Journal: Scientific Reports

Article Title: Dynamics and function of CXCR4 in formation of the granule cell layer during hippocampal development

doi: 10.1038/s41598-017-05738-7

Figure Lengend Snippet: Altered distribution pattern of Prox1-positive granule cell precursors induced by the CXCR4 antagonist AMD3100. AMD3100 or vehicle (control) was injected into the telencephalic lateral ventricle of E15.5 embryos in utero , and the embryos were left to develop until E18.5 before sampling. ( A , B ) Immunoreactivity for CXCR4 and Prox1, a marker for granule cells in the hippocampus at E18.5. ( A ) In the control, cells strongly positive for Prox1 were mainly localized in the dentate gyrus (DG). ( B ) In AMD3100-treated mice, cells strongly positive for Prox1 were observed not only in the DG (arrowhead), but also in the ventricular zone (VZ) and dentate migratory stream (DMS, arrows). ( C ) The number of Prox1-positive cells in the DG, and in the VZ and DMS. The number of Prox1-positive cells in the VZ and DMS is significantly higher in the AMD3100-treated mice than in control mice (*P < 0.05, Student’s t test). No significant difference was observed in the total number of Prox1-positive cells between the two groups. Ten sections were counted from each of 5 (control) or 6 (AMD3100-treated) animals. F, fimbria; HF, hippocampal fissure; V, ventricle. Scale bar = 50 µm in A and B .

Article Snippet: The primary antibodies used were as follows: anti-CXCR4 UMB2 (1:200, rabbit monoclonal, Abcam), anti-GFP (1:1,000, chick polyclonal, Abcam), anti-GM130 (1:500, mouse monoclonal, BD Transduction Laboratories), anti-γ-tubulin (1:200, mouse monoclonal, Sigma), anti-Ki67 (1:500, rabbit polyclonal, Novocastra), anti-LAMP1 (1:1000, rat monoclonal, Abcam), anti-NeuroD (1:200, goat polyclonal, Santa Cruz Biotechnology, Inc.), anti-Prox1 (1:1,000, goat polyclonal, R&D Systems), anti-p73 (1:200, rabbit polyclonal, Santa Cruz Biotechnology, Inc.).

Techniques: Injection, In Utero, Sampling, Marker

Journal: Scientific Reports

Article Title: Dynamics and function of CXCR4 in formation of the granule cell layer during hippocampal development

doi: 10.1038/s41598-017-05738-7

Figure Lengend Snippet: Ectopic positioning of GCPs in the hippocampal fissure-surrounding region (HFSR) induced by the CXCR4 antagonist AMD3100. AMD3100 or vehicle (control) was injected into the lateral ventricle of E15.5 embryos in utero , and the embryos were left to develop until E18.5. ( A,B ) Expression of Gfap -GFP and p73, a marker for Cajal-Retizus cells in the hippocampus of Gfap -GFP mice. The boxed regions in A1 and B1 are enlarged in A2 and B2, respectively. A3 and B3 are 3-dimensional images of A2 and B2, respectively, that were reconstructed from 10 optical slices. p73-positive cells accumulated in the HFSR in both the control ( A1–3 ) and AMD3100-treated ( B1–3 ) mice. In the control, only a few Gfap -GFP+ cells (arrows) were observed in the HFSR ( A1–3 ), although the processes extending from the Gfap -GFP+ cells in the dentate gyrus (DG, open arrowheads) invaded into the HFSR (white arrowheads). In the AMD3100-injected mice, many Gfap -GFP-positive cells accumulated in the HFSR ( B1–3 , arrows). ( C,D ) The proportion of Gfap- GFP+ cells in the HFSR to those in the HFSR and DG. A significant increase in Gfap -GFP+ cells was observed in the AMD3100-treated mice ( C , *P < 0.05; Student t-test). However, no difference was detected between the groups in the number of p73+ cells in the HFSR ( D , P = 0.741, Student t-test). ( E–J ) Properties of Gfap -GFP-expressing cells in the HFSR. In both control and AMD3100-treated mice, Gfap -GFP+ cells (arrows) in the HFSR do not express the granule cell marker Prox1 ( E and I ), although a few GFP-positive cells express the neural progenitor marker NeuroD (arrowheads in F and J ). However, the neural stem cell marker Sox2 ( G and K ) and the proliferation marker Ki67 ( H and L ) are expressed in the majority of Gfap- GFP-positive cells in the HFSR (arrowheads) in both groups. Results of quantitative analysis are shown in M. The number of Gfap -GFP+/Sox2+ cells and Gfap -GFP+/Ki67+ cells increased in the AMD3100-treated mice (* P < 0.05, Student t-test). We counted 10 sections from each of 3 (control) or 4 (AMD3100-treated) animals. DG, dentate gyrus; F, fimbria; V, ventricle. Scale bars = 50 µm in A1, B1 , and E – L ; 20 µm in A2, A3, B2 , and B3 .

