Hand is a direct target of the forkhead transcription factor Biniou during Drosophila visceral mesoderm differentiation

<p>Abstract</p> <p>Background</p> <p>The visceral trunk mesoderm in <it>Drosophila melanogaster </it>develops under inductive signals from the ectoderm. This leads to the activation of the key regulators Tinman, Bagpipe and Biniou that are crucial for specification of the circular visceral muscles. How further differentiation is regulated is widely unknown, therefore it seems to be essential to identify downstream target genes of the early key regulators. In our report we focus on the analysis of the transcriptional control of the highly conserved transcription factor Hand in circular visceral muscle cells, providing evidence that the <it>hand </it>gene is a direct target of Biniou.</p> <p>Results</p> <p>Herein we describe the identification of a regulatory region in the <it>hand </it>gene essential and sufficient for the expression in the visceral mesoderm during embryogenesis. We found that <it>hand </it>expression in the circular visceral mesoderm is abolished in embryos mutant for the FoxF domain containing transcription factor Biniou. Furthermore we demonstrate that Biniou regulates <it>hand </it>expression by direct binding to a 300 bp sequence element, located within the 3^rdintron of the <it>hand </it>gene. This regulatory element is highly conserved in different <it>Drosophila </it>species. In addition, we provide evidence that Hand is dispensable for the initial differentiation of the embryonic visceral mesoderm.</p> <p>Conclusion</p> <p>In the present report we show that cross species sequence comparison of non-coding sequences between orthologous genes is a powerful tool to identify conserved regulatory elements. Combining functional dissection experiments <it>in vivo </it>and protein/DNA binding studies we identified <it>hand </it>as a direct target of Biniou in the circular visceral muscles.</p

The Zic family homologue Odd-paired regulates Alk expression in Drosophila

The Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) plays a critical role in the specification of founder cells (FCs) in the Drosophila visceral mesoderm (VM) during embryogenesis. Reporter gene and CRISPR/Cas9 deletion analysis reveals enhancer regions in and upstream of the Alk locus that influence tissue-specific expression in the amnioserosa (AS), the VM and the epidermis. By performing high throughput yeast one-hybrid screens (Y1H) with a library of Drosophila transcription factors (TFs) we identify Odd-paired (Opa), the Drosophila homologue of the vertebrate Zic family of TFs, as a novel regulator of embryonic Alk expression. Further characterization identifies evolutionarily conserved Opa-binding cis-regulatory motifs in one of the Alk associated enhancer elements. Employing Alk reporter lines as well as CRISPR/Cas9-mediated removal of regulatory elements in the Alk locus, we show modulation of Alk expression by Opa in the embryonic AS, epidermis and VM. In addition, we identify enhancer elements that integrate input from additional TFs, such as Binou (Bin) and Bagpipe (Bap), to regulate VM expression of Alk in a combinatorial manner. Taken together, our data show that the Opa zinc finger TF is a novel regulator of embryonic Alk expression

Functional validation of Opa as a putative regulator of <i>Alk</i>.

<p><b>(A)</b> Schematic overview of <i>AlkEB9</i> element (<i>blue line</i>) and the predicted Opa binding sites, referred to as <i>SELEX_OpaBS</i> and <i>JASPAR_OpaBS</i> (in <i>red</i>). <b>(B)</b> PhastCons analysis of sequence conservation among 21 <i>Drosophila</i> species (in <i>green</i>) along <i>AlkEB9</i>, including the predicted Opa binding sites (marked with dashed boxes (i) and (ii)). Base resolution of the phastCons analysis for <i>SELEX_OpaBS</i> (i) and <i>JASPAR_OpaBS</i> sites (ii) is shown, Opa binding sites are highlighted in <i>yellow</i>. <b>(C, D)</b> Binding affinity of Opa to both <i>SELEX_OpaBS</i> and <i>JASPAR_OpaBS</i> sequences as assessed by EMSA. Opa-induced shifts could be competed by addition of non-labelled probe, but not by unlabeled mutated probes. Sequences of both wild type and mutant probes are indicated, mutated residues are depicted in <i>red</i>. <b>(E)</b> ChIP assay employing either pre-immune serum control (<i>dark grey bars</i>) or anti-Opa serum from the same rabbit (<i>light grey bars</i>) for a control intergenic region (<i>control</i>), an Opa binding region within the <i>slp1</i> enhancer (<i>DESE-Opa</i>) and a 140 bp region containing both the <i>SELEX_Opa-BS</i> and <i>JASPAR_OpaBS</i> sequences within <i>AlkEB9</i> (<i>Alk-OpaBS</i>). Enrichment of the different DNA segments in the immunoprecipitates are reported as a percentage of input DNA, with error bars representing the mean ± SD from three technical replicates of the qPCR.</p

Reporter gene analysis identifies putative regulatory elements responsible for <i>Alk</i> expression.

