Research ArticleDEVELOPMENTAL BIOLOGY

Activating transcription factor 3 coordinates differentiation of cardiac and hematopoietic progenitors by regulating glucose metabolism

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Science Advances  06 May 2020:
Vol. 6, no. 19, eaay9466
DOI: 10.1126/sciadv.aay9466
  • Fig. 1 The expression of atf3 and the identification of the FHF.

    (A) MA plots showing the profiles of differentially expressed genes in the mesodermal cells at E7.5, in HPs at E11.5, and in embryonic cardiac cells at E9.5. Expression levels of genes in the ectoderm were used as control. The white line indicates the differentially expressed genes with twofold expression change. NS, no significance. (B) Genes with enriched expression in mesodermal cells [fold change (FC) ≥2]. Up-regulated genes with expression enrichment in both embryonic cardiac cells and HPs compared with mesodermal cells are highlighted as colored dots (green, yellow, and pink) in the upper right quadrant. Genes labeled by yellow dots were equally enriched in the blood and heart lineages. (C) mRNA expression level of atf3 in nkx2.5+ CPs based on microarray data in zebrafish at 30 hpf. Statistical analysis was carried out using the unpaired two-tailed Student’s t test. Error bars indicate SD, **P < 0.01 (n = 3 per group). (D and E) WISH of atf3 and nkx2.5 at 9 ss. Red arrowheads indicate that the expression position of atf3 was comparable with that of nkx2.5. (F) WISH of hand2 at 9 ss. (G) Colocalization analysis of mRNA expression patterns of hand2 (blue arrowheads) and atf3 (red arrowheads). (H) WISH of tal1 at 9 ss. (I) Colocalization analysis of mRNA expression patterns of tal1 (red arrowheads) and atf3 (blue arrowheads). (J) Fluorescent in situ hybridization of atf3 combined with immunofluorescence staining of Nkx2.5 at 30 hpf. AP, arterial pole; HT, heart tube. (K) Expression enrichment of atf3 in the heart tube (H, white arrowhead), AGM, and CHT (blue arrowheads) at 32 hpf. (L) Fluorescent expression of atf3: RFP2ACre stable line at 32 hpf. (M and N) Colocalization analysis of (M) Atf3 and Cmlc2 and (N) Atf3 and Fli1a using transgenic lines at 60 hpf. V, ventricle; OFT, outflow tract; B, blood. (O) Complementary expression of RFP and eGFP fluorescence in the ventricle in Tg(atf3:RFP2Acre); Tg(ltbp3:eGFP) embryos at 60 hpf. The white arrowhead indicates the ventricular cells derived from the SHF. (P) Schematic of Tg(atf3:RFP2Acre) driver and Tg(ubiquitin:CSY) reporter transgenes. Lineage tracing analysis of atf3+ cells by Tg(atf3:RFP2Acre);Tg(ubiquitin:CSY) at 60 hpf. A, atrium. n ≥ 30 embryos per group for WISH and fluorescence observation. Scale bars, 50 μm.

  • Fig. 2 Loss of atf3 caused dilated atrial cardiomyopathy.

    (A) Genomic sequences of atf3 in the wild-type (WT) and homozygous mutant zebrafish and the schematic diagram of truncated Atf3 protein of the homozygous mutants. Loss of one base pair in exon 2 led to the formation of an early stop codon. Atf3 protein was detected in the WT and mutant embryos by Western blot at 24 hpf. (B) Hematoxylin and eosin staining of transverse sections of hearts (6 μm thick per section, n ≥ 6 embryos per group) at 72 hpf. (C) Confocal images of embryonic hearts in the Tg(cmlc2:dsRednuc) control line and the atf3−/−;Tg(cmlc2:dsRednuc) line at 72 hpf. (D) Statistical analysis of the cell number of cardiomyocytes in the control and atf3−/− embryos in (C) (n ≥ 6). (E) Confocal images of embryonic hearts in the Tg(fli1a:eGFPnuc) control line and the atf3−/−;Tg(fli1a:eGFPnuc) line at 72 hpf. (F) The endocardial cell counts in the control and atf3−/− embryos in (E) (n ≥ 6). (G) Morphology of WT and atf3−/− adult (1-year-old) zebrafish heart (n ≥ 6). (H) The electrocardiography waveform average of 5-min recorded data. Blue arrowhead indicates the P wave. (I) P wave amplitude relative to the QRS amplitude (n ≥ 6 adults per group). (J) Heart rates of the adult WT and atf3−/− adult zebrafish (n ≥ 6). (K) Pulse-wave Doppler image of ventricular inflow of adult (5-month-old) WT and atf3−/− zebrafish. The y axis indicates velocity (mm/s), and the x axis indicates time (ms). (L) The velocity of A-flow (n ≥ 6 fish per group). (M) Assessment of diastolic dysfunction by mitral valve (MV) E/A ratio (n ≥ 6 per group). Statistical analysis was carried out using the unpaired two-tailed Student’s t test. Error bars indicate SD. n.s., P > 0.05, **P < 0.01, ***P < 0.001. Scale bars, 50 μm.