Article Snippet: The primary antibodies used were as follows: anti-CXCR4 UMB2 (1:200, rabbit monoclonal, Abcam), anti-GFP (1:1,000, chick polyclonal, Abcam), anti-GM130 (1:500, mouse monoclonal, BD Transduction Laboratories), anti-γ-tubulin (1:200, mouse monoclonal, Sigma), anti-Ki67 (1:500, rabbit polyclonal, Novocastra), anti-LAMP1 (1:1000, rat monoclonal, Abcam), anti-NeuroD (1:200, goat polyclonal, Santa Cruz Biotechnology, Inc.), anti-Prox1 (1:1,000, goat polyclonal, R&D Systems), anti-p73 (1:200, rabbit polyclonal, Santa Cruz Biotechnology, Inc.).

Techniques: Injection, In Utero, Expressing, Marker

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Deficiency of FABP7 Triggers Premature Neural Differentiation in Idiopathic Normocephalic Autism Organoids.

doi: 10.1002/advs.202406849

Figure Lengend Snippet: Figure 6. MEK2 overexpression causes autistic-like behaviors in mice, and its inhibition can rescue premature differentiation in ASD organoids. A) Schematic of AAV2/9 vectors for expressing MEK2-EGFP. B) Immunostaining images displaying GFP expressed in the hippocampus, including the dentate gyrus (DG) and CA, colabeled with the DG granule cell marker Prox1. Scale bar: 200 μm (left), 50 μm (right). C) Schematic of the open field test (Left) and histograms (Right) presenting the center zoneduration time (AAV-NC: n = 14 mice, AAV-MEK2: n = 15 mice, ns p = 0.86) and center zone entries (AAV-NC: n = 14 mice, AAV-MEK2: n = 15 mice, ns p = 0.31) of the mice in the AAV-NC and AAV-MEK2 groups. D) Schematic of the 3-chambered

