Journal: Nature Communications

Article Title: Notch signal strength controls cell fate in the haemogenic endothelium

doi: 10.1038/ncomms9510

Figure Lengend Snippet: ( a ) Schematic representation of Notch activation history mouse reporters by replacing the intracellular domain of mouse Notch1 with low sensitivity (N1IP::Cre LO ) and high sensitivity (N1IP::Cre HI ) Cre-recombinase. Reporter activation of N1IP::Cre LO requires a high threshold of Notch activity, while N1IP::Cre HI is induced in response to low or high Notch activity. ( b ) Flow cytometry analysis of peripheral blood of adult mice. Cells were stained with Lineage (lin) markers (CD3, B220, Gr1, Mac1 and Ter119) gated on lin+ cells. Numbers indicate the percentage of YPF+ cells. ( c ) Graph represents the percentage of YFP+ cells within haematopoietic cell types in the bone marrow (BM), spleen and thymus of N1IP::Cre LO (grey bars) and N1IP::Cre HI (blue bars) as detected using flow cytometry. ( d ) Representative confocal images of three-dimensional whole-mount immunostaining in N1IP::Cre HI and N1IP::Cre LO embryos (E10.5) detecting YFP (green), c-Kit (cyan) and CD31 (red). General view of the dorsal aorta (left panel) and details of haematopoietic cluster (right panels). White arrows indicate cluster structures. D, dorsal; DA, dorsal aorta, HC, haematopoietic cluster; V, ventral. Scale bars, 100 μm for DA, 25 μm for HC in N1IP::Cre HI and 50 μm in N1IP::Cre Low . See also . ( e , f ) Graphs show the percentage of reconstituted cells in animals transplanted with YFP+ and YFP− fractions of E13-14 fetal liver and BM at 4-month post-transplantation ( e ). Representative dot plots from analysis ( f ). Donor CD45.2 N1IP::Cre HI cell fractions together with 500,000 supporting CD45.1 spleen cells were transplanted into CD45.1/CD45.2 chimeras.

Article Snippet: On transference into scintillation vials containing 100% methanol, embryos were rehydrated and blocked with BSA/PBS-MT solution on ice for 1 h. Primary antibodies were used at the following concentrations: anti-Kit 1:500 (eBioscience 14-1171-81), anti-Jag1 1:50 (Santa Cruz sc-6011), anti-Dll4 1:500 (Abcam ab7280) and biotinylated anti-CD31 1:500 (BD 553371).

Techniques: Activation Assay, Activity Assay, Flow Cytometry, Staining, Immunostaining, Transplantation Assay

Journal: Nature Communications

Article Title: Notch signal strength controls cell fate in the haemogenic endothelium

doi: 10.1038/ncomms9510

Figure Lengend Snippet: ( a ) Representative confocal images of E10.5 embryo transverse section with CD31 (red) and Jag1 (green, left) and Dll4 (green, right). Details of ventral part (lower panels) corresponding to boxed areas. Scale bars, 25 μm. Nuclear staining with 4,6-diamidino-2-phenylindole is shown (D, dorsal; V, ventral). ( b ) Experimental design to test the effects of OP9-Jag1 and OP9-Dll4 on purified CD31 + CD45 − Ter119 − AGM cells after 7 days of culture. ( c ) Quantification of haematopoietic lineage generated from CD31 + CD45 − Ter119 − AGM cells on culture on OP9-Jag1 or OP9-Dll4. Bars represent the total number of cells on 7-day culture; n =4 or more samples of at least two independent experiments. s.e.m. is represented. P value for t -test is indicated. ( d ) Schematic representation for purification of E11.5 AGM CD31 + Kit − CD45 − Ter119 − and CD31 + Kit + CD45 − Ter119 − (Kit− and Kit+). In parallel, CD31 + CD45 − Ter119 − cells were incubated for 2 h in OP9-Jag1 or OP9-Dll4. Cells were resorted on the basis of Kit expression (Kit−J and Kit+J or Kit−D and Kit+D). ( e ) Principal component analysis (PCA) of global gene expression profiles of samples included in the study. Each dot of the same colour represents arrays from replicates of the same sample. Dotted lines arbitrarily reunite replicates of a specific condition. ( f ) Unsupervised hierarchy clustering of transcriptional profiles from selected cell populations. ( g ) Venn diagram displaying the number of genes differentially expressed in cluster-containing versus endothelial populations (Kit+_Kit−) and/or in endothelial population after versus before incubation on OP9-Jag1 (Kit−J_Kit−). Fold change expression levels from MicroArray analysis of 23 angiogenic-related genes are listed. Green-colour grade represents the range of fold change (FC) values on Kit+ or Kit−J populations compared with Kit− cells. All FC values represent statistically significant differences on gene expression ( P value-adjusted<0.05). Grey cells represent genes in which FC is not statistically significant. ( h ) Biological process enrichment analysis of genes differentially expressed in Kit−J_Kit− and Kit+_Kit− comparisons. Only selected GO terms are presented, and all significant terms are given in . ( e – h ) Analysis performed on three independent experiments; one Kit−J sample was excluded for technical reasons.

