The establishment, maintenance, and repair of neuronal connections depend on coordinated neuronal responses to a variety of guidance cues that direct their projections to appropriate targets. My research is focused on understanding how connectivity in the nervous system is initially generated during neural development, how these connections are modified in the adult to provide plasticity at the structural level, and how these or compensatory connections can be re-established following neuronal injury or degeneration. Our goal is to define the cellular and molecular logic underlying the function of neuronal guidance cues during neural development and in the adult nervous system. We also wish to know whether similar mechanisms are used in the development of non-neuronal tissues. To address these issues we use genetic, molecular, cell biological, and biochemical approaches in both invertebrates and vertebrates to understand the underlying molecular basis of neuronal growth cone guidance.
What are the extrinsic and intrinsic factors that mediate repulsive guidance events? Several families of guidance cues and their receptors sculpt developing neural circuits. One of the largest is the semaphorin family of proteins, several members of which are robust neuronal repellents. Our previous work on neural development in the fruit fly Drosophila has allowed us to define the role of semaphorin-mediated repulsion in axonal pathfinding, to understand how the output of semaphorin repulsive receptors is modulated, and to elucidate the intracellular signaling cascades that steer growth cones in response to these guidance cues. Our work on rodent models of neural development and regeneration has helped us to define semaphorin receptors, to understand the logic by which a large and diverse family of guidance cues establishes highly specific neuronal connectivity patterns in a variety of neural systems, and to address the roles played by repellents in the regulation of dendritic morphology and synapse function.
For example, to understand how guidance cues coordinately regulate the assembly of neural connections, we have focused on the relatively simple developing Drosophila nervous system. We found that plexin B (PlexB), one of two Drosophila plexin receptors, plays a key role in both central and peripheral axon pathfinding, functions that are in part complementary to those of the other Drosophila plexin, PlexA. The secreted semaphorin Sema-2a, but not the related protein transmembrane Sema-1a, utilizes PlexB to regulate embryonic axon guidance events.
The two secreted semaphorins in Drosophila, Sema-2a and Sema-2b, define a code that promotes sensory afferent and central nervous system (CNS) interneuron connectivity (Figure 1). Sema-2a and Sema-2b both signal through the PlexB receptor in repulsive and attractive capacities, respectively. Sema-2b attraction directs chordotonal sensory neuron axons to their CNS interneuron targets and assembles these same CNS longitudinal tracts; loss of Sema-2b/PlexB signaling results in vibration response deficits. Therefore, Sema-2b organizes both pre- and postsynaptic components of this sensory circuit. These same sensory and CNS interneuron axons are repelled by Sema-2a, providing a foundation for future work on the molecular basis of plexin attraction and repulsion. Several of our projects are devoted to understanding the intracellular signaling events that transduce these attractive and repulsive semaphorin signaling events, and ongoing work is focused on cytosolic signaling molecules downstream of plexin receptor signaling in both flies and mice.
The organization of complex connections into intricate laminar structures occurs in many neural systems. We find in the mouse that repulsive guidance plays a critical role in organizing select retinal connectivity and in directing overall retinal lamination. A central question is how laminar-specific targeting of retinal neurons within the inner plexiform layer (IPL) arises during development. We have found that the transmembrane repulsive guidance cue Sema6A and its PlexA4 receptor organize retinal projections that normally target the outermost sublaminae of the IPL. Sema6A expression during retinal development defines a zone avoided by PlexA4-expressing amacrine cell neurites (Figure 2). This suggests that complex neuronal lamination develops through initial establishment of broad zones demarcated by guidance cues, here a repellent, that are subsequently refined as the retina develops.
We also find that the mouse transmembrane semaphorins Sema5A and Sema5B also play important roles in regulating retinal laminar organization. These repellents constrain inner retinal neuronal processes to the IPL, separating them from the outer plexiform layer and thereby regulating overall retinal connectivity. These observations and the work of others suggest repulsive and attractive cues act in concert during retinal development to localize circuits to discrete sublaminae. We are interested in determining whether similar mechanisms direct laminar organization elsewhere in the CNS.
It has become clear that classical neuronal guidance cues also can regulate distinct features of dendritic morphology and synapse structure. We have found that this is the case for secreted semaphorins in the mouse, which regulate dendrite growth and the development of excitatory synapses in vivo. In collaboration with David Ginty (HHMI, Johns Hopkins University), we have investigated the roles played by secreted semaphorin guidance cues in CNS synapse formation and neuronal morphology in vivo. We have analyzed mice harboring targeted mutations in Sema3A, Sema3F, and their respective receptors, and we observe dramatic and selective defects in spine morphology and number in both the hippocampus and neocortex. For example, in layer V somatosensory cortical pyramidal neurons we observe pronounced disruptions in spine distribution, increases in spine number along layer V cortical pyramidal neuron apical dendrites, and enlarged dendritic spines (Figure 3).
We also find, in collaboration with Richard Huganir (HHMI, Johns Hopkins University), that Sema3F signaling controls excitatory synapse number and also synaptic transmission in the dentate gyrus and cortex. Neuropilin-2 (Npn-2) receptors are exclusively localized to apical dendrites of cortical neurons with pyramidal morphology in vitro, suggesting that guidance cue receptor subcellular localization to the primary apical dendrite restricts Sema3F signaling to that dendritic compartment. A different secreted semaphorin, Sema3A, affects only basal dendritic arbors of these same neurons, and does so in a positive fashion. Therefore, selective distribution of cell surface receptors within distinct dendritic domains likely underlies distinct in vivo functions of these structurally related extrinsic cues. Genome-wide association studies link certain semaphorin guidance cues and their receptors to autism disorders, further motivating our investigation into guidance cue signaling cascades that define novel functions of these ligands in the regulation of dendritic morphology and synapse structure.
This research is also supported by grants from the National Institutes of Health.
As of May 30, 2012