Our understanding of nerve regeneration can be enhanced by delineating its underlying molecular activities at single-neuron resolution in model organisms such as Caenorhabditis elegans. Existing cell isolation techniques cannot isolate neurons with specific regeneration phenotypes from C. elegans. We present femtosecond laser microdissection (fs-LM), a single-cell isolation method that dissects specific cells directly from living tissue by leveraging the micrometer-scale precision of fs-laser ablation. We show that fs-LM facilitates sensitive and specific gene expression profiling by single-cell RNA sequencing (scRNA-seq), while mitigating the stress-related transcriptional artifacts induced by tissue dissociation. scRNA-seq of fs-LM isolated regenerating neurons revealed transcriptional programs that are correlated with either successful or failed regeneration in wild-type and dlk-1 (0) animals, respectively. This method also allowed studying heterogeneity displayed by the same type of neuron and found gene modules with expression patterns correlated with axon regrowth rate. Our results establish fs-LM as a spatially resolved single-cell isolation method for phenotype-to-genotype mapping.
This is a preview of subscription content, access via your institution
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Get just this article for as long as you need it
Prices may be subject to local taxes which are calculated during checkout
The ChIP–seq datasets used for identifying potential regulators of nerve regeneration are publicly available at https://www.encodeproject.org/ (ENCODE Project75,76). The specific ChIP–seq data used in our study are listed in the Supplementary Table 8. The single-cell gene expression profiles of all six TRNs are used from the publicly available CeNGEN data at https://www.cengen.org/. All RNA-seq data involved in Figs. 1–5 are available on the NCBI SRA repository under study identifier SRP300789. The RNA-seq reads and counts are provided in the Supplementary Table 9. Source data are provided with this paper.
Courtine, G. & Sofroniew, M. V. Spinal cord repair: advances in biology and technology. Nat. Med. 25, 898–908 (2019).
Hutson, T. H. & Di Giovanni, S. The translational landscape in spinal cord injury: focus on neuroplasticity and regeneration. Nat. Rev. Neurol. 15, 732–745 (2019).
Norsworthy, M. W. et al. Sox11 expression promotes regeneration of some retinal ganglion cell types but kills others. Neuron 94, 1112–1120.e1114 (2017).
Duan, X. et al. Subtype-specific regeneration of retinal ganglion cells following axotomy: effects of osteopontin and mTOR signaling. Neuron 85, 1244–1256 (2015).
Yanik, M. F. et al. Functional regeneration after laser axotomy. Nature 432, 822–822 (2004).
Bourgeois, F. & Ben-Yakar, A. Femtosecond laser nanoaxotomy properties and their effect on axonal recovery in C. elegans. Opt. Express 16, 5963 (2008).
Hammarlund, M., Nix, P., Hauth, L., Jorgensen, E. M. & Bastiani, M. Axon regeneration requires a conserved MAP kinase pathway. Science 323, 802 (2009).
Ghosh-Roy, A., Wu, Z., Goncharov, A., Jin, Y. & Chisholm, A. D. Calcium and cyclic AMP promote axonal regeneration in Caenorhabditis elegans and require DLK-1 kinase. J. Neurosci. 30, 3175 (2010).
Nix, P., Hisamoto, N., Matsumoto, K. & Bastiani, M. Axon regeneration requires coordinate activation of p38 and JNK MAPK pathways. Proc. Natl Acad. Sci. USA 108, 10738 (2011).
El Bejjani, R. & Hammarlund, M. Notch signaling inhibits axon regeneration. Neuron 73, 268–278 (2012).
Zou, Y. et al. Developmental decline in neuronal regeneration by the progressive change of two intrinsic timers. Science 340, 372–376 (2013).
Byrne, A. B. et al. Insulin/IGF1 signaling inhibits age-dependent axon regeneration. Neuron 81, 561–573 (2014).
Chuang, M. et al. The microtubule minus-end-binding protein patronin/PTRN-1 is required for axon regeneration in C. elegans. Cell Rep. 9, 874–883 (2014).
Chen, L. et al. Axon injury triggers EFA-6 mediated destabilization of axonal microtubules via TACC and doublecortin like kinase. eLife 4, e08695 (2015).
