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First published online 7 July 2004
doi: 10.1242/dev.01236

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Combinatorial gene regulation by Bmp and Wnt in zebrafish posterior mesoderm formation

Department of Biochemistry, Box 357350, University of Washington, Seattle, WA 98195-7350, USA

View larger version (85K): [in a new window]   Fig. 1. Wnt8 and Bmp2b are required for tbx6 expression. Animal pole views of shield stage embryos with dorsal to the top. The in situ probe is shown at the top of the Figure (ntl, no tail; spt, spadetail) and the genotype of the embryo is shown on the left side. Wnt8 morphants are wild-type embryos injected with a pair of wnt8-specific morpholino oligonucleotides. Note that residual tbx6 expression is only observed ventrally in the wnt8 morphant embryos whereas residual tbx6 expression is present laterally and ventrally in swirl-/- embryos.

View larger version (64K): [in a new window]   Fig. 2. Regulation of tbx6 by Wnt and Bmp. (A-H) Lateral views with the animal pole to the top. (A,B) Expression of endogenous tbx6 in an uninjected embryo (A) and in an embryo injected with 50 pg of noggin RNA (B). The injected embryo shows dramatic reduction of tbx6 expression. (C) An embryo injected with 75 pg of wnt8 RNA shows ectopic expression of tbx6. (D) An embryo coinjected with 75 pg of wnt8 RNA and 50 pg of noggin RNA showing ectopic expression of tbx6 in the animal pole. (E-H) Expression of endogenous tbx6 in an uninjected embryo (E) and in an embryo injected with 150 pg of Xbmp4 RNA (F). The injected embryo shows ectopic expression of tbx6. (G) An embryo injected with wnt8MOs shows decreased expression of tbx6. (H) A zebrafish embryo coinjected with wnt8MOs and 150 pg of Xbmp4 RNA showing strong marginal and ectopic dorsal domains of tbx6 expression.

View larger version (77K): [in a new window]   Fig. 3. tbx6-gfp reporter constructs are correctly expressed in zebrafish embryos. (A) Schematic diagrams of gfp reporter constructs, ptbx6-1.7-gfp and ptbx6-2.0-gfp, which contain 1.7 and 2.0 kb of upstream sequence, respectively. The 1.7 kb region is subdivided into a proximal 900 bp domain and a distal 800 bp domain. The sequences of three putative Tcf binding sites (Tcf-1, Tcf-2 and Tcf-3) are indicated with the arrows showing the orientation of the sites. (B-E) Lateral views with animal pole to the top of shield stage (B,C) and 15-somite (D,E) embryos injected at the one-cell stage with ptbx6-1.7-gfp. Bright field (B,D) and GFP fluorescence (C,E) pictures showing specific GFP fluorescence at the margin at shield stage (C) and in the posterior mesoderm at the 15-somite stage (G). Note that because these embryos are not transgenic, the GFP is expressed mosaically in the embryos from the injected DNA. (F-O) Transgenic embryos containing ptbx6-1.7-gfp. (F,G) Lateral views with anterior to the top. Bright field (F) and fluorescence (G) pictures of a ptbx6-1.7-gfp transgenic embryo at the 15- to 18-somite stage. (H-O) In situ staining for gfp (H-K) and tbx6 (L-O). Embryos shown are at 70% epiboly (H,L; dorsal view with animal pole to the top), 95% epiboly (I,M; posterior view with dorsal to the top), 5-somite stage (J,N; posterior view with dorsal to the top) and 18-somite stage (K,O; lateral view with anterior to the top). The small inserts in K and O show a close-up posterior view of expression in the tip of the tail. Note the tbx6 in situ probe was from the 3' end of the gene and therefore does not bind to the transcripts from the transgene. E, exon sequence.