Article Snippet: CTIP2 Rat IgG 1:500(IF) abcam ab18465 DCX Rabbit IgG 1:1000(IF) Cell Signaling 4604 FABP7 Rabbit IgG 1:2000(WB) 1:500(IP) Cell Signaling Technology 13347S FOXG1 Rabbit IgG 1:1000(IF) abcam ab18259 FOXP2 Rabbit IgG 1:1000(IF) abcam ab16046 GAPDH Mouse IgG 1:5000(WB) affinity T0004 GFP Chicken IgG 1:1000(IF) Millipore ab16901 GFP Rabbit IgG 1:1000(IF) Chemicon AB3080 KI67 Rabbit IgG 1:500(IF) Invitrogen 180191Z MAP4K2 Rabbit IgG 1:2000(WB) abcam ab184169 MEK1/2 Mouse IgG 1:1000(WB) Cell Signaling Technology 4694S NANOG Goat IgG 1:1000(IF) R&D Systems AF1997 NESTIN Goat IgG 1:1000(IF) Santa Cruz SC-21247 PAX6 Rabbit IgG 1:500(IF) convance PRB-278P PHH3 Rat IgG 1:1000(IF) Cell Signaling Technology 9706S PKCλ Mouse IgG 1:1000(IF) BD 610 207 p-MEK1/2 (S217/221) Rabbit IgG 1:1000(WB) Cell Signaling Technology 3958S PROX1 Rabbit IgG 1:300(IF) abcam ab101851 p-Vimentin Mouse IgG 1:1000(IF) MBL D076-3 SOX2 Goat IgG 1:500(IF) R&D Systems AF2018 TBR1 Rabbit IgG 1:500(IF) abcam ab31940 γ-tublin Mouse IgG 1:1000(IF) abcam ab11316 Alexa Fluor 488 Goat anti-Chicken IgG 1:1000(IF) Invitrogen A11039 Alexa Fluor 488 Donkey anti-Rat IgG 1:1000(IF) Invitrogen A21208 Alexa Fluor 488 Donkey anti-Rabbit IgG 1:1000(IF) Invitrogen A21206 Alexa Fluor 488 Donkey anti-Mouse IgG 1:1000(IF) Invitrogen A21202 Alexa Fluor 488 Donkey anti-Goat IgG 1:1000(IF) Invitrogen A11055 Alexa Fluor 546 Donkey anti-Rabbit IgG 1:1000(IF) Invitrogen A10040 Alexa Fluor 546 Donkey anti-Mouse IgG 1:1000(IF) Invitrogen A10036 Alexa Fluor 546 Goat anti-Rat IgG 1:1000(IF) Invitrogen A11081 Alexa Fluor 546 Donkey anti-Goat IgG 1:1000(IF) Invitrogen A11056 Alexa Fluor 647 Donkey anti-Mouse IgG 1:1000(IF) Invitrogen A31571 Alexa Fluor 647 Donkey anti-Rabbit IgG 1:1000(IF) Invitrogen A31573 Alexa Fluor 647 Donkey anti-Goat IgG 1:1000(IF) Invitrogen A21447 Hoechst332258 1:1000(IF) Invitrogen A1339 HRP Goat Anti-Mouse IgG 1:5000(WB) Multi Sciences 70-GAM0072 HRP Goat Anti-Rabbit IgG 1:5000(WB) Multi Sciences 70-GAR0072 was reverse transcribed into cDNA using a PrimeScriptTM RT reagent kit (TaKaRa) and prepared for qPCR using the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific).The primers for qPCR used were as follows: FABP7 forward primer, 5′-TCTCACCTCCTTCCTTCTTCT-3′ and reverse primer, 5′-AGAACCTTG CCAGTGATGTATT-3′; MEK2 forward primer, 5′-CTCACCATCAACCCTAC CATC-3′, and reverse primer, 5′-GCAGGTCCACCAGGTTT-3′; GAPDH forward primer, 5′-TCGACAGTCAGCCGCATCTTCTTT-3′ and reverse primer, 5′-ACCAAATCCGTTGACTCCGACCTT-3′.

Techniques: Over Expression, Inhibition, Expressing, Immunostaining, Marker

Journal: Genes & Development

Article Title: The Prox1–Vegfr3 feedback loop maintains the identity and the number of lymphatic endothelial cell progenitors

doi: 10.1101/gad.216226.113

Figure Lengend Snippet: Vegfr3 is a dosage-dependent target of Prox1. ( A ) H 5 V cells were infected with Prox1-expressing retroviruses. Individual clones were picked following positive selection with antibiotics. RNA extracted from the clones was analyzed by real-time PCR for the expression of Prox1 ( left graph) and Vegfr3 ( right graph). ( B ) Protein levels of Vegfr3 appear to correlate directly with those of Prox1. ( C ) DNA regions located 5 kb upstream of exon 1 of human (Hs), mouse (Mm), rat (Rn), and zebrafish (Dr) Vegfr3 were analyzed for putative Prox1-binding sites by TRANSFAC bioinformatics software. Upward bars indicate the sites in the sense orientation, and downward bars indicate those in the antisense orientation. Colored boxes identify the different Prox1 recognition sites (see Supplemental Table 1). ( D ) ChIP was carried out on mouse LECs collected by flow cytometry. A rabbit polyclonal antibody against Prox1 or control IgG was used to pull down the DNA fragments. Real-time PCR was carried out using primers specific for the various regions along the 5-kb DNA element of mouse Vegfr3 . Substantial Prox1 binding was observed at the indicated sites. These data are from three independent experiments. (*) P < 0.05.

