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Identification of a Kr CRM active in the LTs and VA2 muscles. (A) Chromosome (2R: 25,200,000–25,237,000) showing the Kr genomic region with the transcribed region represented by a blue box and all GMR tested by grey lines; adapted from Flybase GBrowse ( http://flybase.org/ ). The overlapping GMR80H07 and GMR80H11 driving reporter expression in LTs and VA2 muscles are indicted by orange lines. (B, C) Stage 16 Kr GMR80H07 -Gal4; UAS-LacZ (B) and Kr GMR80H11 -Gal4; UAS-LacZ (C) embryos stained for LacZ, illustrating LacZ expression in somatic muscles. (PDF 862 kb)

Separate transcription of col and duf in DA3 syncytial nuclei. (A–C) FISH against nascent duf transcripts (green) coupled to Mef2 (red) and Col (blue), immunostaining of all muscle and DA3 nuclei, respectively, in stage 13, 14 and 15 wt embryos. (D) Double FISH of nascent col (red) and duf (green) transcripts in stage 14 wt embryos immunostained for Col (blue); (D’) Col staining alone; (D”) same as (D), showing DAPI staining (blue) of all nuclei. Single Z sections are shown. The yellow arrow points to a nucleus with low Col protein level transcribing duf and not col. (E) Box plots showing the number of nuclei transcribing either col, duf or both, or either col or sns, relative to the total number of DA3 nuclei. (PDF 2537 kb)

Numbers of nuclei and number of transcriptional dots of duf and realisation genes in DA3 and DT1 muscles at embryonic stages 12 to 16. The numbers of nuclei and transcriptional dots in the DA3 and DT1 muscles were counted in col LCRM -moeGFP; S59-mcd8GFP embryos using FISH with intronic probes, coupled with GFP and Topro staining. For each muscle and stage, the mean number of dots (or nuclei) ± standard deviation, and minimum and maximum numbers of dots (or nuclei) are given (n = 20). The same samples were also used for Additional files 15 and 16: Tables S11 and S12. (PDF 174 kb)

Integrated density of col and S59 transcriptional dots during muscle differentiation. For each muscle and stage, the mean intensity of col transcriptional dots in the DA3 and S59 transcriptional dots in the DT1, LO1, VA2 and VT1 muscles ± standard deviation, and the minimum and maximum intensity are given (n = 20). Same embryo samples as in Additional file 4: Table S3. (PDF 21 kb)

Dynamics of col and S59 transcription during muscle differentiation. In the DA3, the number of nuclei and the number of col transcription dots were counted in col LCRM -moeGFP embryos, using FISH with col intronic probes, coupled with GFP and Col staining. In the DT1, LO1, VA2 and VT1, the number of nuclei and the number of S59 transcription dots were counted in S59-mcd8GFP embryos, using FISH with S59 intronic probes, coupled with GFP and S59 staining. For each muscle and stage, the mean number of dots (or nuclei) ± standard deviation, and minimum and maximum numbers of dots (or nuclei) are given (n = 20). The same samples were also used for Additional file 6: Table S4. (PDF 173 kb)

Integrated density of Kr transcriptional dots during muscle differentiation. For each muscle and stage, the mean intensity of Kr transcriptional dots in the DA1, DO1, LL1, LT2, LT4 and VA2 muscles ± standard deviation, and the minimum and maximum intensity are given. Same embryo samples as in Additional file 7: Table S5. (PDF 21 kb)

Expression of generic differentiation and identity realisation genes in DA3 and DT1. (A) Stage 15 col LCRM -moeGFP embryos stained for GFP (green) and β3-tub (blue) coupled to FISH against Mhc mRNA (red). (A’) red channel only. (B) Numbers of nuclei in the DA3 and DT1 muscles, counted in col LCRM -moeGFP; S59-mcd8GFP embryos stained for GFP and Topro, at stages 12, 13, 14, 15 and 16. For each condition, the mean number of nuclei ± standard deviation is shown (n = 20). The same embryo samples were used for Fig. 6. (C, D, F, H, J) Stage 15 wt embryos stained for β3-tub (green) and Col (blue), coupled to FISH of duf (C), Pax (D), mspo (F), kon (H) and Con (J) mRNA (red); (C’, D’, F’, H’, J’) red channel only. A schematic representation of mRNA expression in DA3 and DT1 is shown on the right, with grey intensity reflecting the level of mRNA accumulation. (E, G, I, K) Stage 14 wt embryos stained for Mef2 (red) and Col (blue) coupled to FISH against nascent Pax (E), mspo (G), kon (I) and Con (K) transcripts (green). The DA3 position is identified by Col staining (blue) and DT1 position is surrounded by a grey dotted line. (PDF 4341 kb)

Supplementary Material and Methods. 5’-3’ sequence of the oligonucleotide primers used for making probes for FISH. (PDF 527 kb)

Dynamics of Kr transcription during muscle differentiation. The number of nuclei and the number of Kr transcription dots were counted in Kr GMR80H11 -Gal4; UAS-mcd8GFP embryos, using FISH with Kr intronic probe coupled with GFP and Kr staining. For each muscle and stage, the mean number of dots (or nuclei) ± standard deviation, and minimum and maximum numbers of dots (or nuclei) are given. The number of nuclei is determined using the Kr staining, whose detection decreases from early stage 14 until the end (n = 12, except for conditions indicated with an asterisk, corresponding to stages where determination of nuclei number is limited due to the loss of Kr staining). The same samples were also used for Additional file 8: Table S6. (PDF 179 kb)

Number of RFP positive nuclei in the DA1, DT1 and DA2 muscles in stage 15 wt and col 1 mutant embryos. The RFP-positive nuclei in the DA3, DA2 and DT1 muscles were counted in col ECRM -H2bRFP and col 1 , col ECRM -H2bRFP embryos, stained for RFP and β3-Tub to visualise muscle shape. In col mutant embryos, the DA3 orients like a DA2 muscle (DA3 > DA2). For each muscle, the average number of nuclei ± standard deviation, the minimum and maximum number of nuclei are given (n = 50). (PDF 9 kb)

Quantifying the repartition of transcriptional dots in DA3 subdomains: The methodology. See Methods for details. (PDF 317 kb)

Dynamics of col, S59 and Kr transcription during muscle differentiation. (A) Stage 14 col LCRM -moeGFP embryo stained for GFP (green) and Col (blue), coupled with FISH of nascent col transcripts (red), four adjacent segments are shown. (B) Intensity of each col transcriptional dots in DA3, at stages 12, 13, 14, 15 and 16. Each dot is represented by one open circle; the bar graphs show the mean values and SDs. (C) Box plots showing the number of S59 transcription dots (orange) and nuclei (grey) in the DT1, LO1, VA2 and VT1 muscles, at stages 12, 13, 14, 15 and 16. (D) Percentage of nuclei transcribing S59 in the DT1, VA2 and VT1 muscles, at stage 15. Bar graphs show the mean percentage of active nuclei and error bars correspond to the SEM; statistical analyses were performed using Pearson’s χ2 test. Asterisks show the significance of variation (ns: not significant; (***): P value