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A critical accomplishment in the rapidly developing field of regenerative medicine will be the ability to foster repair of neurons severed by injury, disease, or microsurgery. In C. elegans, individual visualized axons can be laser-cut in vivo and neuronal responses to damage can be monitored to decipher genetic requirements for regeneration. With an initial interest in how local environments manage cellular debris, we performed femtosecond laser axotomies in genetic backgrounds lacking cell death gene activities. Unexpectedly, we found that the CED-3 caspase, well known as the core apoptotic cell death executioner, acts in early responses to neuronal injury to promote rapid regeneration of dissociated axons. In ced-3 mutants, initial regenerative outgrowth dynamics are impaired and axon repair through reconnection of the two dissociated ends is delayed. The CED-3 activator, CED-4/Apaf-1, similarly promotes regeneration, but the upstream regulators of apoptosis CED-9/Bcl2 and BH3-domain proteins EGL-1 and CED-13 are not essential. Thus, a novel regulatory mechanism must be utilized to activate core apoptotic proteins for neuronal repair. Since calcium plays a conserved modulatory role in regeneration, we hypothesized calcium might play a critical regulatory role in the CED-3/CED-4 repair pathway. We used the calcium reporter cameleon to track in vivo calcium fluxes in the axotomized neuron. We show that when the endoplasmic reticulum calcium-storing chaperone calreticulin, CRT-1, is deleted, both calcium dynamics and initial regenerative outgrowth are impaired. Genetic data suggest that CED-3, CED-4, and CRT-1 act in the same pathway to promote early events in regeneration and that CED-3 might act downstream of CRT-1, but upstream of the conserved DLK-1 kinase implicated in regeneration across species. This study documents reconstructive roles for proteins known to orchestrate apoptotic death and links previously unconnected observations in the vertebrate literature to suggest a similar pathway may be conserved in higher organisms., Competing Interests: The authors have declared that no competing interests exist.

This article describes the fabrication of a microfluidic device for the liquid culture of many individual nematode worms (Caenorhabditis elegans) in separate chambers. Each chamber houses a single worm from the fourth larval stage until death, and enables examination of a population of individual worms for their entire adult lifespans. Adjacent to the chambers, the device includes microfluidic worm clamps, which enable periodic, temporary immobilization of each worm. The device made it possible to track changes in body size and locomotion in individual worms throughout their lifespans. This ability to perform longitudinal measurements within the device enabled the identification of age-related phenotypic changes that correlate with lifespan in C. elegans.

This paper describes the fabrication of a microfluidic device for rapid immobilization of large numbers of live C. elegans for performing morphological analysis, microsurgery, and fluorescence imaging in a high-throughput manner. The device consists of two principal elements: (i) an array of 128 wedge-shaped microchannels, or clamps, which physically immobilize worms, and (ii) a branching network of distribution channels, which deliver worms to the array. The flow of liquid through the device (driven by a constant pressure difference between the inlet and the outlet) automatically distributes individual worms into each clamp. It was possible to immobilize more than 100 worms in less than 15 min. The immobilization process was not damaging to the worms: following removal from the array of clamps, worms lived typical lifespans and reproduced normally. The ability to monitor large numbers of immobilized worms easily and in parallel will enable researchers to investigate physiology and behavior in large populations of C. elegans.