VEGF/Flk1 Signaling Cascade Transactivates <em>Etv2</em> Gene Expression

<div><p>Previous reports regarding the genetic hierarchy between Ets related protein 71 (Er71/Etv2) and Flk1 is unclear. In the present study, we pursued a genetic approach to define the molecular cascade between Etv2 and Flk1. Using a transgenic Etv2-EYFP reporter mouse, we examined the expression pattern of Etv2 relative to Flk1 in the early conceptus. Etv2-EYFP was expressed in subset of Flk1 positive cells during primitive streak stages, suggesting that Flk1 is upstream of Etv2 during gastrulation. Analysis of reporter gene expression in Flk1 and Etv2 mutant mice further supports the hypothesis that Flk1 is necessary for Etv2 expression. The frequency of cells expressing Flk1 in Etv2 mutants is only modestly altered (21% decrease), whereas expression of the Etv2-EYFP transgenic reporter was severely reduced in the Flk1 null background. We further demonstrate using transcriptional assays that, in the presence of Flk1, the Etv2 promoter is activated by VEGF, the Flk1 ligand. Pharmacological inhibition studies demonstrate that VEGF mediated activation is dependent on p38 MAPK, which activates Creb. We identify the VEGF response element in the Etv2 promoter and demonstrate that Creb binds to this motif by EMSA and ChIP assays. In summary, we provide new evidence that VEGF activates Etv2 by signaling through Flk1, which activates Creb through the p38 MAPK signaling cascade.</p> </div

VEGF activation of the Etv2 promoter is dependent on a Creb binding site.

<p>(A) A schematic of the 3.9 kb Etv2 luciferase reporter showing 3 evolutionary conserved Creb binding motifs (CRE1-3) and the mutations schematized in panel B. Numbers reflect genomic position relative to the translational start site of Etv2. (B) A schematic of the truncation and mutation strategy. × indicates a CG to AA mutation of either CRE1 or CRE2. Numbers indicate genomic position relative to the translational start site of Etv2. (C) Transcriptional assays using the constructs shown in B co-transfected with Flk1 cDNA and treated with or without VEGF. (D) Transcriptional assay using the 1 kb Etv2 luciferase cotransfected with increasing amounts of expression vector encoding constitutively phosphorylated Creb cDNA. Data were analyzed using two-way anova (C) or Kruskal-Wallis nonparametric t-test with Dunn’s post test comparison (D). n.s.: not significant; *: p<0.05; **: p<0.01; ****: p<0.0001.</p

Flk1 expression precedes Etv2 expression during embryogenesis.

<p>(A) Diagram of the transgenic construct used in this study. Numbers shown identify the genomic location relative to the translation start site of Etv2. CR-1 represents conserved region one and CR-2 represents conserved region two. (B) Schematic diagram of the embryonic axes and germ layers of a late streak stage embryo. Black lines indicate approximate levels of sections in C. (C) A series of transverse sections of a late streak stage embryo. Representative sections were stained with antibodies to Brachyury (Bry), Flk1 and EYFP (scale bar: 100 microns).</p

VEGF activates the Etv2 promoter.

<p>(A–B) Transcriptional assays using an empty luciferase vector or the 3.9 kb Etv2 luciferase reporter standardized to the CMV-Renilla Luciferase reporter. Flk1 cDNA or a balancer DNA plasmid is co-transfected and the cells are treated with or without VEGF (50 ng/ml) for 2–18 hours as indicated in the panel (A). Flk1 or a balancer DNA is cotransfected and cells are treated for 6 hours with varying amounts of VEGF (0–50 ng/ml) as indicated in the panel (B). (C–D) Transcriptional inhibition assays in which Flk1 cDNA and the 3.9 kb Etv2 Luciferase promoter are co-transfected followed by pretreatment of various doses of SB202190 (C), or GF109203X (D) and a 6 hour treatment of 50 ng/ml VEGF. Data were analyzed by two-way anova (A,C,D) or one-way anova (B). n.s.: not significant; *: p<0.05; ***: p<0.001; ****: p<0.0001.</p

Etv2 is absent in Flk1 null embryos.

