a mixture of rfp+ and rfp– nuclei.
we found that both rfp+ and rfp– nuclei activate col transcription , showing that fcm nuclei can be reprogrammed to a given identity, independent of prior itf expression.
itf gene transcription in syncytial nuclei is both transient and itf-specific
although transcriptional conversion of fcm nuclei to fc identity upon fusion is often presented as a dogma, it has only been documented for col in the da <dig> and s <dig> in the dt <dig>  va <dig> and vt <dig> muscles  <cit> .
to determine the extent and dynamics of transcriptional conversion in different muscles, we reinvestigated the patterns of col and s <dig> transcription, using col- and s59-driven gfp expression to follow muscle development, step by step .
we also investigated the transcription pattern of a third itf gene, kr, in several muscles including dorsolateral ll <dig> .
detection of nascent transcripts was followed by quantification of two parameters, namely the number of hybridisation dots  in the syncytium and the intensity of each transcription dot, as a proxy of the transcription initiation rate.fig.
<dig> dynamics of identity transcription factor transcription during muscle development.
col
lcrm
-moegfp,  s59-mcd8gfp and  kr
gmr80h11
-gal4;uas-mcd8gfp embryos stained for nascent col , s <dig>  or kr  transcripts  and gfp  to visualise muscle shape, and either col , s <dig>  or kr  .
red channel only, with dotted grey lines outlining muscle shape.
kr is transcribed in a single nucleus from stage  <dig> to late stage  <dig>  in all kr-expressing muscles.
schematic view of the col+, s59+ and kr+ muscles colour coded as in fig.
1a.
box plots showing the relative numbers of nuclei and col transcription dots in da <dig> , s <dig> dots in dt <dig> and va <dig> , and kr dots in ll <dig> and va <dig>
simultaneous staining for col, nascent col transcripts and col
lcrm
-moegfp confirmed col active transcription in the da <dig> fc and several syncytial nuclei within growing da <dig> fibres , and stop of transcription at stage  <dig>  quantification of the nuclei number transcribing col, relative to the total number of da <dig> syncytial nuclei, shows, however, that only a fraction transcribe col at a given time , suggesting transient transcription in each nucleus.
the observation of growing da <dig> fibres, in which col is transcribed in virtually all nuclei  shows that all fused fcm nuclei are competent to be converted to the fc identity transcriptional programme.
the variable numbers of transcriptionally active nuclei in adjacent hemisegments  indicate that activation of col transcription in syncytial nuclei is stochastic rather than synchronous.
quantification of the intensity of col transcription dots shows that col is transcribed at a 9-fold higher level in the fc than in syncytial da <dig> nuclei at stage  <dig> .
thus, two different modes of col regulation operate successively.
this correlates with the previous identification of two distinct col mesodermal crms that act sequentially  <cit> .
as for col in da <dig>  only a fraction of nuclei within dt <dig>  va <dig> or vt <dig> syncytia transcribe s <dig> at a given time .
interestingly, this fraction differs between muscles, at approximately 45%, 27% and 23%, in dt <dig>  va <dig> and vt <dig>  respectively, at stage  <dig> , while the integrated density of s <dig> dots is relatively constant from stage  <dig> to  <dig> .
together, these data suggest that the time interval of s <dig> transcription is differentially regulated in dt <dig>  va <dig> and vt <dig>  s <dig> is also expressed in lo <dig> at stage  <dig>  but in this lineage, s <dig> is only transcribed in the fc at stage  <dig> and not at later stages   <cit> .
finally, fish of nascent transcripts shows that kr transcription in kr+ muscles ceases at early stage  <dig> .
unexpectedly, kr transcription is detected at a constant level in a single nucleus per fibre, from stage  <dig> to  <dig> .
the kr
+ nucleus stays at roughly the same position within the muscle throughout the process of fibre growth, suggesting that it is the fc nucleus and that kr transcription is maintained in the fc nucleus during the fusion process without being propagated to other syncytial nuclei.
up to now, two ‘typical’ itf gene transcription patterns have been reported, namely repression at the pc or fc stage  or, alternatively, maintenance and propagation of transcription to other syncytial nuclei .
kr transcription in a single nucleus up to stage  <dig> reveals a novel pattern of itf transcriptional regulation in muscle precursors, namely maintenance in the fc nucleus without propagation to fcm nuclei.
together, the observed patterns of nascent col, kr and s <dig> transcripts show that activation of itf transcription in fcms after fusion is itf- and muscle-specific, and cannot be considered as a general rule.
furthermore, single nucleus kr transcription provides the first evidence that the fc nucleus could maintain a transcriptional programme different from the other syncytial nuclei during muscle development.
transcriptional conversion of fcms to syncytial identity
fusion of fcms with an fc and, subsequently, the derived growing fibre, is based on mutually exclusive expression of the surface proteins duf/rst and sns by all fcs and fcms, respectively  <cit> .
for this to be a reiterative process implies that, upon fusion, fcm nuclei switch off fcm-specific gene transcription  and that the fc-generic duf gene remains expressed.
this process remains to be decrypted at the transcriptional level.
we sought to determine precisely when the fcm to fc/fibre transcription switch occurs, using da <dig> as a paradigm.
fish to duf nascent transcripts confirmed that duf is transcribed in all fcs at stage  <dig>  including the da <dig>  dt <dig> and ll <dig> fcs labelled with col   <cit> .
at stage  <dig>  the increased number of duf transcribing nuclei in da <dig> syncytia correlates with the increased number of recruited fcm nuclei , demonstrating activation of the fc-generic transcriptional programme in fcm nuclei.
duf transcription drops at late stage  <dig>  at the end of the fusion process  <cit>  .
paired fish of nascent duf and col transcripts shows that the da <dig> fc co-transcribes duf and col at stage  <dig> .
however, at stage  <dig>  while 80% of syncytial nuclei transcribe at least one of these two genes, only 50% of these nuclei co-transcribe duf and col , possibly reflecting asynchronous bursts of col and duf transcription.
their dynamics of transcription show that duf and col activation is not synchronised and independent of each other in individual nuclei within a syncytium.
we previously showed that nuclear uptake of col protein precedes activation of col transcription in fused fcm nuclei  <cit> .
the detection of duf dots in nuclei with very low levels of col protein  further supports independent activation of duf and col.fig.
<dig> transcriptional conversion of fused fusion competent myoblast  nuclei to founder cell  identity.
fish against nascent duf  or sns  transcripts , staining for mef <dig> , and col  to visualise the da <dig>  dt <dig> and ll <dig> fc nuclei at stage  <dig>  and the da <dig> muscle at stage  <dig> , wt embryos.
double fish against nascent col  and either duf  or sns  transcripts  and col staining  of the da <dig> fc at stage  <dig>  and da <dig> muscle at stage  <dig> .
double fish against nascent duf  and sns transcripts  and col staining, stage  <dig> da <dig> muscle;  blue channel only.
sns transcription  is detected in "no col" nuclei.
box plots showing the number of nuclei transcribing either col or duf, or col and duf, relative to the total number of da <dig> nuclei , and either col or sns only .
two adjacent hemisegments are shown in , section projections; a single hemisegment in , single confocal sections.
l schematic representation of the transcriptional fcm to generic fc identity switch, post fusion
conversely, nascent sns transcripts are detected in a large number of fcms surroundings fcs at stage  <dig>  but not in fcs themselves .
during the fusion period, only one, if any, sns transcription dot is detected in growing da <dig> myotubes , indicating that switching off the fcm transcriptional programme occurs post fusion.
furthermore, no nuclei co-transcribing sns and col , or sns and duf  were detected, indicating that repression of fcm-specific genes precedes activation of fc-generic and itf transcription.
in support of this, sns dots could only be detected in nuclei displaying no or very low levels of col protein .
in summary , the duf, sns and col transcription patterns show that transcriptional reprogramming of fcms is a dynamic process: after fusion of fcms, transcription of fcm-generic genes  is first turned off, followed by a switch from fcm to fc-generic  and fc-specific  transcriptional programmes.
these latter two aspects of transcriptional activation are asynchronous.
transcription of generic muscle differentiation genes is independent of itf gene transcription
assembly of sarcomeres underlies the formation of contractile muscles.
consistent with mhc being a critical sarcomeric component, mhc mrna level is similar in all muscle fibres at stage  <dig> .
fish of nascent transcripts indicated that mhc transcription is first weakly detected in few nuclei at stage  <dig>  and increases dramatically at stage  <dig>  simultaneously in all developing muscles .
the homogenous distribution of mhc dots suggests that transcription of generic muscle differentiation is independent of muscle identity.
supporting this conclusion, high-level mhc transcription is maintained in muscle syncytia until the end of embryogenesis, when itf transcription has already strongly decreased .
to further assess whether the generic differentiation and muscle identity transcription programmes are coupled, we compared the dynamics of col and mhc transcription in the developing da <dig> muscle.
paired fish of nascent transcripts, coupled to col and dapi staining , confirmed that virtually all da <dig> nuclei transcribe mhc at stage  <dig>  while only roughly a quarter also transcribes col at a given time point .
together, these data support the conclusions that the generic muscle differentiation and identity programmes are uncoupled temporally and independently activated in syncytial nuclei .fig.