Article Snippet: The following primary antibodies were used: rabbit anti-β-gal (MP Biomedicals), rabbit anti-Prox1 (AngioBio and ProteinTech Group), goat anti-Prox1 (R&D Systems), hamster anti-podoplanin (Hybridoma Bank, Developmental Studies, University of Iowa), rat anti-PECAM1 (BD Pharmingen), goat anti-Vegfr3 (R&D Systems), rabbit anti-GFP (Molecular Probes), and chicken anti-GFP (Abcam).

Techniques: Infection, Expressing, Clone Assay, Selection, Real-time Polymerase Chain Reaction, Binding Assay, Software, Flow Cytometry

Journal: Genes & Development

Article Title: The Prox1–Vegfr3 feedback loop maintains the identity and the number of lymphatic endothelial cell progenitors

doi: 10.1101/gad.216226.113

Figure Lengend Snippet: LEC progenitors and LECs are reduced in Prox1 +/GFPCre ;Vegfr3 +/LacZ embryos. Vegfr3 +/LacZ mice were bred with Prox1 +/GFPCre to generate Prox1 +/GFPCre ;Vegfr3 +/LacZ double-heterozygous embryos. ( A–C ) At E13.5 Vegfr3 +/LacZ embryos were phenotypically normal ( A ), Prox1 +/GFPCre embryos had mild edema ( B ), and severe edema and regions of hemorrhagic accumulation were observed in most Prox1 +/GFPCre ;Vegfr3 +/LacZ embryos ( C ). Prox1 +/GFPCre and Prox1 +/GFPCre ;Vegfr3 +/LacZ embryos were collected at E11.5 ( D–F ), E13.5 ( G , H ), or E15.5 ( I , J ) and analyzed by immunohistochemistry for the LEC markers Prox1 and podoplanin together with the pan-EC marker PECAM1. ( D , E , G , H ) Transverse sections, with the neural tube and heart at the right and left , respectively, of the figures. ( I , J ) Whole mounts on the peripheral skin. ( D , E ) Compared with Prox1 +/GFPCre embryos, the expression of Prox1 and podoplanin is reduced in Prox1 +/GFPCre ;Vegfr3 +/LacZ littermates at E11.5. ( F ) Furthermore, the number of Prox1 + LECs is reduced in Prox1 +/GFPCre ;Vegfr3 +/LacZ embryos at E11.5 ( n = 3 for each genotype). At E13.5, while a clear lymph sac (LS) was seen in Prox1 +/GFPCre embryos ( G ), scattered and mispatterned blood-filled structures were seen in Prox1 +/GFPCre ;Vegfr3 +/LacZ embryos ( H ; arrows). At E15.5, lymphatic vessels are seen in Prox1 +/GFPCre embryonic skin ( I ); however, in Prox1 +/GFPCre ;Vegfr3 +/LacZ embryos, the few Prox1 + structures (dashed lines) failed to express podoplanin ( J ). P -values are as follows: (**) P = 0.0068; (*) P = 0.0128. Bars, 100 μm.

Article Snippet: The following primary antibodies were used: rabbit anti-β-gal (MP Biomedicals), rabbit anti-Prox1 (AngioBio and ProteinTech Group), goat anti-Prox1 (R&D Systems), hamster anti-podoplanin (Hybridoma Bank, Developmental Studies, University of Iowa), rat anti-PECAM1 (BD Pharmingen), goat anti-Vegfr3 (R&D Systems), rabbit anti-GFP (Molecular Probes), and chicken anti-GFP (Abcam).

