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First published online November 9, 2007
doi: 10.1242/10.1242/dev.010173

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Divergent functions of two ancient Hydra Brachyury paralogues suggest specific roles for their C-terminal domains in tissue fate induction

¹ Sars International Centre for Marine Molecular Biology, University of Bergen, Thormøhlensgt. 55, 5008 Bergen, Norway.
² Adolf-Butenandt-Institut, Ludwig-Maximilians-Universität, Schillerstrase 44, D-80336 München, Germany.
³ IBDM/LGPD Case 907, Campus de Luminy, 13288 Marseille, France.
⁴ Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile.
⁵ Developmental Biology Center and Developmental and Cell Biology Department, University of California at Irvine, Irvine, CA 92697, USA.

View larger version (44K): [in this window] [in a new window]   Fig. 1. Comparison of protein and DNA sequence conservation of HyBra1 and HyBra2. (A) Alignment of T-domains of HyBra1 and HyBra2 with Brachyury proteins from mouse (M-T), Xenopus (X-bra), Amphioxus (Am-bra-1), the sea urchin Hemicentrotus pulcherrimus (Hp-T), the ascidian Halocynthia roretzi (As-T), Platynereis dumerilii (Pd-bra) and Drosophila melanogaster (Dm-Trg). Amino acid identities are black, partial identities (>60%) in grey. Amino acids marked by grey dots under the sequence are involved in DNA-binding, grey bars indicate amino acids involved in dimerisation (Müller and Herrmann, 1997). Arrows indicate conserved intron sites in HyBra1 and mouse-T. (B) Alignment of the core repression module R1 (Kispert et al., 1994) within the activation domain. HyBra2 and Drosophila Trg do not have this motif. (C) Schematic structures of Xbra, HyBra1 and HyBra2 protein illustrate the degree of amino acid identity in the T-box and the R1 domain (blue box) in HyBra1 and HyBra2 compared with XBra. (D) Comparison of exon-intron structure in Xbra, HyBra1 and HyBra2. Filled boxes mark the ORF, white boxes the UTR. Red boxes indicate conserved exon-intron sites, whereas black boxes indicate non-conserved regions. Black numbers represent the length of introns.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Phylogenetic analysis of Brachyury proteins on the basis of their conserved T-box domain. A group of Tbx6 genes was used as an outgroup for the Brachyury family. Analysis was performed with MrBayes 3.1.2 using the WAG substitution model. Sequences were obtained from GenBank. Aa, Aedes aegypti; Am, Amphioxus (Branchiostoma floridae); As, Halocynthia roretzi; Bf, Branchiostoma floridae; Cj, Clypeaster japonicus; Gg, Gallus gallus; Gm, Grillus maculatus; He, Hydractinia echinata; Hp, Hemicentrotus pulcherrimus; Hs, Homo Sapiens; Lf, Lampetra fluviatilis; Lv, Lytechinus variegatus; Ml, Mnemiopsis leidyi; Mm, Mus musculus; Nem, Nematostella vectensis; Ol, Oikopleura longicauda; Pat, Patella vulgata; Pc, Podocoryne carnea; Pd, Platynereis dumerilii; Pf, Ptychodera flava; Pl, Paracentrotus lividus; Pp, Pleurobrachia pileus; Sr, Scypha raphanus; Ta, Trichoplax adhaerens; Tc, Tribolium castaneum; X, Xenopus laevis; Zf, Zebrafish.

View larger version (83K): [in this window] [in a new window]   Fig. 3. Expression of HyBra1 and HyBra2. HyBra1 and HyBra2 mRNA expression was assessed in adult polyps (A-D) and during budding (E-J). (A,B) In adult hydras HyBra1 is predominantly expressed in the endodermal layer of the hypostome. (C,D) HyBra2 expression was primarily detected in the ectoderm of the hypostome. (B,D) Vibratome cross-sections of the hypostome. (E-J) Both genes are expressed very early during budding, but HyBra1 RNA is predominantly in the endoderm, whereas HyBra2 expression is restricted to the ectoderm (arrows in E and H). (J) Vibratome section of stage 5 bud. (K-M) Switch of HyBra2 expression from the endoderm to the ectoderm during head regeneration. Expression is in the endoderm at 18 hours (K), in both layers at 28 hours (L) and in the ectoderm by 48 hours (M). Arrows indicate the mesoglea, which separates ectoderm from endoderm.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Kinetics of HyBra1 and HyBra2 expression during regeneration. Expression of HyBra1 (dashed line) and HyBra2 (solid line) during head regeneration following bisection at (A) the upper end of the body column, and (B) in the middle of the body column. (C) Expression of the two genes during foot regeneration following bisection in the mid-body column. Data points are means (± s.d.) of 40-80 animals resulting from 2-5 independent experiments. HyBra1 data modified after Technau and Bode (Technau and Bode, 1999).