<dig> independent transcription of identity transcription factors and generic muscle differentiation genes.
a–e wt embryos at various embryonic stages, stained for mef <dig>  and col  coupled to fish against nascent mhc transcripts.
the white arrow points to mhc expression in heart cells, first detected at stage  <dig>  f double fish against nascent col  and mhc  transcripts, and col staining  to visualise the da <dig> nuclei at stage  <dig>  f’ same as , dapi staining  shows all nuclei.
f” col staining only.
single z sections.
the yellow arrow indicates a nucleus with low col protein, which transcribes mhc.
g
box plots showing the number of nuclei transcribing either col, mhc or both, relative to the total number of da <dig> nuclei
fibre-specific transcription of identity realisation genes; multi-level regulation
the current hypothesis is that the itf code specific to each fc controls the differential expression of genes underlying ‘realisation’ of muscle morphological identity.
a search for candidate realisation genes identified two cytoskeletal protein genes, pax and mspo, which display fibre-specific expression levels and are involved in fibre-specific modulation of the fusion rate  <cit> .
two other cell surface proteins, con and kon, were independently implicated in fibre-specific regulation of muscle attachment.
kon encodes a transmembrane protein required in a specific subset of myotubes to recognise and establish stable connections with appropriate tendon cells  <cit> .
kon was also recovered in a screen for fc-specific genes based on transcriptomics and computational analyses under the name of perdido  <cit> .
con encodes a cell surface protein decorating a subset of muscles, including dt <dig>  and the motoneurons which innervate them  <cit> .
we compared the transcription patterns of pax, mspo, kon and con in da <dig> and dt <dig>  which present the advantage of displaying similar numbers of nuclei and fcm recruitment kinetics .
fish of mature transcripts at stage  <dig> showed that all four genes are expressed in both muscles.
yet, while pax is expressed at similar levels in da <dig> and dt <dig>  mspo, kon and con mrnas accumulate at significantly higher level in dt <dig> .
fish of nascent transcripts in stage  <dig> embryos further suggested that differential accumulation of mspo, kon and con mrna reflects differential transcription .
we therefore performed a side by side comparison of the dynamics of transcription of pax, mspo, kon and con between da <dig> and dt <dig>  by both measuring the numbers of hybridisation dots and individual dot intensities for each gene in syncytia, at embryonic stages  <dig>   <dig>   <dig>   <dig> and  <dig>  using duf as internal reference .fig.
<dig> transcription dynamics of identity-realisation genes.
a, e, i, m, q stage  <dig> col
lcrm
-moegfp; s59-mcd8gfp embryos stained for gfp  and topro  to visualise da <dig> and dt <dig> and all nuclei, respectively, coupled with fish of either nascent duf , pax , kon , mspo  or con  transcripts ; a’, e’, i’, m’, q’ red channel only, muscle contours are outlined by dashed grey lines.
b, f, j, n, r number of transcription dots in da <dig> and dt <dig> at different embryonic stages.
bar graphs indicate the mean number of nuclei; error bars, the sd.
c, g, k, o, s intensity  of transcription dots in da <dig> and dt1; each dot is represented by a circle, the bar graphs show the mean value and sd.
d, h, l, p, t cumulative intensity of transcriptional dots  in da <dig> and dt <dig>  the mean value is shown; error bars correspond to the sem
as reported above, the number of duf dots increases between stages  <dig> and  <dig>  in both da <dig> and dt <dig>  and dot intensity is maximal during the muscle elongation phase, stages  <dig> to  <dig>  both in da <dig> and dt <dig> .
the numbers and intensity of pax transcription dots are also similar in da <dig> and dt <dig>  during the fusion phase .
a maximum of  <dig>  dots per fibre at stage  <dig> suggests that transcription is stochastic and/or transient in syncytial nuclei.
the absence of dot detection in fcs at stage  <dig> indicates that pax transcription is activated post fusion.
contrary to pax, kon transcription is detected in the da <dig> and dt <dig> fcs, and the kinetics of kon transcription differs between da <dig> and dt <dig>  the number of kon dots increases between stages  <dig> and  <dig> in dt <dig>  reaching an average of  <dig>  per fibre, while it decreases earlier in da <dig> .
nevertheless, dot intensity indicates that the kon transcription initiation rate is not significantly different between the two muscles .
together, these data indicate that differential transcription of kon between da <dig> and dt <dig> reflects different time intervals of transcription per nucleus.
however, a third scenario is observed for mspo, with higher dot number and intensity in dt <dig> than in da <dig> .
thus, both differences in the time of transcription and transcription initiation rate can contribute to differential mspo transcription in different muscles during the acute fusion phase.
this pattern is even more pronounced for con, where high numbers of high intensity dots are detected in dt <dig>  correlating with con accumulation at the surface of the dt <dig> muscle  <cit> , and rare, low intensity dots are detected in da <dig> .
summary curves corresponding to the mean sum of all dot intensities measured in each fibre underline the differential transcription of kon, mspo and con between da <dig> and dt <dig> .
furthermore, they show that the transcription level of realisation genes is regulated in a fibre-specific manner, via the modulation of the transcription initiation rate and/or time interval of active transcription per syncytial nucleus.
transcriptional activation of realisation genes follows itf gene activation
to determine whether transcription of identity realisation genes is connected to itf activation, we focused on mspo, kon and con, which are differentially expressed in da <dig> versus dt <dig>  double fish of col and either mspo, kon or con nascent transcripts allowed us to measure the number of nuclei, either transcribing one or both genes, in developing da <dig> .
the numbers show that mspo or kon are only transcribed in nuclei also transcribing col. thus, contrary to the asynchronous transcription of itfs with respect to the fc-generic gene duf , transcription of realisation genes is linked to itf transcription in syncytial nuclei.fig.
<dig> transcription of identity-realisation genes is coupled to identity transcription factor transcription.
double fish of nascent col  and either mspo , kon  or con   transcripts, col staining for  visualising the da <dig> nuclei at stage  <dig>  a’, c’, e’ dapi staining  of all nuclei; single z sections.
box plots showing the number of nuclei transcribing either col, mspo or both , and either col, kon or both , relative to the total number of da <dig> nuclei
the fc nucleus remains distinct from the other syncytial nuclei
the variable numbers of syncytial nuclei transcribing different realisation genes raised the additional question of whether functional specialisation of nuclei could exist, according to their position within the syncytial myotube.
at late stage  <dig>  the da <dig> muscle displays an angled shape, reflecting transient tripartite attachment to tendon cells ; the final acute orientation of da <dig> corresponds to stabilisation of the ventral-most anterior, and the dorsal-posterior attachment sites, which critically depends upon appropriate levels of col and nau activity  <cit> .
the da <dig> branch of the intrasegmental nerve contacts the ventral side of the angled da <dig> muscle at a stereotypic position, roughly at its centre  <cit>  .
the position of the neuromuscular junction and the tripartite attachment sites provide morphological landmarks allowing the division of the da <dig> muscle into three subdomains, namely antero-ventral, central and postero-dorsal .
we analysed the topological distributions of nuclei and transcription dots for muscle generic , fc-generic  and identity genes , relative to these three subdomains.
plotting the spatial coordinates of each fish dot and nucleus, relative to the da <dig> shape , showed a roughly homogenous distribution of nuclei and col, duf, kon, mhc, mspo and pax transcriptional dots .
thus, transcriptional activation of these genes does not appear to be influenced by nucleus position.
this could be required for homogeneous distribution of transcripts coding for surface proteins during muscle growth.fig.
