The three Tin-binding motifs in <i>Unc-5-GH</i> element mediate induction of <i>Unc-5</i> transcription by Tin in vivo.

<p>Ectopic expression of <i>tin</i> in <i>engrailed</i> stripes results in the induction of the <i>Unc-5-GH</i> enhancer in a striped pattern (A-A”; arrowheads in A’). Tin expression in ectodermal stripes is labeled with anti-Tin antibody (magenta). Anti-GFP antibody was used to reveal the expression of the reporter (green). As expected, embryos carrying the <i>R8</i>,<i>9</i>,<i>10-GH</i> mutant reporter display little or no GFP induction in the stripes (B-B”; arrowhead-asterisks, and E). (C-C”) The <i>wild-type Unc-5-GH</i> enhancer induces expression of the GFP reporter (green) in all CBs and PCs. Mef2 (red, D or magenta, D’) and Zfh1 (blue, D or magenta D”) antibodies are used to reveal CBs or PCs, respectively. The <i>R8</i>,<i>9</i>,<i>10-GH</i> enhancer generates a GFP expression pattern similar to that of the <i>wild-type Unc-5-GH</i> enhancer in <i>tin-ABD; tin</i>^<i>346</i>/<i>tin</i>^<i>346</i> embryos (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137688#pone.0137688.g004" target="_blank">Fig 4B–4B”</a>) with near complete loss of GFP expression in Tin-positive CBs (D’ and F) and a reduction of expression in PCs (D”). (E and F) Quantification of GFP expression by the mutated <i>Unc-5</i> enhancer (<i>R8</i>,<i>9</i>,<i>10-GH</i>) in ectodermal stripes (E) and CBs (F). Genotypes are indicated on the X axis and fluorescence intensities on the Y axis. For all quantifications GFP expression in sensory neurons (SNs) was used as internal control, as the fluorescence in these cells is not affected. In E, fluorescence is significantly reduced (<i>p</i><1.2 x 10⁻¹⁴) in <i>engrailed</i> stripes of embryos with the mutant reporter compared to those of embryos carrying the <i>wild-type</i> reporter [from 2.8 ± 0.26 s.e.m. for the <i>wild-type Unc-5-GH</i> reporter to 0.25 ± 0.16 s.e.m. for the <i>R8</i>,<i>9</i>,<i>10-GH</i> reporter (with unchanged SNs’ fluorescence of 2.52 ± 0.192 s.e.m and 2.8 ± 0.177 s.e.m, respectively)]. (F) GFP fluorescence is also significantly reduced (<i>p</i><7 x 10⁻¹⁸) in CBs from 1.98 ± 0.127 s.e.m. for the <i>wild-type Unc-5-GH</i> reporter to 0.28 ± 0.052 s.e.m. for the <i>R8</i>,<i>9</i>,<i>10-GH</i> mutant reporter. In F, fluorescence in SNs is not affected with unchanged SNs’ fluorescence of 3.75 ± 0.44 s.e.m in <i>Unc-5-GH</i> embryos and 3.79 ± 0.36 s.e.m in <i>R8</i>,<i>9</i>,<i>10-GH</i> embryos, respectively. All panels are lateral views of stage 14–15 embryos with dorsal side up and anterior to the left. A magnification of the regions delineated by insets is shown for each panel.</p

The Unc-5 Receptor Is Directly Regulated by Tinman in the Developing <i>Drosophila</i> Dorsal Vessel