Techniques: Immunohistochemistry, Marker, Expressing

Journal: Genes & Development

Article Title: The Prox1–Vegfr3 feedback loop maintains the identity and the number of lymphatic endothelial cell progenitors

doi: 10.1101/gad.216226.113

Figure Lengend Snippet: Prox1 +/GFPCre ;Vegfr3 +/LacZ embryos fail to maintain Prox1 expression in LEC progenitors. Prox1 +/GFPCre mice were bred with Vegfr3 +/LacZ ;R26 mT/mG mice, and the resulting E10.5 Prox1 +/GFPCre ;R26 mT/mG and Prox1 +/GFPCre ; Vegfr3 +/LacZ ;R26 mT/mG embryos were analyzed by immunohistochemistry for Prox1 and GFP. Upon Cre-mediated activation, the R26 mT/mG reporter expresses a membrane-tagged GFP that allows the visualization of the cell surface. ( A – C ) In Prox1 +/GFPCre ;R26 mT/mG embryos, numerous Prox1 + cells were seen on the CV. All of these cells and a few Prox1 − cells on the CV were GFP + (white dotted line). ( D–F ) In contrast, in Prox1 +/GFPCre ; Vegfr3 +/LacZ ;R26 mT/mG embryos, fewer Prox1 + cells were seen on the CV, although most of these cells were GFP + (white dotted line). Bars, 100 μm. ( G ) For cell counting, the number of GFP + Prox1 − DAPI + or GFP + Prox1 + DAPI + cells were quantitated in stained sections ( n = 3 embryos for each genotype). Cell counting showed that the number of Prox1 − GFP + LEC progenitors ( P -value = 0.065) was higher in double heterozygous, indicating that the Vegfr3–Prox1 interaction is required to maintain LEC fate.

Article Snippet: The following primary antibodies were used: rabbit anti-β-gal (MP Biomedicals), rabbit anti-Prox1 (AngioBio and ProteinTech Group), goat anti-Prox1 (R&D Systems), hamster anti-podoplanin (Hybridoma Bank, Developmental Studies, University of Iowa), rat anti-PECAM1 (BD Pharmingen), goat anti-Vegfr3 (R&D Systems), rabbit anti-GFP (Molecular Probes), and chicken anti-GFP (Abcam).

Techniques: Expressing, Immunohistochemistry, Activation Assay, Membrane, Cell Counting, Staining

Journal: Genes & Development

Article Title: The Prox1–Vegfr3 feedback loop maintains the identity and the number of lymphatic endothelial cell progenitors

doi: 10.1101/gad.216226.113

Figure Lengend Snippet: Vegfr3 regulates Prox1 expression in LEC progenitors and differentiating LECs. Wild-type (WT) [ Tg(Prox1-tdTomato) ], Prox1 +/− [ Tg(Prox1-tdTomato);Prox1 +/LacZ ], and Vegfr3 +/− [ Tg(Prox1-tdTomato); Vegfr3 +/LacZ ] embryos were collected at E11.5 (one embryo per genotype). Using laser capture microdissection, LEC progenitors (inside the CV) and differentiating LECs (outside the CV) were collected separately, and the levels of Prox1 ( A ) and Vegfr3 ( B ) were analyzed by quantitative real-time PCR. mRNA levels are shown as fold of increase and were normalized to PECAM1 levels. These qPCR results were generated from a single laser capture experiment using pooled mRNA extracted from cells captured from 10-μm sections obtained from the entire embryo for each genotype.

Article Snippet: The following primary antibodies were used: rabbit anti-β-gal (MP Biomedicals), rabbit anti-Prox1 (AngioBio and ProteinTech Group), goat anti-Prox1 (R&D Systems), hamster anti-podoplanin (Hybridoma Bank, Developmental Studies, University of Iowa), rat anti-PECAM1 (BD Pharmingen), goat anti-Vegfr3 (R&D Systems), rabbit anti-GFP (Molecular Probes), and chicken anti-GFP (Abcam).

