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Biochemical Genetics of Signal Transduction
- Mihaela Serpe, PhD, Head, Unit on Cellular Communication
- Young-Jun Kim, PhD, Visiting Fellow
- Peter Nguyen, Biological Laboratory Technician
- Carolyn E. Peluso, PhD, Postdoctoral Fellow
We investigate molecular mechanisms that regulate cell-cell communication during development. During development of all multicellular organisms the identity, movements, and ultimate function of cells must be coordinated with those of their neighbors. These processes are largely guided by highly conserved signaling molecules, which provide positional and temporal information to the developing tissues. The TGF-beta superfamily of growth and differentiation factors, which include Activins and the bone morphogenetic proteins (BMPs), is one of the largest of these families. TGF-beta signaling factors have the ability to function as morphogens, that is, to specify cell fate in a concentration-dependent manner. Also, these signaling factors provide a mechanism for coupling a cell to its environment, so that the cell has the necessary plasticity to respond appropriately to changes in its environment or even to its own state. We use the Drosophila model system and a variety of molecular and biochemical approaches to study genes that modulate the function of TGF-beta–type signals. We are interested in how extracellular availability of the signaling factors is regulated in time and space and in the role of the cell surface in modulating local activation of TGF-beta-type signaling.
The role of enzyme-substrate interactions in formation of BMP morphogen gradients
The Drosophila embryo uses a gradient of Decapentaplegic (Dpp), a homolog of the vertebrate BMP-2/-4, to specify the dorsal structures. In the early embryo, dpp is transcribed uniformly throughout the dorsal domain, yet only about 10% of cells along the dorsal midline receive high levels of signal. The formation of the Dpp gradient occurs at a post-transcriptional level. Dpp is bound in a complex containing Short gastrulation (Sog), a BMP-binding protein secreted from the ventral lateral regions. 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 midline. A critical process 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 of Sog by Tld.
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. Without Sog there is no net movement of Dpp dorsally, the peak signaling domain does not form, amnioserosa—the tissue that requires peak BMP signaling—is not specified and the embryos fail to develop and die. Interestingly, Chordin, the vertebrate ortholog of Sog, can only act as an inhibitor when expressed in Drosophila and cannot promote long-range Dpp signaling. At the molecular level, the difference between Sog and Chordin is that processing of Sog by Tld requires the BMP ligand as an obligatory co-substrate whereas Chordin does not. To determine the source of this difference, we modeled the Tld catalytic domain using the crystal structure of the catalytic domain of human Tld. We purified and sequenced the Sog cleavage fragments and derived a consensus cleavage recognition sequence. We used this peptide to study the enzyme-substrate interactions in Sog and compared them with Chordin sequences. From this modeling, we hypothesized that several residues at the processing site might be responsible for making one substrate, but not the other, dependent on BMP binding for processing.
Our working hypothesis is that Sog’s ability to function in a transport process as a long-range BMP agonist resides, in molecular terms, in the BMP’s co-substrate requirements for Tld-mediated Sog degradation. This hypothesis is supported with computational modeling by David Umulis, our mathematician collaborator. Indeed, modeling suggests that the co-substrate requirement for Sog processing by Tld is critical for proper Dpp gradient formation. In computations that relax this constraint and allow for Sog degradation when not complexed with Dpp, Dpp flux towards the dorsal midline is greatly reduced. To test this working hypothesis, we generated Sog variants that are BMP-independent Tld substrates in vitro (Sog-i variants) and created transgenic lines that express various Sog under the endogenous sog enhancer. Transgenic lines expressing wild-type Sog rescued null sog mutants or trans-heterozygous combinations (sog−/−) to full viability and fertility. In contrast, lines expressing Sog-i variants rescued partially, and only when present in two or more copies.
