XB-ART-37305J Biol Chem April 25, 2008; 283 (17): 11841-9.
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HIF-1alpha signaling upstream of NKX2.5 is required for cardiac development in Xenopus.
HIF-1alpha is originally identified as a transcription factor that activates gene expression in response to hypoxia. In metazoans, HIF-1alpha functions as a master regulator of oxygen homeostasis and regulates adaptive responses to change in oxygen tension during embryogenesis, tissue ischemia, and tumorigenesis. Because Hif-1alpha-deficient mice exhibit a number of developmental defects, the precise role of HIF-1alpha in early cardiac morphogenesis has been uncertain. Therefore, to clarify the role of HIF-1alpha in heart development, we investigated the effect of knockdown of HIF-1alpha in Xenopus embryos using antisense morpholino oligonucleotide microinjection techniques. Knockdown of HIF-1alpha resulted in defects of cardiogenesis. Whole mount in situ hybridization for cardiac troponin I (cTnI) showed the two separated populations of cardiomyocytes, which is indicative of cardia bifida, in HIF-1alpha-depleted embryos. Furthermore, the depletion of HIF-1alpha led to the reduction in cTnI expression, suggesting the correlation between HIF-1alpha and cardiac differentiation. We further examined the expression of several heart markers, nkx2.5, gata4, tbx5, bmp4, hand1, and hand2 in HIF-1alpha-depleted embryos. Among them, the expression of nkx2.5 was significantly reduced. Luciferase reporter assay using the Nkx2.5 promoter showed that knockdown of HIF-1alpha decreased its promoter activity. The cardiac abnormality in the HIF-1alpha-depleted embryo was restored with co-injection of nkx2.5 mRNA. Collectively, these findings reveal that HIF-1alpha-regulated nkx2.5 expression is required for heart development in Xenopus.
PubMed ID: 18303027
Article link: J Biol Chem
Species referenced: Xenopus laevis
Genes referenced: arnt bmp4 gata4 hand1 hand2 hba1 hif1a mhc2-dab (provisional) nkx2-5 odc1 prl.2 smad4 smad4.2 tbx5 tek tnni3
Morpholinos: hif1a MO1 hif1a MO2 nkx2-5 MO1
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|FIGURE 1. Expression of hif-1 during Xenopus development. A, predicted domains of Xenopus HIF-1 . bHLH, basic helix loop helix binding domain; PAS, Per-Arnt-Sim domain; TAD-N, N-terminal activation domain; TAD-C, C-terminal activation domain. B, temporal expression profile of the hif-1 mRNA. RT-PCR analysis was carried out from stage 1 to 25 using a primer set for hif-1 . Quality of RNA at each stage was assessed by amplification of the ubiquitous transcript, ornithine decarboxylase (odc). C, hif-1 expression was examined by whole mount in situ hybridization of embryos at the stage indicated in each panel. a, lateral view of stage 23 embryo; b, lateral view of stage 28 embryo; c– e, Stage 33/34 embryos, lateral view (c), ventral view (d), and higher magnification image (e) of the anterior portion seen in panel d. hif-1 expression in the eyes (yellow arrowhead), brain (yellow arrow), kidneys (red arrow), somites (yellow asterisk), branchial arches (black arrowhead), myocardium (red arrowhead), and the ventral blood island (VBI, black asterisk). f, transversal section of a stage 33/34 embryo at the white line in panel d reveals hif-1 expressed in myocardium.|
|FIGURE 2. The gain- or loss-of-function of HIF-1 . A, effects of injection of hif-1 mRNAs indicated at the top on vascular development. Panels a–d, Xenopus embryos un-injected; panels e–h, embryos injected with hif-1 (P558G) mRNA (750 pg); and panels i and j, embryos injected with hif-1 antisense morpholino 1 (hif-1 MO1) (40 ng).MOormRNAwere injected into one of the two vegetal-dorsal blastomeres at the 8-cell stage. The embryos were assayed at tail-bud stage 33/34 with wholemountdouble in situ hybridization for tie-2 and -globin, endothelial and hematopoietic markers, respectively. Panels a, e, and i, lateral views; and panels c, g, and k, ventral views. Panels b, f, and j, enlarged vascular vitelline network (VVN); and panels b, h, and l, enlarged VBI formation. Positive signal was detected with BM purple (blue) and DAB (brown) for tie-2 and -globin, respectively. VVN meshwork was increased in hif-1 (P558G) mRNA-injected embryos compared with the control. In contrast, knockdown with MO reduced the VVN and VBI regions. B, schematic presentation of 5 UTR-HIF(N)-Venus and the region against which MO are designed. Transcript of 5 UTR-HIF(N)-Venus corresponds to a single transcript of the 5 -UTR (102 bp) and mRNA of hif-1 N-terminal (100 amino acids) andmRNAcoding Venus. C, effect of hif-1 MO1and