Nkx2-5 Regulates Tdgf1 (Cripto) Early During Cardiac Development

Abstract

Congenital Heart Disease (CHD) is the most frequent and deadly birth defect. Patients with CHD that survive the neonatal period often progress to develop advanced heart failure requiring specialized treatment including cardiac transplantation. A full understanding of the transcriptional networks that direct cardiac progenitors during heart development will enhance our understanding of both normal cardiac function and pathological states. These findings will also have important applications for emerging therapies and the treatment of congenital heart disease. Furthermore, a number of shared transcriptional pathways or networks have been proposed to regulate the development and regeneration of tissues such as the heart. We have utilized transgenic technology to isolate and characterize cardiac progenitor cells from the developing mouse heart and have begun to define specific transcriptional networks of cardiovascular development. Initial studies identified Tdgf1 as a potential target of Nkx2-5. To mechanistically dissect the regulation of this molecular program, we utilized an array of molecular biological techniques to confirm that Nkx2-5 is an upstream regulator of the Tdgf1 gene in early cardiac development. These studies further define Nkx2-5 mediated transcriptional networks and enhance our understanding of cardiac morphogenesis.

Keywords: Cardiogenesis; Mouse; Nkx2-5; Tdgf1.

Figure 1. Tdgf1 transcript expression in WT and Nkx2-5 null cardiac progenitor cells

(A) Selected transcripts that were significantly dysregulated at defined developmental stages in the Nkx2-5 null background compared to the age-matched WT cardiac progenitor cells. Note the statistically significant downregulation of Tdgf1. (B) EYFP is directed to the cardiac progenitor cells in the 6kbNkx2-5-EYFP:Nkx2-5-WT and 6kbNkx2-5-EYFP:Nkx2-5- KO littermates at E7.75. Note that the WT and Nkx2-5 mutant embryos are indistinguishable at E7.75. Individual EYFP positive embryos are dissociated and sorted using flow cytometry to collect EYFP positive and negative cells, respectively. RNA is isolated, amplified and qRT-PCR performed from EYFP positive cells of age matched Nkx2-5-WT and Nkx2-5-KO littermates. (C) qRT-PCR of Tdgf1 in WT (open bar) and null (black bar) cardiac progenitors of the cardiac crescent confirming decreased Tdgf1 transcript expression in the Nkx2-5-KO cardiac progenitors vs. the Nkx2-5-WT controls.

Figure 2. Nkx2-5 binds to and activates Tdgf1 expression

(A) Identification of three potential NKEs in the 10kb upstream region of the Tdgf1 gene. (B) Sequence of oligonucleotides used for the EMSA is shown where wild type (WT) and the corresponding mutated (Mut) NKE are in red. Competition studies included increasing amounts of unlabeled WT probe (containing the NKE) or the same unlabeled probe with the NKE mutated (Mut). 32P-labeled WT (NKE) oligonucleotide probe was used for the EMSA and revealed a stable Nkx2-5-DNA (NKE) (lane 2) that could be competed with WT cold competitor (lanes 3–4) but not with Mut cold competitor (lanes 5–6). The complex could be supershifted with anti-myc serum (lane 7). (C) ChIP assay for binding of Nkx2-5 to the Tdgf1 promoter. Anti-myc but not the control antibody is capable of IP of the Tdgf1 promoter from C2C12 cells expressing myc-tagged Nkx2-5. (D) A 1.3 Kb Tdgf1 promoter harboring wild type (WT) or mutated (Mut) NKEs was fused to luciferase (luc) reporter gene and was transfected into C2C12 myoblast cells with or without increased amounts of the Nkx2-5 expressing plasmid. Fold change represents luciferase activity normalized to renilla expression; *, p<0.05, n=3.

Figure 3. Nkx2-5 overexpression in ES/EB system results in increased levels of Tdgf1

(A) Integration of Nkx2-5-expressing vector, pLoxNkx2-5, into the X chromosome of the Ainv15 ES cells to place the cDNA of Nkx2-5 under the control of tetracycline responsive element (TRE). Upon addition of doxycycline (+ Dox), reverse tetracycline transactivator (rtTA) binds to the TRE and induces Nkx2-5 expression. (B) Increased expression of Nkx2-5 RNA and protein in EB cells following the induction with Dox for 48hrs is confirmed; *, p<0.05, n=3. (C) Analyses of fold change in the expression of the Tdgf1 transcript using qRT-PCR following the induction with doxycycline (Dox) for 48hrs (day 2–4) of EB formation; *, p<0.05, n=3. (E) Western blot analysis confirms increased expression of Tdgf1 in EBs overexpressing Nkx2-5 following the induction with Dox for 2 days (day 2–4) vs. control EBs.