<dig> transcription patterns and da <dig> muscle subdomains.
a, b late stage  <dig> col
lcrm
-moegfp embryo stained for gfp  and either col , coupled with fish of nascent col transcripts  , or lacz and fasciclin ii  to visualise tendon cells and the da <dig> innervating motoneuron, respectively.
c subdivision of the da <dig> muscle into ventral, median and dorsal subdomains shows an homogeneous distribution of nuclei; brant and brpost indicate the anterior and posterior da <dig> limits, respectively .
spatial distribution of col , mhc , duf , kon , mspo  and con  transcription dots.
j repartition of hybridisation dots and nuclei  in each da <dig> subdomain
most con transcription dots  map to the central subdomain of the da <dig> muscle.
interestingly, con is the only realisation gene in our study which is, on average, transcribed in a single nucleus throughout da <dig> development, suggesting that this is the fc nucleus, in agreement with the observation that con is expressed in fcs  <cit> .
taken together, single nucleus-restricted kr  and con transcription support the conclusion that the fc nucleus remains located at a stereotypical position and is transcriptionally different from the other syncytial nuclei throughout muscle development.
discussion
in its simplest terms, myogenesis is the differentiation of individual precursor cells, the myoblasts, into syncytial contractile myofibres.
yet, each body wall muscle displays a morphological identity.
understanding how transcriptional regulation of a generic myogenic programme, common to all muscles, and each muscle identity programme are integrated to generate morphological muscle diversity remains a central question in the field.
here, we used drosophila larval muscles to address this question, exploiting hybridisation to nascent transcripts to compare the transcriptional status of a selection of generic and identity genes, in individual nuclei within different syncytial muscles.
our data show that transcriptional reprogramming of fused myoblast nuclei is progressive.
transcriptional activation of identity realisation genes is specific to nuclei expressing itf genes and subject to different modes of regulation in a fibre-specific manner.
conversely, transcription of generic muscle differentiation genes is regulated independently of muscle morphological identity.
in humans, in addition to muscles, there exist two other types of differentiating multinucleated syncytia, the osteoclasts and syncytial trophoblasts.
the transcriptional status of each nucleus in the different types of multinucleated cells and how it contributes to lineage diversity remains a widely open, fundamental field of investigation.
muscle ‘founder’ and naive myoblasts
the development of drosophila larval muscles involves mandatory asymmetric fusion of two types of cells, fcs and fcms, both of which are specified from equivalence groups of myoblasts expressing different itfs, in response to positional cues.
itf expression then becomes restricted to pcs and some fcs   <cit> .
itf activation in equivalence groups raises the legitimate question of whether this influences fcm fate.
to address this question, we exploited the dynamics of col transcription, which is activated in one pmc, maintained in one fc and re-activated in fcm nuclei incorporated into the growing da <dig> myotube  <cit> .
our data showing that fcms recruited in a given myofibre can originate from different equivalence groups and are equally competent to be converted to a specific fc identity, indicate that fused fcms behave as naive myoblasts.
this naive character of fcms finds parallels with the uncommitted character of mammalian satellite cells.
mouse satellite cells specified in different anatomical locations have been shown to contribute to new fibres in a heterotopic location, though retaining distinct molecular signatures reflecting, in part, their developmental history  <cit> .
similarly, grafting of human myogenic progenitors into the dystrophic pharyngeal muscles of oculopharyngeal muscular dystrophy patients shows that ectopic myoblasts isolated from clinically unaffected limbic muscles are able to restore contractility to defective pharyngeal muscles  <cit> .
thus, drosophila fcms and mammalian satellite cells are able to contribute to different muscle fibres, irrespective of their early molecular signature.
in both cases, the syncytial cell is able to reprogramme fusing cells and retain its identity.
transcriptional reprogramming of naive myoblast nuclei, post fusion
previous analyses, performed at the protein expression level, led to the conclusion that muscle lineage-specific itf expression, which determines muscle identity, reflects cross-regulations between itfs at the pc and fc stages  <cit> .
here, the patterns of nascent col, s <dig> and kr transcripts show that propagation of transcriptional identity from the fc to recruited fcm nuclei depends upon the itf and/or muscle lineage, and that there is not one pattern to explain this process.
in the case of col in the da <dig> muscle or tailup/islet <dig> in dorsal muscles, activation of transcription relies upon one crm, while propagation to syncytial nuclei involves another, ‘late’ crm mediating direct autoregulation by the imported itf  <cit> .
in the case of kr, a single muscle crm has been identified.
this fits with the restriction of kr transcription to the fc nucleus and arrest earlier than observed for itf genes displaying auto-regulatory crms.
future characterisation of multiple itf crms differentially active in different muscle lineages should provide a general picture of how pc/fc identity is maintained and/or propagated during muscle development, leading to a robust, stereotyped muscle pattern.
fused fcm nuclei stop transcribing fcm-specific genes, such as sns, before activating generic fc genes such as duf .
one mechanism involved in this transcriptional switch is the active degradation of lmd, present in the fusing fcms, by the ubiqutitin ligase mib <dig>  <cit> .
whether duf activation also requires degradation of ttk <dig>  a repressor of fc fate  <cit> , and/or import of generic fc activator tf, similar to import of specific itfs, remain open questions.
in addition, a potential epigenetic control of nuclei reprogramming in multinucleated cells has been scarcely addressed thus far.
mutants for drosophila sin3a, a chromatin regulator conserved from yeast to humans that serves as a scaffold protein for the sin3/histone deacetylase complex, show muscle identity phenotypes  <cit> , suggesting that sin3a could sensitise certain fcs and muscles to itf activity.
patterns of epigenetic histone markers were found to be identical in all nuclei of mouse multinucleated osteoclasts, whereas some osteoclast-specific genes were only transcribed in a subset of nuclei  <cit> .
further investigation on different types of multinucleated cells is certainly needed for mechanical understanding of the reprogramming of syncytial nuclei, post fusion.fig.
<dig> summary model for transcriptional programming of individual syncytial nuclei in a developing muscle.
before fusion, stage  <dig>  all founder cells  transcribe generic fc genes , and fusion competent myoblasts  transcribe fcm-specific genes .
each fc expresses specific identity transcription factors  and transcribes itf genes .
in a growing muscle syncytium, stage  <dig>  fcm gene transcription is transitorily maintained in newly fused fcm nuclei, before complete switch off and progressive activation of fc-generic transcription.
nuclear uptake of itfs by fused fcms is followed by itf activation.
transcription of identity-realisation genes  is linked to itf activation, while transcription of general muscle differentiation genes , is activated and maintained in all myoblast nuclei, independently of itf expression.
transcription of a subset of genes is restricted to the fc nucleus
transcriptional regulation of muscle-generic and identity genes: two independent programmes
gene expression studies, using either mammalian c2c <dig> cells in culture or in vivo models of muscle regeneration, have shown that abrupt, orderly transcriptional changes occur during myogenesis  <cit> .
however, global transcriptome analysis of cell pools produces a population average that does not distinguish between transcriptional and post-transcriptional regulatory levels.
analyses of muscle cells isolated from drosophila embryos at different time points showed that recruitment of poised rna polymerase ii, which makes genes permissive for future transcription, occurs at many genes de novo, in a stage-specific way  <cit> .
in parallel, systematic genomic-wide analyses of transcription factor binding profiles and chromatin marks has identified temporal signatures of enhancer activity in the drosophila mesoderm  <cit> .
while revealing the importance of timing of enhancer/promoter activity during mesoderm development, these studies did not address the question of the dynamics of gene transcription in individual syncytial nuclei.
our analysis of nascent transcripts in individual nuclei in defined muscles has freed us from post-transcriptional regulation levels that contribute to differential mrna accumulation, as documented for the regulation of mhc by hoi polloi  <cit> .
moreover, our approach allowed the comparison of the transcriptional time interval of a general muscle differentiation gene, mhc, with itfs and identity-realisation genes in individual syncytial nuclei.
in this manner, we observed a mixture of on/off nuclei for itfs and realisation genes, whose relative proportions varied between muscle lineages, suggesting a stochastic mode of activation.
inversely, we found that mhc transcription is synchronously activated in all myoblast nuclei independent of muscle identity, correlating with the previous finding that mhc is expressed in all myoblasts, independent of fusion  <cit> .