hif-1 MO2 on in vitro translated expression of HIF-1 fused to Venus. pCS2 -5 UTRHIF( N)-Venus (0.5 g) was transcribed and translated in vitro in the absence of MOs (lane 1) or in the presence of CMO (lane 2) and hif-1 MO1 and hif-1 MO2 at the concentrations indicated on at the top (lanes 3–6). HIF-1 -Venus was analyzed by immunoblotting (IB) with anti-GFP antibody. Both hif-1 MO1 and hif-1 MO2 inhibit translation of hif-1 in vitro. D, in vivo translation of HIF(N)-Venus (200 pg) is specifically inhibited by hif-1 MOs (40 ng). Embryos (8-cell stage) were injected with pCS2 -5 UTR-HIF(N)-Venus and hif-1 MOs. pCS2 -5 UTR-HIF(N)-Venus plus CMO (left panels), hif-1 MO1 (middle panels), and hif-1 MO2 (right panels) are shown. Embryos were observed by fluorescence microscopy. BF, bright field; GFP, green fluorescence from Venus. E, the lysates from embryos injected with pCS2 -5 UTRHIF( N)-Venus and the MOs as indicated at the top were subjected to immunoblot analysis using anti-GFP antibody to examine the effect of MOs on the transcription of HIF-1 tagged with Venus. -Tubulin was used as loading control. F, the endogenous HIF-1 protein level in Xenopus was reduced in hif-1 MO-injected embryos. Panel a, the lysates obtained from Xenopus embryos were subjected to immunoprecipitation (IP) with the antibody indicated at the top and followed by immunoblotting with anti-HIF-1 antibody. Panel b, Xenopus embryos injected with hif-1 MOs (MO1 or MO2) as described under “Experimental Procedures” was analyzed for expression of HIF-1 byimmunoprecipitationandIBusinganti-HIF-1 antibody. Panel c,quantitative analysis of the results of panel b obtained from three independent experiments. Relative expression of HIF-1 in embryos treated with hif-1 MOto that of those treated with CMO was calculated and expressed as mean S.D.|
|FIGURE 3. Impaired cardiac development in HIF-1-depleted Xenopus embryo. A, a representative result of images obtained from embryos uninjected (control) or injected with hif-1 MO1or hif-1 MO2. Panel a, embryos without injection possessed a beating heart (white arrowhead) anterior to the gut. Panels b and c, embryos injected with either hif-1 MO1or hif-1 MO2(40 ng) had either a tissue-free region where the heart should localize or hearts that did not beat (yellow arrowhead) at stage 42. B, expression of cTnI examined by in situ hybridization in HIF-1-depleted embryos. The embryos injected withCMO(40 ng) (a), hif-1 MO1(40 ng) (b), or hif-1 MO2(40 ng) (c) were analyzed at stage 33/34. Panels show anterior ventral views. Yellow arrowheads point to the side derived from the blastomere where hif-1 MO was injected. In hif-1 MO-injected embryos, cTnI was detected in two separate populations of cardiomyocytes, whereas cTnI was detected at the center in CMO-injected embryos. Representative eosin-stained cross-sections of embryos injected with CMO (b), hif-1 MO1 (d), or hif-1 MO2 (f) are shown. The heart region is indicated by an arrowhead.|
|FIGURE 4. Reduction of the expression of early cardiac markers by depletion of HIF-1 . A, the expression of nkx2.5 (a and b), gata4 (c and d), tbx5 (e and f), hand1 (g and h), hand2 (i and j), and bmp4 (k and l) was analyzed with whole mount in situ hybridization analyses at stage 33/34. The embryos uninjected (a, c, e, g, i, and k) and those injected with hif-1 MO1 (b, d, f, h, j, and l) are shown. The panel show the anterior ventral view. hif-1 MO1-injected embryos showed only faint expression of cardiac markers except BMP4. Yellow arrowheads indicates the side derived from the blastomere where hif-1 MO1 was injected. These results are summarized in Table 3.|
|FIGURE 5. HIF-1 regulates transcription of nkx2.5. A, effects of various transcription factors on nkx2.5 transcriptional activity. Luciferase activity was measured by the lysates from CV1 cells transfected with the Nkx2.5 promoter- Luc plasmid and the expression plasmid indicated at the bottom (control vector (pIRES puro3), mouse Gata4, Smad4, or Hif-1 (P577G)). -Fold differences in relative luciferase activity were calculated as arbitrary luciferase activity. Transfection efficiency was normalized by Renilla luciferase activity derived from null-pRL control plasmid. B, RT-PCR analysis using total RNA from embryos at stage 1 to 25. BothRNAquality and quantity was assessed by amplification of ornithine decarboxylase (odc). C, visualization of Nkx2.5 promoter activity in developing embryos. The Nkx2.5 promoter-Venus (100 pg) was co-injected with 40 ng of CMO or hif-1 MO1. Green fluorescence reflected activation of the Nkx2.5 promoter. Fluorescence was observed around the