<div><p>During early heart morphogenesis cardiac cells migrate in two bilateral opposing rows, meet at the dorsal midline and fuse to form a hollow tube known as the primary heart field in vertebrates or dorsal vessel (DV) in <i>Drosophila</i>. Guidance receptors are thought to mediate this evolutionarily conserved process. A core of transcription factors from the NK2, GATA and T-box families are also believed to orchestrate this process in both vertebrates and invertebrates. Nevertheless, whether they accomplish their function, at least in part, through direct or indirect transcriptional regulation of guidance receptors is currently unknown. In our work, we demonstrate how Tinman (Tin), the <i>Drosophila</i> homolog of the Nkx-2.5 transcription factor, regulates the Unc-5 receptor during DV tube morphogenesis. We use genetics, expression analysis with single cell mRNA resolution and enhancer-reporter assays in vitro or in vivo to demonstrate that Tin is required for Unc-5 receptor expression specifically in cardioblasts. We show that Tin can bind to evolutionary conserved sites within an Unc-5 DV enhancer and that these sites are required for Tin-dependent transactivation both in vitro and in vivo.</p></div

Ectopic expression of Tin induces <i>Unc-5</i> mRNA expression in vivo.

<p>(A-A’) in situ hybridization for endogenous mRNA expression of <i>Unc-5</i> does not show any striped ectodermal signal (magenta, arrowheads with asterisks). (B–B’) ectopic expression of <i>tin</i> in ectodermal stripes (green) from a <i>UAS-tin</i> transgene using <i>engrailed-Gal4</i> induces endogenous <i>Unc-5</i> mRNA in a striped pattern (compare A’ with B’, arrowheads). Engrailed stripes are labelled green by co-expression of Tau-Myc from a <i>UAS-tau-myc</i> construct. All panels are lateral views with anterior to the left. A magnification of the regions delineated by insets is shown for each panel. <i>Unc-5</i> in situ fuorescence in <i>engrailed</i> stripes was quantified (0.33±0.06 s.e.m. or -0.03±0.01 s.e.m. in <i>tin</i>-expressing <i>en</i>-stripes A, A’ or <i>en</i>-stripes not expressing <i>tin</i> respectively B, B’, p<0.005, n = 12). Confocal XZ sections are presented below each panel (location of the sections is indicated in the upper panel a white line) where Unc-5 specific signal can be detected colocalizing with <i>en</i>-stripes when tin is expressed (B, B’) but not in a <i>wild-type</i> embryo (A, A’).</p

<i>tin</i> regulates <i>Unc-5</i> expression in vivo.

<p>(A) Organization and cellular composition and development of the Drosophila dorsal vessel. Schematic represents Drosophila DV at embryonic stages 15 (migrating cells, top) and 17 (tubular heart already formed, bottom) with different cell types color-coded based on the marker TF expressed. Aortic portion is oriented anteriorly and the beating portion (heart) posteriorly. CBs are divided into Tin- (light green) or Svp-expressing SMCs (dark green) subtypes. The three most posterior pairs of Svp-expressing CBs will make the future ostial (inflow valve) cells. Pericardial cells (gray) surround the CBs on their ventrolateral side and fall into two major types; Tin-positive or Tin-negative PCs (not colored). Bottom is a schematic cross section of the heart lumen at stage 17 where CBs on opposite sides take a crescent-like shape after contact, leaving in between them a hollow luminal space. (B) <i>Unc-5</i> mRNA (magenta) is present in the DV in embryos heterozygous for <i>tin</i>^<i>346</i> (B). In <i>tinABD;tin</i>^<i>346</i><i>/tin</i>^<i>346</i> homozygous mutant embryos, however, <i>Unc-5</i> mRNA expression is significantly reduced (C). Anterior side of the embryo is to the left in panels B and C.</p

The <i>Unc-5</i> heart enhancer element is regulated by <i>tin in vivo</i>.