<p><b>(A)</b> Genomic organization of the <i>Alk</i> locus (<i>green</i>) and the neighboring genes <i>CG5065</i> and <i>gprs</i> (<i>light gray</i>). Intron-exon structure of both <i>Alk-RA</i> and <i>Alk-RB</i> transcripts (<i>Alk</i> open reading frame in <i>white</i>) and the analyzed reporter constructs (<i>blue lines</i>) are shown below. The <i>MesoCRM-880</i> and <i>AlkE301</i> CRMs identified in previous ChIP analyses are depicted as <i>grey</i> lines and the 3.6 kb region subjected to Y1H analysis is shown in <i>red</i> (<i>dashed lines</i>). CRISPR/Cas9 deletions disrupting the <i>Alk-RB</i> promoter, <i>Alk</i>^{<i>ΔRB_1</i>.<i>22</i>.<i>2</i>} and <i>Alk</i>^{<i>ΔRB_15</i>.<i>16</i>.<i>2</i>}, are indicated as <i>black dashed lines</i>. <b>(B)</b> <i>AlkEI6</i>.<i>5</i> drives expression in the trunk VM at stage 11; with strongest expression in the founder cells (FCs) (close up; FCs, <i>arrowhead</i>). <b>(C)</b> <i>AlkE4</i> shows a slightly more restricted VM expression pattern compared to that of <i>AlkEI6</i>.<i>5</i>. <b>(D)</b> Similarly, <i>AlkE2</i>.<i>7</i> is expressed in the entire VM with marked stronger expression in the FCs (close up, <i>arrowhead</i>). <b>(E-F)</b> <i>AlkE2</i>.<i>7</i> expression in the FCs is responsive to Alk signaling. <i>lacZ</i> expression in <i>Alk</i>^<i>1</i><i>/Alk</i>^<i>10</i> embryos is weaker when compared to <i>Alk</i>^<i>10</i> heterozygote balanced controls (<i>arrowhead</i>; compare β-gal <i>heatmaps</i> in E and F; note: epidermal β-gal expression in control (E) is due to presence of <i>lacZ</i> balancer; Alk protein is observed in <i>Alk</i>^<i>1</i><i>/Alk</i>^<i>10</i> animals (F) as these alleles encode non-functional Alk protein truncations detected with anti-Alk). <b>(G)</b> Ectopic expression of the Alk ligand Jeb, leads to activation of Alk signaling in all cells of the VM (<i>arrowhead</i>) and is marked by expression of Org-1 in <i>blue</i> resulting in increased <i>lacZ</i> expression from <i>AlkE2</i>.<i>7</i> in all cells of the VM (compare β-gal <i>heatmaps</i> in E and G). Close up regions in E-G are indicated with <i>dashed boxes</i>. Scale bars: 50 μm and 10 μm (embryo and close up, respectively).</p

Contribution of Opa to Alk expression during embryogenesis.

<p><b>(A)</b> Expression of <i>AlkEB9-lacZ</i> (<i>red</i>) in the VM at stage 11 reflects Alk protein expression (<i>green</i>). <b>(B)</b> Loss of expression of <i>AlkEB9-lacZ</i> in the VM of <i>opa</i>^<i>1</i><i>/opa</i>^<i>8</i> mutants (<i>red</i>; compare <i>heatmaps</i> in A’ and B’) along with reduced Alk protein levels in the VM and complete abrogation in epidermis (B). <b>(C)</b> Quantification of Alk protein levels in the VM shows a significant decrease in <i>opa</i>^<i>1</i><i>/opa</i>^<i>8</i> animals (n = 10 animals, p<0.0001). <b>(D-F)</b> Ectopic expression of <i>opa</i>^<i>RNAi</i> with <i>2xPE-GAL4</i> as driver decreases <i>AlkEB9-lacZ</i> reporter expression (compare D’ and E’; quantified in F; n = 10 animals, p<0.0001). <b>(G)</b> Expression of the <i>opa</i>^<i>3D246</i> enhancer trap in Alk-positive cells of the embryonic epidermis (<i>asterisks</i>). <b>(H)</b> The <i>Opa4opt-lacZ</i> reporter, containing Opa binding sites (<i>SELEX_OpaBS</i>), is active in epidermal cells expressing Alk protein (<i>asterisks</i>). <b>(I)</b> Mutation of the <i>SELEX_OpaBS</i> binding sites (<i>Opa4opt-KO-lacZ</i>) results in loss of reporter gene expression in epidermal cells expressing Alk protein (<i>asterisks</i>). Scale bars: 50 μm (A-B’, D-E’) and 10 μm (G-I).</p

The Opa binding site containing CRM is crucial for tissue-specific <i>Alk</i> expression during embryogenesis.