Article Snippet: On transference into scintillation vials containing 100% methanol, embryos were rehydrated and blocked with BSA/PBS-MT solution on ice for 1 h. Primary antibodies were used at the following concentrations: anti-Kit 1:500 (eBioscience 14-1171-81), anti-Jag1 1:50 (Santa Cruz sc-6011), anti-Dll4 1:500 (Abcam ab7280) and biotinylated anti-CD31 1:500 (BD 553371).

Techniques: Staining, Purification, Generated, Incubation, Expressing, Microarray

Journal: Nature Communications

Article Title: Notch signal strength controls cell fate in the haemogenic endothelium

doi: 10.1038/ncomms9510

Figure Lengend Snippet: ( a ) Microarray validation using qRT–PCR. Expression of a panel of endothelial-related genes on E11.5 CD31+CD45−Ter119− AGM cells incubated 2 h on OP9-Jag1. Quantification was performed in Kit−, Kit+, Kit−J and Kit+J. The bars represent the average expression level of four to six replicates from two independent experiments, normalized to Kit− expression. Average fold change (±s.d.) is represented. Student's t -test for comparisons of Kit+, Kit−J and Kit+J populations with Kit− population was performed (* P ≤0.05; ** P ≤0.01; P ≤0.001). ( b ) Expression of haematopoietic genes on E10.5 and E11.5 Kit− and Kit−J populations (2 h on OP9-Jag1). The bars represent the average (±s.e.m.) expression level of six to nine replicates from three or four independent experiments, normalized to Kit− expression. Statistical significance was assessed by Student's t -test (* P ≤0.05; *** P ≤0.001 when significant). nd, not detected.

Article Snippet: On transference into scintillation vials containing 100% methanol, embryos were rehydrated and blocked with BSA/PBS-MT solution on ice for 1 h. Primary antibodies were used at the following concentrations: anti-Kit 1:500 (eBioscience 14-1171-81), anti-Jag1 1:50 (Santa Cruz sc-6011), anti-Dll4 1:500 (Abcam ab7280) and biotinylated anti-CD31 1:500 (BD 553371).

Techniques: Microarray, Quantitative RT-PCR, Expressing, Incubation

Journal: Nature Communications

Article Title: Notch signal strength controls cell fate in the haemogenic endothelium

doi: 10.1038/ncomms9510

Figure Lengend Snippet: ( a ) Representative analysis of population distribution in Jag1 +/+, Jag1 +/− and Jag1 −/− animals. Endothelial population (CD31 + Kit − CD45 − ) in red (frame and graph bars), cluster-containing population (CD31 + Kit + CD45 − ) in orange (frame and graph bars) and mature haematopoietic population (CD31 + Kit − CD45 + ) in blue (frame and graph bars). Bars represent the average population distribution in Jag1 +/+ ( n =6), Jag1 +/− ( n =10) and Jag1 −/− ( n =6) embryos, normalized to Jag1 +/+ ±s.e.m. Significance was assessed by Student's t -test (* P ≤0.05 and ** P ≤0.01 when significant). ( b ) Representative confocal images of transversal sections of E10.5 AGM from Jag1 +/+ and Jag1 −/− stained for CD31 (red) and Kit (green). Nuclear staining with DAPI. White arrows point to cluster-like structures. Scale bar, 25 μm. Asterisks indicate autofluorescent circulating cells. D, dorsal; V, ventral. ( c , d ) Quantification of Kit+ cells ( c ) and Kit+ clusters (group of at least four positive cells; d ) per 100 μm of AGM from Jag1 +/+ and Jag1 −/− embryos. Bars represent the average±s.e.m. of cells per embryo ( n =4). Student's t -test was performed (** P ≤0.01). ( e , f ) FC in the expression levels of the indicated genes as detected using qRT–PCR from E10.5 Jag1−/− compared with Jag1+/+ Kit+ cells ( e ) or in Kit+ compared with Kit− populations in the Jag1+/+ and Jag1−/−. ( f ). Two independent pooled samples for each genotype were analysed. Colour grades reflect the FC values of each gene expression. All FC values represent statistically significant differences on gene expression ( P value≤0.05). Genes that do not show a statistically significant alteration in pairwise comparison are listed in grey. See also . ( g ) CFU-S 11 from E10.5 Jag1 +/+ or Jag1 −/− AGM explant cultures. n represents the number of treated animals per condition. Each mouse was transplanted with one embryo equivalent AGMs on 3 days in explant culture. Results obtained from three independent experiments. Irrad ctrl: irradiated/non-injected control. Bars represent the mean±s.e.m. of colonies per tissue. Significance is assessed by Student's t -test (*** P ≤0.001; not assigned if not significant). ( h ) Percentage of reconstitution at 4 months post transplantation. Each dot represents a single animal. n represents the number of transplanted mice per genotype.

