Like mammalian neurons, C. elegans neurons lose axon regeneration ability as they age, but it is not known why. Here, we report that let-7 contributes to a developmental decline in AVM axon regeneration. In older AVM axons, let-7 inhibits regeneration by down regulating LIN-41, an important AVM axon regeneration-promoting factor. While let-7 inhibits lin-41 expression in older neurons through the lin-41 3'UTR, lin-41 inhibits let-7 expression in younger neurons through Argonaute ALG-1. This reciprocal inhibition ensures axon regeneration is only inhibited in older neurons. These findings show that a let-7-lin-41 regulatory circuit, which was previously shown to control timing of events in mitotic stem cell lineages, is re-utilized in postmitotic neurons to control post-differentiation events.

Probing the neural circuit dynamics underlying behavior would benefit greatly from improved genetically encoded voltage indicators. The proton pump Archaerhodopsin-3 (Arch), an optogenetic tool commonly used for neuronal inhibition, has been shown to emit voltage sensitive fluorescence. Here we report two Arch variants that in response to 655 nm light have 3–5 times increased fluorescence and 55–99 times reduced photocurrents compared to Arch WT. The most fluorescent variant, Archer1, has 25–40% fluorescence change in response to action potentials while using 9 times lower light intensity compared to other Arch-based voltage sensors. Archer1 is capable of wavelength specific functionality as a voltage sensor under red-light and as an inhibitory actuator under green-light. As a proof-of-concept for the application of Arch-based sensors in vivo, we show fluorescence voltage sensing in behaving C. elegans. Archer1’s characteristics contribute to the goal of all-optical detection and modulation of activity in neuronal networks in vivo.

CRISPR-Cas is an efficient method for genome editing in organisms from bacteria to human cells. We describe a transgenefree method for CRISPR-Cas-mediated cleavage in nematodes, enabling RNA-homology-targeted deletions that cause loss of gene function; analysis of whole-genome sequencing indicates that the nuclease activity is highly specific.

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