Cortex IUE

Research Interests

   Understanding the molecular mechanisms that govern nervous system patterning is a central focus of developmental neurobiology and will result in the identification of biological mechanisms relevant to many disease processes. The elaborate and precise patterns of neuronal connections in the mammalian central nervous system (CNS) established during development depend critically upon a vast number of guidance cues. During development, neurons form connections with their appropriate targets by extending processes called axons. However, the mechanisms by which these axons select their correct pathways, find their targets, and form proper connections (synapses) are not clearly understood. Over the past decade, studies in both invertebrates and vertebrates have identified evolutionarily conserved molecules that guide the leading tip of these axons (the growth cone) by diffusible and non-diffusible actions. Molecules that attract growth cones were first, and thus best, characterized include a variety of receptors and ligand adhesion molecules as well as long-range attractants. Equally important are proteins that push away or repel the growth cone, which also play a crucial role in axonal pathfinding and target recognition. Hence, research in our lab centers on characterizing and understanding the different cellular relationships between guidance molecules and their receptors in axonal guidance, target innervation and synapse formation.


   Semaphorins were among the first repulsive guidance cues identified and are one of the largest phylogenetically conserved families of guidance molecules. They are characterized by a well-conserved extracellular ~500 amino acid semaphorin domain that comprises the majority of both secreted and transmembrane semaphorins that bind with high affinity to at least two distinct receptor families, the neuropilins and plexins. Both in vitro and in vivo approaches in vertebrates and invertebrates have demonstrated a repulsive role for semaphorin in aspects of axonal guidance and target recognition. Examples of semaphorin guidance functions in the mammalian nervous systems include, motor neuron pathfinding in the embryonic spinal cord, development of dopaminergic axons and thalamocortical axons in the CNS. Recently, we showed that semaphorins play a key role in negatively regulating the development of tiny specialized structures on dendrites (spines) and excitatory synaptic morphology in the hippocampus and neocortex. Although several downsteam signaling cascades have been identified for semaphorin-mediated axon guidance, little is known about the signaling events for semaphorin-mediated dendritic development, spine morphology and synapse formation. Although, best known for their roles as chemorepellents, many semaphorins and their receptors have diverse functions unrelated to axonal guidance including cardiovascular development, organogenesis, cell migration, neuronal apoptosis, neoplastic transformation, and immune system function. In addition, semaphorins have been implicated in the pathological process of several neurological diseases, such as Autism Spectrum Disorders, Alzheimer's Disease and motor neuron degeneration; and they may play important roles in adult neuronal regeneration.

   Despite these advances, the underlying mechanisms by which semaphorins and their receptors signal to manipulate neuronal connectivity are still poorly understood. Although semaphorins have been localized to subsets of neurons in the vertebrate CNS, their function in specifying distinct axonal pathways, target selection, and synaptogenesis for these subsets of neurons is unclear. To date, two neurophilin and nine plexins genes have been identified in the mammalian genome. Interestingly, the class 3 secreted semaphorins utilize both neuropilins and plexins as ligand binding subunits and signal transducing subunits of holoreceptor complexes, respectively. Downstream intracellular signaling components have been identified and are required for membrane-associated and secreted semaphorins repulsion mediated through binding with their specific receptor complexes. Therefore, a comprehensive survey of the range of specific ligand-receptor interactions between semaphorins and their receptors is crucial to the understanding of how these molecules differentially guide diverse neuronal populations to their appropriate targets and mediate synapse formation.

Synapse Development

   The wiring of neuronal circuits in the mammalian CNS requires the precise formation and subsequent refinement of synapses during postnatal development. The vast majority of excitatory synapses in the mammalian CNS are formed on dendritic spines, tiny protrusions extended from the dendritic membrane. The morphology and distribution of spines are critical determinants of correct circuit function. Although a number of molecular cues are known to promote spine and synapse number, little is known about the molecules that negatively regulate these events. Furthermore, the identity of molecular cues that control spine distribution along dendrites and synaptic structure remain to be defined.

   Accordingly, we are asking the following questions: 1) what are the molecules controlling neural circuit formation? 2) how are these connections maintained throughout life? 3) what are the underlying cellular mechanisms controlling axonal guidance versus synapse formation? To address these questions, we will employ cellular, molecular, genetic and live imaging approaches to analyze both the central and peripheral arms of the mouse nervous system. Our primary experimental approach is to use dissociated, cultured neurons and organotypic slices from the developing neocortex as a model system. The formation of individual synapses in these cultures is studied using the following approaches. First, we use histological and immunocytochemical approaches to identify key molecules involved in the establishment of synaptic contacts in developing cortical neurons. Second, the sequence of molecular events that occur during synapse formation and maturation is investigated by live imaging changes in distribution of pre- and postsynaptic proteins fused to GFP using spinning disk confocal microscopy. Finally, the signals that may guide synapse formation (such as neurotrophins, cell adhesion molecules, or semaphorins) are studied by manipulating them at forming synapses with pharmacological agents, transgenic technology and/or transfection techniques. Taken together, results from our work should provide a platform to study complex neural network formation and further our understanding of the establishment of neuronal circuitry, and how defects in these connections may lead to development of neurological disorders.


**Top left figure: In utero electroporated mouse cortex with EGFP cDNA at E13.5 and processed for GFP immunofluorescence and anti-Ctip2 on P45, revealing labeled layer 5 pyramidal neurons.


In utero electroporated hippocampal dentate gyrus granule cells expressing EGFP.


Merged, anti-GFP, Npn2, PSD95


Dissociated cortical pyramidal neuron labeled with anti-GFP (green), anti-Npn2 (blue), and anti-PSD95 (red).



Fluorescence in situ hybridization revealing a class 3 semaphorin (red) and TrkA (green) mRNA expression in mouse E15 DRG.

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