we conclude that the generic muscle differentiation and morphological identity programmes are regulated independently of each other.
transcriptional control of the generic aspects of skeletal muscle development in vertebrates involves several transcription factors that act as muscle itfs in drosophila, including nautilus/myod, eya, six and col/ebf proteins .
our data in drosophila plead for investigating whether muscle identity and generic differentiation programmes also run in parallel in vertebrates.
transcription of identity realisation genes is linked to itf transcription
how realisation of muscle identity is integrated with generic muscle differentiation is a major question in muscle biology.
here, we compared the transcriptional dynamics of candidate morphological realisation genes in syncytial nuclei between different muscles  <cit> .
our data show that the differential expression of genes encoding surface protein, such as con, kon and mspo, between the da <dig> and dt <dig> muscles is controlled at the transcriptional level, via both regulation of the time interval of active transcription and the transcription initiation rate.
moreover, transcription of these genes in the da <dig> muscle is restricted to nuclei transcribing col, i.e.
reprogrammed to da <dig> identity by imported col protein .
however, not every col
+ nucleus transcribes realisation genes, suggesting either one of three not mutually exclusive possibilities, namely  higher threshold levels of imported col protein are required for activation of realisation genes than for col autoregulation, thus introducing a temporal shift;  the burst frequency of realisation gene transcription is significantly lower than that of col;  other transcription factors, themselves regulated by col, are involved in activation of realisation genes, as documented in the case of col and apterous-expressing neurons .
finally, whether local col mrna translation around col-transcribing nuclei could amplify variations of gene transcription among syncytial nuclei is an interesting question.
we also found that transcription of realisation genes is generally independent of nucleus position within the fibre.
one exception is the con gene, which is transcribed in a single nucleus at a central position that we surmise is the fc nucleus.
the restriction of con transcription to the fc in da <dig> contrasts with its propagation to other syncytial nuclei in dt <dig>  thus, syncytial activation is an active process regulated in a fibre-specific manner.
together with the restricted pattern of kr transcription, da <dig> con transcription also suggests that fc nuclei can keep a transcription programme distinct from the other syncytial muscles throughout muscle development.
whether this could provide molecular cues for positioning of the neuromuscular synapse remains an open question.
the correlation in the severity of da <dig> morphology defects with col mutations of increasing strength, or mutations in other itfs which affect col transcription  <cit> , suggests that proper activation of realisation genes is dependent upon the appropriate levels of itf activity.
our present finding that transcription of realisation genes is linked to itf transcription strongly supports this conclusion.
con, kon and mspo were identified as putative direct col targets by genome-wide chip-seq experiments  <cit> .
this, and other genome wide data  <cit> , suggest that direct binding of different itf combinations is responsible for the differential transcription of identity realisation genes in different muscles.
establishing whether specific crms integrate multiple itf inputs, or multiple crms respond to individual itfs, will be the next step towards deciphering the regulatory logic that confers each muscle its specific morphology.
CONCLUSIONS
in conclusion, our data show that post-fusion reprogramming of naive myoblast nuclei is progressive, such that nuclei within one syncytial fibre are heterogeneous with regards to gene-specific transcription at a given time.
while reprogramming seems independent of nuclear position, preliminary evidence indicates that fc nuclei can remain transcriptionally different from other nuclei throughout muscle development .
this novel view of the dynamics of transcriptional programming of post-mitotic nuclei within syncytial cells provides a new framework for the investigation of transcriptional control of lineage diversity of multinucleated cells.

 the musculature of each animal species is composed of a complex array of body wall muscles that enable precise and stereotypic movements. muscle formation involves fusion of mononucleated myoblasts. the coupling of muscle differentiation with multinucleation raises the general question of transcriptional -programming of nuclei within a syncytium. whereas the transcriptional control of generic aspects of myogenesis has been largely decrypted in vertebrates, elucidating the mechanisms that confer each muscle a specific morphology, i.e. distinctive shape and orientation, number of nuclei, size, skeleton attachment sites and innervation, remains a major challenge in myology research. because of its relative simplicity, the somatic musculature of the drosophila larva is a classical model to approach generic and morphology aspects of muscle formation. every abdominal hemisegment displays approximately distinct body wall muscles, each corresponding to a single multinucleated fibre with a unique morphology. each muscle develops by fusion of one founder cell with a given number of fusion competent myoblasts. fc-fcm fusion is driven by the mutually exclusive expression of ig domain proteins, notably dumbfounded /kirre in fcs and sticks and stones in fcms. thus, drosophila muscle development involves the mandatory asymmetric fusion of two cell types that display intrinsically different gene expression programmes. alongside myoblast fusion, each prospective muscle elongates towards epidermal tendon cells to establish stable junctions with the exoskeleton at specific positions, after which myofibres become contractile. this requires assembly of generic core sarcomeric proteins such as myosin heavy chain. the specific morphology of each muscle reflects the expression of a specific combination of identity transcription factors such as apterous, even-skipped and slouch/s by its fc. fcs originate from asymmetric, terminal division of progenitor cells that are selected from equivalence groups of myoblasts called promuscular clusters, while unselected myoblasts become fcms. specification of the fc itf code integrates positional, temporal and homeotic information as well as extensive cross-regulations between different itf genes at the pc and fc stages. by contrast, all fcms express the lameduck and tramtrack ttk transcription factors. yet, fcms are derived from a pmc that transiently expresses specific itfs. whether this transient itf expression has an impact on fcm fate is a long-standing question. whereas the fc/fcm fusion process has been decrypted in detail, the fate of syncytial fcm nuclei has not received the same attention. expression studies on the s and collier itfs showed that fcm nuclei activate itf gene transcription after fusion, revealing a conversion of syncytial nuclei towards fc-specific identity. the kinetics and selectivity of this conversion, and whether all nuclei in a syncytium behave in the same manner, remain to be established. one current hypothesis is that the fc itf code controls fibre-specific expression of ‘realisation’ genes responsible for the stereotyped morphology of each muscle. how the activation of realisation genes and that of generic differentiation genes common to all body wall muscles articulate with each other also remains unresolved. here, we addressed these different questions by studying the dynamics of transcription of three muscle itf genes ), one fcm-generic gene, one fc-generic gene, the generic muscle differentiation gene, and four putative realisation genes, kon-tiki/perdido, m-spondin and paxillin ), in a group of dorsolateral muscles. using fish with intronic probes to detect nascent transcripts, we describe the temporal window of transcription of each gene in individual nuclei within different syncytia. statistical analyses take into account the variations in signal due to the discontinuous character of transcription, which is comprised of a succession of transcriptional bursts, followed by periods of little or no transcription. we could thus compare the transcriptional status of different genes in individual nuclei, within and between syncytia. our data show that fcms from different pmcs can be incorporated into a given myofibre and are equally competent to be converted to a specific muscle identity. transcriptional re-programming of fcm nuclei is sequential, with the loss of fcm-specific transcription preceding activation of fc genes. fibre-specific expression of realisation genes is only activated in nuclei transcribing itfs, and is regulated at both the level of transcription and the period of transcriptional activity. finally, we show that the muscle generic and identity transcription programmes are regulated independently. our results provide a novel view of the dynamics of transcriptional reprogramming of post-mitotic nuclei within a syncytium, and a new framework for understanding the transcriptional control of muscle identity, a fundamental process in the animal kingdom.  