Col direct control of <i>ap</i> expression in Ap neurons.

<p>(A) Annotation of the Col peak in <i>ap</i>, same representation as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133387#pone.0133387.g002" target="_blank">Fig 2A</a>; 35.8 kb of the <i>ap</i> genomic region are shown (Chr2R: 1.593.000–1.628.800); the previously described apC enhancer is represented by a blue box. (B) <i>ap</i>_Col (GFP) expression in the dAp (yellow arrow) and Tv1-Tv4 neurons (white arrow) in stage 15 embryos, ventral view. (C) <i>ap</i>_Col^mut expression is severely reduced in dAP neurons and Tv neurons. (D,D’) Close up view of 4 segments of stage 16 embryos, showing the specific overlap between Col (red) and <i>ap</i>_Col (green) in the Tv1 and dAp neurons. (E) all Tv neurons express <i>ap</i>_Col and Eya. (F,G) <i>ap_</i>Col^mut expression is lost in dAp and strongly reduced in Tv neurons.</p

Cross-regulation between <i>eya</i> and <i>col</i> is required to specify dorso-lateral muscles.

<p>(A) <i>eya_</i>Col (GFP) expression in the DA3 muscle (arrows) in stage 15 embryos, is not detected for <i>eya_</i>Col^mut (B). (C-E) Triple staining of st11 wt (C), <i>+;col</i>^<i>eCRM</i><i>-lacZ</i> (D) and <i>col</i>^<i>1</i><i>;col</i>^<i>eCRM</i><i>-lacZ</i> (E) embryos for Nau (blue), <i>eya</i> transcripts (red), and either Col (C) or β-galactosidase (D,E) (green), shows co-expression of Col, <i>col</i>^e-CRM-LacZ, Nau and <i>eya</i> in DL muscle PCs (white arrow). (C’-E’) only Nau and <i>eya</i> stainings are shown. <i>eya</i> transcription is specifically lost in DL PCs in <i>col</i>^<i>1</i> mutant embryos (E,E’); dorso-lateral view of the T2 and T3 segments is shown. (F) Schematic drawing of the dorsal, dorso-lateral and lateral transverse muscles in a stage 16 wt embryo, with the DA3 muscle in red and the LL1 muscle indicated by an arrow. (G, H) Staining of stage 16 wt (G) and (H), <i>eya</i>^{<i>cIi-IID</i>}<i>/Df(2L)BSC354</i> (null) mutant embryos for Col (red) and β3-tubuliin (green). Lateral view of 3 segments. In absence of <i>eya</i>, Col expression is lost in most segments, the LL1 muscle is missing (white arrow) and the DA3 muscle (asterisk) malformed. White brackets indicate dorsal, unaffected muscles while the lateral transverse and ventral muscles (yellow brackets) are moderately affected. (I-J) Col immunostaining of st.16 wt (I), and (J) <i>eya</i> mutant embryos, showing the loss of Col muscle expression in most segments (see also G, H), in <i>eya</i> mutants. (K, L) promuscular Col expression, early stage 11, is reduced in <i>eya</i> mutant embryos (L), compared to wt (K).</p

Scheme for Col direct regulation of <i>ap</i> and <i>eya</i> in specific neuron and muscle lineages.

<p>Col directly controls <i>ap</i>_Col and <i>eya</i>_Col CRM activity in the Tv and dAP neurons and <i>eya</i>_Col and <i>col</i>_Col CRM in the DA3 muscle lineage. The two Col binding sites in <i>eya</i>_Col CRM are not functionally equivalent (double arrow). <i>eya</i> and <i>col</i> cross-regulate each other in the DA3 PC. Control of <i>DopR</i> and <i>Nplp1</i> expression in Tv and dAP neurons (Baumgardt et al., 2007) could be indirect.</p

Col direct control of <i>eya</i> expression in Ap neurons.

<p>(A) Annotation of the Col peaks in <i>eya</i>, same representation as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133387#pone.0133387.g002" target="_blank">Fig 2A</a>; 24 kb of the <i>eya</i> genomic region are shown (Chr2L: 6.524.500–6.548.500); the summits of the two ChIP-Col peaks are numbered 1 and 2. (B, C) <i>eya_</i>Col (GFP) expression in the dAp (yellow arrow) and Tv1-Tv4 neurons (white arrow) in stage 16 embryos, ventral view. (C) Close up view of abdominal segments, showing the specific overlap between Col (red) and <i>eya_</i>Col (green) in the dAp (yellow arrow) and Tv1 neurons (white arrow). (D, E) <i>eya_</i>Col^mut expression is lost in dAP neurons and reduced in TV neurons. (F, G) Mutation of the Col binding site 2 (G), but not site 1 (F) eliminates <i>eya_</i>Col (RFP) expression in dAp neurons (yellow arrow).</p

Col control of <i>cnc</i> and <i>Ama</i> expression in the head.

<p>(A) Annotation of the Col peak in <i>cnc</i>, adapted from <i>Gene Browser</i> (GEO submission GSE67805). 39,5 kb of <i>cnc</i> genomic region are shown (Chr3R: 19.009.000–19.048.500) with the Flybase gene annotation indicated by bars (transcribed regions) and intervening blue lines (introns). Black arrows indicate the direction of transcription of <i>cnc</i> and <i>fuzzy onions</i> (<i>fzo</i>), <i>inwardly rectifying potassium channel 1</i> (<i>Irk1</i>). The <i>cnc</i> transcripts coding for the protein isoforms CncA and CncB are indicated. ChIP-seq data for Col (green) substracted from HA (mock) data (red) are shown on the bottom. The Col Dam-ID binding regions [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133387#pone.0133387.ref059" target="_blank">59</a>] are indicated by yellow bars, top line. The summit of the ChIP-Col peak identified by SISSRs and position of the Col binding site(s) identified by MEME are indicated by blue and violet lines, respectively; the position of <i>cnc_</i>Col is represented by a black box; scale is indicated. (B-D) Ventral anterior views of stage 11 embryos. (B) Overlap between <i>cnc_</i>Col (GFP, green) and Col (red) expression in the HL (white arrow). (C) <i>cnc_</i>Col and (D) <i>cnc_</i>Col^mut mRNA expression, showing down-regulation of <i>cnc_</i>Col^mut in the mandibular segment (open arrow). (E,F) <i>Ama</i>_Col, (G,H) <i>Ama</i>_Col^mut expression in stage 10 (E,G) and 11 (F,H) embryos. HL <i>Ama</i>_Col expression (arrow) is lost in <i>Ama</i>_Col^mut (open arrow). (I, J) Overlap between <i>Ama</i> (red), Col (blue), and <i>col</i>2.6–0.9moeGFP (green) expression in the HL (white arrow) in stage 11 wt (I), and <i>col</i>^<i>1</i> mutant embryos (J). Separate signals for <i>Ama</i>, GFP (I, J) and Col (inset in I, left panel) are shown in black and white. <i>Ama</i> expression is specifically lost in the HL in <i>col</i>^<i>1</i> mutants. The asterisk in C, D, F, H, J indicates <i>Ama</i> and <i>cnc</i> expression independent on Col.</p

Candidate Col direct targets and linked CRMs.

<p>n.d.: not detected</p><p>Candidate Col direct targets and linked CRMs.</p