Figure 4
Validation of RNA-seq by qPCR. RNA-seq expression of 22 selected genes was confirmed by independent quantitative RT-PCR (qPCR) method. Expression pattern of four genes (fetub, tgfb1, bmp4, bmp6) involved in transforming growth factor-beta (TGFβ)/bone morphogenic protein (BMP) signaling pathways (A); four genes (dkk2, fos, t7l1a, lrp6) in Wnt signaling pathway (B); three hox genes (hoxb6ab, hox10aa, hoxd3aa) (C); 3 Hedgehog genes (shh, ihhb, twhh) (D); three transcriptional factors (runx3, atf4, sox9) involved in osteochondro-progenitors lineage (E); three fibrillar collagen genes (col2a1, col11a1, col11a2) (F); two calcium binding proteins (sprc, s100a) (G) across all nine notochord libraries (T1A, T1M, T1P, T2A, T2M, T2P, T3A, T3M, T3P) at three developmental stages (T1:510 ± 20 d°, T2: 610 ± 20 d°, T3: 710 ± 20 d°). Of the expression of individual genes in each panel, the X-axis indicates notochord libraries; the Y-axis indicates the gene expression in one library as fold change relative to T1A library (T1A: anterior part of salmon notochord at 510 ± 20 d° stage), measured with RNA-seq (cyan) and qPCR (red). Error bar indicates mean ± SD. Overall, Abbreviations: A (anterior), M (mid), P (posterior). Gene name: Fetuin-B (fetub); Transforming growth factor beta-1 (tgfb1); Bone morphogenetic protein 4 (bmp4); Bone morphogenetic protein 6 (bmp6); Low-density lipoprotein receptor-related protein 6 (lrp6); Dickkopf-related protein 2 (dkk2); Transcription factor 7-like 1-A (t7l1a); Proto-oncogene c-Fos (fos); Homeobox protein HoxB6ab (hoxb6ab); Homeobox protein HoxC10aa (hoxc10aa); Homeobox protein HoxD3aa (hoxd3aa); Indian hedgehog B protein (ihhb); Sonic hedgehog protein A (shh); Tiggy-winkle hedgehog protein (twhh); Transcription factor SOX-9 (sox9); Runt-related transcription factor 3 (runx3); Cyclic AMP-dependent transcription factor ATF-4 (atf4); Protein S100-A1 (s100a); SPARC(sprc); Collagen alpha-1(II) chain (col2a1); Collagen alpha-1(XI) chain (col11a1); Collagen alpha-2(XI) chain (col11a2).