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Blood, Vol. 111, Issue 10, 4965-4972, May 15, 2008
Calcitonin receptor-like receptor guides arterial differentiation in zebrafish
Blood Nicoli et al.
111: 4965
Supplemental materials for: Nicoli et al
Files in this Data Supplement:
- Figure S1. Zebrafish crlr-1 expression and amino acid sequence (JPG, 167 KB)
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Crlr-1 and crlr-2 expression (A) were analysed by semiquantitative RT-PCR on total RNA extracted from zebrafish embryos at different stages of development. GA3PDH was used as a housekeeping gene. Crlr-2 expression is limited to the maternal stage (1 hpf) whereas crlr-1 is expressed at all the zygotic stages analysed (from 12 to 72 hpf). Amino acid sequence of zebrafish crlr-1 is compared to those of the human (hcrlr, AAQ91332) and mouse (mcrlr, NP_061252) species (B). Amino acid residues identical or conserved among crlr proteins are shaded in yellow or blue, respectively. Conserved cysteine residues in the extracellular domain, required for ligand binding, are indicated by asterisks. Seven trans-membrane regions are indicated by blue bars whereas the hormone-binding domain is highlighted by a red bar.
- Figure S2. Expression of fli-1 in crlr morphants (JPG, 69.5 KB)
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Embryos were injected with std-MO (control) or crlr-MO1 (morphant) and analysed for notochord no-tail (nc, red arrow), somitic myoD (s, black arrow), and endothelial progenitor fli-1 (blue arrow) expression by triple ISH at 8-ss. No differences in the expression pattern were observed between control (A) and morphant (B) embryos. At 28 hpf, fli-1 stains the vasculature of the trunk in both control (C) and crlr (D) morphants (D). Note the impaired angiogenic development of fli-1+ ISVs (black arrows) in morphant embryos. Red arrowhead, DA; white arrowhead, PCV.
- Figure S3. Crlr knockdown specifically affects arterial gene expression (JPG, 71.1 KB)
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(A) Std-MO-injected embryos (control) and crlr-MO1-injected embryos (morphant) were analyzed at 26 hpf for the expression of the indicated arterial and venous markers by ISH (lateral views). Note the specific loss of ephrin-B2a, DeltaC, and notch5 expression in the DA of crlr morphants. (B) Total RNA was extracted at 26 hpf from 40 embryos per group and ephrin-B2a and flt4 expression were evaluated by real-time RT-PCR. Data in triplicate were normalized for zebrafish  -actin expression and represent the percentage change in morphant embryos relative to controls. Similar results were obtained in 4 independent experiments (*, p< 0.05 versus controls, Student’s t test).
- Figure S4. The shh pathway guides crlr somite expression (JPG, 35.6 KB)
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Crlr expression was assessed by ISH in 10-ss embryos (all views are dorsal to the top). Note the disappearance of somitic crlr expression in syut4 mutant embryos (B, 6 of 36 embryos from an heterozygous cross) and in wild type cyclopamine-treated embryos (C, 34 of 36 treated embryos) when compared to untreated wild type embryos (A, n=30).
- Figure S5. shh overexpression in crlr morphants (JPG, 60.2 KB)
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Control embryos and crlr morphants were injected with shh mRNA and analyzed for vegf and ephrin-B2a expression at 10 somites (dorsal view) and 26 hpf (lateral view), respectively. Note the strong somitic vegf upregulation (E, arrowhead) and the ectopic ephrin-B2a expression in the PCV (F, arrowhead) induced by shh overexpression in control embryos. In contrast, shh overexpression failed to induce a significant increase in vegf expression (G, arrowhead) and caused only a very limited rescue of ephrin-B2a expression in crlr morphants with no ectopic venous ephrin-B2a expression (H, arrowhead).
- Figure S6. Crlr downregulation inhibits tumor angiogenesis in zebrafish embryo (JPG, 57.2 KB)
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Tumorigenic murine FGF2-T-MAE cells1 were grafted (*) at 48 hpf in the perivitelline space of zebrafish embryos injected with std-MO (A, control) or crlr-MO1 (B, crlr KD) (both at 0.4 pmoles/embryo) as described.2,3 At 72 hpf, whole-mount alkaline-phosphatase staining was performed to visualize the neovessels originating from the sub-intestinal vessel basket (arrows in A, B) and the number of embryos showing a positive angiogenic response was assessed in the two experimental groups (C). Data are the mean ± S.D. of three experiments (*, P<0.01).
REFERENCES
1. Dell’Era P, Coco L, Ronca R, Sennino B, Presta M. Gene expression profile in fibroblast growth factor 2-transformed endothelial cells. Oncogene. 2002;21:2433–2440.
2. Nicoli S, Presta M. The zebrafish/tumor xenograft angiogenesis assay. Nat Protoc. 2007;2:2918–2923.
3. Nicoli S, Ribatti D, Cotelli F, Presta M. Mammalian tumor xenografts induce neovascularization in zebrafish embryos. Cancer Res. 2007;67:2927–2931.
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