heart primordial region at stage 20 embryos. Nkx2.5 promoter activity was suppressed with co-injection with hif-1 MO1, but not with CMO. D, activation of mouse Nkx2.5 promoter (9.0 kbp) in developing Xenopus embryos. Xenopus embryos were injected with the pGL4.10–9kb Nkx2.5 promoter (Nkx2.5 promoter-Luc) or promoter-less luciferase reporter gene (pGL4.10). Dual luciferase assay were performed in the embryos at the neural stage, stage 19–20 when Nkx2.5 mRNA expression peaked. E, the effect of injection of either CMO or hif-1 MO on Nkx2.5 promoter activity. The Nkx2.5 promoter-Luc (100 pg) and pRL-null (2 pg) were co-injected with either 40 ng of CMO or hif-1 MO1. Embryos were harvested at the neural stage, stage 19–20. For each group, 15 pools containing 5 embryos each were used. Luciferase activities are shown, with error bars representing the mean S.E. (n 15 each group). **, p 0.01 versus CMO). F, RT-PCR analysis for examining expression of nkx2.5 mRNA using total RNA from embryos at stage 20. Both RNA quality and quantity was assessed by amplification of ornithine decarboxylase (odc).|
|HIFFIGURE 6. NKX2.5 functions downstream of HIF-1. A, cardia bifida in HIF- 1-depleted embryos was rescued by expression of nkx2.5. Embryos were injected with hif-1 MO1 (40 ng) and nkx2.5 mRNA (0, 125, and 250 pg). The expression pattern of cTnI was analyzed by in situ hybridization at stage 33/34 to detect the precise migration of cardiomyocytes. Cardia bifida caused by depletion of HIF-1 was restored by co-injection of nkx2.5 mRNA. B, depletion of NKX2.5 results in cardia bifida in Xenopus embryos. CMO-injected embryos (a), nkx2.5 MO-injected embryos (b), and hif-1 MO1-injected embryos (c) were examined for cTnI by in situ hybridization at stage 33/34. NKX2.5-depleted embryos showed a similar staining pattern of cTnI to HIF-1-depleted embryos. Yellow arrowheads indicate the side derived from the blastomere where hif-1 MO1 was injected. C, the incidence of cardia bifida in nkx2.5 MO-injected embryos, hif-1 MO-injected embryos, or CMO-injected embryos.|
|Figure 1S. Classification of the embryos showing cardia bifida at stage 33/34. HIF-1α-depleted embryos at showing cardia bifida stage 33/34 in Figure 3B were separated into three groups according to the degree of reduced cardiac troponin I (TnIc) expression in the side derived from the blastomere where HIF-1α MO1 was injected. TnIc mRNAs were visualized using BM purple (blue). TypeI, II, and III reflect faint reduction, mild reduction, and severe reduction, respectively. Reduced expression of TnIc was found in the side (yellow arrowhead) derived from the blastomere where HIF-1α MO1 was injected.|
|Figure 2S. Defect of myocardial cell differentiation and heart development in embryos injected with HIF-1α MO. Whole mount in situ hybridization analysis was carried out at stage 42 to detect cardiac troponin I (TnIc) mRNA expression in embryos injected with 40 ng of CMO (a) or HIF-1α MO1 (b,c) at 8-cell stage. TnIc mRNAs were visualized (blue) on transverse sections of the embryos injected with CMO (d) or HIF-1α MO1 (e,f). TnIc was asymmetrically detected in the embryos injected with HIF-1α MO1 compared to than those injected with CMO. This asymmetrical expression of TnIc reflects the interference of heart development (smaller heart in IF-1α MO1-injected embryos than CMO-injected embryos). nc, notochord; en, endoderm; h, heart|
|Figure 3S. Reduced expression of GATA4 in HIF-1α–depleted embryos. A, RT-PCR analysis using total RNA from embryos at stage 1 to stage 25. Both RNA quality and quantity was assessed by amplification of ornithine decarboxylase (ODC). PCR was carried out using the following primers; 5’-AGTGCTACTGCTGCTACCTC-3’ and 5’-ACTGTAGGAGACCTCTCTGC-3’ (55°C annealing, 30 cycles). B, The effect of injection of either CMO or HIF-1α MO1 on GATA4 promoter activity. Similarly to Nkx2.5, Xenopus embryos were injected with the pGL4.10-1.5kb GATA4 promoter (GATA4 promoter-Luc) and promoter-less luciferase reporter gene (pGL4.10) together with either 40 ng CMO or HIF-1α MO1. **P<0.01 vs. CMO. The mouse GATA4 5’-flanking fragment was isolated from a mouse genome using PCR with the following primer pair: 5’-TCCGGCTTGAAGCTTGCTCCCGGC-3’ and 5’-GGAACCTGCAGGCCCTGATTCTGAC-3’. The 1.5kbp fragment was inserted into pGL4.10 vector with EcoRV and HindIII site.|
|hif1a (hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, lateral view, anterior left, dorsal up.|
|hif1a (hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.|
|hif1a (hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33 and 34, lateral view, anterior left, dorsal up.|