Top Ftz-F1-responsive genes are co-expressed with <i>ftz</i>.

<p>Fluorescent double in situ hybridization was performed against <i>ftz</i> and each target gene, as indicated. <i>ftz</i> stripes (A-J, red); target gene stripes (A’–J’, green); overlay (A”–J”, yellow). Some or all of the target genes’ expression patterns overlap <i>ftz</i> expression during the blastoderm stage for all candidate targets except <i>tal</i>. (A’) <i>ken</i>, (B’) <i>en</i>, (C’) <i>aay</i>, (D’) <i>mid</i>, (E’) <i>tal</i>, (F’) <i>5-HT2A</i>, (G’) <i>trn</i>, (H’) <i>hh</i>, (I’) <i>Antp</i>, (J’) <i>blot</i>. Photographs of embryos from confocal microscopy are shown; anterior, left.</p

Activation of Ftz-F1-Responsive Genes through Ftz/Ftz-F1 Dependent Enhancers

<div><p>The orphan nuclear receptor Ftz-F1 is expressed in all somatic nuclei in <i>Drosophila</i> embryos, but mutations result in a pair-rule phenotype. This was explained by the interaction of Ftz-F1 with the homeodomain protein Ftz that is expressed in stripes in the primordia of segments missing in either <i>ftz-f1</i> or <i>ftz</i> mutants. Ftz-F1 and Ftz were shown to physically interact and coordinately activate the expression of <i>ftz</i> itself and <i>engrailed</i> by synergistic binding to composite Ftz-F1/Ftz binding sites. However, attempts to identify additional target genes on the basis of Ftz-F1/ Ftz binding alone has met with only limited success. To discern rules for Ftz-F1 target site selection in vivo and to identify additional target genes, a microarray analysis was performed comparing wildtype and <i>ftz-f1</i> mutant embryos. Ftz-F1-responsive genes most highly regulated included <i>engrailed</i> and nine additional genes expressed in patterns dependent on both <i>ftz</i> and <i>ftz-f1</i>. Candidate enhancers for these genes were identified by combining BDTNP Ftz ChIP-chip data with a computational search for Ftz-F1 binding sites. Of eight enhancer reporter genes tested in transgenic embryos, six generated expression patterns similar to the corresponding endogenous gene and expression was lost in <i>ftz</i> mutants. These studies identified a new set of Ftz-F1 targets, all of which are co-regulated by Ftz. Comparative analysis of enhancers containing Ftz/Ftz-F1 binding sites that were or were not bona fide targets in vivo suggested that GAF negatively regulates enhancers that contain Ftz/Ftz-F1 binding sites but are not actually utilized. These targets include other regulatory factors as well as genes involved directly in morphogenesis, providing insight into how pair-rule genes establish the body pattern.</p></div

Summary of Ftz/Ftz-F1 dependent target genes and CREs.

<p>Summary of Ftz/Ftz-F1 dependent target genes and CREs.</p

Target gene CREs direct <i>ftz</i>-dependent striped expression patterns.

<p>Expression of enhancer-<i>lacZ</i> reporter constructs, as indicated, in transgenic embryos is shown. (A-G) Early transgene expression. (A’-G’) Late transgene expression. Note that β-galactosidase is stable and accumulates in embryos such that expression appears stronger at late germband extension. Expression of <i>ken-lacZ</i>, <i>mid-lacZ</i>, <i>trn-lacZ</i>, <i>hh-lacZ</i>, <i>Antp-lacZ</i> and <i>blot-lacZ</i> was similar to the corresponding endogenous gene. <i>5-HT2A-lacZ</i> was expressed weakly. (A”-G”) Expression of enhancer-<i>lacZ</i> reporter transgenes (brown) in <i>ftz</i> mutant embryos. To identify <i>ftz</i> mutants, in situ hybridization was used to detect <i>en</i> (blue), which is expressed in 14 stripes in wildtype embryos and 7 stripes in <i>ftz</i> embryos.</p

Transcription factor binding motifs within the confirmed and false enhancer groups.

<p>Transcription factor binding motifs within the confirmed and false enhancer groups.</p

Identification of candidate Ftz/Ftz-F1 dependent enhancers.

