The Mesoderm-Forming Gene brachyury Regulates Ectoderm-Endoderm Demarcation in the Coral Acropora digitifera  Yuuri Yasuoka, Chuya Shinzato, Noriyuki Satoh  Current Biology  Volume 26, Issue 21, Pages 2885-2892 (November 2016) DOI: 10.1016/j.cub.2016.08.011 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Adi_bra Is Continuously Expressed around the Blastopore and Pharynx in Coral Embryos, and Wnt/β-Catenin Signaling Activates Adi_bra Expression Only in Ectoderm (A–F) Whole-mount in situ hybridization of Adi_bra during embryonic development: early gastrula (A and B), late gastrula (C), early planula (D), late planula (E), and early polyp (F). (G–J) Control embryos for chemical treatments. (K–P) Adi_bra expression is activated in Wnt/β-catenin signaling-activated embryos. AZ, azakenpaullone. (M and P) Hemisections of late-gastrula embryos along the oral-aboral axis. Adi_bra is not activated in endoderm. (Q–T) Inhibition of Adi_bra expression in Wnt/β-catenin signaling-inhibited embryos. Embryos are shown in blastoporal view (A, F, G–J, K, L, N, O, Q–T) or lateral view (B–E). Hemisections are shown with the blastopore at the top. (U) Vista plots reveal conservation of sequences around brachyury in anthozoans. The sequence of A. digitifera is used as a reference. Ate, Acropora tenuis; Pau, Porites australiensis; Nve, Nematostella vectensis. A region of ∼1 kb upstream of brachyury is conserved among anthozoans, as indicated by the orange box. This corresponds to the sequence analyzed in reporter assays. (V) Responses of the Adi_bra 1-kb upstream region to Adi_Tcf and Wnt8 in the luciferase assay using Xenopus embryos. See text for a detailed description of the results. Bars represent the mean ± SEM of luciferase activities relative to the mean value obtained from a reporter alone. ∗∗p < 0.01; ∗∗∗p < 0.001 (t test, two-tailed). Combinations of injected mRNA are indicated at bottom. VP, VP16 fusion construct of Adi_Tcf. mRNAs for Adi_Tcf and Wnt8 were injected with 100 pg per embryo and 25 pg per embryo, respectively. See also Figures S1 and S2. Current Biology 2016 26, 2885-2892DOI: (10.1016/j.cub.2016.08.011) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Microinjection Experiments Using Coral Embryos Demonstrate that Adi_bra Is Required for Pharynx Formation in Planula Larvae (A) Fertilized Acropora eggs restrained between glass capillaries. (B) A magnified image of aligned eggs. (C) A schematic figure showing microinjection of Acropora eggs. (D–I) Injection phenotypes of A. digitifera embryos at the planula stage (D–F) and (G–I) injection phenotypes of A. tenuis embryos at the planula stage are shown. (D and G) Control morphants. (E and H) Adi_bra morphants. (F and I) Uninjected controls. Adi_bra morphants showed pharynxless phenotypes in both A. digitifera and A. tenuis. Magenta indicates nuclei stained with propidium iodide; green indicates plasma membrane stained with CellMask. Numbers indicate the frequency of the phenotype observed. For A. digitifera (D–F), reproducible results from two biologically independent experiments were summed. Scale bars, 100 μm. See also Figure S3. Current Biology 2016 26, 2885-2892DOI: (10.1016/j.cub.2016.08.011) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Adi_Bra Regulates Germ Layer Demarcation in the Vicinity of the Blastopore (A) Procedures for transcriptomic analysis. RNA-seq data of morphants and corresponding uninjected control embryos at the late-gastrula stage were analyzed with EBSeq to identify differentially expressed genes. (B) Venn diagrams of differentially expressed genes in Adi_bra morphants and control morphants. These data indicate that 206 and 678 genes were specifically down- and upregulated in Adi_bra morphants, respectively. (C–N) Expression patterns of Adi_bra morphants specifically downregulated genes in normal embryos. (O–X) Expression patterns of Adi_bra morphants specifically upregulated genes in normal embryos. In (F), (H), (J), (L), (N), (P), (R), (T), (V), and (X), hemisections of embryos along the oral-aboral axis are shown. Embryos are shown in blastoporal view in (C)–(E), (G), (I), (K), (M), (O), (Q), (S), and (U). Hemisections are shown with the blastopore at the top. EG, early gastrula; LG, late gastrula. See the main text for detailed explanations of expression patterns of each gene. See also Figure S4 and Tables S1 and S2. Current Biology 2016 26, 2885-2892DOI: (10.1016/j.cub.2016.08.011) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 The Ectoderm-Brachyury Model of Ectoderm-Endoderm Demarcation in Acropora Embryos Suggests a Possible Origin for Vertebrate Mesoderm (A) Summary of this study. In Acropora gastrula embryos, brachyury expression is induced by Wnt/β-catenin signaling around the blastopore. Brachyury regulates demarcation of ectoderm and endoderm by activating ectodermal and blastoporal genes and by repressing endodermal genes. (B) An evolutionary perspective on the origin of vertebrate mesoderm. In a eumetazoan ancestor, brachyury appears to have been expressed at the ectodermal margin of the blastopore during gastrulation. Our results suggest that Brachyury maintains the ectoderm-endoderm boundary in cnidarians. In addition to its ancestral role, Brachyury may have acquired the new function of segregating mesoderm from ectoderm in vertebrates. Schematic figures of embryos are shown with their blastopores at the top. Asterisks indicate blastopores. Green hatched lines indicate brachyury expression domains. See also Table S3. Current Biology 2016 26, 2885-2892DOI: (10.1016/j.cub.2016.08.011) Copyright © 2016 Elsevier Ltd Terms and Conditions

Current Biology 2016 26, 2885-2892DOI: (10.1016/j.cub.2016.08.011) Copyright © 2016 Elsevier Ltd Terms and Conditions