Li, C., Hisamoto, N. & Matsumoto, K. Axon regeneration is regulated by Ets-C/EBP transcription complexes generated by activation of the cAMP/Ca2+ signaling pathways. PLoS Genet. 11, e1005603 (2015).
Neumann, B. et al. EFF-1-mediated regenerative axonal fusion requires components of the apoptotic pathway. Nature 517, 219–222 (2015).
Alam, T. et al. Axotomy-induced HIF-serotonin signalling axis promotes axon regeneration in C. elegans. Nat. Commun. 7, 10388 (2016).
Byrne, A. B. et al. Inhibiting poly(ADP-ribosylation) improves axon regeneration. eLife 5, e12734 (2016).
Chung, S. H. et al. Novel DLK-independent neuronal regeneration in Caenorhabditis elegans shares links with activity-dependent ectopic outgrowth. Proc. Natl Acad. Sci. USA 113, E2852–E2860 (2016).
Han, S. M., Baig, H. S. & Hammarlund, M. Mitochondria localize to injured axons to support regeneration. Neuron 92, 1308–1323 (2016).
Abay, Z. C. et al. Phosphatidylserine save-me signals drive functional recovery of severed axons in Caenorhabditis elegans. Proc. Natl Acad. Sci. USA 114, E10196 (2017).
Cartoni, R. et al. The mammalian-specific protein Armcx1 regulates mitochondrial transport during axon regeneration. Neuron 94, 689 (2017).
Hisamoto, N. et al. Phosphatidylserine exposure mediated by ABC transporter activates the integrin signaling pathway promoting axon regeneration. Nat. Commun. 9, 3099 (2018).
Kim, K. W. et al. A neuronal piRNA pathway inhibits axon regeneration in C. elegans. Neuron 97, 511–519.e516 (2018).
Linton, C. et al. Disruption of RAB-5 increases EFF-1 fusogen availability at the cell surface and promotes the regenerative axonal fusion capacity of the neuron. J. Neurosci. 39, 2823–2836 (2019).
Tang, N. H. et al. The mRNA Decay Factor CAR-1/LSM14 regulates axon regeneration via mitochondrial calcium dynamics. Curr. Biol. 30, 865–876.e867 (2020).
Yan, D., Wu, Z., Chisholm, A. D. & Jin, Y. The DLK-1 kinase promotes mRNA stability and local translation in C. elegans synapses and axon regeneration. Cell 138, 1005–1018 (2009).
Bounoutas, A. et al. Microtubule depolymerization in Caenorhabditis elegans touch receptor neurons reduces gene expression through a p38 MAPK pathway. Proc. Natl Acad. Sci. USA 108, 3982–3987 (2011).
Li, C. et al. The growth factor SVH-1 regulates axon regeneration in C. elegans via the JNK MAPK cascade. Nat. Neurosci. 15, 551–557 (2012).
Yan, D. & Jin, Y. Regulation of DLK-1 kinase activity by calcium-mediated dissociation from an inhibitory isoform. Neuron 76, 534–548 (2012).
Malinow, R. A. et al. Functional dissection of C. elegans bZip-protein CEBP-1 reveals novel structural motifs required for axon regeneration and nuclear import. Front Cell Neurosci. 13, 348 (2019).
Kaletsky, R. et al. The C. elegans adult neuronal IIS/FOXO transcriptome reveals adult phenotype regulators. Nature 529, 92–96 (2016).
Spencer, W. C. et al. Isolation of specific neurons from C. elegans larvae for gene expression profiling. PLoS ONE 9, e112102 (2014).
van den Brink, S. C. et al. Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations. Nat. Methods 14, 935–936 (2017).
O’Flanagan, C. H. et al. Dissociation of solid tumor tissues with cold active protease for single-cell RNA-seq minimizes conserved collagenase-associated stress responses. Genome Biol. 20, 210 (2019).
Emmert-Buck, M. R. et al. Laser capture microdissection. Science 274, 998–1001 (1996).
Schwarz, E. M., Kato, M. & Sternberg, P. W. Functional transcriptomics of a migrating cell in Caenorhabditis elegans. Proc. Natl Acad. Sci. USA 109, 16246–16251 (2012).
Nath, R. D., Chow, E. S., Wang, H., Schwarz, E. M. & Sternberg, P. W. C. elegans stress-induced sleep emerges from the collective action of multiple neuropeptides. Curr. Biol. 26, 2446–2455 (2016).