fcms are naive myoblasts one key step in generating the stereotyped drosophila skeletal muscle pattern is the specification of muscle pc and fc identity. pcs and fcms both derive from equivalent groups of cells, called pmcs. all myoblasts in a given cluster express some itfs in response to positional information. itf expression is maintained in pcs and some fcs, while lost in fcms. hence, a long-standing question in the field is whether transient activation of specific itf in prospective fcms biases their fate?fig. tracking fusion competent myoblasts derived from the col+ promuscular cluster. a schematic representation of dorso-lateral muscle formation, with embryonic stages indicated. the da3/do ll1/do and dt1/do progenitor cells are selected from a promuscular cluster expressing col; unselected myoblasts become fcms. each pc generates two founder cells that fuse with fcms to form syncytial fibres, which attach to tendon cells to form contractile muscles. the ll and dt fcs express kr and s respectively. col expression is maintained in the da muscle. out-group dorsal da lateral lt and ventral va muscles are circled in blue, other muscles in black. b–b’ stage col ecrm -h2brfp; duf-lacz embryo stained for rfp to identify the col+ promuscular cluster nuclei, lacz and lmd to visualise all fcs and all fcms, respectively. b external and internal layers where fcs and fcms are located, respectively; c–f repartition of rfp+ fcm nuclei at stage box plots indicate the numbers of rfp+ relative to mef2+ nuclei in da dt lt and va; col ecrm -h2brfp; col lcrm -moegfp, col ecrm -h2brfp; s59-mcd8gfp and col ecrm -h2brfp; kr gmr80h11 -gal uas-mcd8gfp embryos stained for rfp and gfp to outline the da, dt and va, and lt and ll contours; mef staining visualises all myoblast nuclei. lateral views of embryos, dorsal up, anterior left; two adjacent abdominal hemisegments in b, b’, three in d-f; scale bar: 20 μm. in c, bar graphs indicate the mean, and error bars the sd in order to explore this question, we used as a paradigm the col-expressing pmc from which the da3/do dt1/do and ll1/do pcs are selected, after which only the da fc and muscle maintain col expression. in order to specifically label col-expressing pmc myoblasts and follow their fate, we expressed h2b-rfp under the control of the pmc col cis-regulatory module, col ecrm. lacz expression under the control of cis-regulatory elements of the fc-specific gene duf and lmd served to visualise all fcs and fcms, respectively, at stage as expected, h2b-rfp staining was detected in nuclei of fcs and fcms. due to its stability and nuclear retention, h2b-rfp expression allowed tracking of the repartition of myoblasts derived from the col pmc in developing myofibres up to stage it showed that rfp+ nuclei were mainly incorporated in dorsolateral muscles and occasionally found in more dorsal as well as ventral-lateral muscles. this repartition suggests that fcm repartition follows neighbouring cues. we precisely quantified the fraction of rfp+ nuclei within da and dt each of the four lateral transverse muscles, and the ventral va muscle. for this quantification, all myoblast nuclei were visualised by mef staining. the da and dt plus va contours were identified by col lcrm- and s59-driven gfp expression, respectively, and the contours of lts by mcd8-gfp expression under control of a kr muscle crm, which we identified by systematically testing kr-linked gmr lines. in either da or dt dorsolateral muscles, approximately 50% of nuclei were rfp+. a similar proportion was observed in ll a third dorsolateral muscle. in contrast, both lateral lts, or ventral va muscles contained few rfp+ nuclei, supporting the conclusion that fcm recruitment follows local cues. recruitment of both rfp+ and rfp– fcms into da and dt further shows that fcms are not pre-determined to contribute to a given muscle by early itf expression. to further strengthen this conclusion, we analysed the repartition of h2b-rfp nuclei in col mutant embryos expressing col ecrm -h2b-rfp, with col ecrm activity being independent of col. in col embryos, the da muscle is transformed into a more dorsal, da2-like muscle, while dt morphology is unchanged. we found that the average number of rfp+ nuclei incorporated into dt was unchanged. however, it was lower in the dorsalised da3 > da muscle than in da, while remaining higher than in dorsal da. these data show that the recruitment of fcms in distinct muscles during the elongation process follows topological rules.fig. promuscular col expression does not impact fusion competent myoblast fate. stage col ecrm -h2b-rfp and col 1,col ecrm -h2b-rfp embryos stained for rfp and β3-tub to visualise the nuclei of col+ pmc myoblasts and all muscles, respectively; red channel only; close up of three abdominal hemisegments. c number of rfp+ nuclei in da da and dt at stage in col ecrm -h2brfp and da da3 > da and dt in col 1,col ecrm -h2brfp embryos. the da transformation into a da2-like muscle in col embryos is schematised on the right. d fish of nascent col transcripts in stage col ecrm -h2brfp embryos stained for rfp and col; col is transcribed in a fraction of both rfp+ and rfp– da nuclei. scale bar: 20 μm finally, we investigated whether fused fcms were differentially competent to transcribe fc itf, depending upon their pmc origin by analysing col transcription in da muscles of col ecrm -h2b-rfp embryos, which contain a mixture of rfp+ and rfp– nuclei. we found that both rfp+ and rfp– nuclei activate col transcription, showing that fcm nuclei can be reprogrammed to a given identity, independent of prior itf expression. itf gene transcription in syncytial nuclei is both transient and itf-specific although transcriptional conversion of fcm nuclei to fc identity upon fusion is often presented as a dogma, it has only been documented for col in the da and s in the dt va and vt muscles. to determine the extent and dynamics of transcriptional conversion in different muscles, we reinvestigated the patterns of col and s transcription, using col- and s59-driven gfp expression to follow muscle development, step by step. we also investigated the transcription pattern of a third itf gene, kr, in several muscles including dorsolateral ll. detection of nascent transcripts was followed by quantification of two parameters, namely the number of hybridisation dots in the syncytium and the intensity of each transcription dot, as a proxy of the transcription initiation rate.fig. dynamics of identity transcription factor transcription during muscle development. col lcrm -moegfp, s59-mcd8gfp and kr gmr80h11 -gal4;uas-mcd8gfp embryos stained for nascent col, s or kr transcripts and gfp to visualise muscle shape, and either col, s or kr. red channel only, with dotted grey lines outlining muscle shape. kr is transcribed in a single nucleus from stage to late stage in all kr-expressing muscles. schematic view of the col+, s59+ and kr+ muscles colour coded as in fig. 1a. box plots showing the relative numbers of nuclei and col transcription dots in da, s dots in dt and va, and kr dots in ll and va simultaneous staining for col, nascent col transcripts and col lcrm -moegfp confirmed col active transcription in the da fc and several syncytial nuclei within growing da fibres, and stop of transcription at stage quantification of the nuclei number transcribing col, relative to the total number of da syncytial nuclei, shows, however, that only a fraction transcribe col at a given time, suggesting transient transcription in each nucleus. the observation of growing da fibres, in which col is transcribed in virtually all nuclei shows that all fused fcm nuclei are competent to be converted to the fc identity transcriptional programme. the variable numbers of transcriptionally active nuclei in adjacent hemisegments indicate that activation of col transcription in syncytial nuclei is stochastic rather than synchronous. quantification of the intensity of col transcription dots shows that col is transcribed at a 9-fold higher level in the fc than in syncytial da nuclei at stage. thus, two different modes of col regulation operate successively. this correlates with the previous identification of two distinct col mesodermal crms that act sequentially. as for col in da only a fraction of nuclei within dt va or vt syncytia transcribe s at a given time. interestingly, this fraction differs between muscles, at approximately 45%, 27% and 23%, in dt va and vt respectively, at stage, while the integrated density of s dots is relatively constant from stage to. together, these data suggest that the time interval of s transcription is differentially regulated in dt va and vt s is also expressed in lo at stage but in this lineage, s is only transcribed in the fc at stage and not at later stages. finally, fish of nascent transcripts shows that kr transcription in kr+ muscles ceases at early stage. unexpectedly, kr transcription is detected at a constant level in a single nucleus per fibre, from stage to. the kr + nucleus stays at roughly the same position within the muscle throughout the process of fibre growth, suggesting that it is the fc nucleus and that kr transcription is maintained in the fc nucleus during the fusion process without being propagated to other syncytial nuclei. up to now, two ‘typical’ itf gene transcription patterns have been reported, namely repression at the pc or fc stage or, alternatively, maintenance and propagation of transcription to other syncytial nuclei. kr transcription in a single nucleus up to stage reveals a novel pattern of itf transcriptional regulation in muscle precursors, namely maintenance in the fc nucleus without propagation to fcm nuclei. together, the observed patterns of nascent col, kr and s transcripts show that activation of itf transcription in fcms after fusion is itf- and muscle-specific, and cannot be considered as a general rule. furthermore, single nucleus kr transcription provides the first evidence that the fc nucleus could maintain a transcriptional programme different from the other syncytial nuclei during muscle development. transcriptional conversion of fcms to syncytial identity fusion of fcms with an fc and, subsequently, the derived growing fibre, is based on mutually exclusive expression of the surface proteins duf/rst and sns by all fcs and fcms, respectively. for this to be a reiterative process implies that, upon fusion, fcm nuclei switch off fcm-specific gene transcription and that the fc-generic duf gene remains expressed. this process remains to be decrypted at the transcriptional level. we sought to determine precisely when the fcm to fc/fibre transcription switch occurs, using da as a paradigm. fish to duf nascent transcripts confirmed that duf is transcribed in all fcs at stage including the da dt and ll fcs labelled with col. at stage the increased number of duf transcribing nuclei in da syncytia correlates with the increased number of recruited fcm nuclei, demonstrating activation of the fc-generic transcriptional programme in fcm