Kreeger, L., Arur, S., ZHAO, P., Ben-Yakar, A., Trimmer, K., Messing, R., Ma, K., Martin, C., Zemelman, B., Jiang, N., Maiya, R.
[
International Worm Meeting,
2019]
C. elegans has become a versatile system for studying in vivo nerve regeneration since the advent of precise laser axotomy method for severing specific axons. Through mutant and RNAi screening, a number of regeneration regulator genes have been identified. Nevertheless, their downstream effectors remain elusive. As a complementary approach, we propose to perform single-cell RNA-sequencing on regrowing neurons to capture the genome-wide dynamics underlying nerve regeneration. However, it has been technically unfeasible to isolate regrowing neurons from living C. elegans. The prevalent isolation method uses FACS to sort neurons of interest from chemo-mechanically dissociated animals, thus requires thousands of animals with synchronized nerve injury, which cannot be obtained even with state-of-the-art automated microfluidic systems. We developed a new femtosecond laser microdissection (fs-LM) method to rapidly and precisely isolate single cells directly from living tissue or organisms by leveraging femtosecond laser ablation as a high-precision cutting tool. Compared to traditional laser capture microdissection, our method provides a few crucial advantages. 1) fs-LM yields intact single cells without sample sectioning, freezing, or fixing, thus preventing sample degradation or contamination. 2) compared to the dissociation and sorting method, fs-LM induces less stress response in isolated cells. 3) fs-LM preserves the spatial and phenotypic information of the collected neurons. In addition, by correlating gene expression to the context-dependent regeneration phenotypes, it is possible to further dissect the genetic activities encoding nerve regeneration. 4) fs-LM does can isolate unlabeled cells. We isolated regrowing posterior lateral microtubule (PLM) neurons from larval 4 stage animals. Single cell RNA-sequencing on the isolated neurons identified gene expression patterns underlying axon regeneration. To demonstrate the versatility of our method, we have also dissected and sequenced single C. elegans oocytes and mammalian brain neurons.
[
International Worm Meeting,
2005]
Understanding the basics of nerve regeneration can be used to identify novel therapies for neurological diseases. Animal models of nerve regeneration are mostly limited to vertebrate organisms. Establishment of axotomy models in invertebrate organisms such as C. elegans is highly desired because of remarkable research potential promised by genetics and screening methods. We reported a surgical technique based on femtosecond (fs) laser pulses to severe nerve processes (0.2-0.4 ? in radius) in the worm (Nature 432:822 (2004)). In this technique, amplified fs pulses were used in a low-energy processing regime to achieve axotomy with minimal surrounding damage. Anesthetized animals were mounted on a slide and 100 laser pulses with 10-40 nanojoule energy and 200 fs short duration at 1 kHz repetition rate were tightly focused on the fluorescent nerve processes. By cutting circumferential axons of D-type motor neurons, we were able to establish a nerve injury and regeneration model. Operated animals showed uncoordinated backward motion, a manifestation of D neuron deficiency (Nature 364:337-41 (1993)). Lesioned axons recovered in one day and the behavioral defect improved over the same time period. Invertebrate animals have differing capacities for natural recovery after nerve injury, probably regulated by permissive environment and intrinsic qualities of neurons. Since the energy deposited by laser pulses induces structural damage, the assessment of damage extent in the tissue after laser operation becomes important to understand tissue permissivity factors regarding nerve regeneration. To assess surrounding damage by laser pulses, we used a strain where both nerve processes and muscle cell membrane were labeled by GFP. We severed the nerve process and measured ensuing surrounding damage by loss of GFP signal in the lesion periphery. Unwanted damage by laser pulses seemed to be proportional to laser pulse intensity which provides a controllable parameter to curb damage spread, and, thus, facilitate the investigation on the permissive environmental factors. We extended the axotomy model to sensory neurons to determine if intrinsic qualities of neurons confer differential responses to nerve injury. The results, technique, and its potential applications will be discussed.