Techniques: Expressing, Laser Capture Microdissection, Real-time Polymerase Chain Reaction, Generated

Journal: Genes & Development

Article Title: The Prox1–Vegfr3 feedback loop maintains the identity and the number of lymphatic endothelial cell progenitors

doi: 10.1101/gad.216226.113

Figure Lengend Snippet: Prox1 and Vegfr3 levels are reduced in Prox1 +/GFPCre ;Vegfr3 +/LacZ embryos. ( A–H′ ) Immunohistochemistry for Prox1, Vegfr3, and PECAM1 was carried out in E11.5 wild-type (WT) ( A , A′ , E , E′ ), Vegfr3 +/LacZ ( B , B ′, F , F ′), Prox1 +/GFPCre ( C , C′ , G , G′ ), and Prox1 +/GFPCre ;Vegfr3 +/LacZ ( D , D′ , H , H′ ) embryos. The expression levels of Prox1, Vegfr3, and PECAM1 in venous LEC progenitors ( A–D′ ) and differentiating LECs outside of the CV ( E–H′ ) were quantified using the Slidebook image analysis software ( I , J ). Prox1 and Vegfr3 expression levels were significantly reduced in Prox1 +/GFPCre ;Vegfr3 +/LacZ embryos compared with their single heterozygous littermates. At least three embryos for each genotype were used for quantification. Bars: A–H , 100 μm.

Article Snippet: The following primary antibodies were used: rabbit anti-β-gal (MP Biomedicals), rabbit anti-Prox1 (AngioBio and ProteinTech Group), goat anti-Prox1 (R&D Systems), hamster anti-podoplanin (Hybridoma Bank, Developmental Studies, University of Iowa), rat anti-PECAM1 (BD Pharmingen), goat anti-Vegfr3 (R&D Systems), rabbit anti-GFP (Molecular Probes), and chicken anti-GFP (Abcam).

Techniques: Immunohistochemistry, Expressing, Software

Journal: Genes & Development

Article Title: The Prox1–Vegfr3 feedback loop maintains the identity and the number of lymphatic endothelial cell progenitors

doi: 10.1101/gad.216226.113

Figure Lengend Snippet: Feedback loop regulation between Prox1 and Vegfr3 is at the RNA and protein levels. ( A ) hLECs were transfected with siRNAs and cultured in conditioned medium. After 48 hm mRNA was extracted, and qPCR was carried out to quantify Prox1 and Vegfr3 expression levels. siProx1 down-regulates both Prox1 and Vegfr3 levels ( P < 0.0001 and P = 0.0108, respectively). Likewise, siVegfr3 down-regulates both Vegfr3 and Prox1 levels ( P = 0.0003 and P = 0.0051, respectively). These data are the result of three independent experiments. ( B ) hLECs transfected with siRNA were cultured and immunostained for Prox1. Prox1 expression was down-regulated in those cells that were successfully transfected with fluorescently labeled siRNAs directed against either Prox1 or Vegfr3 (arrowheads).

Article Snippet: The following primary antibodies were used: rabbit anti-β-gal (MP Biomedicals), rabbit anti-Prox1 (AngioBio and ProteinTech Group), goat anti-Prox1 (R&D Systems), hamster anti-podoplanin (Hybridoma Bank, Developmental Studies, University of Iowa), rat anti-PECAM1 (BD Pharmingen), goat anti-Vegfr3 (R&D Systems), rabbit anti-GFP (Molecular Probes), and chicken anti-GFP (Abcam).