  • Fig. 3 Deficiency of atf3 impaired differentiation of both CPs and HPs.

    (A and B) WISH of atrial (amhc) and ventricular (vmhc) lineage markers in the WT and atf3−/− embryos at 22 ss (n = 21 for WT and n = 16 for atf3−/− embryos in the WISH of amhc; n = 23 for WT and n = 21 for atf3−/− embryos in the WISH of vmhc). (C) Statistical analysis of the expression area of amhc and vmhc (n = 16 per group), using the unpaired two-tailed Student’s t test. **P < 0.01. (D) Relative mRNA expression levels of amhc and vmhc in WT and atf3−/− embryos at 22 ss (n = 6). Statistical analysis was carried out using the unpaired two-tailed Student’s t test. **P < 0.01, ***P < 0.001. (E) WISH of the ALPM marker gata4 (n = 29 for WT, n = 15 for atf3−/− embryos), the heart-forming region marker hand2 (n = 29 for WT, n = 38 for atf3−/− embryos), and the CP marker nkx2.5 (n = 20 for WT, n = 15 for atf3−/− embryos) at 9 ss. Arrowheads indicate the specific expression patterns of the markers. The brackets indicate the width of the expression pattern in the ALPM. (F) The width of the expression region of gata4, hand2, and nkx2.5 in the ALPM (n = 15 per group). Statistical analysis was carried out using the unpaired two-tailed Student’s t test. Error bars indicate SD. **P < 0.01. (G) The relative mRNA expression levels of gata4, hand2, and nkx2.5 in WT and atf3−/− embryos at 9 ss (n = 6). Statistical analysis was carried out using the unpaired two-tailed Student’s t test. n.s., P > 0.05, ***P < 0.001. (H) Expression of tal1 at 9 ss (n = 30 per group). The red brackets indicate the length of the tal1 expression region in the ALPM. (I) Expression of pu.1 (n = 30 per group). (J) Statistical analysis of the length of the expression region of tal1 in (H) (n ≥ 6 per group) using the unpaired two-tailed Student’s t test. ***P < 0.001. (K) Counts of pu.1+ cells in (I) (n ≥ 6 per group). (L) Expression pattern of mpx at 32 hpf (n = 30 per group). (M) Counts of mpx+ cells in (L) (n = 30 per group). Statistical analysis was carried out using the unpaired two-tailed Student’s t test. ***P < 0.001. (N) Wright-Giemsa staining of (1-year-old) KM cells after cytospin process (n = 6 per group). Green arrowheads indicate mature myeloid cells, red arrowheads indicate immature myeloid cells, blue arrowheads indicate precursors, and hollow arrowheads indicate other cells, such as lymphoid cells. (O) Ratios of different blood cell types in the KM of WT and atf3−/− zebrafish (n = 6 per group). Statistical analysis was carried out using the Kruskal-Wallis test with adjustments for multiple comparisons. **P < 0.01. SS-A, side scatter-axis; FS-A, forward scatter-axis. (P) Flow cytometry analysis of the KM cells of adult (5-month-old) WT and atf3−/− zebrafish (n = 6 per group). Scale bars, 50 μm.

