Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jun 9;15(1):48.
doi: 10.1186/s12915-017-0386-2.

Dynamics of transcriptional (re)-programming of syncytial nuclei in developing muscles

Laetitia Bataillé  1 , Hadi Boukhatmi  1   2 , Jean-Louis Frendo  3 , Alain Vincent  4
Affiliations

Dynamics of transcriptional (re)-programming of syncytial nuclei in developing muscles

Laetitia Bataillé et al. BMC Biol. .

Abstract

Background: 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 (FC) with multiple fusion competent myoblasts (FCMs). The specific morphology of each muscle, i.e. distinctive shape, orientation, size and skeletal attachment sites, reflects the specific combination of identity transcription factors (iTFs) 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?

Results: 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.

Conclusions: 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 (re)programming of post-mitotic nuclei within syncytial cells provides a new framework for understanding the transcriptional control of the lineage diversity of multinucleated cells.

Keywords: Drosophila; Muscle identity; Myogenesis; Realisation genes; Syncytia; Transcriptional regulation.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Tracking fusion competent myoblasts (FCMs) derived from the Col+ promuscular cluster. A Schematic representation of dorso-lateral muscle formation, with embryonic stages indicated. The DA3/DO5, LL1/DO4 and DT1/DO3 progenitor cells (PCs) are selected from a promuscular cluster expressing Col (red hatched); unselected myoblasts become FCMs (grey). Each PC generates two founder cells (FCs) that fuse with FCMs to form syncytial fibres, which attach to tendon cells to form contractile muscles. The LL1 and DT1 FCs express Kr and S59, respectively (colour coded). Col expression is maintained in the DA3 muscle (red). Out-group dorsal DA2, lateral LT2 and ventral VA2 muscles are circled in blue, other muscles in black. B–B’ Stage 12 col ECRM -H2bRFP; duf-LacZ embryo stained for RFP (red) to identify the Col+ promuscular cluster (PMC) nuclei, LacZ (green) and Lmd (blue) to visualise all FCs and all FCMs, respectively. B External and (B’) internal layers where FCs and FCMs are located, respectively; C–F Repartition of RFP+ FCM nuclei at stage 15. Box plots indicate the numbers of RFP+ relative to Mef2+ nuclei in DA3, DT1, LT2 and VA2 (C); col ECRM -H2bRFP; col LCRM -moeGFP (D), col ECRM -H2bRFP; S59-mcd8GFP (E) and col ECRM -H2bRFP; Kr GMR80H11 -Gal4,UAS-mcd8GFP (F) embryos stained for RFP (red) and GFP (green) to outline the DA3 (D), DT1 and VA2 (E), and LT2 and LL1 (F) contours; Mef2 staining (blue) 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
Fig. 2
Fig. 2
Promuscular col expression does not impact fusion competent myoblast (FCM) fate. Stage 15 col ECRM -H2B-RFP (A) and col 1 ,col ECRM -H2B-RFP (B) embryos stained for RFP (red) and β3-tub (green) to visualise the nuclei of Col+ PMC myoblasts and all muscles, respectively; (A’, B’) red channel only; (A”, B”) close up of three abdominal hemisegments. C Number of RFP+ nuclei in DA2, DA3 and DT1 at stage 15 in col ECRM -H2bRFP and DA2, DA3 > DA2 and DT1 in col 1 ,col ECRM -H2bRFP embryos. The DA3 transformation into a DA2-like muscle (DA3 > DA2) in col 1 embryos is schematised on the right. D FISH of nascent col transcripts (green) in stage 14 col ECRM -H2bRFP embryos stained for RFP (red) and Col (blue); col is transcribed in a fraction of both RFP+ and RFP– DA3 nuclei. Scale bar: 20 μm
Fig. 3
Fig. 3
Dynamics of identity transcription factor transcription during muscle development. (A–E’) col LCRM -moeGFP, (G–K’) S59-mcd8GFP and (M–Q’) Kr GMR80H11 -Gal4;UAS-mcd8GFP embryos stained for nascent col (A–E’), S59 (G–K’) or Kr (M–Q’) transcripts (red) and GFP (green) to visualise muscle shape, and either Col (A–E’), S59 (G–K’) or Kr (M–Q’) (blue). (A’–E’, G’–K’, M’–Q’) red channel only, with dotted grey lines outlining muscle shape. Kr is transcribed in a single nucleus from stage 12 to late stage 14, in all Kr-expressing muscles. (F, L, R) Schematic view of the Col+, S59+ and Kr+ muscles colour coded as in Fig. 1A. (S–U) Box plots showing the relative numbers of nuclei and col transcription dots in DA3 (S), S59 dots in DT1 and VA2 (T), and Kr dots in LL1 and VA2 (U)
Fig. 4
Fig. 4
Transcriptional conversion of fused fusion competent myoblast (FCM) nuclei to founder cell (FC) identity. FISH against nascent duf (A, C) or sns (E, G) transcripts (green), staining for Mef2 (red), and Col (blue) to visualise the DA3, DT1 and LL1 FC nuclei at stage 12 (A, E) and the DA3 muscle at stage 14 (C, G), wt embryos. Double FISH against nascent col (red) and either duf (B, D) or sns (F, H) transcripts (green) and Col staining (blue) of the DA3 FC at stage 12 (B, F) and DA3 muscle at stage 14 (D, H). (I, I’) Double FISH against nascent duf (red) and sns transcripts (green) and Col staining, stage 14 DA3 muscle; (I’) blue channel only. (H, I, I’) sns transcription (yellow arrow) is detected in "no Col" nuclei. (J, K) Box plots showing the number of nuclei transcribing either col or duf, or col and duf, relative to the total number of DA3 nuclei (J), and either col or sns only (K). Two adjacent hemisegments are shown in (A, C, E, G), section projections; a single hemisegment in (B, D, F, H, I), single confocal sections. L Schematic representation of the transcriptional FCM to generic FC identity switch, post fusion