<p>(A–A”) Reporter gene (green) is expressed in all CBs and PCs in <i>wild-type</i> embryos. (B–B”) In <i>tin-ABD</i>; <i>tin</i>^<i>346</i>/<i>tin</i>^<i>346</i> mutant embryos, where <i>tin</i> is only absent in the DV, reporter gene expression is almost absent in CBs while it is only partially downregulated in PCs. Note the unchanged GFP expression in sensory neurons (arrowheads in A” and B”) in <i>wild-type</i> and mutant backgrounds. Quantification of GFP expression in CBs (D) or PCs (E). Genotypes of embryos are indicated on the X axis and fluorescence on the Y axis. GFP expression in SNs is used as internal control and their fluorescence is not affected in <i>tin-ABD</i>; <i>tin</i>^<i>346</i>/<i>tin</i>^<i>346</i> mutant background (3.86 ± 0.4 s.e.m and 3.82 ± 0.29 s.e.m, in <i>wild-type</i> or <i>tin</i> mutants respectively). However, the signal is significantly (<i>P</i><6 x 10⁻¹⁴) reduced in CBs, from 1.98 ± 0.128 to 0.173 ± 0.04 (D). PCs show a slight reduction in signal (E), from 3.4 ± 0.2 in <i>wild-type</i> to 2.47 ± 0.21 in <i>tin-ABD;tin</i>^<i>346</i> (<i>P</i><6.2 x 10⁻⁴). All panels are dorsal views with anterior to the left. A magnification of the regions delineated by insets is shown for each panel. All embryos are aged from early to late stage 15. CB cardioblast, PC pericardial cell, SMC Svp positive myocardial cells, SN sensory neuron.</p

The three Tin-binding motifs in the <i>Unc-5-GH</i> element mediate induction of <i>Unc-5</i> transcription by Tin in vitro.

<p>Mutating the three Tin-binding motifs (singly or in combination) results in reduction of the Tin transcriptional activity as observed in our luciferase assays. Each mutation on a Tin-binding site is represented as a red cross, over the corresponding site (represented as a box) at the left of the graph. Error bars represent the standard deviation and the significance of pairwise comparisons is indicated by *** (p<0.005).</p

The <i>Unc-5-GH</i> reporter is expressed in all cardioblasts and pericardial cells.

<p>(A) Schematic representation of the positions and the relative sizes of the dissected fragments from the <i>Unc-5</i> locus. Reporter constructs were generated by fusing each fragment to a GFP open reading frame. Examination of GFP expression in transgenic lines carrying any of these reporters indicates potential enhancer activity of each fragment. All reporters containing the smallest (bottom) fragment revealed activity in the DV. The smallest fragment (GH-reporter) was chosen for further examinations. (B-D”) We used specific markers to label CBs: Mef2, (B and B’, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137688#pone.0137688.ref029" target="_blank">29</a>]), PCs: all PCs with Zfh1 (B and B”,[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137688#pone.0137688.ref030" target="_blank">30</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137688#pone.0137688.ref031" target="_blank">31</a>]) or subsets with Eve and Odd (C-C”, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137688#pone.0137688.ref032" target="_blank">32</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137688#pone.0137688.ref033" target="_blank">33</a>]); and a Svp-LacZ reporter for labelling a set of Tin-negative myocardial cells, also known as Seven Up (Svp)-positive myocardial cells (SMCs) (D-D”, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137688#pone.0137688.ref017" target="_blank">17</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137688#pone.0137688.ref034" target="_blank">34</a>]). GFP expression (green) is present in all CBs (B’; magenta) and PCs (B”; magenta). (C–C”) Odd- (C’; magenta) and Eve-positive (C”; magenta) PCs express GFP (green) driven by <i>Unc-5-GH</i> enhancer fragment. (D–D”) <i>Unc-5-GH</i> reporter also drives expression in Tin-positive CBs and Svp myocardial cells (SMC). (E-E”) Correlation between GH enhancer expression pattern and endogenous <i>Unc-5</i> mRNA expression in the DV was examined by in situ hybridization. Colocalization of Tau-Myc expression pattern (labeled in green), driven by the enhancer (<i>Unc-5 GH-Gal4</i>), and <i>Unc-5</i> mRNA (magenta) indicates perfect overlap between the two. XZ an YZ sections are presented at the bottom and right of the main panel (E) and white lines indicate their location. CB cardioblast, PC pericardial cell. All panels are dorsal views with anterior to the left. A magnification of the regions delineated by insets is shown for each panel. All embryos are stage 15.</p