  • Fig. 4 atf3 autonomously regulated differentiation and mediated the interaction between nkx2.5 and tal1.

    (A) Top: Schematic diagram showing a control fraction of atf3 (Ctrl Δ) and a truncated form of atf3 bereft of the C-terminal activation domain (Atf3 Δzip) acting as a DN inhibitor, based on previous reports. Bottom: tol2-mediated transgenesis of tal1 and nkx2.5 promoting atf3△zip-eGFP and mCherry expression, respectively. (B) Morphological analysis of the embryonic heart in Tg(nkx2.5:ZsYellow) embryos with control atf3∆ mRNA injection and Tg(nkx2.5:ZsYellow) embryos with atf3∆zip mRNA injection at 48 hpf (n = 30 per group). (C) Expression pattern of fluorescence in ICM of the Tg(scl:atf3△zip-eGFP) stable line at 22 hpf (n = 30). (D) Expression pattern of fluorescence in the ventricle and PAAs of the Tg(nkx2.5:atf3△zip-mCherry) stable line at 48 hpf (n = 30). (E) Expression patterns of tal1 in WT, Tg(tal1: atf3△zip2AeGFP), and Tg(nkx2.5: atf3△zip2AmCherry) embryos at 9 ss (n ≥ 30 per group). (F) Expression patterns of nkx2.5 in WT, Tg(tal1: atf3△zip2AeGFP), and Tg(nkx2.5: atf3△zip2AmCherry) embryos at 9 ss (n ≥ 30 per group). (G) Statistical analysis of the length of the expression pattern of tal1 in (E) (n ≥ 9 for each group), using the Kruskal-Wallis test. Error bars indicated SD. **P < 0.01. (H) Statistical analysis of the width of the expression region of nkx2.5 in (F) (n ≥ 9 per group), using the unpaired two-tailed Student’s t test. **P < 0.01. (I) Expression region of nkx2.5 in the control embryos, atf3−/− embryos, and atf3−/− embryos with MOtal1 injection at 9 ss (n ≥ 35 per group). (J) Statistical analysis of the width of the expression region of nkx2.5 (n = 16 per group), using the unpaired two-tailed Student’s t test. **P < 0.01. (K and L) Morphological analysis of the embryonic heart in the control embryos (1), atf3−/−;Tg(nkx2.5:ZsYellow) embryos (2), Tg(nkx2.5:ZsYellow) embryos with MOtal1 injection (3), Tg(nkx2.5:ZsYellow) embryos with MOnkx2.5 (4), Tg(nkx2.5:ZsYellow) embryos with MOnkx2.5 and mRNAatf3 coinjection (5), and atf3−/−;Tg(nkx2.5:ZsYellow) embryos with mRNAnkx2.5 injection (6) at 72 hpf (n ≥ 30 per group). The dashed line outlines the atrium. (M) Statistical analysis of the atrial area in (K, 1 to 3) and (L, 4 to 6) (n = 20 per group), using the unpaired Student’s two-tailed t test. Error bars indicate SD. n.s., P > 0.05, **P < 0.01. (N) WISH of atf3 in the control and embryos with MOnkx2.5 injection at 9 ss (n = 40 per group). (O) Percentage of the differential expression of atf3 (n = 6, 30 embryos per test). Statistical analysis was carried out using the Kruskal-Wallis test with adjustments for multiple comparisons. **P < 0.01. (P) Binding enrichment region at the atf3 locus as indicated by primers P1 to P6. The minimal DNA binding consensus for Nkx2.5 contained a 5′-TNNAGTG-3′ sequence motif, indicated by the red arrowhead. The ChIP analysis was performed with anti-Nkx2.5 antibody for the promoter region of atf3 in WT embryos at 9 ss (n = 6, 100 embryos per group per experiment). Error bars indicate SD. Statistical analysis was carried out using the unpaired two-tailed Student’s t test. n.s., P > 0.05, **P < 0.01. Scale bars, 50 μm. IgG, immunoglobulin G.

  • Fig. 5 atf3 targeted cebpγ, negatively regulating its expression.

