ZFIN ID: ZDB-FIG-160926-20
Monteiro et al., 2016 - Transforming Growth Factor β Drives Hemogenic Endothelium Programming and the Transition to Hematopoietic Stem Cells. Developmental Cell   38(4):358-70 Full text @ Dev. Cell
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Fish:
Conditions:
Anatomical Terms:
Stage Range: 26+ somites to Prim-5
PHENOTYPE:
Fish:
Conditions:
Observed In:
Stage Range: 26+ somites to Prim-5

Fig. 4

Vegf Signaling Is Required for tgfb1a and tgfb1b Expression in the Dorsal Aorta

(A and B) Wild-type embryos were treated from 10 hpf with DMSO (control), Vegf inhibitor DMH4 (20 µM), and Notch inhibitor DAPM (100 µM) and collected at 22 hpf or 28 hpf. Embryos were collected and analyzed for (A) kdrl expression at 22 hpf and (B) runx1 expression at 28 hpf.

(C) Tg(Fli1:EGFP) embryos were treated from 10 to 26 hpf with DMSO or DMH4 (20 µM). DMH4-treated embryos showed a severe loss of intersomitic vessels but ECs are still present in the trunk, and circulation was detected in a majority of embryos at 48 hpf (data not shown).

(D) Expression of kdrl, tgfb1a, tgfb1b by qPCR in 26 hpf sorted Fli1:EGFP+ ECs. All three genes were downregulated after DMH4 treatment. Results are shown as averages ± SD of 4-5 biological replicates.

(E) Wild-type embryos were treated from 10 hpf with DMSO (control), Vegf inhibitor DMH4 (20 µM), and Notch inhibitor DAPM (100 µM) and collected at 22 hpf for analysis of tgfb1a, tgfb1b, tgfb3, and tgfbR2 by in situ hybridization at 22 hpf.

(F) Schematic representation of the experimental results.

Black arrows indicate the location of the DA; yellow arrowheads indicate the location of runx1 expression in the floor of the DA. The numbers of embryos are shown in each panel as the number of embryos with phenotype/total number analyzed. Arterial EC, arterial endothelial cell.

See also Figure S4.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
EGFP y1Tg control Prim-5 trunk vasculature IFL
y1Tg chemical treatment: vascular endothelial growth factor receptor antagonist Prim-5 trunk vasculature IFL
kdrl WT control 26+ somites trunk vasculature ISH
26+ somites vascular endothelium ISH
WT chemical treatment: EC 3.4.23.46 (memapsin 2) inhibitor 26+ somites trunk vasculature ISH
26+ somites vascular endothelium ISH
WT chemical treatment: vascular endothelial growth factor receptor antagonist 26+ somites trunk vasculature ISH
y1Tg chemical treatment: vascular endothelial growth factor receptor antagonist Prim-5 blood vessel endothelial cell RTPCR
runx1 WT control Prim-5 ventral wall of dorsal aorta hematopoietic multipotent progenitor cell ISH
WT chemical treatment: EC 3.4.23.46 (memapsin 2) inhibitor Prim-5 ventral wall of dorsal aorta hematopoietic multipotent progenitor cell ISH
WT chemical treatment: vascular endothelial growth factor receptor antagonist Prim-5 ventral wall of dorsal aorta hematopoietic multipotent progenitor cell ISH
tgfb1a WT control 26+ somites dorsal aorta ISH
26+ somites vascular endothelium ISH
WT chemical treatment: EC 3.4.23.46 (memapsin 2) inhibitor 26+ somites dorsal aorta ISH
26+ somites vascular endothelium ISH
WT chemical treatment: vascular endothelial growth factor receptor antagonist 26+ somites dorsal aorta ISH
y1Tg chemical treatment: vascular endothelial growth factor receptor antagonist Prim-5 blood vessel endothelial cell RTPCR
tgfb1b WT control 26+ somites dorsal aorta ISH
26+ somites vascular endothelium ISH
WT chemical treatment: EC 3.4.23.46 (memapsin 2) inhibitor 26+ somites dorsal aorta ISH
26+ somites vascular endothelium ISH
WT chemical treatment: vascular endothelial growth factor receptor antagonist 26+ somites dorsal aorta ISH
y1Tg chemical treatment: vascular endothelial growth factor receptor antagonist Prim-5 blood vessel endothelial cell RTPCR
tgfb3 WT control 26+ somites dorsal aorta ISH
26+ somites vascular endothelium ISH
WT chemical treatment: EC 3.4.23.46 (memapsin 2) inhibitor 26+ somites dorsal aorta ISH
26+ somites vascular endothelium ISH
WT chemical treatment: vascular endothelial growth factor receptor antagonist 26+ somites dorsal aorta ISH
26+ somites vascular endothelium ISH
tgfbr2b WT control 26+ somites dorsal aorta ISH
26+ somites vascular endothelium ISH
WT chemical treatment: EC 3.4.23.46 (memapsin 2) inhibitor 26+ somites dorsal aorta ISH
26+ somites vascular endothelium ISH
WT chemical treatment: vascular endothelial growth factor receptor antagonist 26+ somites dorsal aorta ISH
26+ somites vascular endothelium ISH
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
WT chemical treatment: EC 3.4.23.46 (memapsin 2) inhibitor Prim-5 ventral wall of dorsal aorta hematopoietic multipotent progenitor cell decreased amount, abnormal
Prim-5 ventral wall of dorsal aorta hematopoietic multipotent progenitor cell runx1 expression decreased amount, abnormal
WT chemical treatment: vascular endothelial growth factor receptor antagonist 26+ somites dorsal aorta tgfb1b expression decreased amount, abnormal
26+ somites dorsal aorta tgfb1a expression decreased amount, abnormal
26+ somites intersegmental vessel aplastic, abnormal
26+ somites trunk vasculature kdrl expression decreased amount, abnormal
Prim-5 ventral wall of dorsal aorta hematopoietic multipotent progenitor cell runx1 expression decreased amount, abnormal
Prim-5 ventral wall of dorsal aorta hematopoietic multipotent progenitor cell decreased amount, abnormal
y1Tg chemical treatment: vascular endothelial growth factor receptor antagonist Prim-5 blood vessel endothelial cell kdrl expression decreased amount, abnormal
Prim-5 blood vessel endothelial cell tgfb1b expression decreased amount, abnormal
Prim-5 blood vessel endothelial cell tgfb1a expression decreased amount, abnormal
Prim-5 intersegmental vessel aplastic, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Developmental Cell for permission to reproduce figures from this article. Please note that this material may be protected by copyright.

Reprinted from Developmental Cell, 38(4), Monteiro, R., Pinheiro, P., Joseph, N., Peterkin, T., Koth, J., Repapi, E., Bonkhofer, F., Kirmizitas, A., Patient, R., Transforming Growth Factor β Drives Hemogenic Endothelium Programming and the Transition to Hematopoietic Stem Cells, 358-70, Copyright (2016) with permission from Elsevier. Full text @ Dev. Cell