<p>(A–C) FACS analysis of the Etv2-EYFP transgenic reporter crossed into the Flk1 wildtype and mutant backgrounds at E7.75 and E8.0. Representative FACS profiles are shown (A). (B–C) Data from all experiments are compiled graphically to show the percent of EYFP positive cells in Flk1 wildtype, heterozygous, and null embryos (B) and the mean fluorescent intensity of the EYFP positive cells (C). (D–E) Quantitative PCR for Flk1 (D) and Etv2 (E) was performed on RNA isolated from Flk1 wildtype and null embryos at E7.75, E8.0, and E9.0. (F) Quantitative PCR for Etv2 was performed on FACS isolated Etv2-EYFP positive cells. Each bar represents triplicate measurements from independent samples (single embryos). Asterisks indicate the following: **p<0.01, *p<0.05.</p

Creb1 binds to the CRE2 motif in the Etv2 promoter.

<p>(A–B) Creb1 specifically interacts with the CRE2 motif in the Etv2 promoter as shown by ChIP assay. EBs were collected 3.5 days after initiating mesoderm differentiation. A region in the Gapdh gene was used as a negative control (Control) and 1% of the total chromatin DNA before the immunoprecipitation was used as a positive control (Input). The PCR products were run on a gel for direct visualization (A) and qPCR was performed for quantitation (B). (C) Creb1 binds CRE2 directly <i>in vitro</i>. Creb1 was synthesized <i>in vitro</i>. The oligonucleotides harboring CRE2 motif is labeled with 32P. Synthesized Creb1, non-radioactive oligonucleotides, and the antibodies were incubated with radioactive oligonucleotide probes as indicated in the figure. The interaction between Creb1 and the CRE2 motif was analyzed on a 4% TBE gel.</p

Etv2 is coexpressed with Flk1.

<p>(A–Q) Expression analysis of Etv2-EYFP and Flk1 in E7.0 to E9.5 embryos. Sections were stained with the GFP antibody (A, B, D, E, G–I, K, L, N, P; shown in green) or the Flk1 antibody (A, C, D, F–H, J, K, M, O, Q; shown in red). DAPI nuclear staining is shown in blue (N–Q). Yellow indicates overlap of green and red channels (A, D, G, H, K). A–C: No bud stage. D–F: Early-bud stage. G–M: Early head-fold stage. N–Q: E9.5. The boxed area in A is enlarged in B–C; the boxed area in D is enlarged in E–F; the boxed area in G is enlarged in H–J, and the boxed area in K is enlarged in L–M. Arrowheads in B, C point to progenitors of blood islands coexpressing Flk1 and EYFP. Arrows and arrowheads in H–M indicate endothelial lineages co-expressing Flk1 and EYFP. Asterisks in A, H–M indicate regions in which either gene is expressed alone. Arrowheads in P indicate developing intersomitic vessels. Structures are designated as follows (al: allantois, bi: blood island, da: dorsal aorta, h: heart, ec: endocardium, hf: head fold, nt: neural tube, ph: pharynx, s: somite). Bars in A, D, G, K, N, and P indicate 200 µm. Bars in C, F, J, and M indicate 50 µm.</p

Smyd1 Facilitates Heart Development by Antagonizing Oxidative and ER Stress Responses

<div><p>Smyd1/Bop is an evolutionary conserved histone methyltransferase previously shown by conventional knockout to be critical for embryonic heart development. To further explore the mechanism(s) in a cell autonomous context, we conditionally ablated <i>Smyd1</i> in the first and second heart fields of mice using a knock-in (KI) <i>Nkx2</i>.<i>5-cre</i> driver. Robust deletion of <i>floxed-Smyd1</i> in cardiomyocytes and the outflow tract (OFT) resulted in embryonic lethality at E9.5, truncation of the OFT and right ventricle, and additional defects consistent with impaired expansion and proliferation of the second heart field (SHF). Using a transgenic (Tg) <i>Nkx2</i>.<i>5-cre</i> driver previously shown to not delete in the SHF and OFT, early embryonic lethality was bypassed and both ventricular chambers were formed; however, reduced cardiomyocyte proliferation and other heart defects resulted in later embryonic death at E11.5-12.5. Proliferative impairment prior to both early and mid-gestational lethality was accompanied by dysregulation of transcripts critical for endoplasmic reticulum (ER) stress. Mid-gestational death was also associated with impairment of oxidative stress defense—a phenotype highly similar to the previously characterized knockout of the Smyd1-interacting transcription factor, skNAC. We describe a potential feedback mechanism in which the stress response factor Tribbles3/TRB3, when directly methylated by Smyd1, acts as a co-repressor of Smyd1-mediated transcription. Our findings suggest that Smyd1 is required for maintaining cardiomyocyte proliferation at minimally two different embryonic heart developmental stages, and its loss leads to linked stress responses that signal ensuing lethality.</p></div

Deletion of <i>Smyd1</i> by <i>Tg-Nkx2</i>.<i>5-cre</i> leads to a delayed embryonic lethal cardiac phenotype.

