Yasuoka Y et al. (2014), Occupancy of tissue-specific cis-regulatory mod...

XB-ART-49342

Nat Commun 2014 Jul 09;5:4322. doi: 10.1038/ncomms5322.

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Occupancy of tissue-specific cis-regulatory modules by Otx2 and TLE/Groucho for embryonic head specification.

Yasuoka Y , Suzuki Y , Takahashi S , Someya H , Sudou N , Haramoto Y , Cho KW , Asashima M , Sugano S , Taira M .

Abstract
Head specification by the head-selector gene, orthodenticle (otx), is highly conserved among bilaterian lineages. However, the molecular mechanisms by which Otx and other transcription factors (TFs) interact with the genome to direct head formation are largely unknown. Here we employ ChIP-seq and RNA-seq approaches in Xenopus tropicalis gastrulae and find that occupancy of the corepressor, TLE/Groucho, is a better indicator of tissue-specific cis-regulatory modules (CRMs) than the coactivator p300, during early embryonic stages. On the basis of TLE binding and comprehensive CRM profiling, we define two distinct types of Otx2- and TLE-occupied CRMs. Using these devices, Otx2 and other head organizer TFs (for example, Lim1/Lhx1 (activator) or Goosecoid (repressor)) are able to upregulate or downregulate a large battery of target genes in the head organizer. An underlying principle is that Otx marks target genes for head specification to be regulated positively or negatively by partner TFs through specific types of CRMs.

PubMed ID: 25005894
PMC ID: PMC4805429
Article link: Nat Commun
Grant support: [+]

Species referenced: Xenopus tropicalis
Genes referenced: acta1 actc1 admp bmp4 cer1 chrd.1 crebbp crx cyp26a1 dkk1 en2 eomes ep300 ezh2 fgf8 foxa4 foxb1 foxd4l1.1 frzb frzb2 fzd8 gsc jarid2 ldb1 lhx1 meis1 meis3 mmut nog not otx2 pbx1 prdm1 ssbp3 tbxt tle1 tle2 tle4 vegt ventx1 ventx2.2 wnt11 wnt8a
GO keywords: head development
Antibodies: Acetylated H3f3a Ab20 Tle1 Ab1 ep300 Ab1 methyl-H3f3a Ab13
Morpholinos: crx MO1 gsc MO1 lhx1 MO2 otx2 MO1