Techniques: Transfection, Cell Culture, Expressing, Labeling

Journal: Genes & Development

Article Title: The Prox1–Vegfr3 feedback loop maintains the identity and the number of lymphatic endothelial cell progenitors

doi: 10.1101/gad.216226.113

Figure Lengend Snippet: Prox1 and Vegfc genetically interact with each other to regulate the number of LEC progenitors and LECs. ( A–D ) Prox1 +/GFPCre and Vegfc +/− mice were bred with each other, and embryos were collected at E11.5 and analyzed by immunohistochemistry for the LEC markers Prox1 and podoplanin together with the pan-EC marker PECAM1. Compared with wild-type (WT) embryos ( A ), Vegfc +/− ( B ) and Prox1 +/GFPCre ( C ) embryos had fewer Prox1 + LEC progenitors and LECs. ( D ) Prox1 +/GFPCre ;Vegfc +/− embryos had even fewer LECs. ( E ) Quantification of the cell numbers revealed that the differences in differentiating LEC numbers are statistically significant ([***] P ≤ 0.001; n = 3 embryos for each genotype). Regarding LEC progenitors, there is no significant difference; however, there is a trend that shows that the number of LECs decreases in the double-heterozygous embryos ( P = 0.0925). Bar, 100 μm.

Article Snippet: The following primary antibodies were used: rabbit anti-β-gal (MP Biomedicals), rabbit anti-Prox1 (AngioBio and ProteinTech Group), goat anti-Prox1 (R&D Systems), hamster anti-podoplanin (Hybridoma Bank, Developmental Studies, University of Iowa), rat anti-PECAM1 (BD Pharmingen), goat anti-Vegfr3 (R&D Systems), rabbit anti-GFP (Molecular Probes), and chicken anti-GFP (Abcam).

Techniques: Immunohistochemistry, Marker

Journal: Genes & Development

Article Title: The Prox1–Vegfr3 feedback loop maintains the identity and the number of lymphatic endothelial cell progenitors

doi: 10.1101/gad.216226.113

Figure Lengend Snippet: Model of the feedback loop regulating the specification of Prox1-expressing LEC progenitors in the embryonic veins. Between E9.75 and E13.5, in some venous EC cells with low or no Notch signaling, CoupTFII is up-regulated; then, in combination with Sox18, it induces Prox1 expression such that LEC progenitors start to be specified. Notch signaling is repressed in the specified LEC progenitors by Coup-TFII. Prox1 in turn activates the expression of Vegfr3 in a dosage-dependent manner. Activation of Vegfr3 signaling by Vegfc will also maintain Prox1 expression in LEC progenitors and differentiating LECs. Coup-TFII also interacts with Prox1 to maintain Prox1 expression. Coup-TFII and Prox1 also likely maintain Notch signaling at low levels in LEC progenitors at this stage. Prox1 + LEC progenitors will subsequently bud off from the CV and intersomitic vessels (ISV) and start to express differentiation markers such as podoplanin. After E13.5, Notch signaling levels are increased in venous ECs such that Notch will suppress Coup-TFII to prevent further specification of Prox1 + LEC progenitors. At the same time, Notch signaling also likely inhibits Vegfr3 expression via Hey1/2, thereby short-circuiting the Prox1–Vegfr3 feedback loop and stopping the generation of LEC progenitors in the veins. It could be speculated that most likely the Prox1–Vegfr3 feedback loop does not operate in differentiated LECs.

Article Snippet: The following primary antibodies were used: rabbit anti-β-gal (MP Biomedicals), rabbit anti-Prox1 (AngioBio and ProteinTech Group), goat anti-Prox1 (R&D Systems), hamster anti-podoplanin (Hybridoma Bank, Developmental Studies, University of Iowa), rat anti-PECAM1 (BD Pharmingen), goat anti-Vegfr3 (R&D Systems), rabbit anti-GFP (Molecular Probes), and chicken anti-GFP (Abcam).

Techniques: Expressing, Activation Assay

Journal: Neuroscience Insights

Article Title: Dentate Gyrus Proliferative Responses After Traumatic Brain Injury and Binge Alcohol in Adult Rats

doi: 10.1177/2633105520968904

Figure Lengend Snippet: Summary of antibodies used for immunofluorescence and immunohistochemistry.

