Organizer-drived Bmp Signal Is Required For Zebrafish Embryonic Dorsoventral Pattering

In vertebrate embryos, the Bmp activity gradient, indicated by p-Smad1/5/8gradient, is established along the ventral to dorsal axis and controls dorsoventralpatterning as a critical morphogen. This study, using the zebrafish model system, aimsto explore how an appropriate Bmp activity gradient is formed and maintained duringgastrulation.The formation of the Bmp activity gradient during dorsoventral patterning of earlyembryos mainly relies on synthesis of Bmp ligands and their antagonists. However, thedistribution of endogenous Bmp proteins and their antagonists in embryos remainselusive. In the zebrafish embryo, Bmp2b and the antagonist Chordin (Chd) playimportant roles in dorsoventral patterning. Their distribution patterns are uncovered inthis study by immunostaining using specific antibodies. In early gastrulae, Bmp2bforms a ventral to dorsal gradient, outside the organizer field, with the highest level inthe ventralmost region, but it is also enriched in the organizer; Chd diffuses far beyondits synthesis place to establish a dorsal (high) to ventral (low) gradient, suggesting along range diffusion ability; and p-Smad1/5/8shows a single ventral (high) to dorsal(low) gradient, but it does exist in nuclei within the organizer. Knockdown of chdcauses a drastical increase of Bmp2b levels along the whole ventrodorsal axis and amore flatterned p-Smad1/5/8gradient due to an increase mainly in the dorsal side,supporting the idea that repression of Bmp signaling by Chd in the dorsal region isnecessary for forming an appropriate Bmp activity gradient.Bmp2b is present in the organizer, but its function is unknown. In this study, theorganizer-derived Bmp2b (osBmp2b) is intervened by specific expression of tBr, adominant negative form of type I Bmp receptor, in the organizer usingorganizer-specific promoters. When osBmp2b is blocked in embryos, both Bmp2b andp-Smad1/5/8exhibit a significant decrease throughout the ventrodorsal axis at the shieldstage, indicating that osBmp2b ligands contribute to the overall Bmp signal activity inthe whole embryo.Interfering with osBmp2b results in a remarkable ventral expansion of chd mRNAexpression domain and consequently a universal rise of Chd protein levels in early gastrulae, suggesting that osBmp2b acts to repress chd transcription. The promoter ofchd is found to contain putative Smad1/5binding sites. These sites are bound by Smad1in embryos and their mutations cause a loss of response to Bmp/Smad repression, whichindicate a direct role of Bmp/Smad in repressing chd transcription. Furthermore,interfering with osBmp2b signaling alters dorsoventral patterns of gastrulas and causesdorsalized phenotypes during pharyngula stages, indicating an important role ofosBmp2b in embryonic dorsoventral patterning.Finally, the mathematic simulation of the reaction-diffusion dynamics of the Bmpactivity gradient, based on two Bmp sources, i.e., ventral and dorsal Bmps, supports thenotion that the dorsal source of Bmp (Bmp2b) plays an essential role in establishing andmaintaining an appropriate Bmp activity gradient along the ventrodorsal axis duringearly gastrulation.