<p><b>(A)</b> Overview of the CRISPR/Cas9 deletions generated (<i>dashed lines</i>) within the <i>AlkEB9</i> and <i>AlkEB8</i> enhancer region of the <i>Alk</i> locus (<i>blue lines</i>). Predicted binding sites shown in <i>red</i>. <b>(B, C)</b> Alk protein is normally expressed in the VM and adjacent epidermis of wild-type embryos at stage 11. Alk activation drives expression of the <i>HandC-GFP</i> reporter in FCs of wild-type embryos (C, inset, stage 11 embryo, <i>green</i>), resulting in midgut formation (C, stage 16, <i>arrowhead</i>). <b>(D, E)</b> <i>Alk</i>^{<i>ΔRB1</i>.<i>22</i>.<i>2</i>} mutants are indistinguishable from <i>Alk</i> null alleles in the VM, exhibiting loss Alk protein and FC specification (E inset, stage 11 <i>Alk</i>^{<i>ΔRB1</i>.<i>22</i>.<i>2</i>} mutant embryo) and lack of midgut formation (E, stage 16, <i>arrowhead</i>). <b>(F-I)</b> Both <i>Alk</i>^{<i>ΔOpaBS_10</i>.<i>36</i>.<i>1</i>} and <i>Alk</i>^{<i>ΔOpaBS_10</i>.<i>28</i>.<i>3</i>} mutants lack Alk protein in epidermis and display reduced levels of Alk protein in the VM (F, H, stage 11; quantified in N), although Alk levels in the VM are sufficient to drive FC specification (G, I, insets represent stage 11 <i>Alk</i>^{<i>ΔOpaBS_10</i>.<i>36</i>.<i>1</i>} and <i>Alk</i>^{<i>ΔOpaBS_10</i>.<i>28</i>.<i>3</i>} mutant embryos). <b>(J, K)</b> <i>Alk</i>^<i>ΔEB8</i> mutants show reduced Alk protein levels in the VM (J, stage 11; quantified in N), while epidermal expression of Alk appears to be unaffected (J, stage 11). Neither FC specification (K, inset, stage 11 <i>Alk</i>^<i>ΔEB8</i> mutant embryo) nor midgut formation (K, stage 16, <i>arrowhead</i>) are impaired in <i>Alk</i>^<i>ΔEB8</i> mutants. <b>(L, M)</b> <i>Alk</i>^{<i>ΔOpaBS+EB8</i>} behaves as an <i>Alk</i> null allele in the VM, lacking detectable Alk protein in the VM and epidermis (L, stage 11), and failing to specify FCs (M, inset, stage 11 <i>Alk</i>^{<i>ΔOpaBS+EB8</i>} mutant embryo) or develop a midgut (M, stage 16, <i>arrowhead</i>). <b>(N)</b> <i>Alk</i>^{<i>ΔOpaBS_10</i>.<i>36</i>.<i>1</i>}, <i>Alk</i>^{<i>ΔOpaBS_10</i>.<i>28</i>.<i>3</i>} and <i>Alk</i>^<i>ΔEB8</i> mutants show a significant decrease in Alk protein in the VM when compared to control embryos (n = 10 animals per genotype, **** p≤0.0001). Scale bars: 50 μm.</p

<i>In vivo</i> Opa binding sites analysis in the <i>AlkEB9</i> element.

<p><b>(A-C’)</b><i>AlkEB9</i> drives <i>lacZ</i> reporter expression in the VM (stage 11, <i>dashed box</i> in A indicates area of close ups in B, B’) and epidermis (stage 14, <i>asterisk</i>). <b>(D-F’)</b> The 154 bp <i>AlkEB9_OpaBS-lacZ</i> reporter drives expression in a pattern similar to that of <i>AlkEB9-lacZ</i>, and is slightly weaker in the VM when compared to <i>AlkEB9</i> (stage 11, compare B’ and E’, <i>dashed box</i> in D indicates area of close ups in E, E’) but intact in the epidermis (F, F’, compare with C, C’; stage 14; <i>asterisks</i>). The wild-type <i>SELEX_OpaBS</i> and <i>JASPAR_OpaBS</i> sequences are shown for <i>AlkEB9-lacZ</i> and <i>AlkEB9_OpaBS-lacZ</i> reporters with the mutated nucleotides in the <i>AlkEB9_OpaKO-lacZ</i> indicated in <i>red</i>. <b>(G-I’)</b> Mutation of the Opa binding sites within <i>AlkEB9_OpaBS</i> (<i>AlkEB9_OpaKO-lacZ</i>) decreases <i>lacZ</i> reporter activity in the VM (stage 11, compare E’ and H’, <i>dashed box</i> in G indicates area of close ups in H, H’), but abolishes it in the epidermis (I, I’, compare with F, F’; stage 14; <i>asterisks</i>). Scale bars: 50 μm and 10 μm (embryo and close ups, respectively). Quantification of <i>lacZ</i> activity in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006617#pgen.1006617.s007" target="_blank">S7 Fig</a>.</p

