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Notch signaling is required for arterial-venous differentiation during embryonic vascular development

Nathan D. Lawson1, Nico Scheer2, Van N. Pham1, Cheol-Hee Kim1, Ajay B. Chitnis1, Jose A. Campos-Ortega2 and Brant M. Weinstein1,*

1 Laboratory of Molecular Genetics, NICHD, NIH, Bethesda, MD, 20892, USA
2 Institut für Entwicklungsbiologie, Universität zu Köln, 50923 Cologne, Germany



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  		Fig. 1. In situ hybridization of 30-somite stage wild-type embryos using markers of arterial-venous specification. (A) Line drawing of a 30-somite stage embryo. The boxed region corresponds to the area shown in B,D,F and H (Kimmel et al., 1995). (B,D,F,H) Lateral views of (B) ephrinB2, (D) notch3, (F) flt4 and (H) fli1 expression in the trunk vessels. (C,E,G,I) Thick sections (~35-50 µm) through the trunk of embryos showing expression of (C) ephrinB2, (E) notch3, (G) flt4, and (I) fli1. (B-I) The dorsal aorta (DA) is indicated by a red arrowhead and the posterior cardinal vein (PCV) by a blue arrowhead. (E) Pronephric duct expression of notch3 is indicated by black arrowheads. NT, neural tube; NC, notochord. Scale bars, (A) 100 µm, (B,D,F,H) 40 µm, (C,E,G,I) 30 µm.

 


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  		Fig. 2. Microinjection of EGFPSuDN perturbs arterial differentiation. (A,B) In situ hybridization of ephrinB2 in injected embryos fixed at 24 hpf. (A) Embryos injected with 800 pg of EGFP express ephrinB2 in the DA (red arrowhead). (B) In embryos injected with 800 pg of EGFPSuDN there is reduction of vascular ephrinB2. (C,D) In situ hybridization of notch3 in injected embryos fixed at 24 hpf. (C) Embryos injected with 1 ng of EGFP express notch3 within the neural tube (black arrow) and DA (red arrowhead). (D) notch3 expression within the neural tube (black arrow) and the DA (red arrowhead) is reduced following injection of 1 ng of EGFPSuDN. (E,F) In situ hybridization of grl in injected embryos fixed at 24 hpf. (E) Embyros injected with 800 pg EGFP express grl within the DA; (F) grl expression is not affected by injection with 800 pg EGFPSuDN. (A-F) All embryos were injected with indicated amount of mRNA at the 1-cell stage. (G, H) Transmitted light images of living embryos at 36 hpf. (G) Wild-type embryo injected with 800 pg of EGFP mRNA. (H) Wild-type embryo injected with 800 pg of EGFPSuDN exhibiting trunk curvature. (A-F) Lateral views, anterior to the left. (A-F) Scale bars (A-F) 100 µm, (G, H) 200 µm.

 


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  		Fig. 3. In situ hybridization of artery markers in 30-somite stage mibta52b mutant and wild-type sibling embryos. (A) In wild-type siblings, ephrinB2 expression is apparent in the DA (red arrowheads in A-J). (B) In mibta52b mutant embryos this expression is absent. (C) In wild-type sibling embryos notch3 is expressed within the DA. (D) In mibta52b mutant embryos notch3 expression is absent from both vascular and non-vascular tissues. (E) In wild-type sibling embryos grl is expressed specifically in the DA. (F) In mibta52b mutant embryos the expression of grl is not affected. (G) In wild-type sibling embryos deltaC is expressed within the DA. (H) In mibta52b mutant embryos expression of deltaC within the DA is decreased in approximately half of the mutant embryos. Excess neuronal expression is apparent (black arrow). (I) In wild-type siblings the T-box transcription factor tbx20 is expressed normally within the roof of the DA. (J) In mibta52b mutant embryos this expression is normal. Scale bar (A-J) 100 µm.

 


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  		Fig. 4. Ectopic expression of venous markers in mibta52b mutant embryos. (A) In wild-type siblings flt4 expression is restricted to the PCV by the 30-somite stage. (B) In mibta52b mutant embryos expression persists within both the PCV (blue arrow) and the DA (red arrowhead) at this stage. (C) In wild-type siblings rtk5 is expressed within the hypochord (black arrow), and is detected at a higher level within the PCV (blue arrow) than the DA (red arrowhead). (D) In mibta52b mutant embryos rtk5 expression is absent from the PCV, and regions of ectopic rtk5 expression are noted in the DA (red arrowhead). (E,F) Transmitted light images of (E) wild-type and (F) mibta52b mutant living sibling embryos at 36 hpf. (F) The curled tail and other characteristics (see text) are apparent in the mutant embryo and are similar to embryos injected with EGFPSuDN (see Fig. 2H). Scale bars (A,B) 100 µm, (C,D) 50 µm, (E,F) 200 µm.

