Assistant Professor, Department of Biology
Ph.D. University of Oregon
Postdoc, Fred Hutchinson Cancer Research Center
Research Interests: Neural circuit wiring, synapse formation, and electrical synaptogenesis in zebrafish.
Overview: The human brain contains more connections between neurons than the Milky Way has stars! The brain is wired at a gross level into stereotyped neural circuits that underlie sensation, information processing, motor output, and ultimately, consciousness. Disrupted neural circuitry has been linked to many neurodevelopmental disorders, such as autism, epilepsy, and schizophrenia. How do the neurons of the brain connect and wire up into circuits? The goal of the research in the lab is to integrate genetics, biochemistry, cell biology, circuit function, and behavior, to understand how the brain creates functioning neural networks.
Neural circuits are defined by the connections made between neurons, and connections, termed synapses, come in two flavors: chemical, where transmission is mediated by neurotransmitters and receptors, and electrical, where neurons directly communicate with one another through gap junction channels. While the last decade has provided much insight into the developmental genetic mechanisms of building chemical synapses, electrical synapse formation is still not understood. However, it is known that electrical synapses are used by all animals both during development and in adulthood, and are found in sensory, central, and motor circuits. The goal of this project is to unlock the molecular mechanisms underlying electrical synaptogenesis.
Using zebrafish as a model system we have performed a forward genetic screen to identify mutations that cause defects in electrical synapse formation. Mapping mutations from forward genetic screens is challenging, particularly in large vertebrate genomes, but we have developed methods using on next generation sequencing which facilitate the identification of mutated genes (Genome Research). One of the mutations identified in the screen disrupted the autism-associated gene neurobeachin and we found that it was required for both electrical and chemical synapse formation, placing this gene as a critical lynchpin in all of synapse formation (Current Biology). We have also developed a novel CRISPR-based reverse genetic screening method to identify genes required for development – this was the first example that such an approach could be taken in a vertebrate (Nature Methods). The screen identified structural proteins that create the gap junction channel between the neurons and scaffolding that stabilize the synaptic structure. Ongoing work has revealed that electrical synapses can be asymmetric, with unique proteins on each side of the junction. This molecular asymmetry may underlie functional asymmetry and provide differential substrates for altering electrical synapse function.
Current projects focus on several diverse, but related, areas of electrical synaptogenesis:
1) Electrical synapse asymmetry – biochemistry, molecular biology, and genetics
How do the proteins of the synapse function at the molecular level to form the connection? What proteins interact and how do those interactions build the synapse? What other proteins are present at the synapse?
2) Electrical synapse formation – cell biology, development, and genetics
How are proteins trafficked to the synapse? How are they captured and stabilized once present? What are the cytoskeletal structures and motor proteins that facilitate movement? How long do proteins remain at the synapse and are they responsive to neuronal activity?
3) Electrical synapse function – behavior and physiology
Does the composition of the electrical synapse change based on circuit activity? Do molecular asymmetries produce effects on synapse function? How are molecular asymmetries integrated into circuit level function and behavioral output?
4) Electrical and chemical synapse interactions – physiology, development, and genetics
Are early-forming electrical synapses required for subsequent chemical synapse formation? What gap junction channels and scaffolds mediate early circuit activity? How are some early-forming electrical synapses removed as neural circuits mature? How are others retained?
Targeted candidate gene screens using CRISPR/Cas9 technology.
Methods Cell Biol. 2016;135:89-106
Authors: Shah AN, Moens CB, Miller AC
In the postgenomic era, the ability to quickly, efficiently, and inexpensively assign function to the zebrafish proteome is critical. Clustered regularly interspaced short palindromic repeats (CRISPRs) have revolutionized the ability to perform reverse genetics because of its simplicity and broad applicability. The CRISPR system is composed of an engineered, gene-specific single guide RNA (sgRNA) and a Cas9 enzyme that causes double-stranded breaks in DNA at the targeted site. This simple, two-part system, when injected into one-cell stage zebrafish embryos, efficiently mutates target loci at a frequency such that injected embryos phenocopy known mutant phenotypes. This property allows for CRISPR-based F0 screening in zebrafish, which provides a means to screen through a large number of candidate genes for their role in a phenotype of interest. While there are important considerations for any successful genetic screen, CRISPR screening has significant benefits over conventional methods and can be accomplished in any lab with modest molecular biology experience.
PMID: 27443921 [PubMed - indexed for MEDLINE]