<p><b>A.</b><i>Smyd1</i>^{<i>flox/flox</i>}; <i>Tg-Nkx2</i>.<i>5-cre</i> (Tg-CKO) embryos die at midgestation. Table numbers are total recovered embryos of each genotype. The number of dead or abnormal embryos is given in parentheses. <b>B, C.</b><i>Smyd1</i> mRNA expression at E10.5 assayed by RT-PCR (B) and real-time PCR (C). <b>D.</b> H&E-stained transverse sections of E11.5 control (Cx) and Tg-CKO embryos showing pericardial edema, thinned pericardium and decreased trabeculation. <b>E.</b> Decreased proliferation was observed in the hearts of E10.5 <i>Smyd1</i> Tg-CKO embryos. Representative images of Cx and Tg-CKO hearts stained with H&E (upper panels) and the mitosis marker phospho-histone H3 serine 10 (p-H3) (lower panels). <b>F.</b> Quantification of p-H3 positive cells within the heart from three sections for three independent embryos (n = 3). <b>G.</b> Comparison of <i>skNAC</i> knockout and <i>Smyd1 Tg-CKO</i> heart gene expression by real-time PCR at E11.5. Data, focused primarily on oxidative response deregulation, are presented as mean for each genotype (<i>skNAC</i>^-/-, n = 5; <i>Smyd1</i> Tg-CKO, n = 4). Error bars indicate SEM. <b>H.</b> Genes encoding mediators of ER stress are deregulated by loss of Smyd1. Real-time PCR data represents average of 3 biological replicates each with 3 technical replicates; error bars indicate SEM. Data were analyzed by Student’s t-test (*P < 0.05, **P <0.01, ***P < 0.001, ****P < 0.0001).</p

Loss of <i>Smyd1</i> using <i>Ki-Nkx2</i>.<i>5</i>^<i>cre/+</i> disrupts looping morphogenesis and chamber formation through perturbation of the SHF and activation of ER stress.

<p><b>A.</b> Gross morphological comparison of control (Cx: <i>Nkx2</i>.<i>5</i>^<i>+/+</i>; <i>Smyd1</i>^{<i>Flox/Flox</i>}) and <i>Smyd1</i> Ki-CKO (CKO: <i>Nkx2</i>.<i>5</i>^<i>cre/+</i>; <i>Smyd1</i>^{<i>Flox/Flox</i>}) hearts at E9.5. Scale bar = 200 μm. <b>B, C</b>. The lengths of the outflow tract (OFT) (B) and right ventricle (RV) (C) were significantly reduced in Ki-CKO embryos at E9.5 (n = 6/group). <b>D.</b> Representative results of microarray gene expression comparison of transcripts critical to SHF and chamber formation at E9.5. Data are presented as expression values of 2 independent biological replicas of each genotype averaged from 2 technical replicas. <b>E.</b> Confirmation of microarray for deregulated transcripts critical to SHF and chamber formation by real-time PCR using RNA from E9.5 heart/pharyngeal mesoderm (n = 9/group). <b>F</b>. Whole mount <i>in situ</i> hybridization comparison of selected SHF and chamber formation transcripts deregulated and/or mislocalized in CKO hearts at E9.5 (n = 3/group). Arrows denote areas of differential expression. <b>G.</b> Comparison of cell proliferation in the outflow tract of Control (Cx) and Ki-CKO by BrdU immunohistochemistry. PE, Pharyngeal Endoderm. OFT, outflow tract. V, ventricle. Scale bar = 100 μm. <b>H.</b> Quantification of anti-BrdU staining in the outflow tract (n = 6/group). <b>I.</b> Loss of Smyd1 leads to deregulation of genes critical to anti-proliferative responses to ER stress. Data are presented as a heat map with expression values of 2 independent biological replicas of each genotype averaged from 2 technical replicas plotted as log² expression values. For B, C, E and H, data were analyzed by Student’s t-test (*P < 0.05).</p