Alvaro Sagasti

email:  sagasti@mcdb.ucla.edu
phone:  206-6147
office:  450B BSRB
homepage:  http://www.mcdb.ucla.edu/Research/Sagasti/index.html

Research Interests

Studies in my lab address two fundamental aspects of nervous system wiring: how axon arbors choose their innervation territories, and how structural plasticity of axons is regulated. We have developed a zebrafish model, and a unique suite of tools and techniques, to study arborization of peripheral sensory neurons in the skin, allowing us to address these questions with unprecedented resolution. Part of my lab studies the morphogenesis of sensory axon arbors during development, and part focuses on the re-establishment of axon arbor territories after injury. 1. Development Molecules and pathways that mediate in axon-axon interactions (Fang Wang) In previous work I showed that branches of an arborizing sensory axon repel one another (isoneuronal repulsion), promoting axon arbor expansion. Similarly, neighboring sensory axons repel one another to partition their territories (heteroneuronal repulsion), in a process known as “tiling�. Together, these interactions promote comprehensive but non-overlapping innervation of the skin. To identify repellent molecules involved in these processes, we screened 81 genes by whole-mount in situ hybridization for expression in somatosensory neurons during arborization stages. Six genes emerged as candidates for mediating isoneuronal and heteroneuronal repulsion, including three genes related to the LAR receptor tyrosine phosphatase, which is involved in axonal repulsion in leech, and one gene related to DSCAM, which is involved in isoneuronal repulsion in Drosophila and mouse. We are currently knocking down the activity of each of these genes with morpholino antisense oligonucleotides. Preliminary studies suggest that the DSCAM-like gene may play roles in both isoneuronal and heteroneuronal interactions. Knock-down of one LAR homolog exhibits an unexpected skin blistering phenotype, which we are currently characterizing in more detail. Knock-down and phenotypic analysis of the other two LAR homologs is underway. To detect defects in axonal repulsion, we created a transgenic line expressing a photoconvertible protein (KikGR) in somatosensory neurons. By photoconverting all of the cells in one of the two bilaterally symmetric trigeminal ganglia, we can easily visualize tiling interactions at the dorsal midline of the head. We are currently using this assay in a small molecule screen to identify new pathways involved in axonal repulsion. Identifying trigeminal sensory neuron subsets (Ana Marie Palanca) We have made two observations indicating that the zebrafish somatosensory system is made up of at least two distinct subclasses of neurons. First, time-lapse movies reveal that although all sensory arbor territories are limited by repulsive interactions, each arbor engages in repulsive interactions only with a distinct subset of neighboring arbors, but is impervious to others. Second, we have discovered that each somatosensory cell extends a central axon into the brain that terminates in one of two distinct regions of the CNS, the spinal cord or the hindbrain. We hypothesize that functionally distinct sensory neuron subtypes mediate different behaviors by activating distinct downstream targets, and that their peripheral arbors tile independently of one another. To test these hypotheses, we have created ten transgenic reporter lines that may be expressed in distinct sensory subtypes, using putative promoters of Trk receptors and TRP channels. At least one of these transgenes (TrkA-GFP) appears to highlight a set of neurons whose central projections terminate exclusively within the spinal cord. We are currently making a stable transgenic line to test whether the peripheral arbors of TrkA-GFP sensory neurons tile independently of other sensory axons. In parallel, we are conducting a morphometric analysis, in collaboration with Matteo Pelligrini, to determine whether subclasses of sensory neurons possess distinct axon branching morphologies. We are also collaborating with a neuronal physiologist (Petronella Ketunnen) to assess whether different sensory neuron subtypes mediate distinct behaviors. 