 


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  		Fig. 5. Activation of Notch signaling represses venous cell identity. (A,B) Expression of grl in hsp70:Gal4;UAS:notch1a-intra heat shocked embryos. (A) grl expression in heat shocked embryos not expressing Notch1a-intra. (B) grl is not ectopically induced within the vasculature by Notch1a-intra. (C-F) flt4 expression in hsp70:Gal4; UAS:notch1a-intra embryos heat shocked at the 18-somite stage. flt4 is expressed within venous vessels of the head (C) and trunk (E) at the 28- to 30-somite stages in embryos that do not express Notch1a-intra. (D,F) Notch1a-intra represses flt4 expression in venous blood vessels of the head (D) and trunk (F). (G,H) Targeted expression of Notch3 ICD to the endothelium using the fli1 promoter, represses flt4 expression. (G) An embryo injected with pfliMTN3ICD showing normal flt4 expression within the midcerebral vein (blue arrowhead) in the absence of ectopic Notch3 ICD; anterior is to the right. (H) Opposite side of embryo in G, showing a myc-positive cell (blue arrowhead) within the midcerebral vein that fails to express flt4; anterior is to the left. (I-L) Expression of tie1 in hsp70:Gal4;UAS:notch1a-intra heat shocked embryos. (I,K) In embryos not expressing ectopic Notch1a-intra, tie1 is expressed in all blood vessels at the 28- to 30-somite stage. (J,L) Tie1 expression within all vessels is maintained in embryos expressing Notch1a-intra. (C, D) Embryos are shown prior to staining with anti-myc antibody. (A,B,E-L) Embryos were subjected to whole-mount immunostaining with an anti-myc antibody to visualize ectopic expression of the (A,B,E,F,I-L) Notch1-intra or (G,H) Notch 3 ICD. (E,F,I,J) Arrows and arrowheads indicate the positions of the primordial midbrain channel and mid-cerebral vein, respectively. (A,B,K,L) Red arrowheads and blue arrows indicate the position of the DA and PCV, respectively. Scale bar (A,B,K,L) 50 µm, (C,D,I,J) 100 µm, (G,H) 200 µm.

 


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  		Fig. 6. Vascular defects in mibta52b embryos. (A) Head of a wild-type mibta52b sibling at 60 hpf. (B) Cranial hemorrhage in a mibta52b mutant embryo at 60 hpf (black arrows). (C) Schematic drawing of the trunk circulatory loop in a zebrafish embryo. Red and blue are arterial and venous vessels respectively, with the direction of blood flow indicated. The black arrow shows the position of the shunt in the mutant embryos in (E,G). (D) DIC image of the mid-trunk of a live wild-type mibta52b sibling at 55 hpf. A well-formed DA (red bracket) and PCV (blue bracket) are visible (see Movie 1, available on-line). The black arrow points to the endothelial cell walls clearly demarcating the boundary between the DA and PCV. (E) DIC image of a mutant mibta52b embryo displaying a shunt (black arrow) between the DA and PCV (see Movie 2, available on-line). (F) Longitudinal histological section through the trunk of a wild-type embryo showing the DA (red bracket) and PCV (blue bracket). Blood cells are present within the lumen of each vessel. The black arrow indicates the endothelial cell wall between the DA and PCV. (G) Longitudinal histological section through the trunk of the same mibta52b mutant embryo shown in E, also in the region of the shunt (black arrow). (H) DIC image of a mibta52b embryo showing a disorganized boundary between the DA and PCV (arrows) and lack of a properly remodeled PCV. (I) Confocal microangiogram of the trunk of a wild-type mibta52b sibling at 55 hpf. Intersomitic vessels appear regularly at the vertical myosepta. (J) Confocal microangiogram of the trunk of a mibta52b mutant at 55 hpf. Disorganization of the normal intersomitic vessel structure is apparent. (K) Higher magnification confocal microangiogram of the trunk of the mibta52b mutant in J. Ectopic sprouts from the main branches of the intersomitic vessels are visible (white arrows) penetrating the somite. Scale bars (A,B,I,J) 50 µm, (D-H,K) 25 µm. DA, dorsal aorta; CA, caudal artery; ACV, anterior cardinal vein; PCV, posterior cardinal vein; CV, caudal vein.

 

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