View larger version (103K): [in this window] [in a new window]   Fig. 5. Expression of HyBra1 and HyBra2 during regeneration in the presence or absence of cycloheximide. Animals were pretreated for 15 minutes in 5 µg/ml cycloheximide, decapitated and allowed to regenerate for the times indicated. The first two hours of regeneration were carried out in the presence of the cycloheximide. (A-C) HyBra1 expression under control conditions. (D-F) The onset of HyBra1 is not affected by cycloheximide. HyBra2 expression in cycloheximide-treated animals (J-L) starts later than in control animals (G-I).

View larger version (155K): [in this window] [in a new window]   Fig. 6. Ectopic expression of HyBra1 and HyBra2 in response to alsterpaullone treatment. Animals were exposed to 5 µM alsterpaullone (AP) for 24 hours and subsequently transferred to Hydra media (HM) for 15 hours and 40 hours respectively. HyBra1 (A-D), HyBra2 (E-H) and Wnt (I-L) are affected by the GSK-3 inhibitor alsterpaullone. Ectopic HyBra1 and HyBra2 expression starts 24 hours after AP treatment in single cells (B,F, high-magnification inset) whereas normal hypostome expression declines rapidly and is shifted to the tentacle bases (compare A,B with E,F); elevated levels of expression are seen over the uniform endodermal expression (compare A,E with B,F). In later stages, the single cell expression in the body column begins to cluster (arrows in C and G) and subsequently peaks in a stochastic pattern of spots of 10-15 epithelial cells (arrows in G and H). HyWnt hypostome expression is lost 15 hours after completion of AP treatment and clusters along the body column in ectopic expression domains are narrower than those of HyBra1 and HyBra2.

View larger version (76K): [in this window] [in a new window]   Fig. 7. Distinct morphological phenotypes induced in Xenopus animal caps by Hydra Brachyury proteins. (A) Schematic view of the microinjected constructs (AD, activation domain; C-T, C-terminal domain). (B) Sketch of the animal cap experiment (st, stage). (C-E) Control specimens: (C) wt sibling embryo at NF36, (D) uninjected and (E) GFP-injected animal caps with default epidermal morphology. Animal caps injected with Xbra (F), HyBra1 (G) or H2TXA (I) with elongated morphology, indicating mesoderm formation. (H) HyBra2-injected animal cap with two darkly pigmented cement gland areas. (J) XTH2A-injected explant with single cement gland area. (K) Statistical overview of morphological characteristics [uninduced, elongated and cement gland celltype-specific (cg cells)] induced by different Brachyury paralogues (3-10 independent injections). RNA doses were titrated from 0.1 to 1.0 ng/embryo, resulting in consistent, qualitatively different phenotypes represented by the displayed specimen.

View larger version (82K): [in this window] [in a new window]   Fig. 8. Molecular analysis of the differentiated animal cap phenotypes induced by HyBra proteins. Marker analysis by double (A,C,E,G,I-L) or single (B,D,F,H) RNA in situ hybridisation; probes are listed within panels, black letters indicate BM-Purple stain, red letters indicate Fast-Red stain (n3 independent repeats). (A,B) GFP-injected control explants showed no staining of tested marker genes. Injections of Xbra (C) and HyBra1 (E) induced muscle actin, but not xcg-1 expression, whereas HyBra2 (G) triggers the converse gene expression pattern. Furthermore, mRNA of the differentiated neuronal marker ß-tubulin was often present in HyBra2- (H), but never in HyBra1- (F), and only rarely in Xbra-injected explants (D). (I-L) Double in situ staining of animal caps injected with the Hybra2/Xbra chimerae. (I,J) H2TXA-injected animal caps show no xcg-1, ß-tubulin induction and very limited muscle actin expression. (K,L) By contrast, XTH2A-injected caps clearly show strong induction of xcg-1 and ß-tubulin, but no muscle actin mRNA. (M) Statistical overview of induced markers (xcg-1, muscle actin, ß-tubulin) by injection of particular mRNAs (2-6 independent single and/or double in situ hybridisation experiments).