research

The broad interest of the lab is to understand how cell-cell signaling molecules act to establish spatial pattern in the nervous system, particularly in axon guidance. Approaches include identification of novel signaling molecules, and characterization of their functions by molecular, cellular, genetic and embryological methods.

Identification of novel cell-cell signaling molecules

An approach that has been productive for us is to identify novel ligands of orphan receptors, using soluble receptor techniques we have developed (1, 9, 10). Much of our current work, described further below, stems from identification of a new family of ligands, the ephrins (1, 6). Our methods using soluble receptor and ligand fusion proteins are now widely used to characterize ligand/receptor biology. We are continuing to pursue methods development in this area, and are using these techniques to identify other novel ligands and receptors that should open up new areas of cellular and developmental biology.

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Identifying new receptors and ligands. The Eph receptors were initially all identified as orphan receptors. Using soluble receptor methods developed in the lab, we identified several members of a corresponding ligand family, the ephrins, allowing elucidation of their functions.

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Soluble receptors or ligands as probes for cognate binding partners. Here, a soluble Eph receptor probe was used to identify the distribution of corresponding ligands in a mouse embryo (viewed laterally or dorsally in the two images)(1). This approach can be used to clone novel binding partners, or to obtain unique types of biological information.

Wiring up the nervous system: molecular cues for axon pathway selection and neural mapping

The functioning of the nervous system depends on the development of a precise and complex spatial order in its connections. First, projecting axons must select the correct pathways on the way to their target regions. Then, within the target, axons typically form topographic maps, where the spatial arrangement of the projecting neurons is maintained in the order of their connections. We are interested in understanding molecular cues for both pathway selection and neural map formation.

The existence of labels for neural mapping was initially proposed by Sperry in the 1940s, but their molecular identity remained elusive for several decades. Work by our lab and others has now identified ephrins as topographic mapping labels. We have studied ephrin function by a variety of approaches, including expression patterns, molecular binding analysis, in vitro guidance assays, in vivo gain of function using viral vectors, and loss of function by gene knockout (2-4, 6, 8, 12). This work has identified novel roles for ephrins in pathway selection, and as neural mapping labels throughout the nervous system, including higher maps in the cortex. This work has led to new qualitative and quantitative insights into the basic principles of neural map formation.

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Time lapse microscopy of growing axons in vitro. A cluster of 4 neurons toward the right of the picture send out axons, tipped by highly motile growth cones. One of them can be seen interacting in the center with a target cell transfected with a repellent molecule, which induces growth cone collapse and axon withdrawal (11).

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Time lapse microscopy of growing axons in vivo. The growth of commissural axons across the spinal cord midline is one of the best studied examples of axon pathway selection. Here, axons were labeled by electroporation with a green fluorescent protein marker. Two axons are seen by time-lapse microscopy extending within a spinal cord open book explant, from the top downwards toward the spinal cord midline (14).

How are extracellular guidance cues converted to an appropriate response? Molecular signaling mechanisms in the axon.

A major focus of the lab is to investigate molecular mechanisms that are used by axons to convert extracellular signals into an appropriate response. Some examples of lab projects are as follows.

How is high-affinity binding reconciled with axon repulsion?

Axon guidance cues can be either diffusible, or linked to cell surfaces. The use of cell surface cues allows high precision in guidance. However, it also creates a potential problem, especially in the case of axon repellents: since ligand and receptor bind to one another with high affinity, one would expect the interaction to be adhesive, so how is this reconciled with axon motility and efficient repulsion? We have found that ephrins are cleaved from the cell surface by a tightly regulated proteolysis mechanism that is triggered by ligand-receptor binding (11). These results provide a mechanism to facilitate contact mediated axon repulsion, and more generally can provide a means to reconcile cell contact mediated signaling with cell movement.

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Regulated cleavage of a contact mediated axon repellent. Ephrins form a complex with the protease Kuzbanian. Eph receptor binding triggers ligand cleavage in a localized reaction specific to the bound ligand, providing a mechanism to facilitate contact mediated axon repulsion (11).

Bi-directional signaling by cell surface receptors and ligands

Ephrins are cell surface ligands, providing the potential for both a 'forward' signal through the receptor and a 'reverse' signal through the ligand. We identified a cytoplasmic protein, PDZ-RGS3, which can mediate reverse signaling from the ephrin-B cytoplasmic tail (13). This protein has an RGS domain that can regulate heterotrimeric G protein signaling, and it can selectively regulate responsiveness to a G protein coupled chemoattractant. This study reveals a pathway that links reverse signaling to cellular guidance, uncovers a novel mechanism for extracellular signals to control heterotrimeric G protein pathways, and demonstrates a mechanism to selectively regulate responsiveness to neuronal guidance cues.

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Reverse signaling through ephrins, mediated by a novel transduction pathway. Reverse signaling through ephrins acts via a protein with PDZ and RGS domains, and can regulate a heterotrimeric G protein pathway. This mechanism allows selective regulation of responsiveness to neuronal guidance cues (13).

RNA-based regulatory mechanisms in axons

The idea that axons can locally sythesize proteins has been proposed for more than 40 years, but has been controversial. Experiments using RNA-based vectors, and reporters that can be localized subcellularly, provide direct evidence that cut axons are capable of translating proteins, and also exporting them to the surface membrane (14). Recent studies by us and other groups indicate that such mechanisms have several important roles in axon guidance. In one study, we found that specific motifs in the 3'UTR of an axonal receptor mRNA can specify upregulation of protein expression in axons as they reach an intermediate guidance target (14). Recent studies such as this open up a wide field of interesting questions about RNA-based regulation in axons that have barely begun to be addressed, including the spectrum of RNA motifs involved, upstream regulatory pathways, downstream target mRNAs and biological roles.

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RNA-based regulation in axons. The receptor EphA2 is upregulated on axons as they reach the spinal cord midline. Here, axons were electroporated with a green fluorescent protein (GFP) reporter, linked to a highly conserved sequence from the 3'UTR of EphA2 mRNA. This RNA sequence specifies midline upregulation. In the photograph, an axon is traced with an RFP reporter (red), with GFP expression (green, or yellow where red fluorescence overlaps) seen in the cell body (toward the left), and in the growth cone (toward the right) as it reaches the spinal cord midline (darker band within the blue nuclear counterstaining) (14). Local translation and export are likely to have important roles in several aspects of axon guidance.