Transactivation in Drosophila of human enhancers by human transcription factors involved in congenital heart diseases

Abstract

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.

Fig. 1

GATA4, Nkx2.5, and TBX5 transactivate the ANF300 enhancer in Drosophila embryos. Detection of GFP expression driven by the ANF300 enhancer by in situ hybridization using HRP staining. All embryos are shown with the anterior end to the left. Left column: Stage 3–14 embryos. Right column: Stage 15–16 embryos (dorsal side up). All embryos carry the ANF-hs43-nGFP reporter and the Hand-GAL4 driver. In wild-type embryos (A, B), no GFP is detected. In embryos carrying also the UAS-GFP (C, D), UAS-GATA4 (E, F), UAS-Nkx2.5 (G, H), or UAS-TBX5 (I, J) transgene, GFP is detected in the cardiac tube (block arrows) and in the visceral mesoderm (arrows). Staining with the sense GFP probe did not produce any signal except for salivary glands (arrowheads, data not shown).

Fig. 2

GATA4, Nkx2.5, and TBX5 transactivate the MYH6 enhancer in Drosophila embryos. Detection of GFP expression driven by the MYH6 enhancer by in situ hybridization using HRP staining. Left column: Stage-13–14 embryos. Right column: Stage-15–16 embryos (dorsal side up). All embryos carry the MYH6-hs43-nGFP reporter and the Hand-GAL4 driver. In wild-type embryos (A, B), no GFP is detected. In embryos carrying also the UAS-GFP (C, D), UAS-GATA4 (E, F), UAS-Nkx2.5 (G, H), or UAS-TBX5 (I, J) transgene, GFP is detected in the cardiac tube (block arrows) and in the visceral mesoderm (arrows). Staining with the sense GFP probe did not produce any signal except for salivary glands (arrowheads, data not shown).

Fig. 3

Detection of GFP by RT-PCR. RT-PCR was performed on the same embryos as those of Figure 1. A: RT-PCR detection of GFP. B: RT-PCR detection of the rp49 housekeeping gene. 1, mock PCR; 2, white; 3, Hand-GAL4; ANF300MYH6-hs43-nGFP; 4, Hand-GAL4; ANF300MYH6-hs43-nGFP + UAS-GATA4; 5, Hand-GAL4; ANF300MYH6-hs43-nGFP + UAS-Nkx2.5; 6, Hand-GAL4; ANF300MYH6-hs43-nGFP + UAS-TBX5; 7, Hand-GAL4; ANF300MYH6-hs43-nGFP + UAS-GFP. A specific GFP band is detected only in GATA4, Nkx2.5, and TBX5 overexpressing embryos (lanes 4–6) and in the positive control (lane 7).

Fig. 4

tin overexpression transactivates the ANF300 and MYH6 enhancers. Detection of GFP expression driven by the ANF300 and MYH6 enhancers upon tin overexpression by in situ hybridization. Left two columns: Germ band–extended embryos. Right two columns: Germ band–retracted embryos. Embryos carrying only the reporter and GAL4 transgenes (top row) do not show GFP expression. Embryos overexpressing GFP (second row), Nkx2.5 (third row), or tin (fourth row) through the 24B-GAL4 driver show GFP expression in the somatic and visceral (arrows) mesoderm. Overexpression of tin is detected by immunohistochemistry using anti-Tin antibodies (bottom row).

Fig. 5

Endogenous Drosophila TFs are not mis-expressed by the over-expression of GATA4, Nkx2.5, or TBX5. Detection of tin protein by immunohistochemistry and of pnr, Doc1, and mid transcripts by in situ hybridization in control embryos (top) and in embryos overexpressing GATA4, Nkx2.5, or TBX5 (see left) in the whole mesoderm through the 24B-GAL4 driver. A: Germ band–extended embryos. B: Germ band–retracted embryos. Each gene is expressed at normal levels in all embryos.

Fig. 6

Developmental defects caused by GAT4, Nkx2.5, or TBX5 over-expression in eyes and in wings. Reduced eyes and wings caused by the overexpression of GATA4 (C, D), Nkx2.5 (E, F) or TBX5 (G, H) in the eye through the GMR-GAL4 driver (C, E, G) or in the wing through the vg-GAL4 driver (D, F, H). Top: Control flies carrying only the GMR-GAL4 (A) or vg-GAL4 (B) drivers. All flies were raised at 27°C, but phenotypic effects were visible also at 18°C (data not shown and Supp. Fig. S2).