Fig 1: ATP6AP2 is critical for sprouting angiogenesis.(A) Images of IB4+ vessels from P7 control and Atp6ap2iECKO retinas, with white arrowheads indicating sprouts at the vascular front. Scale bars: 100 µm. (B) Quantification of the number of sprouts in control and Atp6ap2iECKO mice at P7 (n = 4). (C) Images of IB4+ vessels at high magnification showing filopodia in sprouts (white asterisks) at the vascular front . Scale bars: 10 µm. (D) Quantification of the number of filopodia per sprout in control and Atp6ap2iECKO mice at P7 (n = 4). (E) Western blot analysis of ATP6AP2 and ß-actin in control-siRNA and Atp6ap2 siRNA–treated TeloHAECs. (F) Densitometric quantification of ATP6AP2 levels in E normalized to ß-actin in TeloHAECs following siRNA treatments (n = 3). (G) qPCR analysis of control-siRNA and Atp6ap2 siRNA–treated TeloHAECs for Atp6ap2 mRNA levels normalized to GAPDH transcripts (n = 3; triplicates for each sample). (H) Representative images of TeloHAEC sprouting bead assays embedded in 3D fibrinogen gel at 120 hours following control and Atp6ap2-siRNA treatments. scale bars: 100 µm. Black arrowheads indicate vessel bifurcations. (I) Quantification of the number of sprouts per bead (n = 18). (J) Images of IB4+ vessels at the vascular front of control and Atp6ap2iECKO P7 retinas immunolabeled for Ki67 and the EC-specific nuclear marker ETS transcription factor ERG. Scale bars: 50 µm. (K) Quantification of Ki67+ERG+ proliferative ECs in control and Atp6ap2iECKO mice at P7 (n = 4). (L) Images of ERG+ EC nuclei and IB4+ vessels at the vascular front. Scale bars: 25 µm. (M) Quantification of the number of ERG+ ECs in control and Atp6ap2iECKO P7 mice at the vascular front (n = 4). Data are shown as mean ± SD; 2-tailed unpaired t test. **P < 0.01, ****P < 0.0001.
Fig 2: ATP6AP2 is required for proper revascularization during pathological angiogenesis in the OIR mouse model.(A) Schematic showing the timeline of the OIR protocol with hyperoxia phase from P7 to P12, tamoxifen administration from P12 to P14, and retina analysis at P17. (B) Whole-mount IB4 stained retinas showing the avascular area (yellow) and neovascular tuft (NVT) area (red) in control OIR and Atp6ap2iECKO OIR mice at P17. Scale bars: 1,000 µm. (C and D) Quantification of the avascular area and NVT area in control OIR (n = 8) and Atp6ap2iECKO OIR mice (n = 9) at P17. (E) Images of IB4+ vessels, ERG+ nuclei of ECs, and GM130+ Golgi apparatuses at the neovascularization edge in control OIR and Atp6ap2iECKO OIR P17 retinas. Scale bars: 50 µm. The respective insets (white dashed-line boxes) show magnified views of tip cells. White arrowheads indicate the Golgi apparatus position in respect to the nucleus of tip ECs. White arrows indicate direction of revascularization toward the avascular area. Data are shown as mean ± SD; 2-tailed unpaired t test. ****P < 0.0001.
Fig 3: The Tg(iSuRe-Cre) allele enables multiple gene deletions in single cells or tissues. a The schemes illustrate the Dll4 and Kdr floxed alleles, showing inter-LoxP-site genetic distance, which is significantly larger in the Kdr allele. All four alleles must be deleted to achieve full dual gene loss-of-function. Kdr and Dll4 proteins are expressed in most liver ECs (ERG+, nuclei) of Dll4flox/flox/Kdrflox/flox animals injected with tamoxifen on 3 consecutive days. b Adult mice carrying in addition the Tg(Cdh5-CreERT2) and Tg(iSuRe-Cre) alleles and treated with the same high-dose tamoxifen for 3 consecutive days show very pronounced deletion of Dll4, but not Kdr, in liver MbTomato-/ERG + ECs (yellow arrowheads). However, MbTomato+ cells (white arrowheads) have complete deletion of both genes. c Quantification of the immunostaining signals for ERG, Dll4, Kdr, and MbTomato in large liver sections of the indicated animals. d Illustration of the Myc, Mycn, and Rbpj-floxed alleles showing the genetic distances between the LoxP sites. e Genotypes of control and mutant adult mice injected once with 1 mg of tamoxifen and used for gene-deletion quantification by PCR. The control PCR band provides a DNA input quantitative control for the PCR, since it corresponds to a wild-type genomic sequence, present in all DNA samples (see supplementary table 2 for primer sequences). Animals containing the Tg(Cdh5-CreERT2) and Tg(iSuRe-Cre) alleles have deletion of the six floxed alleles only in FACS-sorted MbTomato+ cells, as detected by semi-quantitative competitive PCR. Weak Mycn and Rbpj-floxed bands in the MbTomato + sample PCR may result from incomplete gene-deletion or contamination of this sample with MbTomato-negative cells, or their DNA, during the FACS protocol. f Image J quantification of the relative intensity of the floxed and control PCR gel bands shown in e, providing an estimate of the degree of the indicated floxed gene deletion. Scale Bars 65 µm. Error bars indicate StDev. **p < 0.001. NS nonsignificant. One-way ANOVA with Tukey’s post hoc test. Source data are provided as a Source Data file