Lockhead, D. et al. The tubulin repertoire of C. elegans sensory neurons and its context-dependent role in process outgrowth. Mol. Biol. Cell 27, 3717–3728 (2016).
Cadwell, C. R. et al. Electrophysiological, transcriptomic and morphologic profiling of single neurons using Patch-seq. Nat. Biotechnol. 34, 199–203 (2016).
Barrett, A. et al. Integrating bulk and single cell RNA-seq refines transcriptomic profiles of specific C. elegans neurons. Preprint at bioRxiv https://doi.org/10.1101/2022.04.05.487209 (2022).
Gökçe, S. K. et al. A fully automated microfluidic femtosecond laser axotomy platform for nerve regeneration studies in C. elegans. PLoS ONE 9, e113917 (2014).
Gokce, S. K. et al. A multi-trap microfluidic chip enabling longitudinal studies of nerve regeneration in Caenorhabditis elegans. Sci. Rep. 7, 9837 (2017).
Ben-Yakar, A. & Bourgeois, F. Ultrafast laser nanosurgery in microfluidics for genome-wide screenings. Curr. Opin. Biotechnol. 20, 100–105 (2009).
Spaeth, C. S., Boydston, E. A., Figard, L. R., Zuzek, A. & Bittner, G. D. A model for sealing plasmalemmal damage in neurons and other eukaryotic cells. J. Neurosci. 30, 15790–15800 (2010).
Wu, A. R. et al. Quantitative assessment of single-cell RNA-sequencing methods. Nat. Methods 11, 41–46 (2014).
Ziegenhain, C. et al. Comparative analysis of single-cell RNA sequencing methods. Mol. Cell 65, 631–643.e634 (2017).
Yosef, N. & Regev, A. Impulse control: temporal dynamics in gene transcription. Cell 144, 886–896 (2011).
Bahrami, S. & Drabløs, F. Gene regulation in the immediate-early response process. Adv. Biol. Regul. 62, 37–49 (2016).
Taylor, S. R. et al. Molecular topography of an entire nervous system. Cell 184, 4329–4347.e4323 (2021).
Neumann, B. & Hilliard, M. A. Loss of MEC-17 leads to microtubule instability and axonal degeneration. Cell Rep. 6, 93–103 (2014).
Savage, C. et al. Mutations in the Caenorhabditis elegans beta-tubulin gene mec-7: effects on microtubule assembly and stability and on tubulin autoregulation. J. Cell Sci. 107, 2165–2175 (1994).
Duan, H. et al. Transcriptome analyses reveal molecular mechanisms underlying functional recovery after spinal cord injury. Proc. Natl Acad. Sci. USA 112, 13360 (2015).
Teoh, J.-S., Wong, M. Y.-Y., Vijayaraghavan, T. & Neumann, B. Bridging the gap: axonal fusion drives rapid functional recovery of the nervous system. Neural Regen. Res. 13, 591–594 (2018).
Qiu, X. et al. Reversed graph embedding resolves complex single-cell trajectories. Nat. Methods 14, 979–982 (2017).
Tedeschi, A. et al. The calcium channel subunit Alpha2delta2 suppresses axon regeneration in the adult CNS. Neuron 92, 419–434 (2016).
Celniker, S. E. et al. Unlocking the secrets of the genome. Nature 459, 927–930 (2009).
Kudron, M. M. et al. The ModERN resource: genome-wide binding profiles for hundreds of Drosophila and Caenorhabditis elegans transcription factors. Genetics 208, 937 (2018).
Wu, Z. et al. Caenorhabditis elegans neuronal regeneration is influenced by life stage, ephrin signaling, and synaptic branching. Proc. Natl Acad. Sci. USA 104, 15132–15137 (2007).
Ma, T. C. & Willis, D. E. What makes a RAG regeneration associated? Front Mol. Neurosci. 8, 43 (2015).
Schmitt, A. B. et al. Identification of regeneration-associated genes after central and peripheral nerve injury in the adult rat. BMC Neurosci. 4, 8 (2003).
Chandran, V. et al. A systems-level analysis of the peripheral nerve intrinsic axonal growth program. Neuron 89, 956–970 (2016).
Chen, B. K. et al. Axon regeneration through scaffold into distal spinal cord after transection. J. Neurotrauma 26, 1759–1771 (2009).