nuclei. duf transcription drops at late stage at the end of the fusion process. paired fish of nascent duf and col transcripts shows that the da fc co-transcribes duf and col at stage. however, at stage while 80% of syncytial nuclei transcribe at least one of these two genes, only 50% of these nuclei co-transcribe duf and col, possibly reflecting asynchronous bursts of col and duf transcription. their dynamics of transcription show that duf and col activation is not synchronised and independent of each other in individual nuclei within a syncytium. we previously showed that nuclear uptake of col protein precedes activation of col transcription in fused fcm nuclei. the detection of duf dots in nuclei with very low levels of col protein further supports independent activation of duf and col.fig. transcriptional conversion of fused fusion competent myoblast nuclei to founder cell identity. fish against nascent duf or sns transcripts, staining for mef, and col to visualise the da dt and ll fc nuclei at stage and the da muscle at stage, wt embryos. double fish against nascent col and either duf or sns transcripts and col staining of the da fc at stage and da muscle at stage. double fish against nascent duf and sns transcripts and col staining, stage da muscle; blue channel only. sns transcription is detected in "no col" nuclei. box plots showing the number of nuclei transcribing either col or duf, or col and duf, relative to the total number of da nuclei, and either col or sns only. two adjacent hemisegments are shown in, section projections; a single hemisegment in, single confocal sections. l schematic representation of the transcriptional fcm to generic fc identity switch, post fusion conversely, nascent sns transcripts are detected in a large number of fcms surroundings fcs at stage but not in fcs themselves. during the fusion period, only one, if any, sns transcription dot is detected in growing da myotubes, indicating that switching off the fcm transcriptional programme occurs post fusion. furthermore, no nuclei co-transcribing sns and col, or sns and duf were detected, indicating that repression of fcm-specific genes precedes activation of fc-generic and itf transcription. in support of this, sns dots could only be detected in nuclei displaying no or very low levels of col protein. in summary, the duf, sns and col transcription patterns show that transcriptional reprogramming of fcms is a dynamic process: after fusion of fcms, transcription of fcm-generic genes is first turned off, followed by a switch from fcm to fc-generic and fc-specific transcriptional programmes. these latter two aspects of transcriptional activation are asynchronous. transcription of generic muscle differentiation genes is independent of itf gene transcription assembly of sarcomeres underlies the formation of contractile muscles. consistent with mhc being a critical sarcomeric component, mhc mrna level is similar in all muscle fibres at stage. fish of nascent transcripts indicated that mhc transcription is first weakly detected in few nuclei at stage and increases dramatically at stage simultaneously in all developing muscles. the homogenous distribution of mhc dots suggests that transcription of generic muscle differentiation is independent of muscle identity. supporting this conclusion, high-level mhc transcription is maintained in muscle syncytia until the end of embryogenesis, when itf transcription has already strongly decreased. to further assess whether the generic differentiation and muscle identity transcription programmes are coupled, we compared the dynamics of col and mhc transcription in the developing da muscle. paired fish of nascent transcripts, coupled to col and dapi staining, confirmed that virtually all da nuclei transcribe mhc at stage while only roughly a quarter also transcribes col at a given time point. together, these data support the conclusions that the generic muscle differentiation and identity programmes are uncoupled temporally and independently activated in syncytial nuclei.fig. independent transcription of identity transcription factors and generic muscle differentiation genes. a–e wt embryos at various embryonic stages, stained for mef and col coupled to fish against nascent mhc transcripts. the white arrow points to mhc expression in heart cells, first detected at stage f double fish against nascent col and mhc transcripts, and col staining to visualise the da nuclei at stage f’ same as, dapi staining shows all nuclei. f” col staining only. single z sections. the yellow arrow indicates a nucleus with low col protein, which transcribes mhc. g box plots showing the number of nuclei transcribing either col, mhc or both, relative to the total number of da nuclei fibre-specific transcription of identity realisation genes; multi-level regulation the current hypothesis is that the itf code specific to each fc controls the differential expression of genes underlying ‘realisation’ of muscle morphological identity. a search for candidate realisation genes identified two cytoskeletal protein genes, pax and mspo, which display fibre-specific expression levels and are involved in fibre-specific modulation of the fusion rate. two other cell surface proteins, con and kon, were independently implicated in fibre-specific regulation of muscle attachment. kon encodes a transmembrane protein required in a specific subset of myotubes to recognise and establish stable connections with appropriate tendon cells. kon was also recovered in a screen for fc-specific genes based on transcriptomics and computational analyses under the name of perdido. con encodes a cell surface protein decorating a subset of muscles, including dt and the motoneurons which innervate them. we compared the transcription patterns of pax, mspo, kon and con in da and dt which present the advantage of displaying similar numbers of nuclei and fcm recruitment kinetics. fish of mature transcripts at stage showed that all four genes are expressed in both muscles. yet, while pax is expressed at similar levels in da and dt mspo, kon and con mrnas accumulate at significantly higher level in dt. fish of nascent transcripts in stage embryos further suggested that differential accumulation of mspo, kon and con mrna reflects differential transcription. we therefore performed a side by side comparison of the dynamics of transcription of pax, mspo, kon and con between da and dt by both measuring the numbers of hybridisation dots and individual dot intensities for each gene in syncytia, at embryonic stages and using duf as internal reference.fig. transcription dynamics of identity-realisation genes. a, e, i, m, q stage col lcrm -moegfp; s59-mcd8gfp embryos stained for gfp and topro to visualise da and dt and all nuclei, respectively, coupled with fish of either nascent duf, pax, kon, mspo or con transcripts; a’, e’, i’, m’, q’ red channel only, muscle contours are outlined by dashed grey lines. b, f, j, n, r number of transcription dots in da and dt at different embryonic stages. bar graphs indicate the mean number of nuclei; error bars, the sd. c, g, k, o, s intensity of transcription dots in da and dt1; each dot is represented by a circle, the bar graphs show the mean value and sd. d, h, l, p, t cumulative intensity of transcriptional dots in da and dt the mean value is shown; error bars correspond to the sem as reported above, the number of duf dots increases between stages and in both da and dt and dot intensity is maximal during the muscle elongation phase, stages to both in da and dt. the numbers and intensity of pax transcription dots are also similar in da and dt during the fusion phase. a maximum of dots per fibre at stage suggests that transcription is stochastic and/or transient in syncytial nuclei. the absence of dot detection in fcs at stage indicates that pax transcription is activated post fusion. contrary to pax, kon transcription is detected in the da and dt fcs, and the kinetics of kon transcription differs between da and dt the number of kon dots increases between stages and in dt reaching an average of per fibre, while it decreases earlier in da. nevertheless, dot intensity indicates that the kon transcription initiation rate is not significantly different between the two muscles. together, these data indicate that differential transcription of kon between da and dt reflects different time intervals of transcription per nucleus. however, a third scenario is observed for mspo, with higher dot number and intensity in dt than in da. thus, both differences in the time of transcription and transcription initiation rate can contribute to differential mspo transcription in different muscles during the acute fusion phase. this pattern is even more pronounced for con, where high numbers of high intensity dots are detected in dt correlating with con accumulation at the surface of the dt muscle, and rare, low intensity dots are detected in da. summary curves corresponding to the mean sum of all dot intensities measured in each fibre underline the differential transcription of kon, mspo and con between da and dt. furthermore, they show that the transcription level of realisation genes is regulated in a fibre-specific manner, via the modulation of the transcription initiation rate and/or time interval of active transcription per syncytial nucleus. transcriptional activation of realisation genes follows itf gene activation to determine whether transcription of identity realisation genes is connected to itf activation, we focused on mspo, kon and con, which are differentially expressed in da versus dt double fish of col and either mspo, kon or con nascent transcripts allowed us to measure the number of nuclei, either transcribing one or both genes, in developing da. the numbers show that mspo or kon are only transcribed in nuclei also transcribing col. thus, contrary to the asynchronous transcription of itfs with respect to the fc-generic gene duf, transcription of realisation genes is linked to itf transcription in syncytial nuclei.fig. transcription of identity-realisation genes is coupled to identity transcription factor transcription. double fish of nascent col and either mspo, kon or con transcripts, col staining for visualising