Article Snippet: On transference into scintillation vials containing 100% methanol, embryos were rehydrated and blocked with BSA/PBS-MT solution on ice for 1 h. Primary antibodies were used at the following concentrations: anti-Kit 1:500 (eBioscience 14-1171-81), anti-Jag1 1:50 (Santa Cruz sc-6011), anti-Dll4 1:500 (Abcam ab7280) and biotinylated anti-CD31 1:500 (BD 553371).

Techniques: Staining, Expressing, Quantitative RT-PCR, Irradiation, Injection, Transplantation Assay

Journal: Nature Communications

Article Title: Notch signal strength controls cell fate in the haemogenic endothelium

doi: 10.1038/ncomms9510

Figure Lengend Snippet: ( a ) Schematic of experimental design. E10.5 AGM CD31 + CD45 − Ter119 − cells were sorted and incubated in the medium with anti-Dll4- or anti-Jag1-blocking antibodies or irrelevant Ig for 7 days. ( b ) Graphs represent the average FC of total cell number obtained after culturing with anti-Dll4 or anti-Jag1 compared with each Ig control±s.e.m. from three independent experiments. Student's t -test was used to assess the significance (** P ≤0.01; not assigned if not significant). ( c ) Relative number of CFC haematopoietic progenitors obtained in b . Average results from three independent experiments ±s.d. determination. Student's t -test was used to assess the significance (* P ≤0.05; ** P ≤0.01). ( d ) E11.5 CD31 + Kit − CD45 − Ter119 − cells were sorted and incubated for 5 h on the medium supplemented with anti-Dll4-blocking antibody or irrelevant Ig control. Cells were injected in 3-Gy-irradiated SCID-Beige mice or resorted for mRNA extraction and qRT–PCR analyses. ( e ) Quantification of spleen colonies (CFU-S 11 ). Bars represent the average of six replicates from three independent experiments (coloured dots) ±s.e.m. Student's t -test was used to assess significance. ( f ) FC in expression levels of a panel of angiogenic- and haematopoietic-related genes using qRT–PCR. Colour grades reflect the fold change values of each gene expression (normalization to expression levels in control conditions). All coloured FC values represent statistically significant differences on gene expression ( P ≤0.05, using Student's t -test). Grey is shown for non-statistical significant differences.

Article Snippet: On transference into scintillation vials containing 100% methanol, embryos were rehydrated and blocked with BSA/PBS-MT solution on ice for 1 h. Primary antibodies were used at the following concentrations: anti-Kit 1:500 (eBioscience 14-1171-81), anti-Jag1 1:50 (Santa Cruz sc-6011), anti-Dll4 1:500 (Abcam ab7280) and biotinylated anti-CD31 1:500 (BD 553371).

Techniques: Incubation, Blocking Assay, Injection, Irradiation, Quantitative RT-PCR, Expressing

Journal: Nature Communications

Article Title: Transcriptional regulation of endothelial cell behavior during sprouting angiogenesis

doi: 10.1038/s41467-017-00738-7

Figure Lengend Snippet: RiboTag analysis of retinal ECs during postnatal development. a Diagram depicting retinal EC-specific RiboTag transcriptome analysis. Inducible (Tmx controlled) recombination of the RiboTag allele ( Rpl22 HA/HA ) led to expression of HA-tagged L22 ribosomal protein specifically in ECs. Ribosome-bound transcripts were immunoprecipitated (IP) from homogenized whole retinas with anti-HA antibody-coupled magnetic beads. Extracted mRNAs were analyzed by RT-qPCR or RNA-Seq analysis. b P6 Rpl22 HA/HA (control) and Pdgfb-iCre Rpl22 HA/HA retinas stained with Isolectin B4 (IB4, red ) and anti-HA antibody ( green ). Scale bars represent 300 μm. c RT-qPCR analysis of transcripts expressed in retinal ECs. EC-specific markers, Pecam1 and Emcn , and Acta2 negative control were normalized to Actb levels using ΔΔCT method. Anti-HA immunoprecipitated RNAs were compared to whole retinal lysate input. Error bars represent mean ± s.e.m., n = 6, *** P < 0.0001; unpaired Student’s t -test. d IB4 staining of whole retinas at P6, P10, P15, P21, and P50 showing vascularization during postnatal development. Scale bars represent 0.5 mm. e Unsupervised hierarchical clustering of 15 retinal EC RNA-Seq data sets with three replicates per stage. f Two-dimensional principal component analysis (PCA) plot showing the discrimination of gene expression profiles between retinal ECs from different ages

Article Snippet: We performed immunostaining using following antibodies: biotinylated IB4 (B-1205; VectorLabs), rat anti-HA (3F10; Roche), rabbit anti-MafB (IHC-00351-1; Bethyl Laboratories), rabbit anti-Erg1 (EPR3863; Abcam), rabbit anti-ColIV (2150-1470; Serotec), goat anti-Dll4 (AF1389; R&D), goat anti-Pdgfrb (AF1042; R&D), and streptavidin Alexa Fluor-488 (Invitrogen).

Techniques: Expressing, Immunoprecipitation, Magnetic Beads, Quantitative RT-PCR, RNA Sequencing Assay, Staining, Negative Control