Examination of Dpp gradient profile (visualized as activated/phosphorylated effector of the BMP signaling pathway P-Mad), and cell fate (amnioserosa cells) revealed the biological consequences of replacing Sog by Sog-i. Wild-type embryos have a narrow and sharp P-Mad–positive domain, which is wider and reduced in intensity (by 30%) in heterozygous (sog+/−) embryos. Due to the widening of their P-Mad domain, sog+/− heterozygous embryos have an average of 314 amnioserosa cells—about 50% more than the wild-type embryos (197 cells). When sog-wt transgenes replaced the endogenous sog, the profile of the BMP gradient as well as the cell fate were fully rescued, in a concentration-dependent manner: sog−/− bearing one sog-wt copy resembled sog+/− embryos, while sog−/− bearing two sog-wt copies had a wild-type P-Mad profile and amnioserosa field. However, when sog-i transgenic lines were similarly tested, addition of 2 sog-i copies to any sog−/− background produced wide, and irregularly shaped P-Mad domains and also increased amnioserosa fields (294 cells).
Taken together, our results indicate that mutations that render Sog co-substrate independent for processing by Tld alter the range of the Sog-i–Dpp complexes in vivo and thus the formation of the Dpp morphogen gradient. Like Chordin, Sog-i retained signal-blocking capabilities but had diminished abilities to facilitate the diffusion of BMP-type ligands. Also, similar to developmental systems using Chordin, but not Sog, we found that sog-i bearing embryos required the activity of Crossveinless-2 (Cv-2) for the early patterning. Thus, any positive contribution for Cv-2 to the Dpp gradient formation in the Drosophila early embryo appeared to have been previously masked by the Sog-mediated long-range ligand diffusion. As we interfered with the ability of Sog to facilitate ligand diffusion and replaced it with a Chordin-like form, we uncovered a highly conserved function for Cv-2 in vertebrates/Chordin using systems.
Characterization of mechanisms that spatially restrict Tolloid activities
Drosophila has two highly homologous enzymes of the BMP-1/Tld family: Tld and Tolloid-related (Tlr). In the embryo, secreted Tld acts cell-autonomously. Tlr is a circulatory enzyme that poses the risk of promiscuous proteolysis when inappropriately activated. We proposed that cell tethering and cell-surface catalysis constitutes an important level of regulation for Tld-type proteins, and we initiated a project to identify and characterize component(s) that act to localize Tld activities.
In mammals, one function of BMP-1/Tld enzymes is to process the inhibitory pro-peptides of certain TGF-beta–type ligands such as myostatin and GDF11, which activates the ligand in the circulatory system. In Drosophila, we found that Tlds can cleave and destabilize the pro-domains of several TGF-beta ligands including activin (Act), activin-like protein dawdle (Daw), and myoglianin (Myo). In the case of Daw, pro-peptide cleavage enhances the signaling activity of the ligand. Daw activation by Tlr is required for normal guidance of motor axons en route to their muscle targets. Null mutants of daw, as well as mutations in components of the canonical activin signaling pathway downstream of daw, all exhibit axon guidance defects that are similar to but less severe than null tlr mutants. The Daw signaling pathway is activated in motor neurons by ligand secreted from muscles and/or glial cells. An important question is how this pathway is locally activated by Tlr. To address this issue, we generated Tlr-transmembrane domain chimeras that remained tethered to the extracellular membranes in vitro and in vivo. We are using these reagents and the excellent genetic tools available in Drosophila to determine the spatial and temporal requirements for Tlr enzymatic activities at the tissue and single-cell levels. Future studies will focus on characterization of cell-specific component(s) and mechanisms that help localize Tlr activity.
Publication
- Serpe M, Umulis D, Ralston A, Chen J, Olson DJ, Avanesov A, Othmer H, O’Connor MB, Blair SS. The BMP-binding protein Crossveinless-2 is a short-range, concentration-dependent, biphasic modulator of BMP signaling in Drosophila. Dev Cell 2008 14:940-953.
Collaborators
- David Umulis, PhD, Purdue University, West Lafayette, IN
- Chi-Hon Lee, PhD, Program in Cellular Regulation and Metabolism, NICHD, Bethesda, MD
- Seth S. Blair, PhD, University of Wisconsin, Madison, WI
- Michael B. O’Connor, PhD, University of Minnesota, Minneapolis, MN
Contact
For more information, email serpemih@mail.nih.gov or visit ucc.nichd.nih.gov.