Bmp inhibition is necessary for post-gastrulation patterning and morphogenesis of the zebrafish tailbud

Abstract

Intricate interactions between the Wnt and Bmp signaling pathways pattern the gastrulating vertebrate embryo using a network of secreted protein ligands and inhibitors. While many of these proteins are expressed post-gastrula, their later roles have typically remained unclear, obscured by the effects of early perturbation. We find that Bmp signaling continues during somitogenesis in zebrafish embryos, with high activity in a small region of the mesodermal progenitor zone at the posterior end of the embryo. To test the hypothesis that Bmp inhibitors expressed just anterior to the tailbud are important to restrain Bmp signaling we produced a new zebrafish transgenic line, allowing temporal cell-autonomous activation of Bmp signaling and thereby bypassing the effects of the Bmp inhibitors. Ectopic activation of Bmp signaling during somitogenesis results in severe defects in the tailbud, including altered morphogenesis and gene expression. We show that these defects are due to non-autonomous effects on the tailbud, and present evidence that the tailbud defects are caused by alterations in Wnt signaling. We present a model in which the posteriorly expressed Bmp inhibitors function during somitogenesis to constrain Bmp signaling in the tailbud in order to allow normal expression of Wnt inhibitors in the presomitic mesoderm, which in turn constrain the levels of canonical and non-canonical Wnt signaling in the tailbud.

Fig. 1. Bmp signaling is tightly regulated in the tailbud during early somitogenesis

(A,B) Bmp signaling is active in the mesodermal progenitor zone. Expression of no tail is shown by FISH in green in 3-somite stage embryos marking both the mesodermal progenitor zone and the notochord (n). Bmp signaling is indicated by antibody staining for phosphorylated Smad proteins (red in B-D). Bmp inhibitors are expressed just anterior to the activated Smad zone. The Bmp inhibitors chordin, noggin1 and noggin2 are shown by FISH in green and the Smad staining is shown in red in 3-somite (C) and 12-somite (D) embryos. All embryos are shown from a dorsal view with anterior to the top.

Fig. 2. A new transgenic line allows temporal cell-autonomous activation of Bmp signaling

(A) Schematic diagram of construct used to create the new line. A multimerized minimal heat shock-responsive promoter is flanked by genes for GFP and a constitutively active mutant form of the Type Ib Bmp receptor. Tol2 elements enhance transgenesis efficiency. (B) Early heat shock of a transgenic embryo at dome stage recapitulates the expected Bmp ventralized phenotype. (C) eve1 expression is rapidly induced by ectopic Bmp signaling. Embryos were heat-shocked at dome stage and fixed at shield stage.

Fig. 3. Ectopic Bmp signaling during early somitogenesis alters tailbud morphogenesis

(A-D) Transgenic embryos (right) and wild-type controls were heat shocked at the 3-somite stage. No morphological change is visible at the 9-somite stage (B). By the 16-somite stage the tailbud and posterior somites display clear alterations (D). (E, F) At approximately 42 hpf transgenic embryos (F) display a shortened body axis and the epidermal fin fold has failed to form normally.

Fig. 4. Ectopic Bmp signaling during early somitogenesis alters patterning and morphogenesis

(A, B) Expression of no tail is expanded in the tailbud of transgenic embryos at the 9-somite stage. (C-E) Cell migration, as shown by photoconverted NLS-Kikume fluorescence (red), is disrupted as a result of ectopic Bmp signaling. The graph shows the most anteriorward migration of labeled cells from the posterior end of the embryo in individual embryos. (F, G, H) Cell migration defects are a non-cell-autonomous result of ectopic Bmp signaling. Cell transplants were performed as diagrammed in (F). Cells from a transgenic donor (H) contribute to the same cell types and anterior extent as those from a wild-type donor (G). Embryos were heat shocked at the 3-somite stage.

Fig. 5. Ectopic Bmp signaling enhances Wnt signaling

(A-D) Ectopic Bmp signaling represses expression of secreted Wnt inhibitors sfrp1a and wif1. (E-L) Ectopic Bmp signaling expands the region of canonical Wnt signaling in the tailbud. Nuclear β-catenin accumulates in a larger region in transgenic embryos after heat shock.

Fig. 6. Ectopic Wnt5a expression recapitulates ectopic Bmp signaling in the tailbud

Cell migration, as shown by photoconverted NLS-Kikume fluorescence (red), is disrupted as a result of ectopic Wnt5a expression. The graph shows the most anterior contribution of labeled cells to presomitic mesoderm in individual embryos.

Fig. 7. Model of the role of the Bmp inhibitors in the tailbud

Bmp inhibitors are required to restrict Bmp signaling to the embryonic tailbud since Bmp signaling represses expression of the Wnt inhibitors, which are required to modulate the levels of Wnt signaling. Canonical Wnts (cWnt) maintain mesoderm progenitors while non-canonical Wnts (ncWnt) regulate morphogenesis.