2. Regeneration Molecular pathways that limit axon plasticity (Georgeann O’Brien) We have optimized a technique to sever precisely peripheral axons with a laser at defined locations in live animals. Following axotomy, we image recovering axons with time-lapse confocal imaging. We have used these techniques to characterize the behavior of severed axon arbors and their intact neighboring arbors after peripheral injury at several developmental stages. At early stages, regenerating and uninjured axons both grow into the denervated region, where they compete with one another for territory. At later stages, the ability of uninjured arbors to grow into denervated territory is dramatically diminished. Although severed axons do reinitiate growth at these later stages, they specifically avoid innervating their former territories. These observations suggest that defined critical periods control the ability of both severed and intact axons to innervate denervated territory. In the CNS, inhibitors of regeneration prevent axon growth by activating the Nogo Receptor pathway and the small GTPase RhoA. By monitoring regeneration of axons expressing dominant negative transgenes, we have shown that several members of this pathway (Nogo Receptor, Lingo-1, RhoA, and CRMP2) are also required for the inhibition of trigeminal peripheral axon reinnervation in the skin; expressing these transgenes in trigeminal neurons allowed their peripheral axons to reinnervate former territory. By pharmacologically inhibiting another protein in the pathway, Rho kinase, we demonstrated that this pathway is required for inhibition during a specific time window, and that it does not limit the sprouting of intact axons. Regulation of sensory axon plasticity in the skin thus shares molecular similarities with the regulation of axon regeneration in the CNS. We are currently using our axotomy and imaging techniques to assess whether other pathways that influence regeneration in the CNS also influence sensory reinnervation of the skin. Specifically, we are using a combination of pharmacological, genetic, and embryological approaches to examine the roles of chondroitin sulfate proteoglycans (CSPGs), cyclic AMP signaling, and prostaglandin signaling in regenerative sensory axon arborization. Mechanism and function of axonal degeneration (Seanna Martin) Following laser axotomy of a peripheral axon branch, the distal portion of that branch degenerates rapidly, in a process known as “Wallerian degeneration�. Our live time-lapse techniques have allowed us to perform a detailed kinetic analysis of degeneration in a single severed axon. We are assessing the physiological role of Wallerian degeneration by manipulating pathways that affect the rate of degeneration. By expressing WLDS in sensory neurons, a protein that leads to overproduction of NAD and slows Wallerian degeneration in mice, we have delayed axon degeneration in zebrafish trigeminal neurons by more than ten-fold. We have found that delaying degeneration of a severed axon interferes with regeneration of the axon, and have identified a developmental role for Wallerian degeneration in eliminating axon fragments created by naturally-occurring local pruning. We are currently testing whether manipulating another pathways that regulates Wallerian degeneration (Ubiquitin/proteosome degradation) also affects trigeminal regeneration and local pruning. In parallel, we are attempting to identify that phagocytic cell types that mediate Wallerian degeneration to dissect the function of phagocytosis in the degeneration process. In other systems phagocytic blood cells and glial cells are the major cell types that clear axonal debris, but with genetic methods we have shown that neither cell type plays a role in the degeneration of sensory axons in the zebrafish skin. We are currently testing the hypothesis that epidermal cells are responsible for axon phagocytosis. In collaboration with Miguel Allende’s lab at the University of Chile, we will examine axonal degeneration in another peripheral sensory organ, the posterior lateral line (pLL). By comparing degeneration in trigeminal and pLL neurons we will test the hypothesis that axonal degeneration and phagocytosis can be mechanistically different in different cell types. Interdependence of sensory innervation and fin regeneration (Sandra Rieger) Studies of limb regeneration in amphibians have identified a critical role for sensory innervation in the regeneration process. We have found that genetically ablating sensory neurons inhibits fin regeneration in larval zebrafish. We are currently testing whether continuous axonal innervation is required for this effect. We are also using a panel of markers to identify which step in the fin regeneration process requires sensory innervation. In a complementary set of experiments, we have found that damage to fin tissue is required for sensory reinnervation after peripheral axotomy. We are currently testing whether an autonomous or diffusible factor is responsible for the affect of fin damage on axon innervation, and are examining the possibility that inflammation plays a role in this phenomenon.

Selected Publications

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