<p> A screen shot from Flybase GBrowse showing genomic regions around Ftz-F1-responsive genes. Published BDTNP data available at <a href="http://bdtnp.lbl.gov/Fly-Net/browseChipper.jsp" target="_blank">http://bdtnp.lbl.gov/Fly-Net/browseChipper.jsp</a> was downloaded and all interval coordinates along with the peak binding positions were remapped from release 4 to release 6 of the <i>Drosophila</i> genome using the Coordinate Converter provided by Flybase. The Ftz binding data was then uploaded into Flybase GBrowse for visualization. Candidate enhancers (red arrows) were identified using three major criteria: 1) Location near Ftz-F1-responsive target gene (light blue); 2) Ftz ChIP-chip binding, yellow arrow (1% FDR) or orange arrow (25% FDR); 3) candidate Ftz-F1 binding sites (thin purple arrows). Open chromatin (green arrows), was also found at blastoderm stage at all enhancers. Enhancer chromosomal locations (flybase v. 6) are: (A) <i>ken</i> 2R:23868513..23869466, (B) <i>en</i> 2R:11524814..11525958, (C) <i>aay</i> 3L:9416225..9417230, (D) <i>mid</i> 2L:5473726..5474732, (E) <i>5-HT2A</i> 3R:4612279..4614294, (F) <i>trn</i> 3L:13073977..13075024, (G) <i>hh</i> 3R:23138154..23139164, (H) <i>Antp</i> 3R:6948758..6949766, (I) <i>blot</i> 3L:17409206..17410251.</p

Ftz-F1-responsive genes are regulated by Ftz and Ftz-F1.

<p>(A-I) The expression patterns of nine of the top candidate Ftz-F1 target genes from the microarray are shown. All are expressed in stripes in early embryos. (A) <i>ken</i>, (B) <i>aay</i>, (C) <i>mid</i>, (D) <i>tal</i>, (E) <i>5-HT2A</i>, (F) <i>trn</i>, (G) <i>hh</i>, (H) <i>Antp</i>, (I) <i>blot</i>. (A’–I’) Expression was examined in <i>ftz</i> mutant embryos. (A”–I”) Expression was examined in <i>ftz-f1</i> germline clone (GLC) embryos.</p

Microarray identification of Ftz-F1-responsive genes in <i>Drosophila</i> embryos.

<p>RNA was extracted from control or <i>ftz-f1</i> mutant embryos at stages 5, 6, and 8, as indicated, and used to synthesize cDNA for hybridization to Affymetrix <i>Drosophila</i> 2.0 expression arrays. The dendrogram shows Pearson correlation coefficients of mean expression levels across probesets under assayed conditions. The heatmap shows expression levels of probesets that increased between stages 5 and 6 in wildtype embryos and were expressed at lower levels in <i>ftz-f1</i> mutant embryos than control. Genes were sorted from highest to lowest average fold change in expression between control and <i>ftz-f1</i> mutants after cellularization.</p

Top Ftz-F1-responsive genes are co-expressed with <i>ftz</i>.

<p>Fluorescent double in situ hybridization was performed against <i>ftz</i> and each target gene, as indicated. <i>ftz</i> stripes (A-J, red); target gene stripes (A’–J’, green); overlay (A”–J”, yellow). Some or all of the target genes’ expression patterns overlap <i>ftz</i> expression during the blastoderm stage for all candidate targets except <i>tal</i>. (A’) <i>ken</i>, (B’) <i>en</i>, (C’) <i>aay</i>, (D’) <i>mid</i>, (E’) <i>tal</i>, (F’) <i>5-HT2A</i>, (G’) <i>trn</i>, (H’) <i>hh</i>, (I’) <i>Antp</i>, (J’) <i>blot</i>. Photographs of embryos from confocal microscopy are shown; anterior, left.</p

Ftz-F1 binding sites are overrepresented in Ftz genomic binding peaks.

<p>(A) Genomic positions of Ftz binding based on Ftz ChIP-chip data from BDTNP is shown schematically mapped to the four <i>Drosophila</i> chromosomes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163128#pone.0163128.ref039" target="_blank">39</a>]. Red lines represent strong Ftz binding; green lines represent weak Ftz binding. The genomic loci of candidate Ftz-F1 targets from the microarray are indicated in pink. <i>ftz</i>-responsive genes not found in this study are indicated in black. Positions of <i>FRT[2A]</i> and <i>ftz-f1</i>, used to generate <i>ftz-f1</i> germline clones, are indicated in blue. (B,C) Over-represented sequences in the Ftz ChIP-chip binding data, identified by (B) DREME and (C) MEME. Ftz-F1 candidate binding sites (core, AAGG) are overrepresented in genomic regions where Ftz binds DNA.</p