Associate Professor, Department of Biology
Ph.D. Universita di Padova, Italy
B.Sc. Imperial College London, UK
Research Interests: Molecular mechanisms of synapse formation
Overview: Information is exchanged between neurons at synapses, which are essentially specialized sites of cell-cell adhesion . A mature synapse is defined as an accumulation of synaptic vesicles within the axon, in close apposition to a dendritic membrane studded with receptors (see figure)which are held in place by a submembranous scaffold (Sheng and Kim, 2002). The formation of such an intercellular structure requires spatially and temporally controlled changes in morphology and molecular content at sites of contacts. Recent advances in subcellular fluorescence microscopy have revealed that this process involves the rapid recruitment and stabilization of both pre- and postsynaptic elements. These studies have shown that major components of the synaptic vesicle and active zone machinery travel in clusters together with other presynaptic proteins, such as calcium channels, and are rapidly recruited to new sites of contact (Ahmari et al., 2000; Zhai et al., 2001; Washbourne et al., 2002) .
On the postsynaptic side, receptor subunits and components of the scaffold or post-synaptic density (PSD) are recruited separately and with distinct time courses within minutes to hours after initial contact (Friedman et al., 2000; Bresler et al., 2001; Washbourne et al., 2002; Bresler et al., 2004)
Despite these advances the basic mechanisms by which synapse formation is induced at discrete locations and by which the molecular machinery is recruited to sites of contact remain elusive. We are currently using both mammalian primary neuronal cultures and zebrafish embryos to investigate molecules that are involved in the mechanisms of synapse formation. Techniques currently employed are live confocal imaging of fluorescently-tagged synaptic components, electron microscopy, biochemistry and molecular biology.
Grxcr1 Promotes Hair Bundle Development by Destabilizing the Physical Interaction between Harmonin and Sans Usher Syndrome Proteins.
Cell Rep. 2018 Oct 30;25(5):1281-1291.e4
Authors: Blanco-Sánchez B, Clément A, Fierro J, Stednitz S, Phillips JB, Wegner J, Panlilio JM, Peirce JL, Washbourne P, Westerfield M
Morphogenesis and mechanoelectrical transduction of the hair cell mechanoreceptor depend on the correct assembly of Usher syndrome (USH) proteins into highly organized macromolecular complexes. Defects in these proteins lead to deafness and vestibular areflexia in USH patients. Mutations in a non-USH protein, glutaredoxin domain-containing cysteine-rich 1 (GRXCR1), cause non-syndromic sensorineural deafness. To understand the deglutathionylating enzyme function of GRXCR1 in deafness, we generated two grxcr1 zebrafish mutant alleles. We found that hair bundles are thinner in homozygous grxcr1 mutants, similar to the USH1 mutants ush1c (Harmonin) and ush1ga (Sans). In vitro assays showed that glutathionylation promotes the interaction between Ush1c and Ush1ga and that Grxcr1 regulates mechanoreceptor development by preventing physical interaction between these proteins without affecting the assembly of another USH1 protein complex, the Ush1c-Cadherin23-Myosin7aa tripartite complex. By elucidating the molecular mechanism through which Grxcr1 functions, we also identify a mechanism that dynamically regulates the formation of Usher protein complexes.
PMID: 30380418 [PubMed - in process]
4.1Ba is necessary for glutamatergic synapse formation in the sensorimotor circuit of developing zebrafish.
PLoS One. 2018;13(10):e0205255
Authors: Fierro J, Haynes DR, Washbourne P
During the process of synapse formation, thousands of proteins assemble at prospective sites of cell-cell communication. Although many of these proteins have been identified, the roles they play in generating functional connections during development remain unknown. 4.1 scaffolding proteins have been implicated in synapse formation and maturation in vitro, but in vivo studies for some family members have suggested these proteins are not important for this role. We examined the role of family member 4.1B because it has been implicated in glutamatergic synaptogenesis, but has not been described in vivo. We identified two 4.1B genes in zebrafish, 4.1Ba and 4.1Bb, by sequence comparisons and synteny analysis. In situ hybridization shows these genes are differentially expressed, with 4.1Ba expressed primarily in the nervous system and 4.1Bb expressed in the nervous system and muscle, but not the spinal cord. We focused our studies on 4.1Ba in the spinal cord. 4.1Ba knockdown reduced the number of glutamatergic synapses at caudal primary motor neurons and caused an increase in the duration of touch-evoked coiling. These results suggest 4.1Ba is important for the formation of functional glutamatergic synapses in the developing zebrafish spinal cord.
PMID: 30286167 [PubMed - in process]
Forebrain Control of Behaviorally Driven Social Orienting in Zebrafish.
Curr Biol. 2018 Jul 21;:
Authors: Stednitz SJ, McDermott EM, Ncube D, Tallafuss A, Eisen JS, Washbourne P
Deficits in social engagement are diagnostic of multiple neurodevelopmental disorders, including autism and schizophrenia . Genetically tractable animal models like zebrafish (Danio rerio) could provide valuable insight into developmental factors underlying these social impairments, but this approach is predicated on the ability to accurately and reliably quantify subtle behavioral changes. Similarly, characterizing local molecular and morphological phenotypes requires knowledge of the neuroanatomical correlates of social behavior. We leveraged behavioral and genetic tools in zebrafish to both refine our understanding of social behavior and identify brain regions important for driving it. We characterized visual social interactions between pairs of adult zebrafish and discovered that they perform a stereotyped orienting behavior that reflects social attention . Furthermore, in pairs of fish, the orienting behavior of one individual is the primary factor driving the same behavior in the other individual. We used manual and genetic lesions to investigate the forebrain contribution to this behavior and identified a population of neurons in the ventral telencephalon whose ablation suppresses social interactions, while sparing other locomotor and visual behaviors. These neurons are cholinergic and express the gene encoding the transcription factor Lhx8a, which is required for development of cholinergic neurons in the mouse forebrain . The neuronal population identified in zebrafish lies in a region homologous to mammalian forebrain regions implicated in social behavior such as the lateral septum . Our data suggest that an evolutionarily conserved population of neurons controls social orienting in zebrafish.
PMID: 30057306 [PubMed - as supplied by publisher]