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First published online 13 April 2005
doi: 10.1242/dev.01806

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Transgenic zebrafish reveal stage-specific roles for Bmp signaling in ventral and posterior mesoderm development

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

View larger version (23K): [in a new window]   Fig. 1. A method for attenuating Bmp signaling in transgenic zebrafish. Diagram of a wild-type cell receiving a Bmp signal (left) and a transgenic cell receiving a Bmp signal after heatshock (right). In a wild-type cell, the Bmp ligand binds to a Type I/Type II receptor complex, which then phosphorylates and activates a receptor SMAD (rSMAD), leading to target gene activation. After heatshock of the transgenic cell, the truncated Type I Bmp receptor containing GFP in place of the kinase domain displaces the endogenous Type I receptor, preventing the receptor complex from activating rSMADs. As a result, downstream target genes are not activated, and the cell is reduced in its capacity to respond to Bmp signals. KD, kinase domain; LB, ligand-binding domain; P, phosphate; TM, transmembrane domain.

View larger version (70K): [in a new window]   Fig. 2. Phenotypes caused by reducing Bmp signaling at various developmental stages. Embryos from a transgenic outcross were heatshocked in an air incubator at 3 hpf (A-C) or shield stage (D-F), and the GFP-positive embryos were sorted and scored for dorsalized phenotypes at 30 hpf. Note the fully formed tail in the wild-type sibling (whole embryo in A, tail view in D). Transgenic embryos heatshocked at 3 hpf display both C4 (B) and C3 dorsalized (C) phenotypes. Note the curled trunk and tail in B and the curled tail in C. Transgenic embryos heatshocked at shield stage display either a C1 phenotype (E; arrow denotes loss of the ventral tail fin) or a C1 phenotype coupled with the formation of an ectopic tail (F; arrowhead denotes second tail). ET, ectopic tail.

View larger version (72K): [in a new window]   Fig. 3. eve1 expression is dependent on Bmp signaling both during and after gastrulation. Embryos were heatshocked in an air incubator at the indicated stages, and the embryos containing the transgene were sorted by GFP fluorescence. (A,B) Vegetal views of embryos heatshocked at shield stage and assayed for eve1 expression at 70% epiboly. Note the light staining on the left (ventral) side of the transgenic embryo (B) compared with the wild-type sibling (A). (C,D) Posterior views of embryos heatshocked at bud stage and assayed for eve1 expression at the 7-somite stage. Note the darker staining of eve1 in the wild-type embryo (C), with expression extending farther anteriorly than in the transgenic embryo (D). (E,F) Posterior views of embryos heatshocked at the 3-somite stage, then assayed for eve1 expression at the 9-somite stage. Note the darker staining of eve1 in the wild-type embryo (E) than in the transgenic embryo (F). Insets in C-F show lateral views of the tailbud. HS, heatshock.

View larger version (60K): [in a new window]   Fig. 4. High levels of Bmp signaling are important for ventral mesoderm patterning during gastrulation but not afterward. Embryos were heatshocked in an air incubator at 3 hpf for 1 hour, or heatshocked continuously from bud stage, sorted for GFP fluorescence and fixed at the 12-somite stage. Expression of flk-1 (A-C), gata1 (D-F) or pax2.1 (G-I) was then examined by in-situ hybridization. Non-transgenic embryos heatshocked under either protocol were identical, so only a single example is shown for the –GFP samples (C,F,I). Expression of all three markers was reduced in transgenic embryos (A,D,G) compared with wild-type siblings (C,F,I) after 3 hpf heatshocks. Gene expression was unaltered following post-gastrula heatshocks (B,E,H). Arrows in A-C denote flk-1-expressing cells. Arrowheads in G-I denote pax2.1 expression in the otic placode, while brackets indicate pax2.1 expression in the pronephric ducts and tubules. Views in A-C and G-I are dorsal, with anterior to the top. (D-F) Posterior views of embryos.

View larger version (98K): [in a new window]   Fig. 5. Analysis of gene expression in the ectopic tails. Embryos were heatshocked from shield to bud stage to maximize ectopic tail formation, then photographed live at 3 dpf (A), or fixed at either 21 hpf (B) or 30 hpf (C-I). Expression of eve1 (B,C), no tail (ntl; D), myod (E), collagen 2a (coll2a; F), caudal (G), neurogenin1 (H), or sonic hedgehog (shh; I). Arrows in each panel indicate the location of the ectopic tail. In A note the presence of both fin and pigment tissue in the secondary tail. In all other panels except H note the expression of each corresponding gene in the ectopic tails. ntl expression in D was localized only to the tailbud of the ectopic tail. All images except I are representative of the majority of ectopic tails examined. We observed sonic hedgehog-expressing cells in 44% (n=16) of ectopic tails.

View larger version (39K): [in a new window]   Fig. 6. Dynamic growth of secondary tails. Confocal microscopic examination of live truncated Bmp receptor-GFP/tbx6-gfp transgenic embryos during secondary tail growth. (A) Time points were started at the 25-somite stage, then taken every hour afterwards, corresponding to approximately one somite per time point at room temperature. Note the growth of the GFP-expressing secondary tail and the maturation of the secondary tail's presomitic mesoderm into somitic blocks. Arrows indicate the second tail, which grows along the primary axis. The asterisk indicates the first fully formed ectopic tail somite at the 25-somite stage. (B) In a separate experiment, truncated Bmp receptor-GFP/tbx6-gfp embryos were mounted at the 16-17-somite stage, then monitored by confocal microscopy to detect the emergence of a second tail. Confocal slices from two representative embryos are shown. Arrows denote the tip of the second tailbud, and `ye' indicates the yolk extension. In a and c, note the ventral patch of GFP expression corresponding to an emerging second tail. (b,d) The extending second tail about 4 hours later. The ectopic tail is growing into the plane of the picture in b and out of the plane in d.

View larger version (58K): [in a new window]   Fig. 7. Only cells with reduced Bmp signaling can populate ectopic tails. (A) Diagram of the transplant scheme used. Fluorescein-labeled donor cells (wild type or transgenic) were transplanted at dome stage to the ventral side of shield-stage transgenic hosts. Hosts were immediately heatshocked, then analyzed at 24 hpf (after the transgenic GFP faded) for presence of fluorescent cells in the ectopic tails. (B) Wild-type donor cells in a transgenic host. Note the absence of fluorescein-labeled donor cells in the ectopic tail (denoted by an arrow). (C) Transgenic donor cells in a transgenic host. Note the presence of fluorescein-labeled donor cells in the ectopic tail (denoted by an arrow) as well as in the primary tail.

View larger version (37K): [in a new window]   Fig. 8. A model for the distinct temporal roles of Bmp signaling during posterior mesoderm development. Embryos at different stages of development are shown, with posterior mesodermal Bmp signaling denoted in red. Below is a timeline of development and the roles of Bmp signaling in ventral mesoderm and tail tissues at various stages.