    (A) Genomic distribution regions of Atf3 binding peaks in the immunoprecipitated (IP) sample at 32 hpf. (B) Signaling pathway enrichment analysis of atf3 binding by gene set enrichment analysis. RV, right ventricular. (C) Peak enrichment of atf3 binding genes in IP samples in a violin-box plot. The red arrowheads indicate the top-ranked genes (duplicates per group). (D) Track analysis of Atf3 binding profiles in the promoter regions of cebpγ and atf3. (E) Major genes of the chronic myeloid leukemia signaling pathway were analyzed by the unsupervised hierarchy clustering, shown in the heat map. (F and G) Expression of cebpγ in the WT and atf3−/− embryos at 9 ss and 24 hpf (n = 35 per group). Blue arrowheads indicate HP region, and red arrowheads indicate CP region. (H) Relative mRNA expression level of cebpγ in the WT and atf3−/− embryos (n = 30 per group). Statistical analysis was carried out using the unpaired two-tailed Student’s t test. Error bars indicate SD. **P < 0.01, ***P < 0.001. (I) Immunofluorescent staining of Nkx2.5 in the control embryos, atf3−/− embryos, WT embryos with mRNAcebpγ injection, and atf3−/− embryos with MOcebpγ injection at 9 ss (n = 30 per group). (J) Morphological analysis of the embryonic heart in the control embryos, atf3−/−;Tg(nkx2.5:ZsYellow) embryos, Tg(nkx2.5:ZsYellow) embryos with mRNAcebpγ injection, and atf3−/−; Tg(nkx2.5:ZsYellow) embryos with MOcebpγ injection at 72 hpf (n = 30 per group). (K) Expression of tal1 in the control embryos, atf3−/− embryos, WT embryos with mRNAcebpγ injection, and atf3−/− embryos with MOcebpγ injection at 9 ss (n = 56 per group). (L) Statistical analysis of the length of the Nkx2.5 staining region in (I) (n = 20), using the unpaired two-tailed Student’s t test. Error bars indicate SD. n.s., P > 0.05, **P < 0.01. (M) Statistical analysis of the expression area of Nkx2.5 in the atrium in (J) (n ≥ 25 per group), using the unpaired two-tailed Student’s t test. Error bars indicate SD. **P < 0.01. (N) Statistical analysis of the length of the expression region of tal1 in (K) (n ≥ 6 for each group), using the Kruskal-Wallis test. Error bars indicate SD, n.s., P > 0.05, ***P < 0.001. a3−/−, atf3−/−; OEceb, mRNAcebpγ; MOceb, MOcebpγ. Scale bars, 50 μm.

  • Fig. 6 atf3-cebpγ regulated glucose metabolism by targeting glut1 and provided cells with adaptive capacity to high glucose levels.