the da nuclei at stage a’, c’, e’ dapi staining of all nuclei; single z sections. box plots showing the number of nuclei transcribing either col, mspo or both, and either col, kon or both, relative to the total number of da nuclei the fc nucleus remains distinct from the other syncytial nuclei the variable numbers of syncytial nuclei transcribing different realisation genes raised the additional question of whether functional specialisation of nuclei could exist, according to their position within the syncytial myotube. at late stage the da muscle displays an angled shape, reflecting transient tripartite attachment to tendon cells; the final acute orientation of da corresponds to stabilisation of the ventral-most anterior, and the dorsal-posterior attachment sites, which critically depends upon appropriate levels of col and nau activity. the da branch of the intrasegmental nerve contacts the ventral side of the angled da muscle at a stereotypic position, roughly at its centre. the position of the neuromuscular junction and the tripartite attachment sites provide morphological landmarks allowing the division of the da muscle into three subdomains, namely antero-ventral, central and postero-dorsal. we analysed the topological distributions of nuclei and transcription dots for muscle generic, fc-generic and identity genes, relative to these three subdomains. plotting the spatial coordinates of each fish dot and nucleus, relative to the da shape, showed a roughly homogenous distribution of nuclei and col, duf, kon, mhc, mspo and pax transcriptional dots. thus, transcriptional activation of these genes does not appear to be influenced by nucleus position. this could be required for homogeneous distribution of transcripts coding for surface proteins during muscle growth.fig. transcription patterns and da muscle subdomains. a, b late stage col lcrm -moegfp embryo stained for gfp and either col, coupled with fish of nascent col transcripts, or lacz and fasciclin ii to visualise tendon cells and the da innervating motoneuron, respectively. c subdivision of the da muscle into ventral, median and dorsal subdomains shows an homogeneous distribution of nuclei; brant and brpost indicate the anterior and posterior da limits, respectively. spatial distribution of col, mhc, duf, kon, mspo and con transcription dots. j repartition of hybridisation dots and nuclei in each da subdomain most con transcription dots map to the central subdomain of the da muscle. interestingly, con is the only realisation gene in our study which is, on average, transcribed in a single nucleus throughout da development, suggesting that this is the fc nucleus, in agreement with the observation that con is expressed in fcs. taken together, single nucleus-restricted kr and con transcription support the conclusion that the fc nucleus remains located at a stereotypical position and is transcriptionally different from the other syncytial nuclei throughout muscle development. discussion in its simplest terms, myogenesis is the differentiation of individual precursor cells, the myoblasts, into syncytial contractile myofibres. yet, each body wall muscle displays a morphological identity. understanding how transcriptional regulation of a generic myogenic programme, common to all muscles, and each muscle identity programme are integrated to generate morphological muscle diversity remains a central question in the field. here, we used drosophila larval muscles to address this question, exploiting hybridisation to nascent transcripts to compare the transcriptional status of a selection of generic and identity genes, in individual nuclei within different syncytial muscles. our data show that transcriptional reprogramming of fused myoblast nuclei is progressive. transcriptional activation of identity realisation genes is specific to nuclei expressing itf genes and subject to different modes of regulation in a fibre-specific manner. conversely, transcription of generic muscle differentiation genes is regulated independently of muscle morphological identity. in humans, in addition to muscles, there exist two other types of differentiating multinucleated syncytia, the osteoclasts and syncytial trophoblasts. the transcriptional status of each nucleus in the different types of multinucleated cells and how it contributes to lineage diversity remains a widely open, fundamental field of investigation. muscle ‘founder’ and naive myoblasts the development of drosophila larval muscles involves mandatory asymmetric fusion of two types of cells, fcs and fcms, both of which are specified from equivalence groups of myoblasts expressing different itfs, in response to positional cues. itf expression then becomes restricted to pcs and some fcs. itf activation in equivalence groups raises the legitimate question of whether this influences fcm fate. to address this question, we exploited the dynamics of col transcription, which is activated in one pmc, maintained in one fc and re-activated in fcm nuclei incorporated into the growing da myotube. our data showing that fcms recruited in a given myofibre can originate from different equivalence groups and are equally competent to be converted to a specific fc identity, indicate that fused fcms behave as naive myoblasts. this naive character of fcms finds parallels with the uncommitted character of mammalian satellite cells. mouse satellite cells specified in different anatomical locations have been shown to contribute to new fibres in a heterotopic location, though retaining distinct molecular signatures reflecting, in part, their developmental history. similarly, grafting of human myogenic progenitors into the dystrophic pharyngeal muscles of oculopharyngeal muscular dystrophy patients shows that ectopic myoblasts isolated from clinically unaffected limbic muscles are able to restore contractility to defective pharyngeal muscles. thus, drosophila fcms and mammalian satellite cells are able to contribute to different muscle fibres, irrespective of their early molecular signature. in both cases, the syncytial cell is able to reprogramme fusing cells and retain its identity. transcriptional reprogramming of naive myoblast nuclei, post fusion previous analyses, performed at the protein expression level, led to the conclusion that muscle lineage-specific itf expression, which determines muscle identity, reflects cross-regulations between itfs at the pc and fc stages. here, the patterns of nascent col, s and kr transcripts show that propagation of transcriptional identity from the fc to recruited fcm nuclei depends upon the itf and/or muscle lineage, and that there is not one pattern to explain this process. in the case of col in the da muscle or tailup/islet in dorsal muscles, activation of transcription relies upon one crm, while propagation to syncytial nuclei involves another, ‘late’ crm mediating direct autoregulation by the imported itf. in the case of kr, a single muscle crm has been identified. this fits with the restriction of kr transcription to the fc nucleus and arrest earlier than observed for itf genes displaying auto-regulatory crms. future characterisation of multiple itf crms differentially active in different muscle lineages should provide a general picture of how pc/fc identity is maintained and/or propagated during muscle development, leading to a robust, stereotyped muscle pattern. fused fcm nuclei stop transcribing fcm-specific genes, such as sns, before activating generic fc genes such as duf. one mechanism involved in this transcriptional switch is the active degradation of lmd, present in the fusing fcms, by the ubiqutitin ligase mib. whether duf activation also requires degradation of ttk a repressor of fc fate, and/or import of generic fc activator tf, similar to import of specific itfs, remain open questions. in addition, a potential epigenetic control of nuclei reprogramming in multinucleated cells has been scarcely addressed thus far. mutants for drosophila sin3a, a chromatin regulator conserved from yeast to humans that serves as a scaffold protein for the sin3/histone deacetylase complex, show muscle identity phenotypes, suggesting that sin3a could sensitise certain fcs and muscles to itf activity. patterns of epigenetic histone markers were found to be identical in all nuclei of mouse multinucleated osteoclasts, whereas some osteoclast-specific genes were only transcribed in a subset of nuclei. further investigation on different types of multinucleated cells is certainly needed for mechanical understanding of the reprogramming of syncytial nuclei, post fusion.fig. summary model for transcriptional programming of individual syncytial nuclei in a developing muscle. before fusion, stage all founder cells transcribe generic fc genes, and fusion competent myoblasts transcribe fcm-specific genes. each fc expresses specific identity transcription factors and transcribes itf genes. in a growing muscle syncytium, stage fcm gene transcription is transitorily maintained in newly fused fcm nuclei, before complete switch off and progressive activation of fc-generic transcription. nuclear uptake of itfs by fused fcms is followed by itf activation. transcription of identity-realisation genes is linked to itf activation, while transcription of general muscle differentiation genes, is activated and maintained in all myoblast nuclei, independently of itf expression. transcription of a subset of genes is restricted to the fc nucleus transcriptional regulation of muscle-generic and identity genes: two independent programmes gene expression studies, using either mammalian c2c cells in culture or in vivo models of muscle regeneration, have shown that abrupt, orderly transcriptional changes occur during myogenesis. however, global transcriptome analysis of cell pools produces a population average that does not distinguish between transcriptional and post-transcriptional regulatory levels. analyses of muscle cells isolated from drosophila embryos at different time points showed that recruitment of poised rna polymerase ii, which makes genes permissive for future transcription, occurs at many genes de novo, in a stage-specific way. in parallel, systematic genomic-wide analyses of transcription factor binding