Article Snippet: Rabbit anti-PROX1 polyclonal , Prospero homeobox 1 , Synthetic , PROX1 fusion protein Ag1543 , Proteintech 11067-2-AP [RRID: AB_2268804] , 1:500.

Techniques: Immunofluorescence, Immunohistochemistry, Binding Assay, Purification

Journal: Neuroscience Insights

Article Title: Dentate Gyrus Proliferative Responses After Traumatic Brain Injury and Binge Alcohol in Adult Rats

doi: 10.1177/2633105520968904

Figure Lengend Snippet: Binge alcohol and TBI altered the distribution of newly generated cells within different layers of the dentate gyrus, (A-D) BrdU DAB IHC of the contralesional DG from the 6 week time point. Inserts in C and D are a magnified view of the boxed area, and red arrows point to BrdU + cells in the Hilus 2 zone, (E-H) Plots of BrdU + cell number and percent of total BrdU + cells over 2 coronal brain sections per rat encompassing the dorsal hippocampus in different layers of the DG at 1 week and 6 weeks post-TBI. (I, J) Representative Prox1 (green) and BrdU (red) immunofluorescence of contralesional dentate gyrus at the 6 weeks time point. (I) schematic of hilar ectopic granule cell quantification method. The GCL was sub-divided into 2 zones, GCL1 and GCL2. The dentate hilar area adjacent to the GCL was also sub-divided into the Hilus 1 and Hilus 2. Each zone was approximately 30 μm in thickness. Immunolabeled sections were processed for DAB and BrdU + cells in each zone were manually counted. Scale bars: A = 60 μm, insert = 30 μm, I = 30 μm, and (J) alcohol TBI group showed more Prox1 + cells in the ectopic hilar region. White arrowheads point to single-labeled hilar Prox1 + cells, and yellow arrows point to double-labeled cells (hilar subdivisions not shown). Two-way ANOVA, post-hoc Dunn’s multiple comparison, * denotes P ⩽ .05, Error bars = SEM.

Article Snippet: Rabbit anti-PROX1 polyclonal , Prospero homeobox 1 , Synthetic , PROX1 fusion protein Ag1543 , Proteintech 11067-2-AP [RRID: AB_2268804] , 1:500.

Techniques: Generated, Immunofluorescence, Immunolabeling, Labeling, Comparison

Journal: Neuroscience Insights

Article Title: Dentate Gyrus Proliferative Responses After Traumatic Brain Injury and Binge Alcohol in Adult Rats

doi: 10.1177/2633105520968904

Figure Lengend Snippet: Representative immunofluorescence of the ipsilesional DG from a vehicle treated rat showing BrdU + cells within the GCL at 6 weeks post TBI expressing makers of matured neurons but very few expressed glial markers. BrdU + cells (red nuclei) can be seen co-labeled with Prox1 (A), NeuN (B) and calbindin (C), which are markers of matured neurons in the GCL. Very few BrdU + cells are seen co-labeled with the microglial maker Iba1 (D) and none can be seen co-labeled with the matured astrocyte marker S100B (E). White arrowheads point to double-labeled cells, yellow dash lines outline the SGZ, scale bar in A = 20 μm, and in magnified panels = 10 μm. Abbreviations: GCL, granule cell layer; ML, molecular layer.

Article Snippet: Rabbit anti-PROX1 polyclonal , Prospero homeobox 1 , Synthetic , PROX1 fusion protein Ag1543 , Proteintech 11067-2-AP [RRID: AB_2268804] , 1:500.

Techniques: Immunofluorescence, Expressing, Labeling, Marker

Journal: Cells

Article Title: Regional Variation of Gap Junctional Connections in the Mammalian Inner Retina

doi: 10.3390/cells10092396

Figure Lengend Snippet: List of key resources.

Article Snippet: rabbit polyclonal anti-Prox1 , Merck Hungary, Budapest, Hungary , ABN 278.

Techniques: Plasmid Preparation, Laser-Scanning Microscopy, Software