Using CRISPR/Cas9 to identify genes required for mechanosensory neuron development and function.

Tunicates are marine, non-vertebrate chordates that comprise the sister group to the vertebrates. Most tunicates have a biphasic lifecycle that alternates between a swimming larva and a sessile adult. Recent advances have shed light on the neural basis for the tunicate larva's ability to sense a proper substrate for settlement and initiate metamorphosis. Work in the highly tractable laboratory model tunicate Ciona robusta suggests that sensory neurons embedded in the anterior papillae transduce mechanosensory stimuli to trigger larval tail retraction and initiate the process of metamorphosis. Here, we take advantage of the low-cost and simplicity of Ciona by using tissue-specific CRISPR/Cas9-mediated mutagenesis to screen for genes potentially involved in mechanosensation and metamorphosis, in the context of an undergraduate 'capstone' research course. This small screen revealed at least one gene, Vamp1/2/3, which appears crucial for the ability of the papillae to trigger metamorphosis. We also provide step-by-step protocols and tutorials associated with this course, in the hope that it might be replicated in similar CRISPR-based laboratory courses wherever Ciona are available.

Using CRISPR/Cas9 to identify genes required for mechanosensory neuron development and function.

Tunicates are marine, non-vertebrate chordates that comprise the sister group to the vertebrates. Most tunicates have a biphasic lifecycle that alternates between a swimming larva and a sessile adult. Recent advances have shed light on the neural basis for the tunicate larva's ability to sense a proper substrate for settlement and initiate metamorphosis. Work in the highly tractable laboratory model tunicate Ciona robusta suggests that sensory neurons embedded in the anterior papillae of transduce mechanosensory stimuli to trigger larval tail retraction and initiate the process of metamorphosis. Here, we take advantage of the low-cost and simplicity of Ciona by using tissue-specific CRISPR/Cas9-mediated mutagenesis to screen for genes potentially involved in mechanosensation and metamorphosis, in the context of an undergraduate "capstone" research course. This small screen revealed at least one gene, Vamp1/2/3 , that appears crucial for the ability of the papillae to trigger metamorphosis. We also provide step-by-step protocols and tutorials associated with this course, in the hope that it might be replicated in similar CRISPR-based laboratory courses wherever Ciona are available.

Heparan sulfate proteoglycans mediate prion-like α-synuclein toxicity in Parkinson's in vivo models.

Parkinson's disease (PD) is a debilitating neurodegenerative disorder characterized by progressive motor decline and the aggregation of α-synuclein protein. Growing evidence suggests that α-synuclein aggregates may spread from neurons of the digestive tract to the central nervous system in a prion-like manner, yet the mechanisms of α-synuclein transmission and neurotoxicity remain poorly understood. Animal models that are amenable to high-throughput investigations are needed to facilitate the discovery of disease mechanisms. Here we describe the first Caenorhabditis elegans models in which feeding with α-synuclein preformed fibrils (PFFs) induces dopaminergic neurodegeneration, prion-like seeding of aggregation of human α-synuclein expressed in the host, and an associated motor decline. RNAi-mediated knockdown of the C. elegans syndecan sdn-1, or other enzymes involved in heparan sulfate proteoglycan synthesis, protected against PFF-induced α-synuclein aggregation, motor dysfunction, and dopamine neuron degeneration. This work offers new models by which to investigate gut-derived α-synuclein spreading and propagation of disease.