profiles and chromatin marks has identified temporal signatures of enhancer activity in the drosophila mesoderm. while revealing the importance of timing of enhancer/promoter activity during mesoderm development, these studies did not address the question of the dynamics of gene transcription in individual syncytial nuclei. our analysis of nascent transcripts in individual nuclei in defined muscles has freed us from post-transcriptional regulation levels that contribute to differential mrna accumulation, as documented for the regulation of mhc by hoi polloi. moreover, our approach allowed the comparison of the transcriptional time interval of a general muscle differentiation gene, mhc, with itfs and identity-realisation genes in individual syncytial nuclei. in this manner, we observed a mixture of on/off nuclei for itfs and realisation genes, whose relative proportions varied between muscle lineages, suggesting a stochastic mode of activation. inversely, we found that mhc transcription is synchronously activated in all myoblast nuclei independent of muscle identity, correlating with the previous finding that mhc is expressed in all myoblasts, independent of fusion. we conclude that the generic muscle differentiation and morphological identity programmes are regulated independently of each other. transcriptional control of the generic aspects of skeletal muscle development in vertebrates involves several transcription factors that act as muscle itfs in drosophila, including nautilus/myod, eya, six and col/ebf proteins. our data in drosophila plead for investigating whether muscle identity and generic differentiation programmes also run in parallel in vertebrates. transcription of identity realisation genes is linked to itf transcription how realisation of muscle identity is integrated with generic muscle differentiation is a major question in muscle biology. here, we compared the transcriptional dynamics of candidate morphological realisation genes in syncytial nuclei between different muscles. our data show that the differential expression of genes encoding surface protein, such as con, kon and mspo, between the da and dt muscles is controlled at the transcriptional level, via both regulation of the time interval of active transcription and the transcription initiation rate. moreover, transcription of these genes in the da muscle is restricted to nuclei transcribing col, i.e. reprogrammed to da identity by imported col protein. however, not every col + nucleus transcribes realisation genes, suggesting either one of three not mutually exclusive possibilities, namely higher threshold levels of imported col protein are required for activation of realisation genes than for col autoregulation, thus introducing a temporal shift; the burst frequency of realisation gene transcription is significantly lower than that of col; other transcription factors, themselves regulated by col, are involved in activation of realisation genes, as documented in the case of col and apterous-expressing neurons. finally, whether local col mrna translation around col-transcribing nuclei could amplify variations of gene transcription among syncytial nuclei is an interesting question. we also found that transcription of realisation genes is generally independent of nucleus position within the fibre. one exception is the con gene, which is transcribed in a single nucleus at a central position that we surmise is the fc nucleus. the restriction of con transcription to the fc in da contrasts with its propagation to other syncytial nuclei in dt thus, syncytial activation is an active process regulated in a fibre-specific manner. together with the restricted pattern of kr transcription, da con transcription also suggests that fc nuclei can keep a transcription programme distinct from the other syncytial muscles throughout muscle development. whether this could provide molecular cues for positioning of the neuromuscular synapse remains an open question. the correlation in the severity of da morphology defects with col mutations of increasing strength, or mutations in other itfs which affect col transcription, suggests that proper activation of realisation genes is dependent upon the appropriate levels of itf activity. our present finding that transcription of realisation genes is linked to itf transcription strongly supports this conclusion. con, kon and mspo were identified as putative direct col targets by genome-wide chip-seq experiments. this, and other genome wide data, suggest that direct binding of different itf combinations is responsible for the differential transcription of identity realisation genes in different muscles. establishing whether specific crms integrate multiple itf inputs, or multiple crms respond to individual itfs, will be the next step towards deciphering the regulatory logic that confers each muscle its specific morphology.  in conclusion, our data show that post-fusion reprogramming of naive myoblast nuclei is progressive, such that nuclei within one syncytial fibre are heterogeneous with regards to gene-specific transcription at a given time. while reprogramming seems independent of nuclear position, preliminary evidence indicates that fc nuclei can remain transcriptionally different from other nuclei throughout muscle development. this novel view of the dynamics of transcriptional programming of post-mitotic nuclei within syncytial cells provides a new framework for the investigation of transcriptional control of lineage diversity of multinucleated cells. 
distinctive shape, orientation, size and skeletal attachment sites, reflects the specific combination of identity transcription factors expressed by its fc. this comprehensive view of the dynamics of transcriptional programming of post-mitotic nuclei within syncytial cells provides a new framework for understanding the transcriptional control of the lineage diversity of multinucleated cells. a stereotyped array of body wall muscles enables precision and stereotypy of animal movements. is transcription of generic myogenic and identity realisation genes coordinated during muscle differentiation? the generic muscle differentiation programme is activated independently. transcription reprogramming of fused myoblast nuclei is progressive, such that nuclei within a syncytial fibre at a given time point during muscle development are heterogeneous with regards to specific gene transcription. the tracking of nuclei in developing muscles shows that fcm nuclei are competent to be transcriptionally reprogrammed to a given muscle identity, post fusion. myogenesissyncytiamuscle identityrealisation genestranscriptional regulationdrosophilahttp://dx.doi.org/ /501100002915fondation pour la recherche médicaledeq20090515429vincent alain http://dx.doi.org/ /501100001665agence nationale de la recherche13-bsve2-0010vincent alain http://dx.doi.org/ /100007393association française contre les myopathieshttp://dx.doi.org/ /501100004794centre national de la recherche scientifiquehttp://dx.doi.org/ /501100004562ministère de l'education nationale, de l'enseignement superieur et de la rechercheissue-copyright-statement© the author 2017 transcription of identity realisation genes is linked to itf activation and regulated at levels of both transcription initiation rate and period of transcription. in drosophila, each syncytial muscle forms via fusion of one founder cell with multiple fusion competent myoblasts. in situ hybridisation to nascent transcripts for fcm, fc-generic and itf genes shows that this reprogramming is progressive, beginning by repression of fcm-specific genes in fused nuclei, with some evidence that fc nuclei retain specific characteristics. here, we addressed three questions: are fcm nuclei naive? the specific morphology of each muscle, i.e.
 a stereotyped array of body wall muscles enables precision and stereotypy of animal movements. in drosophila, each syncytial muscle forms via fusion of one founder cell with multiple fusion competent myoblasts. the specific morphology of each muscle, i.e. distinctive shape, orientation, size and skeletal attachment sites, reflects the specific combination of identity transcription factors expressed by its fc. here, we addressed three questions: are fcm nuclei naive? what is the selectivity and temporal sequence of transcriptional reprogramming of fcms recruited into growing syncytium? is transcription of generic myogenic and identity realisation genes coordinated during muscle differentiation?  the tracking of nuclei in developing muscles shows that fcm nuclei are competent to be transcriptionally reprogrammed to a given muscle identity, post fusion. in situ hybridisation to nascent transcripts for fcm, fc-generic and itf genes shows that this reprogramming is progressive, beginning by repression of fcm-specific genes in fused nuclei, with some evidence that fc nuclei retain specific characteristics. transcription of identity realisation genes is linked to itf activation and regulated at levels of both transcription initiation rate and period of transcription. the generic muscle differentiation programme is activated independently.  transcription reprogramming of fused myoblast nuclei is progressive, such that nuclei within a syncytial fibre at a given time point during muscle development are heterogeneous with regards to specific gene transcription. this comprehensive view of the dynamics of transcriptional programming of post-mitotic nuclei within syncytial cells provides a new framework for understanding the transcriptional control of the lineage diversity of multinucleated cells. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords myogenesissyncytiamuscle identityrealisation genestranscriptional regulationdrosophilahttp://dx.doi.org/ /501100002915fondation pour la recherche médicaledeq20090515429vincent alain http://dx.doi.org/ /501100001665agence nationale de la recherche13-bsve2-0010vincent alain http://dx.doi.org/ /100007393association française contre les myopathieshttp://dx.doi.org/ /501100004794centre national de la recherche scientifiquehttp://dx.doi.org/ /501100004562ministère de l'education nationale, de l'enseignement superieur et de la rechercheissue-copyright-statement© the author 2017 