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ZIRC
ZFIN ID: ZDB-FIG-170315-10
Zhang et al., 2015 - G protein-coupled receptor 183 facilitates endothelial-to-hematopoietic transition via Notch1 inhibition. Cell Research   25:1093-107 Full text @ Cell Res.
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Fish:
Condition:
Knockdown Reagents:
Anatomical Terms:
Stage Range: Prim-5 to Prim-25
PHENOTYPE:
Fish:
Condition:
Knockdown Reagents:
Observed In:
Stage Range: Prim-15 to Prim-25

Fig. 4

Gpr183 cooperates with Arrb1 and Nedd4 to downregulate Notch signaling. (A) Internalization of Gpr183-GFP in 90% epiboly embryos (n = 6 for each treatment). White arrowheads denote the internalized Gpr183-GFP in cytoplasm. (B) Gpr183 interacts with Arrb1 in 7α-25-OHC-stimulated cells. 293T cells were transfected with Gpr183-Flag and/or Arrb1-Myc and stimulated with 7α-25-OHC for 0.5 h. Cell lysates were subjected to immunoprecipitation using anti-Myc followed by western blot analysis. (C) Expression of runx1, cmyb and hey2 in arrb1 morphants. Red arrowheads label the staining signals in AGM. (D) NICD expression in zebrafish embryos treated with 7α-25-OHC (from 1-cell stage to 36 hpf) or MG132 (from 26 to 36 hpf). Right panel shows the quantification analysis. (E) MS analysis of Notch1-interacting proteins in control or 7α-25-OHC-treated AGM cells. Mouse E10.5 AGM cells were cultured in the presence of hematopoietic cytokines for three days, and then treated with DMSO or 7α-25-OHC for 1 h. (F) Polyubiquitination of NICD-Flag (zebrafish) induced by zebrafish Nedd4a-GST (Nedd4a(Z)) or mouse Nedd4-GST (Nedd4(M)). (G) GST pull-down assay showing the interaction between Nedd4-GST and NICD-Flag. (H) runx1 expression is reduced in nedd4a morphants and increased in nedd4a-overexpressing embryos. (I) fli1a+tp1+ cells (white arrowheads) are increased in nedd4a morphants and are reduced in nedd4a-overexpressing embryos. Right panel shows the quantification result. (J) NICD expression in nedd4a morphants or nedd4a-overexpressing embryos. Right panel shows the quantification result. (K) HE in Tg(flk1:mCherry/runx1:en-GFP) embryos after nedd4a knockdown or 7α-25-OHC treatment. White arrowheads indicate HE in the AGM region at 36 hpf. Error bar represents SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
EGFP jh11Tg; y1Tg control Prim-25 ventral wall of dorsal aorta IFL
jh11Tg; y1Tg + MO1-nedd4a control Prim-25 ventral wall of dorsal aorta IFL
GFP ioz1Tg; s896Tg control Prim-25 angioblastic mesenchymal cell IFL
ioz1Tg; s896Tg chemical treatment by injection: 7alpha,25-dihydroxycholesterol Prim-25 angioblastic mesenchymal cell IFL
ioz1Tg; s896Tg + MO1-nedd4a control Prim-25 angioblastic mesenchymal cell IFL
ioz1Tg; s896Tg + MO1-nedd4a chemical treatment by injection: 7alpha,25-dihydroxycholesterol Prim-25 angioblastic mesenchymal cell IFL
hey2 TU control Prim-15 ventral wall of dorsal aorta ISH
TU + MO1-arrb1 control Prim-15 ventral wall of dorsal aorta ISH
mCherry ioz1Tg; s896Tg control Prim-25 ventral wall of dorsal aorta IFL
ioz1Tg; s896Tg chemical treatment by injection: 7alpha,25-dihydroxycholesterol Prim-25 ventral wall of dorsal aorta IFL
ioz1Tg; s896Tg + MO1-nedd4a control Prim-25 ventral wall of dorsal aorta IFL
ioz1Tg; s896Tg + MO1-nedd4a chemical treatment by injection: 7alpha,25-dihydroxycholesterol Prim-25 ventral wall of dorsal aorta IFL
jh11Tg; y1Tg control Prim-25 ventral wall of dorsal aorta IFL
jh11Tg; y1Tg + MO1-nedd4a control Prim-25 ventral wall of dorsal aorta IFL
myb TU control Prim-15 ventral wall of dorsal aorta ISH
TU + MO1-arrb1 control Prim-15 ventral wall of dorsal aorta ISH
runx1 TU control Prim-5 ventral wall of dorsal aorta ISH
Prim-15 hematopoietic multipotent progenitor cell ISH
TU + MO1-arrb1 control Prim-5 ventral wall of dorsal aorta ISH
TU + MO1-nedd4a control Prim-15 hematopoietic multipotent progenitor cell ISH
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
TU + MO1-arrb1 control Prim-15 ventral wall of dorsal aorta myb expression decreased amount, abnormal
Prim-15 ventral wall of dorsal aorta hey2 expression increased amount, abnormal
TU + MO1-nedd4a control Prim-15 hematopoietic multipotent progenitor cell runx1 expression decreased amount, abnormal
ioz1Tg; s896Tg chemical treatment by injection: 7alpha,25-dihydroxycholesterol Prim-25 angioblastic mesenchymal cell GFP expression increased amount, abnormal
ioz1Tg; s896Tg + MO1-nedd4a control Prim-25 angioblastic mesenchymal cell GFP expression decreased amount, abnormal
ioz1Tg; s896Tg + MO1-nedd4a chemical treatment by injection: 7alpha,25-dihydroxycholesterol Prim-25 angioblastic mesenchymal cell GFP expression amount, ameliorated
jh11Tg; y1Tg + MO1-nedd4a control Prim-25 ventral wall of dorsal aorta mCherry expression increased amount, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Cell Research for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Cell Res.