XB-IMG-155178
Xenbase Image ID: 155178
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Figure 6. Cp110 levels in MCCs are controlled by ciliary transcription factors and miR-34/449 microRNAs.(A) cp110 expression in MCCs is regulated through the MCC signaling/transcriptional cascade. Embryos were injected with Su(H)-dbm to stimulate MCC induction (green) or with Su(H)-dbm and dominant-negative multicilin (dn-mci) to prevent MCC induction (red). RNA-sequencing (RNA-seq) was performed at MCC specification stage (st. 16). Normalized counts are shown as bar graphs. n = 2. Related to Figure 6—figure supplement 1A. (B) cp110 expression is activated by ciliary transcription factors. Chromatin immunoprecipitation and DNA-sequencing (ChIP-seq; upper five lanes) and RNA-seq (bottom two lanes) at stage 16. Embryos were injected with Notch-icd to inhibit MCC induction or together with multicilin (mci) to induce MCCs. ChIP-seq using antibodies to mark active chromatin (Histone H3 lysine tri-methylation, H3K4me3; Histone H3 lysine acetylation, H3K27ac), E2F4 binding (E2F4), RFX2 binding (RFX2), and Foxj1 binding (Foxj1) are shown. A gene model is shown in bottom lane. ChIP-seq peaks are indicated by a yellow background. (C) cp110 levels at ciliogenesis stage (st. 25) are controlled by miR-34/449 miRNAs. For quantitative RT-PCR analysis (qPCR), manipulations were performed as described in (A) (green and red bars). Additionally, miR-34/449s were knocked down (miR-34/449MO, blue bar). The uninjected control was set to 1. n = 2. (D) miR-34/449 family members are regulated through the conserved MCC signaling/transcriptional cascade. qPCR analysis for miR-34/449 expression was performed as described (C). ND, not detected. n = 2. (E–F) Expression of miRNAs miR-34b/c and miR-449a-c is activated by ciliary transcription factors. ChIP-seq and RNA-seq was performed as described in (B). miRNA location in (E) is indicated by red box. miR-449a-c are expressed from cdc20b intron 2. Related to Figure 6—figure supplement 1B. The foxj1 expression analysis confirmed successful manipulation in (A, C) and (D). Error bars represent s.e.m. in (C) and (D).DOI:
http://dx.doi.org/10.7554/eLife.17557.023Figure 6—figure supplement 1. Cp110 levels in MCCs are controlled by ciliary transcription factors and miR-34/449 microRNAs.(A) Related to Figure 6A. cp110 expression in MCCs is regulated through the conserved MCC signaling/transcriptional cascade. Embryos were injected with Notch-icd to inhibit MCC induction (red) or with Notch-icd together with multicilin (mci) to stimulate MCC induction (green). RNA-sequencing (RNA-seq) was performed on extracts from mucociliary organoids at MCC specification stage (st. 16). Normalized counts are shown as bar graphs. The foxj1 expression analysis confirmed successful manipulation. (B) Related to Figure 6B,E–F. Expression of miRNA miR-34a is not activated by ciliary transcription factors. ChIP-seq and RNA-seq was performed as described in Figure 6B. miRNA location is indicated by red box.DOI:
http://dx.doi.org/10.7554/eLife.17557.024 Image published in: Walentek P et al. (2016) © 2016, Walentek et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View |