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 - indexed for MEDLINE]
Forebrain Control of Behaviorally Driven Social Orienting in Zebrafish.
Curr Biol. 2018 Aug 06;28(15):2445-2451.e3
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 - in process]
Correction: A MultiSite Gateway Toolkit for Rapid Cloning of Vertebrate Expression Constructs with Diverse Research Applications.
PLoS One. 2017;12(4):e0176543
Authors: Fowler DK, Stewart S, Seredick S, Eisen JS, Stankunas K, Washbourne P
[This corrects the article DOI: 10.1371/journal.pone.0159277.].
PMID: 28426753 [PubMed]
Redundant Postsynaptic Functions of SynCAMs 1-3 during Synapse Formation.
Front Mol Neurosci. 2017;10:24
Authors: Fowler DK, Peters JH, Williams C, Washbourne P
Investigating the roles of synaptogenic adhesion molecules during synapse formation has proven challenging, often due to compensatory functions between additional family members. The synaptic cell adhesion molecules 1-3 (SynCAM1-3) are expressed both pre- and postsynaptically, share highly homologous domains and are synaptogenic when ectopically presented to neurons; yet their endogenous functions during synaptogenesis are unclear. Here we report that SynCAM1-3 are functionally redundant and collectively necessary for synapse formation in cultured hippocampal neurons. Only triple knockdown (KD) of SynCAM1-3 using highly efficient, chained artificial microRNAs (amiRNAs) reduced synapse density and increased synapse area. Electrophysiological recordings of quantal release events supported an increase in synapse size caused by SynCAM1-3 depletion. Furthermore, a combinatorial, mosaic lentiviral approach comparing wild type (WT) and SynCAM1-3 KD neurons in the same culture demonstrate that SynCAM1-3 set synapse number and size through postsynaptic mechanisms. The results demonstrate that the redundancy between SynCAM1-3 has concealed their synaptogenic function at the postsynaptic terminal.
PMID: 28197078 [PubMed]
A MultiSite Gateway Toolkit for Rapid Cloning of Vertebrate Expression Constructs with Diverse Research Applications.
PLoS One. 2016;11(8):e0159277
Authors: Fowler DK, Stewart S, Seredick S, Eisen JS, Stankunas K, Washbourne P
Recombination-based cloning is a quick and efficient way to generate expression vectors. Recent advancements have provided powerful recombinant DNA methods for molecular manipulations. Here, we describe a novel collection of three-fragment MultiSite Gateway cloning system-compatible vectors providing expanded molecular tools for vertebrate research. The components of this toolkit encompass a broad range of uses such as fluorescent imaging, dual gene expression, RNA interference, tandem affinity purification, chemically-inducible dimerization and lentiviral production. We demonstrate examples highlighting the utility of this toolkit for producing multi-component vertebrate expression vectors with diverse primary research applications. The vectors presented here are compatible with other Gateway toolkits and collections, facilitating the rapid generation of a broad range of innovative DNA constructs for biological research.
PMID: 27500400 [PubMed - indexed for MEDLINE]
Transcriptomes of post-mitotic neurons identify the usage of alternative pathways during adult and embryonic neuronal differentiation.
BMC Genomics. 2015 Dec 23;16:1100
Authors: Tallafuss A, Kelly M, Gay L, Gibson D, Batzel P, Karfilis KV, Eisen J, Stankunas K, Postlethwait JH, Washbourne P
BACKGROUND: Understanding the mechanisms by which neurons are generated and specified, and how they integrate into functional circuits is key to being able to treat disorders of the nervous system and acute brain trauma. Much of what we know about neuronal differentiation has been studied in developing embryos, but differentiation steps may be very different during adult neurogenesis. For this reason, we compared the transcriptomes of newly differentiated neurons in zebrafish embryos and adults.
RESULTS: Using a 4tU RNA labeling method, we isolated and sequenced mRNA specifically from cells of one day old embryos and adults expressing the transgene HA-uprt-mcherry under control of the neuronal marker elavl3. By categorizing transcript products into different protein classes, we identified similarities and differences of gene usage between adult and embryonic neuronal differentiation. We found that neurons in the adult brain and in the nervous system of one day old embryos commonly use transcription factors - some of them identical - during the differentiation process. When we directly compared adult differentiating neurons to embryonic differentiating neurons, however, we found that during adult neuronal differentiation, the expression of neuropeptides and neurotransmitter pathway genes is more common, whereas classical developmental signaling through secreted molecules like Hedgehog or Wnt are less enriched, as compared to embryonic stages.
CONCLUSIONS: We conclude that both adult and embryonic differentiating neurons show enriched use of transcription factors compared to surrounding cells. However, adult and embryonic developing neurons use alternative pathways to differentiate. Our study provides evidence that adult neuronal differentiation is distinct from the better characterized embryonic neuronal differentiation process. This important insight and the lists of enriched genes we have identified will now help pave the way to a better understanding of the mechanisms of embryonic and adult neuronal differentiation and how to manipulate these processes.
PMID: 26699284 [PubMed - indexed for MEDLINE]
Improved knockdown from artificial microRNAs in an enhanced miR-155 backbone: a designer's guide to potent multi-target RNAi.
Nucleic Acids Res. 2016 Mar 18;44(5):e48
Authors: Fowler DK, Williams C, Gerritsen AT, Washbourne P
Artificial microRNA (amiRNA) sequences embedded in natural microRNA (miRNA) backbones have proven to be useful tools for RNA interference (RNAi). amiRNAs have reduced off-target and toxic effects compared to other RNAi-based methods such as short-hairpin RNAs (shRNA). amiRNAs are often less effective for knockdown, however, compared to their shRNA counterparts. We screened a large empirically-designed amiRNA set in the synthetic inhibitory BIC/miR-155 RNA (SIBR) scaffold and show common structural and sequence-specific features associated with effective amiRNAs. We then introduced exogenous motifs into the basal stem region which increase amiRNA biogenesis and knockdown potency. We call this modified backbone the enhanced SIBR (eSIBR) scaffold. Using chained amiRNAs for multi-gene knockdown, we show that concatenation of miRNAs targeting different genes is itself sufficient for increased knockdown efficacy. Further, we show that eSIBR outperforms wild-type SIBR (wtSIBR) when amiRNAs are chained. Finally, we use a lentiviral expression system in cultured neurons, where we again find that eSIBR amiRNAs are more potent for multi-target knockdown of endogenous genes. eSIBR will be a valuable tool for RNAi approaches, especially for studies where knockdown of multiple targets is desired.
PMID: 26582923 [PubMed - indexed for MEDLINE]