    (A) The mRNA expression levels of Slc2a1–4/Glut1–4 family members in the mouse mesoderm, the embryonic heart, and CD45+CD144+ HPs as analyzed by RNA sequencing (RNA-seq), relative to the expression in ectodermal cells (n = 3 per group). RPKM, reads per kilo base per million mapped reads. (B) Expression of slc2a1–3/glut1–3 family members in nkx2.5+ CPs and control cells in zebrafish at 9 ss (n = 9). (C) Relative mRNA expression levels of glut1a, glut1b, and pck1 in the control, atf3−/− embryos, and WT embryos with cebpγ mRNA injection at 9 ss (n = 6). (A to C) Statistical analysis was carried out using the unpaired two-tailed Student’s t test. Error bars indicate SD. n.s., P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. (D) ChIP-qPCR analysis of Cebpγ binding in the promoter region of glut1a at 9 ss. The binding enrichment region at the glut1a locus was identified by primers P1 and P2 (fig. S7). The ChIP analysis with anti-Cebpγ for the promoter region of glut1a in the WT and atf3−/− embryos at 9 ss (n = 6, 100 embryos per group per experiment). Statistical analysis was carried out using analysis of variance (ANOVA) with multiple comparison post hoc test. **P < 0.01, ***P < 0.001. (E) Difference in expression of genes encoding signaling proteins between the scramble and sh-Atf3 H9c2 cell lines, as analyzed by RNA-seq (n = 3 per group). (F) Heat map of genes encoding proteins involved in the signaling pathways (n = 3 per group). (G) In situ hybridization analysis of the expression pattern of atf3 in embryos treated with different doses of glucose at 9 ss (n = 30 per group). (H) The relative mRNA expression level of atf3 in (G) (n = 6). Statistical analysis was carried out using the unpaired two-tailed Student’s t test. Error bars indicate SD. *P < 0.05, ***P < 0.001. (I and K) In situ hybridization analysis of the expression pattern of (I) tal1 and (K) hand2 in the control, atf3−/− embryos, atf3−/− embryos treated with glucose, and atf3−/− embryos treated with slc2a1a MO injection at 9 ss (n = 20 per group). (J) Statistical analysis of the length of the expression pattern of tal1 in (I) (n ≥ 6), using the Kruskal-Wallis test. **P < 0.01, ***P < 0.001. a3−/−, atf3−/−; MOslc, MOslc2a1a. (L) Statistical analysis of the width of the expression region of hand2 in (K) (n ≥ 6), using the unpaired two-tailed Student’s t test. Error bars indicate SD. **P < 0.01. a3−/−, atf3−/−; MOslc, MOslc2a1a. (M) Morphological analysis of the embryonic heart in the control, atf3−/−; Tg(nkx2.5:ZsYellow) embryos, atf3−/−; Tg(nkx2.5:ZsYellow) embryos with 2% glucose treatment, and atf3−/−; Tg(nkx2.5:ZsYellow) embryos with slc2a1a MO injection at 72 hpf (n = 30 per group). (N) Statistical analysis of the expression area of Nkx2.5 in the atrium in (M) (n = 25 per group), using the unpaired two-tailed Student’s t test. Error bars indicate SD. **P < 0.01. a3−/−, atf3−/−; MOslc, MOslc2a1a. (O) Oxygen consumption rates (OCR) (oxidative phosphorylation) and extracellular acidification rates (ECAR) (glycolysis) were examined in WT cells, atf3−/− cells, and atf3−/− cells treated with 10 μM of the slc2a1 inhibitor STF-31 at 9 ss, as analyzed by seahorse assay (n = 8). Statistical analysis was carried out using the unpaired two-tailed Student’s t test. Error bars indicate SD. n.s., P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. A, oligomycin; B, FCCP; C, rotenone/antimycin A; a, glucose; b, oligomycin; c, 2-deoxyglucose. (P and R) Relative mRNA expression levels of oxidative and ER stress markers in the control and atf3−/− embryos at 9 ss (n = 6). (Q) NADP/NADPH ratios at 9 ss, measured by the Sigma NADP/NADPH Quantification Kit (n = 6). (P to R) Statistical analysis was carried out using the unpaired two-tailed Student’s t test. Error bars indicate SD. n.s., P > 0.05, *P < 0.05, **P < 0.01. (S) Perk protein levels in the control embryos, atf3−/− embryos, and WT embryos treated with 2 mM H2O2 at 9 ss, as analyzed by Western blot. (T) Immunofluorescence staining of Nkx2.5 in the control, atf3−/− embryos, WT embryos treated with 2 mM H2O2, and atf3−/− embryos with catalase injection (350 ng per embryo) at 12 hpf (n = 30 per group). (U) pu.1+ myeloid progenitors in the control embryos, Tg(pu.1:eGFP);atf3−/− embryos, Tg(pu.1:eGFP) embryos treated with H2O2, and Tg(pu.1:eGFP);atf3−/− embryos with catalase injection at 22 hpf (n = 30 per group). (V) Schematic representation of the role of atf3 in the regulation of differentiation of CPs and HPs. Scale bars, 50 μm.

Supplementary Materials

  • Supplementary Materials

    Activating transcription factor 3 coordinates differentiation of cardiac and hematopoietic progenitors by regulating glucose metabolism

    Hui-Min Yin, Li-Feng Yan, Qian Liu, Zheng Peng, Chi-Yuan Zhang, Yu Xia, Dan Su, Ai-Hua Gu, Yong Zhou

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