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Fig. 7. RNF220-like ubiquitin ligaseK00726is a β-catenin-responsive dorsal marker. (A) (i) Genomic snapshot of DNA occupancy of β-catenin (Nakamura et al., 2016) at the K00726 locus in early gastrula embryos. (ii) Luciferase assay for indicated constructs containing K00726 promoter, putative cis-regulatory modules (pCRM) 1–3 or negative control region (NCR). Genomic position of these genomic elements are shown in (i). Reporter activity was normalised to basal activity of the K00726 promoter. Error bars, SEM of biological duplicates. Student's two-tailed t-test: ***, p < 0.01; n.s, not significant. (B) RT-qPCR of K00726 in animal caps derived from embryos injected with and without 500 pg of β-catenin mRNA. Error bars, SEM of biological triplicates. Student's two-tailed t-test: **, p < 0.05. (C) Representation of Rnf220 and K00726 protein including known domains. Comparison of the domain sequences revealed that the E3 ubiquitin ligase and zinc-finger RING-type domain have 35% and 75% conservation, respectively. (D) Morphological outcomes of the secondary axis induction assay: AP defect, smaller or kinked antero-posterior axis; partial, secondary axis formed without cement gland; full, secondary axis formed with cement gland. Amount of injected mRNA: β-catenin**, 250–500 pg; β-catenin*, 5–25 pg (subthreshold); β-Gal, K00726 and Rnf220, 500–1000 pg. At the top of each bar is the total number of analysed embryos. Image published in: Monteiro RS et al. (2018) © 2018 The Authors. 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

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