Figure S2. Transcriptional Misregulation of Xbra Target Genes Induced by the Single and Combined Knockdown of Xbra and Xbra3 Suggests Functional Redundancy between these Brachyury Paralogues and Reflects the Severity of Corresponding Phenotypes, Related to Figure 2(A) Genomic architecture of Xbra and Xbra3 in X. tropicalis including the position of the T-box within the first four exons (red). Black bars, position of splice- (MO1) and translation-blocking (MO2) morpholinos. Black ‘fish-hook’ arrows, position of PCR primers used to assess the efficiency of MO1. The length of the first intron of Xbra3 is unknown because of a sequencing gap in the genome (JGI4.1).(B) Efficiency of blocking splicing of Xbra (i) and Xbra3 (ii) by the injection of corresponding MO1 at stage 10.5 (Xbra) and 12 (Xbra3). In contrast to 25 ng control MO, 10 or 20 ng Xbra MO1 caused cryptic splicing, which created multiple bands by diagnostic RT-PCR (i). odc1, loading control. -RT, PCR without reverse transcription. Efficiency of disrupting correct splicing of Xbra3 was quantified by RT-qPCR taking into account melting curves to discriminate wild-type from cryptic templates. Compared to 25ng control MO, 10 or 20 ng of Xbra3 MO1 reduced wild-type splicing to about 5%–10% (ii). Relative concentrations of Xbra3 were normalized with odc1. Error bars, s.d. of biological duplicates.(C) Semiquantitative immunoblotting of endogenous Xbra protein levels at stage 11 upon injection of MO1 or MO2 at different doses (10 and 20 ng) compared to controls (uninjected and injected with 25 ng control MO) (i). Loading control, β-actin. Synthesis of 200 pg Xbra3-HA was inhibited in vivo by Xbra3 MO2 compared to control MO (ii). ChIP-grade Xbra antibody did not recognize Xbra3-HA. Loading control, α-tubulin.(D) The embryo was consistently truncated along its antero-posterior axis, more strongly by the double Xbra/Xbra3 knock-down (KD) than the single Xbra KD. No defects were observed for Xbra3 KD embryos. By tadpole stage, Xbra/Xbra3 morphants were severely truncated to the extent that the tail was hardly elongating. Statistics given here in the top right corner of every picture were from one single experiment recorded at stage 37. Scale bar, 0.5 mm.(E) Developmental time course of embryos injected with MO1 and MO2 (5 ng each) to knock down Xbra and Xbra3 individually and simultaneously. Scale bar, 0.5 mm.(F) Clustered differential expression profile (Xbra, Xbra3 and Xbra/Xbra3 KD versus control) for some Xbra target genes: (i) Transcriptional fold changes induced by the KD of Xbra, Xbra3 or Xbra/Xbra3 were determined at stage 13 (early neurula), 26 (mid-tailbud) and 32 (early tadpole) by RT-qPCR (n = 3), logarithmised, clustered and visualized as a heat map (red, downregulated; blue, upregulated). ≥ 1.5-fold transcriptional mis-regulation: ∗, FDR < 10%; ∗∗, FDR < 1%; ∗∗∗, FDR < 0.1%. (ii) If available, the fold change of transcription is indicated for Xbra/Xbra3 (B/B3) KD compared to control embryos at stage 32 calculated by RT-qPCR and RNA-Seq/DESeq (DE-Seq). Floating-point numbers preceded by a minus symbol indicate a downregulation. NA indicates that differential expression data could not be obtained from the RNA-Seq experiment since the transcriptome assembly (JGI4.1) used for the alignment did not contain the sequences for esr5, myos, ripply2.1, ripply2.2 and tbx6. Differential expression data was subsequently complemented with these missing genes. A comparison of 70 genes indicated that RNA-Seq followed by DESeq (Anders and Huber, 2010) resulted in an average 30% (s.d. +/−28.4%) greater positive fold change than RT-qPCR. (iii, iv) Xbra binding levels and positions per selected target gene at gastrula stage. (iii) Quantification of binding per target gene (sum [-log p]). Error bar, s.d. of biological duplicates. (iv) Xbra binding pattern closest to selected target genes. DNA occupancies were binned at 400-bp intervals between 10 kb up- and downstream from the corresponding TSS. Beyond this range Xbra binding events were collected into single bins. DNA occupancy was visualized as a heat map according to the natural logarithm (ln) of [-log p + 1].
Image published in: Gentsch GE et al. (2013)
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