Bone Morphogenetic Proteins (BMPs) act as a morphogen to pattern dorsoventral (DV) axial tissues in invertebrates and vertebrates. The shape of the BMP gradient spatially across the DV axis is critical: precise amounts of BMP signaling at discreet DV positions specify distinct cell fates. DV patterning occurs from late blastula through gastrula stages, so the BMP gradient is also maintained temporally during gastrulation. Gastrulation and dorsal convergence generate the embryonic germ layers and body axis by rapidly reorganizing a large number of cells, challenging the mechanisms that shape the BMP gradient in space and time. To determine the shape of the BMP gradient, we developed a quantitative immunofluorescence assay of nuclear phosphorylated Smad1/5, the direct intracellular readout of BMP signaling. Using this assay, we discovered that the shape of the BMP gradient significantly changes during gastrulation: the BMP gradient steepens both dramatically and rapidly between mid- and late gastrulation stages.
Extracellular modulators of BMP ligand availability shape the BMP gradient. An essential extracellular modulator is Chordin, a BMP antagonist that binds BMP to inhibit signaling. Chordin is central to generating the initial BMP signaling gradient and continually regulates the BMP gradient during gastrulation. Chordin itself is regulated by two key classes of proteins: (i) the highly homologous metalloproteases Tolloid and Bmp1a, which cleave and inactivate Chordin, and (ii) the metalloprotease inhibitor Sizzled.
We discovered that tolloid (tld) and sizzled (szl) mutants display distinct alterations in the shape of the BMP gradient at the end of gastrulation, suggesting region-specific roles for Tld and Szl. Our data indicate that Tld maintains the steepness of the late BMP signaling gradient, while Szl restricts BMP signaling in lateral regions. These results support a novel requirement for metalloprotease regulation during late gastrulation to steepen the BMP gradient and correctly pattern posterior tissues of the zebrafish embryo. Unlike tld, bmp1a is maternally deposited. We found that while MZ-bmp1a mutants have no DV patterning defects, MZ-bmp1a embryos depleted of Tld are severely dorsalized. The severity of dorsalization indicates that Tld and Bmp1a are redundant and essential to establish the BMP gradient at late blastula stages. These data support fundamental, stage-specific roles for Bmp1a/Tld to differentially shape the BMP gradient, putting forth spatiotemporal metalloprotease regulation as a central mechanism for the spatiotemporal regulation of BMP signaling.