GEO Series: GSE172076: NCBI

Article Images: [+] show captions

	Figure 1 \| Phenotypes of loss- and gain-of-function of head organizer TFs in frogs. (a) Expression patterns of otx2, otx5, lim1 and gsc in Xenopus gastrula embryos. Schematic representation shows co-expression of lim1, otx2, otx5 and gsc in the head organizer4–9,16,22,23,56 and lim1 expression in the trunk organizers16,56. A double-headed arrow indicates the dorsal (D) and ventral (V) axis. Co-expression of these genes in X. tropicalis early gastrula embryos was shown with in situ hybridization using hemisections. In anterior neuroectoderm, otx2 is weakly expressed, while otx5 is more strongly expressed. Arrowhead, dorsal blastopore; open arrowhead, expression in the anterior neuroectoderm; and dashed line, boundary of ectoderm and mesoderm. (b) Loss- of-function analysis in X. tropicalis. Phenotypes of normal embryos (control morphants) and head-reduced embryos (lim1/otx2/otx5/gsc morphants) are shown. Sagittal section and whole mount in situ hybridization of the telencephalon marker (eomes) and the midbrain–hindbrain boundary marker (en2) showed that forebrain, midbrain and foregut were shrunk in head-reduced embryos. Note that the notochord reached the anterior-most region in the morphant. Tailbud embryos are shown in lateral (anterior to the left; three left panels) or dorsoanterior (right-most panels) views. fb, forebrain; fg, foregut; no, notochord. (c) Gain-of-function analysis in X. laevis. Left panels show lateral views of a secondary head with one eye generated after ventral injection (inj.) of a cocktail of mRNAs as indicated. Right panels show immunostaining of head organizer cocktail-injected embryos against notochord by MZ15 (dorsal view of the same embryo as shown in the left panel) and somites by 12/101 (lateral view), respectively. Scale bar, 250 mm.
	Supplementary Figure 2. Loss of function analysis using Xenopus tropicalis embryos (A) Sequence alignment of MOs and mRNAs. WT, wild type; mut, MO resistant mRNAs. Start codons are underlined. (B) Specificity of MOs. Translation of WT mRNA, but not mut mRNA for lim1, otx2, or HA-tagged otx5 was inhibited by the corresponding MO. Western blotting was performed using lysates from X. laevis embryos, which were first injected with MO (5 pmol/embryo), followed by a second injection of lim1, otx2 or HA-tagged otx5 mRNA (200 pg/embryo) to avoid interactions between MO and mRNA prior to injection. AntiYLim1, antiYOtx2, and anti-HA antibodies were used for detection. (C) Morphant phenotypes in X. tropicalis. Five phenotypes were defined as indicated, (+++) being the most severe phenotypes. Tailbud embryos were shown in lateral views. Combinations of injected MOs demonstrated synergisms among lim1, otx2, otx5, and gsc. Phenotypes are summarized as bar graphs. Scale bar is 250 Pm. n, the number of embryos; exp, the number of independent experiments. (D) Rescuing morphant phenotypes in X. tropicalis. mRNAs were injected into the dorsal equatorial region of two blastomeres at 4Ycell stage embryos which had been injected with MOs at 2Ycell stage. Head defects were partially rescued by injection of MO-resistant mRNAs for lim1, otx2, and otx5 in a dose-dependent manner. Significance was examined by chi-square test. (E) Rescue experiments for morphant gene expression. Reduction of gsc and chordin expression by triple MOs was recovered by injection of MO-resistant mRNAs for lim1, otx2, and otx5 in a dose dependent manner. RNA samples 1 to 4 were prepared from embryos corresponding to (D). RTYqPCR was performed to quantify changes in mRNA levels. Relative expression changes compared to control morphants are shown. Normalization was done with the expression level of ef1D. k, P<0.05 (t-test, two-tailed).
	Supplementary Figure 1. Summary of this paper (A) Schematic presentation of gene expression patterns in Xenopus mid-gastrula embryos. Expression patterns of head organizer genes (1, 2) and non-head organizer genes (3Y6) are shown in head, notochord, and posterior mesoderm (indicated by dotted lines) or neural genes (7) in dorsal ectoderm. Some expression patterns were referred to Xenbase (http://www.xenbase.org/common/). (B) Model for Otx2 functions as a molecular landmark of the head. Otx2 is involved in activation of anterior genes (head organizer genes) while repressing the expression of the posterior/ventral genes (trunk genes) within the same cells. Otx2 and Lim1 together with indicated transcriptional factors bind to cis-regulatory modules (CRMs) containing Lim1Ybinding motifs (type I CRMs), which are found among head organizer genes, such as gsc, cerberus, chordin and crescent, as suggested by reporter gene assays 1,2,3, animal cap assays4,5 and this study. Otx2 binding to chordin and crescent CRMs is probably indirect because there are no Otx2 binding motifs (bicoid/P3C) in these CRMs. The co-activator, p300, binds to the CRMs, but the factor directly binding to p300 is unknown. Otx2, Gsc, and TLE bind to CRMs containing multiple bicoid/P3C binding motifs (type II CRMs) located within the trunk genes. Otx2 and Gsc possibly form a heterodimer on P3C sites,6 and bind directly to the co-repressor, TLE/Groucho.7,8 Thus, Otx2 influences expression of a battery of genes, each of which interprets the input for activation or repression via various CRMs to provide head identify. HD, homeodomain; RD, transrepression domain; AD, transactivation domain.

References [+] :

Acampora, Forebrain and midbrain regions are deleted in Otx2-/- mutants due to a defective anterior neuroectoderm specification during gastrulation. 1995, Pubmed