Functional analysis of <i>Alk-RB</i> by CRISPR/Cas9 mediated deletion.

<p><b>(A)</b> Schematic overview indicating the two independent deletions generated to disrupt the <i>Alk-RB</i> promoter (<i>dashed lines</i>), referred as <i>Alk</i>^{<i>ΔRB_1</i>.<i>22</i>.<i>2</i>} and <i>Alk</i>^{<i>ΔRB_15</i>.<i>16</i>.<i>2</i>}. The <i>Alk</i> locus is shown in <i>green</i> and the first exon of <i>Alk-RB</i> in <i>black</i>. <b>(B)</b> FasIII staining of the chambered midgut (<i>arrowhead</i>) of <i>Alk</i>^{<i>ΔRB_1</i>.<i>22</i>.<i>2</i>} heterozygotes in stage 16 wild-type embryos. Close up of a stage 11 <i>Alk</i>^{<i>ΔRB_1.22.2</i>} heterozygote embryo showing <i>HandC-GFP</i> reporter as a marker for FC specification in the VM. <b>(C)</b> <i>Alk</i>^{<i>ΔRB1</i>.<i>22</i>.<i>2</i>} mutants fail to specify FCs from VM precursors FCs (inset, close up of a stage 11 <i>Alk</i>^{<i>ΔRB1</i>.<i>22</i>.<i>2</i>} mutant embryo), resulting in stage 16 embryos lacking a FasIII positive midgut (<i>arrowhead</i>), phenocopying <i>Alk</i> null mutants. FasIII is still present in the foregut and hindgut (<i>asterisks</i>). <b>(D-F)</b> <i>Alk</i> mRNA is expressed in the presumptive AS (D, stage 6), the VM (E, stage 10) and the CNS (F, stage 16). <b>(G-L)</b> <i>Alk</i>^{<i>ΔRB1</i>.<i>22</i>.<i>2</i>} mutant embryos lack Alk protein in the presumptive AS (J, stage 9, <i>asterisk</i>; compare with G), the VM (K, stage 10, <i>arrowhead</i>, compare with H) and the epidermis (K, stage 10, <i>asterisks</i>, compare with H). In contrast to the lack of <i>Alk</i> mRNA and protein in the AS, VM and epidermis, <i>Alk</i>^{<i>ΔRB1</i>.<i>22</i>.<i>2</i>} embryos display normal Alk protein levels in the CNS (stage 16, compare L with I). Scale bars: 50 μm.</p

<i>In vivo</i> characterization of <i>Alk</i> locus five prime regulatory regions.

<p><b>(A)</b> Schematic representation of the 5’ region of the <i>Alk</i> locus together with the regions covered by transgenes employed for reporter activity analysis, namely <i>AlkEB8</i> and <i>AlkEB9</i> (<i>black lines</i>). <i>AlkE2</i>.<i>7</i> is shown for comparison (<i>blue</i>). <b>(B, D)</b> <i>AlkEB8-lacZ</i> and <i>AlkEB9-lacZ</i> transgenic reporter flies showed VM activity (<i>arrowheads</i>, <i>dashed boxes</i> in B’ and D’ indicate close ups in B” and D”), with stronger expression of <i>AlkEB9-lacZ</i> (compare B, B” and D, D”; quantified in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006617#pgen.1006617.s005" target="_blank">S5 Fig</a>). <i>AlkEB9-lacZ</i> also displays reporter activity in the epidermis (E compare with C, stage 14; <i>asterisks</i>). <b>(F-H)</b> The gut phenotype of <i>Alk</i> loss of function mutants (G, <i>arrowhead</i>) can be rescued by ectopic expression of Alk driven by <i>AlkEB9-GAL4</i> (H), indicating that <i>AlkEB9</i> contains sufficient regulatory information to drive expression in the VM during embryogenesis. Scale bars: 50 μm and 10 μm (embryo and close up, respectively).</p