ZFIN ID: ZDB-FIG-160926-21
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:
Knockdown Reagent:
Anatomical Terms:
Stage Range: Prim-5 to Prim-15
PHENOTYPE:
Fish:
Knockdown Reagent:
Observed In:
Stage Range: Prim-5 to Prim-15

Fig. 5

Multiplex Analysis of Gene Expression Shows that jag1a Is a Downstream Target of TGFβ Signaling

(A) Schematic representation of the trunk dissection experiment for isolation of mRNA for hybridization with the NanoString Probe CodeSet. Six groups of independent wild-type (wt1-6) and tgfbR2MO1-injected embryos (mo1-6) were used in this analysis.

(B) Volcano plot depicting differential gene expression between wild-type and tgfbR2 morphants in log2-fold change with a significance level of p < 0.05. Vertical broken lines limit the absolute logFC larger than 0.5-fold change range, whereas the horizontal broken line represents the false discovery rate (FDR) threshold set at FDR < 0.1. The genes where FDR < 0.1 are shown as orange dots. The size of the dots is proportional to mRNA expression levels.

(C) Hierarchical clustering of genes expressed with FDR < 0.1 in each of the six biological replicates analyzed (wild-type, wt1 to wt6; tgfbR2MO1, mo1 to mo6). Results are normalized and presented as Z scores from -2 (downregulated) to 2 (upregulated).

(D) Schematic representation of the sorting of kdrl:GFP+ cells in wild-type and tgfbR2 morphants (MO) by fluorescence-activated cell sorting to isolate mRNA and validate the NanoString results by qPCR.

(E and F) qPCR of p53, cdkn1a, rspo1, and bax in (E) kdrl:GFP+ cells and (F) kdrl:GFP- cells of wild-type and tgfbR2 morphants (MO) at 28 hpf. Taz was omitted from the analysis as its fold induction <2.

(G and G′) TUNEL-stained apoptotic cells in uninjected (control) kdrl:GFP embryos at 30 hpf.

(H and H′) Apoptotic cells in tgfbR2MO1-injected kdrl:GFP embryos at 30 hpf. White arrows, apoptotic endothelial cells; outline arrows, apoptotic non-endothelial cells.

(I and J) qPCR for jag1a in (I) kdrl:GFP- cells and (J) in kdrl:GFP+ cells at 28 hpf.

See also Figure S5 and Table S1.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
baxa s843Tg + MO1-tgfbr2b standard conditions Prim-5 trunk RTPCR
cdkn1a s843Tg + MO1-tgfbr2b standard conditions Prim-5 trunk RTPCR
Prim-5 trunk endothelial cell RTPCR
EGFP s843Tg standard conditions Prim-15 trunk vasculature IHC
s843Tg + MO1-tgfbr2b standard conditions Prim-15 trunk vasculature IHC
jag1a s843Tg + MO1-tgfbr2b standard conditions Prim-5 trunk RTPCR
Prim-5 trunk endothelial cell RTPCR
tp53 s843Tg + MO1-tgfbr2b standard conditions Prim-5 trunk RTPCR
Prim-5 trunk endothelial cell RTPCR
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
s843Tg + MO1-tgfbr2b standard conditions Prim-5 trunk baxa expression increased amount, abnormal
Prim-5 trunk cdkn1a expression increased amount, abnormal
Prim-5 trunk jag1a expression decreased amount, abnormal
Prim-5 trunk tp53 expression increased amount, abnormal
Prim-5 trunk endothelial cell cdkn1a expression increased amount, abnormal
Prim-5 trunk endothelial cell jag1a expression decreased amount, abnormal
Prim-5 trunk endothelial cell tp53 expression increased amount, abnormal
Prim-15 caudal vein plexus apoptotic process increased occurrence, 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