Vizualizing the morphogen adsorption gradient in the Xenopus laevis embryo using fluorescently labeled heparin-binding motif of BMP4 morphogen

Abstract

Spatial distribution of heparan sulfates in the embryonic extracellular matrix at midgastrula stage has been demonstrated in the Xenopus laevis embryo model. Towards this end, fluorescently labeled fusion protein EGFP-hbmBMP4 made up by green fluorescent protein (EGFP) and heparin-binding motif of Bone Morphogenetic Protein 4 (BMP4) was produced in the E. coli expression system. Xenopus laevis embryos at midgastrula stage (stage 11) were fixed and cut along the anteroposterior axis and then incubated with EGFP-hbmBMP4. The fluorescently labeled samples were analyzed in fluorescence microscope. The spatial distribution of fluorescence intensity reflecting BMP4 adsorption on the embryonic extracellular matrix proved to be similar to the corresponding distribution pattern for the Noggin1 heparin-binding motif obtained previously. The highest intensity zone was detected around the dorsal blastopore lip; another high intensity zone, although slightly less prominent, was observed in the ventral blastopore lip region. Since on one hand, heparin-binding sites significantly differ in their organization in BMP4 and Noggin1 proteins and, on the other hand, spatial adsorption distribution patterns for these proteins coincide in the embryo, it appears that all secreted morphogens containing a heparin-binding site share a single adsorption gradient in the embryonic extracellular space.