Kadoya, K. et al. Spinal cord reconstitution with homologous neural grafts enables robust corticospinal regeneration. Nat. Med. 22, 479–487 (2016).
Langfelder, P. & Horvath, S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinf. 9, 559 (2008).
Lovatt, D. et al. Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue. Nat. Methods 11, 190–196 (2014).
Reynoso, M. A. et al. Translating Ribosome Affinity Purification (TRAP) followed by RNA sequencing technology (TRAP-SEQ) for quantitative assessment of plant translatomes. Methods Mol. Biol. 1284, 185–207 (2015).
Lee, J. H. et al. Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues. Nat. Protoc. 10, 442–458 (2015).
Wang, X. et al. Three-dimensional intact-tissue sequencing of single-cell transcriptional states. Science 361, eaat5691 (2018).
Hammarlund, M., Hobert, O., Miller, D. M. 3rd & Sestan, N. The CeNGEN Project: the complete gene expression map of an entire nervous system. Neuron 99, 430–433 (2018).
Chen, L. et al. Axon regeneration pathways identified by systematic genetic screening in C. elegans. Neuron 71, 1043–1057 (2011).
Jin, Y., Jorgensen, E., Hartwieg, E. & Horvitz, H. R. The Caenorhabditis elegans gene unc-25 encodes glutamic acid decarboxylase and is required for synaptic transmission but not synaptic development. J. Neurosci. 19, 539–548 (1999).
Schuske, K., Beg, A. A. & Jorgensen, E. M. The GABA nervous system in C. elegans. Trends Neurosci. 27, 407–414 (2004).
Nass, R., Hall, D. H., Miller, D. M. 3rd & Blakely, R. D. Neurotoxin-induced degeneration of dopamine neurons in Caenorhabditis elegans. Proc. Natl Acad. Sci. USA 99, 3264–3269 (2002).
Davis, C. A. et al. The encyclopedia of DNA elements (ENCODE): data portal update. Nucleic Acids Res. 46, D794–D801 (2017).
Consortium, E. P. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).
This work was supported by the National Institutes of Health (NIH) grant nos. R21-NS109821 (A.B.-Y.) and RO1-NS060129 (A.B.-Y.), by the training grant no. EB007507 (C.M.), and by a grant from The University of Texas System Neuroscience and Neurotechnology Research Institute (A.B.-Y. and R.O.M.). We are grateful to B. Zemelman, L. Kreeger and N. Golding (The University of Texas at Austin) for sharing insights and brain slice samples from PV-Cre;Ai14 mice and gerbils injected with two adeno-associated viruses during the early-stage development of this method. We acknowledge A. Battenhouse (The University of Texas at Austin) for ChIP–seq analysis, Y. Li (Dell Pediatric Research Institute) for assistance during FACS experiments, Y. Jin (University of California San Diego) for C. elegans strains used in this study (CZ10175 and CZ11327), C. Williams (The University of Texas at Austin) for preparing micropipette and E. Hegarty (The University of Texas at Austin) for help with animal maintenance and insights in experimental design. Bioinformatics analysis support was provided by the Bioinformatics Consulting Group at UT Austin, Center for Biomedical Research Support (grant no. RRID:SCR_022688). We thank the ENCODE Consortium, in particular M. Snyder, V. Reinke and K. White for providing the ChIP–seq datasets. Some strains were provided by the Caenorhabditis Genetic Center, which is funded by the NIH Office of Research Infrastructure Programs (grant no. P40 OD010440).
The authors declare no competing interests.
Peer review information
Nature Methods thanks Itai Yanai and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Nina Vogt, in collaboration with the Nature Methods team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Schematic of the fs-LM setup for single-cell microdissection of C. elegans neurons.