View larger version (67K): [in a new window]   Fig. 4. Regulation of ptbx6-1.7-gfp and endogenous tbx6 by Wnt and Bmp. (A-J) Lateral views with the animal pole to the top. (A-D) Overexpression of Wnt8. (A,B) Expression of endogenous tbx6 in an uninjected embryo (A) and in an embryo injected with 75 pg of wnt8 RNA (B). The injected embryo shows ectopic expression of tbx6. (C,D) Bright field (C) and fluorescence (D) pictures of a zebrafish embryo coinjected with ptbx6-1.7-gfp and 75 pg of wnt8 RNA showing ectopic GFP fluorescence in the animal pole. (E-H) Expression of endogenous tbx6 in an uninjected embryo (E) and in an embryo injected with 150 pg of Xbmp4 RNA (F). The injected embryo shows ectopic expression of tbx6. (G,H) Bright field (G) and fluorescence (H) pictures of a zebrafish embryo coinjected with ptbx6-1.7-gfp and 150 pg of Xbmp4 RNA at the shield stage showing ectopic GFP fluorescence in the animal pole. (I,J) Bright field (I) and fluorescence (J) pictures of a zebrafish embryo coinjected with ptbx6-1.7-gfp and 25 pg of TVGR RNA showing ectopic GFP fluorescence in the animal pole. TVGR is a constitutively active form of the Tcf transcription factor (Darken and Wilson, 2001). The in situ hybridizations are at shield stage whereas the bright field and fluorescence images are at 50% epiboly.

View larger version (83K): [in a new window]   Fig. 5. A dual fluorescence reporter for examining promoter expression in zebrafish embryos. (A) Schematic diagram of the dual fluorescence reporter construct, ptbx6-2.0-gfp/pXex-bfp. The black box shows the tbx6 first intron. (B-G) Pictures of embryos injected with the ptbx6-2.0-gfp/pXex-bfp promoter in bright field (B,E), showing GFP fluorescence (C,F) and BFP fluorescence (D,G). (B-D) Lateral views with animal pole to the top at 70% epiboly. (E-G) Ventral views with anterior to the top at the 16-somite stage. E, exon sequence.

View larger version (58K): [in a new window]   Fig. 6. Tcf sites are required for normal tbx6 expression. Embryos were injected with the tbx6 promoter containing a mutation in Tcf site 1 (mut1-2.0-gfp/Xex-bfp; A-F) or a promoter with mutations in Tcf sites 1 and 2 (mut1,2-2.0-gfp/Xex-bfp; G-L). Embryos were viewed in bright field, for GFP fluorescence or for BFP fluorescence as indicated on the right side of the panels. Embryos were at 70-90% epiboly (A-C,G-I) or at the 5-somite stage (D-F,J-L).

View larger version (18K): [in a new window]   Fig. 7. Regulation of the tbx6 promoter. (A) Luciferase activities of the indicated constructs measured at 50% epiboly, with and without the coinjection of Xbmp4 (bmp). dr800 is the distal 800 bp and intron is the first intron of the tbx6 promoter. (B) Luciferase activities at 50% epiboly of the indicated constructs coinjected with Xbmp4, showing a 65 bp region of the tbx6 promoter is fully capable of responding to Bmp activation. (C) Sequence and schematic diagram of the 65 bp region. Division of this region into domains of 25 bp [(1) to (5)] causes a complete loss of Bmp responsiveness.

View larger version (20K): [in a new window]   Fig. 8. Model for the combinatorial regulation of tbx6 by Bmp and Wnt. (A) In the wild-type embryo, tbx6 promoter utilizes two distinct domains to integrate the signaling inputs of both Bmp and Wnt (gray box, Bmp response domain; white box with three oval circles, Wnt response domain; oval circles, Tcf binding sites; black gradient, Bmp activity gradient; blue region, wnt8 expression domain; blue filled oval circle, occupied Tcf site; pink color, tbx6 gene and transcripts). wnt8 expressed at the margin works combinatorially with bmps expressed ventrally to activate the tbx6 promoter throughout the ventrolateral region. (B) In a bmp mutant or noggin-injected embryo, endogenous Wnt8 only weakly activates expression throughout the margin. (C) In a wnt8 mutant or in an embryo injected with wnt8MOs, endogenous Bmp signaling activates tbx6 only in the most ventral region where Bmp activity is the highest. When Wnt8 signaling is inhibited, Bmp signaling is confined to a more ventral domain than in wild-type embryos.