Fig. 5
Fig. 5
Independent transcription of identity transcription factors and generic muscle differentiation genes. A–E wt embryos at various embryonic stages, stained for Mef2 (red) and Col (blue) coupled to FISH against nascent Mhc transcripts. The white arrow points to Mhc expression in heart cells, first detected at stage 15. F Double FISH against nascent col (red) and Mhc (green) transcripts, and Col staining (blue) to visualise the DA3 nuclei at stage 15. F’ same as (F), DAPI staining (blue) 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 DA3 nuclei
Fig. 6
Fig. 6
Transcription dynamics of identity-realisation genes. A, E, I, M, Q Stage 14 col LCRM -moeGFP; S59-mcd8GFP embryos stained for GFP (green) and Topro (blue) to visualise DA3 and DT1 and all nuclei, respectively, coupled with FISH of either nascent duf (A), Pax (E), kon (I), mspo (M) or Con (Q) transcripts (red); 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 DA3 and DT1 at different embryonic stages. Bar graphs indicate the mean number of nuclei; error bars, the SD. C, G, K, O, S Intensity (IntDen) of transcription dots in DA3 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 (Total IntDen per fibre) in DA3 and DT1. The mean value is shown; error bars correspond to the SEM
Fig. 7
Fig. 7
Transcription of identity-realisation genes is coupled to identity transcription factor transcription. Double FISH of nascent col (red) and either mspo (A), kon (C) or Con (E) (green) transcripts, Col staining for (blue) visualising the DA3 nuclei at stage 14. A’, C’, E’ DAPI staining (blue) of all nuclei; single Z sections. Box plots showing the number of nuclei transcribing either col, mspo or both (B), and either col, kon or both (D), relative to the total number of DA3 nuclei
Fig. 8
Fig. 8
Transcription patterns and DA3 muscle subdomains. a, b Late stage 14 col LCRM -moeGFP embryo stained for GFP (green) and either Col (blue), coupled with FISH of nascent col transcripts (red) (a), or LacZ and Fasciclin II (FascII) to visualise tendon cells and the DA3 innervating motoneuron, respectively. c Subdivision of the DA3 muscle into ventral, median and dorsal subdomains shows an homogeneous distribution of nuclei; BRant and BRpost indicate the anterior and posterior DA3 limits, respectively (Additional file 17: Figure S5). Spatial distribution of col (d), Mhc (e), duf (f), kon (g), mspo (h) and Con (i) transcription dots. j Repartition of hybridisation dots and nuclei (hatched bar) in each DA3 subdomain
Fig. 9
Fig. 9
Summary model for transcriptional (re)programming of individual syncytial nuclei in a developing muscle. Before fusion, stage 12, all founder cells (FCs) transcribe generic FC genes (duf, blue dot), and fusion competent myoblasts (FCMs) transcribe FCM-specific genes (sns, green dot). Each FC expresses specific identity transcription factors (iTFs; e.g. Col, grey) and transcribes iTF genes (e.g. col, red dot). In a growing muscle syncytium, stage 14, 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 (e.g. Pax, mspo, kon, orange dot) is linked to iTF activation, while transcription of general muscle differentiation genes (Mhc), is activated and maintained in all myoblast nuclei, independently of iTF expression. Transcription of a subset of genes is restricted to the FC nucleus (e.g. Con, orange dot circled by a red line)
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bate M. The embryonic development of larval muscles in Drosophila. Development. 1990;110(3):791–804. - PubMed
    1. Ruiz-Gómez M, Coutts N, Price A, Taylor MV, Bate M. Drosophila dumbfounded: a myoblast attractant essential for fusion. Cell. 2000;102(2):189–98. doi: 10.1016/S0092-8674(00)00024-6. - DOI - PubMed
    1. Bour BA, Chakravarti M, West JM, Abmayr SM. Drosophila SNS, a member of the immunoglobulin superfamily that is essential for myoblast fusion. Genes Dev. 2000;14(12):1498–511. - PMC - PubMed
    1. Kim JH, Jin P, Duan R, Chen EH. Mechanisms of myoblast fusion during muscle development. Curr Opin Genet Dev. 2015;32:162–70. doi: 10.1016/j.gde.2015.03.006. - DOI - PMC - PubMed
    1. Schweitzer R, Zelzer E, Volk T. Connecting muscles to tendons: tendons and musculoskeletal development in flies and vertebrates. Development. 2010;137(17):2807–17. doi: 10.1242/dev.047498. - DOI - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources

-