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A recurrent theme in biology is that cells communicate with each other using a handful of conserved families of signaling molecules. The TGF-β superfamily of growth and differentiation factors, including TGF-βs/ Activins and the bone morphogenetic proteins (BMPs), is one of the largest of these families.
TGF-β signaling factors have the ability to function as morphogens, that is to specify cell fate in a concentration dependent manner. In the early Drosophila embryo, decapentaplegic (dpp), a BMP-type ligand, is transcribed uniformly throughout the dorsal domain, yet only about 20 cells along the dorsal midline receive high levels of signal. In the pupal wing, Dpp diffuses from the longitudinal veins into the posterior crossvein competent zone and creates a corridor of peak signaling that is perpendicular to the source of morphogen. In both instances, the formation of the Dpp gradient occurs at a post-transcriptional level and involves modulation by additional secreted gene products.
In the early embryo, Dpp is bound in a complex
containing Short gastrulation (Sog). This complex inhibits binding of
Dpp to its receptors in lateral regions and facilitates long-range
ligand diffusion, shuttling Dpp from the lateral domain towards the
Download the dpp movie.
Figure 1. The Dpp transcription domain exists along the top hemisphere of the egg known as the dorsal ectoderm
Click image to enlarge, opens in new window. A key component that helps create flux is the processing of Sog by Tolloid (Tld), a metalloprotease of the BMP-1 family. The net movement of Dpp dorsally is generated by reiterated cycles of complex formation, diffusion and destruction by Tld. Similarly Sog is required for the long range Dpp signaling during the specification of the Drosophila posterior crossvein (PCV). In this case, dpp is expressed in the adjacent longitudinal veins (L4 and L5) and moves into the PCV region.
Our work and others showed that these two processes share a related set of extracellular factors and a similar strategy to achieve peak level of BMP signaling in a spatially restricted domain. As in the embryo, signaling in the PCV requires a Tolloid family protease, in this case Tolloid-related (Tlr), a Tsg family member, formation of BMP heterodimers, and, finally, positive feedback to sharpen the peak signaling domains (Figure 1).
Tld and Tlr are highly homologous enzymes expressed in similar patterns in the early embryos. They both process Sog, yet they cannot substitute for each other during development. We have shown that their developmental specificity is regulated by enzyme kinetics. Tlr processes Sog at least an order of magnitude slower that does Tld. The fast embryonic development precludes the use of the slow Tlr; in contrast, formation of the posterior crossvein is a slow process. The Tlr catalytic properties fulfill the temporal constrains of the pupal wing development while the more rapid kinetics of Tld are matched to the rapid development of the embryo.
In both processes, Sog plays both positive and negative roles in regulating BMP activity, a phenomenon previously referred to as the “Sog paradox”. The negative role comes from blocking access of ligands to receptors. The positive effect comes from its ability to facilitate Dpp diffusion. By studying the enzyme-substrate interactions we have begun to understand the molecular basis for Sog’s function in a facilitated diffusion process. We are currently testing the hypothesis that Sog’s ability to mediate a BMPs transport process resides, in molecular terms, in the BMPs co-substrate requirements for Tolloid-mediated Sog degradation.
The shaping of BMP gradients in the early embryo and in the pupal wing share many similar features but differ in their spatial constrains and in their requirement for another BMP binding molecule, Crossveinless-2 (Cv-2). Loss of Cv-2 causes loss of BMP signaling in the developing crossveins, while high levels of Cv-2 sequester ligands. Like Sog, Cv-2 exhibits both BMP agonistic and antagonist activities. Unlike Sog, Cv-2 does not mediate their long-range transport. Cv-2 binds to the cell surface and acts locally within the crossvein itself. Cv-2 forms a complex with the BMP signaling molecule and the type I receptor. Through this complex, Cv-2 can antagonize BMP ligands with high affinity for the receptor, whilst it facilitates the recruitment of low affinity ligands in close proximity to the receptors, potentiating their signaling. Consequently, Cv-2 can distinguish between various BMP ligands and differentially modulate their signaling. In addition, Cv-2 is a transcriptional target of BMP signaling and its activity helps refine an otherwise shallow gradient of BMP in the developing wing.
Besides crossvein specification, a non-essential function for fly viability, both Tlr and Cv-2 play additional roles during nervous system development. We are interested in understanding the role of Cv-2 and Tlr in the nervous system development and functioning.
Figure 2. The guidance defects of tlr mutants start in late embryogenesis and persist until later stages
We have already discovered that lack of Tlr enzymatic activity during embryogenesis leads to axon guidance defects that start in late embryos (Figure 2, upper panels) and persist until larval stages (lower panels). Tlr is expressed in the muscles, central nervous system and ring gland. In addition, a significant amount of the enzyme is found circulating in the hemolymph. In mammals one function of BMP-1/Tld enzymes is to process the inhibitory pro-peptides of certain TGF-β-type ligands therefore activating the ligands in the circulatory system. We found that the Drosophila enzymes could similarly process inhibitory pro-peptides, releasing the ligand from latent complexes. Starting in late embryogenesis, Tlr is required, at least in part, to activate an activin-like ligand, Dawdle, and the downstream canonical activin signaling pathway. Our work and that from other laboratories suggest that this pathway is activated in motor neurons by ligand secreted from muscles and/or glia cells. An important question is to determine if and how this pathway is locally activated by Tlr, a circulatory enzyme. The role of cell surface catalysis in the activation of TGF-β during wound healing suggests one possible mechanism. Tethering the Tlr enzymatic activity by interaction of Tlr with a cell surface component located only on motor neurons could provide the required specificity. We propose that cell tethering and cell surface catalysis constitutes an important level of regulation for Tld-type proteins. We are currently in the process of identifying and characterizing component(s) that act to localize Tld and Tlr activities in the Drosophila embryo.