Transactivation in Drosophila of Human Enhancers by Human Transcription Factors Involved in Congenital Heart Diseases

Background: The human transcription factors (TFs) GATA4, NKX2.5 and TBX5 form part of the core network necessary to build a human heart and are involved in Congenital Heart Diseases (CHDs). The human natriuretic peptide precursor A (NPPA) and α-myosin heavy chain 6 (MYH6) genes are downstream effectors involved in cardiogenesis that have been demonstrated to be in vitro targets of such TFs. Results: To study the interactions between these human TFs and their target enhancers in vivo, we overexpressed them in the whole Drosophila cardiac tube using the UAS/GAL4 system. We observed that all three TFs up-regulate their natural target enhancers in Drosophila and cause developmental defects when overexpressed in eyes and wings. Conclusions: A strong potential of the present model might be the development of combinatorial and mutational assays to study the interactions between human TFs and their natural target promoters, which are not easily undertaken in tissue culture cells because of the variability in transfection efficiency, especially when multiple constructs are used. Thus, this novel system could be used to determine in vivo the genetic nature of the human mutant forms of these TFs, setting up a powerful tool to unravel the molecular genetic mechanisms that lead to CHDs. Developmental Dynamics 241:190–199, 2012. © 2011 Wiley Periodicals, Inc

Ndae1 expression and regulation in Drosophila embryos.

The construction and prediction of cell fate maps at the whole embryo level require the establishment of an accurate atlas of gene expression patterns throughout development and the identification of the corresponding cis-regulatory sequences. However, while the expression and regulation of genes encoding upstream developmental regulators such as transcription factors or signaling pathway components have been analyzed in detail, up to date the number of cis-regulatory sequences identified for downstream effector genes, like ion channels, pumps and exchangers, is very low. The control and regulation of ion homeostasis in each cell, including at blastoderm stages, are essential for normal embryonic development. In this study, we analyzed in detail the embryonic expression pattern and cis-regulatory modules of the Drosophila Na+-driven anion exchanger 1 (Ndae1) gene, involved in the regulation of pH homeostasis. We show that Ndae1 is expressed in a tight and complex spatial-temporal pattern. In particular, we report that this downstream effector gene is under the control of the canonical dorsal-ventral patterning cascade through dorsal, Toll, twist and snail at early embryogenesis. Moreover, we identify several cis-regulatory modules, some of which control discrete and non-overlapping aspects of endogenous gene expression throughout development

<i>Ndae1</i> expression in mutants of the DV pathway.

<p>(A) <i>dl¹/dl⁴</i> embryo. (B) <i>Tl^10B</i> embryo. (C) <i>twi¹</i> embryo. (D) <i>sna¹⁸</i> embryo. <i>Ndae1</i> is not expressed in <i>dl</i>, <i>twi</i> and <i>sna</i> mutants, while it is expressed ectopically in <i>Tl</i> mutants. (E) Overstained <i>dl¹/dl⁴</i> embryo in which residual anterior expression is observed. All embryos are at cellular blastoderm (stage 5).</p

<i>Cis-</i>regulatory sequence analysis of <i>Ndae1</i>.

<p>(A) Genomic region of <i>Ndae1</i> (2L:7223328..7249343 in the R5.50 <i>Drosophila</i> genome sequence). Two of the nine splicing variants of <i>Ndae1</i> transcripts are represented. Genomic fragments cloned in reporter constructs are indicated by black or green lines corresponding to <i>lacZ</i> or <i>GFP</i> reporter constructs, respectively. Below each fragment there are representative stained embryos. Crossed lines indicate those fragments that do not drive any expression. Reporter constructs expression patterns are detected by immunohistochemistry. (B, C) β-Galactosidase (B) and lacZ mRNA (C) expression directed by pMC024 in rapidly changing cells from stage 10 until stage 15, likely being hemocytes. pMC028 (D) and pMC035 (E) drive expression in the CNS, in lateral cells (arrowheads) and in a dorsal line of amnioserosa cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092956#pone-0092956-g005" target="_blank">figure 5</a>). Fragments pMC025 (F), pMC027 (G) and pMC029 (H) drive the same expression pattern in the anal pads.</p

GFP expression driven by the fourth <i>Ndae1</i> intron.

<p>All embryos carry the pMC035 reporter construct and are stained by immunofluorescence with anti-GFP antibodies (green). (A) Magnification of a dorsal view of a stage 14 embryo double-stained with anti-Mef2 antibodies (red), labeling muscle cells including the cardiac tube. GFP is expressed in amnioserosa cells. (B) Lateral view of a stage 16 embryo double labeled with anti Mef-2 antibodies (red). (C) Magnification of a boxed portion of embryo in (B). The GFP-positive cells do not overlap the Mef-2-positive cells. (D) Lateral view of a stage 16 embryo double labeled with 22C10 antibodies (red). (E) Magnification of a boxed portion of embryo in (D). The GFP-positive cells lay below the 22C10-positive neurons.</p

<i>Ndae1</i> expression in early embryogenesis.

<p><i>Ndae1</i> expression patterns in embryos detected by <i>in situ</i> hybridization with TSA amplification followed by histochemistry (same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092956#pone-0092956-g002" target="_blank">figures 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092956#pone-0092956-g003" target="_blank">3</a>). (A) Ventral expression in a ventral view of a stage 5 embryo. (B) Ventral expression in a lateral view of a stage 5 embryo. (C) Lateral view of the posterior end of a stage 5 embryo. <i>Ndae1</i> is not expressed in pole cells (arrow). (D) Lateral view of the posterior end of a stage 6 embryo. No <i>Ndae1</i> transcripts are observed in pole cells (arrow). (E) Lateral view of a gastrulating stage 6 embryo showing strong expression in the head and weak in the tail. (F) Lateral view of a stage 10 embryo. <i>Ndae1</i> is likely expressed in yolk cells. (G) Stage 12 embryo with amnioserosa expression (arrows) and the beginning of anal pad expression (block arrow). (H) Lateral view of a stage 14 embryo showing expression in lateral cells (arrowhead) and in the anal pads (block arrow).</p

<i>Ndae1</i> expression in stage 15–16 embryos.

<p>(A) Lateral view of a stage 15 embryo showing expression in the CNS, in lateral cells and in the anal pads. (B) Ventral view of a stage 16 embryo expressing <i>Ndae1</i> in the CNS, in lateral cells and in the anal pads. (C) Magnification of a ventral view of a stage 16 embryo expressing <i>Ndae1</i> in the CNS and in lateral cells. (D) Magnification of the posterior end of a stage 16 embryo showing strong expression in the anal pads. Arrowheads: lateral cells. Block arrows: anal pads.</p