Spatially non-uniform distributions of secreted morphogens guide tissue development in a highly reproducible and robust manner. In early embryonic patterning of insects and chordates, BMPs guide dorsal/ventral patterning through a complex network that relies on the spatial distribution of a BMP ligand (Dpp, BMP2/4) and a BMP antagonist (Sog/Chordin), but the regulation of Sog in Drosophila and Chordin in vertebrates have acquired different characteristics. We have used biochemical, genetic and computational modeling studies to explore the molecular etiology of these differences. In the early Drosophila embryo, Decapentaplegic (Dpp), a homologue of the vertebrate BMP-2/-4, is key in assigning identity to all dorsal structures. Dpp is transcribed uniformly throughout the dorsal domain, yet it forms an activity gradient via a cascade of extracellular regulation that restricts Dpp availability laterally, while simultaneously amplifying Dpp activity near the dorsal midline. The BMP shuttling mechanism is highly conserved throughout the animal kingdom and relies on the spatial distribution of a BMP ligand (Dpp/ BMP2/4) and a BMP antagonist (Short gastrulation (Sog)/ Chordin). The BMP signaling domains are further refined by positive feedback and additional secreted BMP modulators, such as Crossveinless-2 (Cv-2), which binds to the cell surface and acts over a short range. However, the biochemical properties of pathway components can vary according to species- and stage-specific developmental requirements. For example, Tolloid (Tld)-like metalloproteases cleave vertebrate Chordins constitutively, while the Drosophila ortholog Sog is only cleaved efficiently when bound to BMPs. We have identified Sog characteristics responsible for making its cleavage dependent on BMP binding. Chordin-like variants that are processed independently of BMPs changed the steep BMP gradient found in Drosophila embryos to a shallower profile, analogous to that observed in some vertebrate embryos. This change affected cell fate allocation and tissue size and resulted in increased variability of patterning. Thus, the acquisition of BMP-dependent Sog processing during evolution facilitated long-range ligand diffusion and formation of the robust, bi-stable BMP morphogen gradients required for early Drosophila patterning. The BMP-1/Tolloid proteases are developmentally important enzymes regulated at multiple levels. Drosophila has two enzymes of this family, Tld and Tolloid-related (Tlr). Tlr is expressed in the muscles, central nervous system and in the ring gland, a secretory organ. Tlr circulates in the hemolymph and can degrade inhibitory pro-peptides and liberate active ligands from TGF-beta latent complexes. We found that Tlr activity is required on the neuronal surface for normal guidance of motor axons and development of synapses at the neuromuscular junction (NMJ). To assess the spatial requirements of Tlr we generated membrane tethered Tlr variants (Tlr-TM). We found that the Tlr-TM can rescue the axon defects of tlr mutants only when expressed on the motor neurons, but not when expressed on the target muscles. Furthermore, restriction of Tlr-TM on the muscles caused severe defects in axon guidance and synapse morphology. This indicates that Tlr, albeit endogenously expressed in the striated muscle, must be actively relocated and concentrated on the motor neurons surface for proper axon guidance and synapse development.
