Figure 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 tgfbR2^MO1-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; tgfbR2^MO1, 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 tgfbR2^MO1-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.