Fig 4: The Tg(iSuRe-Cre) allele increases the efficiency of inducible genetic modifications. a–c Representative confocal micrographs of P6 retina vessels labelled with IsolectinB4 (endothelial surface) and anti-ERG antibody (endothelial nuclei), obtained from animals with the genotype indicated to the left and induced with high-dose tamoxifen from P1 to P3. Images represent the observed phenotypic variability; EC number for each image is depicted in chart c (yellow dots). 20 vs 24 retina microscopic fields were analysed (n = 5 vs n = 6 animals per group). d Linear regression showing the high correlation (r2) between total Erg + EC number (phenotype) and the number of Erg + /MbTomato- cells. e, f Representative confocal micrographs of P6 retina vessels from animals with the genotype indicated to the left and induced with high-dose tamoxifen from P1 to P3. Images represent the phenotypic variability of animals with the same genotype. EC number and reporter expression frequency is indicated below the figures. A comparison of EC number and reporter expression frequency is depicted in chart for 24 vs 34 retina microscopic fields (n = 6 animals per group). Yellow dots in chart f represent the values for images in b and e. g qRT-PCR analysis of Kdr mRNA levels from FACS-sorted liver ECs (n = 5 animals), or immunostaining analysis of liver sections of animals (n = 3) with the indicated genotype (see also Supplementary Fig. 2b). h Semi-quantitative competitive PCR showing the efficiency of Kdr, Rbpj and Nmyc deletion in FACS-sorted cells of adult mice with the indicated genotypes and induced with tamoxifen. Note that some cross-contamination of samples and DNA may occur during tissue dissociation and FACS of mutant and wild-type cells. i Semi-quantitative competitive PCR for the Notch1 floxed allele and a control genomic sequence showing the efficiency of the Ubc-CreERT2 induced Notch1 deletion in Tomato+ and Tomato- cells of the liver. j Semi-quantitative competitive PCR showing the efficiency of Rbpj gene inducible deletion in the Tomato− and Tomato+ cells of several distinct organs from Rosa26-CreERT2 mice induced with tamoxifen. k Epas1 mRNA relative levels (qRT-PCR) in LysM-Cre-reporter-expressing bone marrow-derived macrophages (n = 4). Scale Bars 200 μm. Error bars indicate StDev; **p < 0.001; ***p < 0.0001. Two-tailed unpaired t-test (4c) or ANOVA (4 g and 4k). Source data are provided as a Source Data file
Fig 5: The Tg(iSuRe-Cre) allele is ubiquitously expressed and is a reliable reporter of recombination of other reporter alleles. a Analysis of Tg(iSuRe-Cre) expression in the absence of Cre activity reveals its expression (N-PhiM+) in most cells of mouse embryos. The allele does not self-recombine in embryos (MbTomato-negative). b, c FACS analysis reveals that the Tg(iSuRe-Cre) allele does not self-recombine in blood, unlike its ROSA26 gene-targeted version (each dot in the chart represents quantification of one animal). d, e Confocal micrographs of postnatal day (P) 6 retina vessels from animals with the alleles depicted above the panels and induced with tamoxifen from P1 to P3. All endothelial cells (ECs; nuclei, ERG+) expressing MbTomato-2A-Int-Cre also recombined the reporter allele ROSA26LSL-YFP. f Quantification of the different recombination events/reporters in retinal vessels with low and high frequencies of tamoxifen-induced CreERT2 recombination (n = 4 full retinas per group). g FACS analysis of liver ECs from tamoxifen-induced adult animals with the genotype indicated in d. All induced MbTomato+ cells also recombined the reporter allele ROSA26LSL-YFP. The MbTomato reporter from the Tg(iSuRe-Cre) allele is easier to separate from baseline autofluorescence. h, i Six-channel confocal micrograph of P6 retina vessels from animals (n = 3) with the genotype indicated above panels and induced with tamoxifen at P3. All cells expressing the MbTomato reporter recombined the two reporter alleles (ROSA26LSL-YFP and ROSA26iChr2-Mosaic), resulting in expression of EYFP and one of the three possible nuclear-localized proteins (H2B-Cherry, H2B-EGFP, or HA-H2B-Cerulean). In contrast, only a small fraction of EYFP+ cells recombined and expressed this reporter in their chromatin/nuclei. Scale Bars 150 μm. Error bars indicate StDev; ***p < 0.0001; Two-tailed unpaired t-test (3c). Data in 3i indicate the mean frequencies obtained in the indicated cell groups
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