a, Schematic of the optical setup. The diagram shows a femtosecond laser (fs-laser), beam splitter (BS), mirror (M), shutter (S), flip mirror (FM), neutral density filter (ND), photodetector (PD), hot mirror, tube lens (TL), filter set, fluorescence source (FL Lamp), motorized stage, condenser, and bright-field light source (BF Lamp). The microdissection is performed with a 60× 1.0 NA water dipping objective. The sample manipulation is performed with a 10× 0.3NA air objective with a 10 mm working distance. A glass microneedle is mounted on a 3-axis manipulator and suction pressure is controlled using a precise pneumatic puller. The pi-chart shows the isolation of n = 123 PLM neurons (32.0% out of n = 384 total PLM neurons). The pie chart also depicts the unsuccessful situations and classifies them under different categories based on the step at which we lost the neuron. Most failures (incomplete resection, n = 88 neurons) occurred primarily when the neurons were torn into pieces during the release process through the cuticle (Extended Data Fig. 2e-l). b, Priming steps for the glass micropipette before it can be used for cell manipulation. c, fs-LM isolation steps including C. elegans immobilization, single-cell dissection, cell collection in the glass micropipette, and cell lysis for single-cell transcriptomic analysis.
Extended Data Fig. 2 Successful and unsuccessful fs-LM isolation of C. elegans PLM neurons.
a-d, Example of successful fs-LM isolation of neurons. The resected neuron was released through the incision smoothly without damage, as evidenced by consistent volume and cytoplasmic GFP intensity (n = 123 out of total 384 attempts). e-h, Incomplete resection of neuron causing it to remain in place after the incision was made (n = 88 out of total 384 attempts). As a result, the neuron was torn by the outflow of surrounding tissue. i-l, A resected neuron was fragmented when migrating through a small cuticle incision. In this case, the neuron was moving towards the incision, which indicated complete resection. Scale bar, 50 μm.
Extended Data Fig. 3 Quantification of amplified cDNA libraries from fs-LM isolated single C. elegans neurons.
a, Example of libraries of good quality. Inset: the isolated neurons retained GFP intensity until being deposited into the lysis buffer. b, Example of libraries that failed quality check, and were thus excluded from this study. Inset: the isolated neuron lysed before being deposited into the lysis buffer. Although the cellular content of the neuron supposedly remained in the micropipette, degradation, and attachment to the inner wall of the micropipette affected reverse transcription (RT)-PCR, resulting in libraries dominated by short fragments that were mostly primer dimers. c, Example of NTC controls.
Extended Data Fig. 4 Isolation of C. elegans touch receptor neurons by the dissociation-FACS method.
a-d, FACSAria II gate settings for isolating living GFP-labeled C. elegans touch receptor neurons from dissociated animals. Prior to FACS, the cell suspension was filtered through a 5 μm syringe filter to prevent clogging. SSC-A, SSC-W, and FSC gates were set up to exclude debris and clusters of cells. We stained the dead neurons with propidium iodide. GFP + PI- events were sorted into 96 well plates or 1.5 mL conical tubes containing lysis buffer. The GFP threshold was determined in reference to the autofluorescence of cells from dissociated N2 worms. The PI threshold was determined by test sorting a small number of PI-stained cells and observing two distinct populations of live/dead cells. e, Histogram of GFP levels of the isolated neurons. f, Cell suspension prior to sorting and g, example of a collected neuron. Green: GFP. Red: PI. Multiple locations were imaged to confirm GFP + neurons using the fluorescent microscope (confirmed with 3 independent sample preparation). Scale bar: 5 μm. h, Quantification of cDNA library prepared from one of the GFP + sorted cells using FACS method.
Extended Data Fig. 5 RNA-sequencing of fs-LM and dissociation-FACS isolated neurons.
a, b, Number of genes detected at different sequencing depth obtained by subsampling 19 million total reads from bulk (each bulk sample has >5,000 cells) and single neuron samples. c, Cumulative distribution of sequencing depth among all single-neuron samples collected with fs-LM and verified using scRNA-seq library quality checks. Single-neuron samples below the red dotted line were discarded due to insufficient sequencing depth. d, Number of genes identified in fs-LM isolated single neurons (n = 46 uninjured PLM neurons) and single TRNs isolated using dissociation-FACS method (n = 23 single TRNs from WT animals). e, f, Top tissue enrichment analysis terms of fs-LM isolated single neurons (e) and dissociation-FACS isolated single neurons (f). Data presented as mean + standard error of mean (SEM). g, Correlation of gene expression profiles between the ensembles of single TRNs (isolated using FACS-dissociation method, n = 23) and the single PLM neurons (isolated using the fs-LM method, n = 40).
Extended Data Fig. 6 Gene expression profiles for the neurons collected using fs-LM and dissociation-FACS methods.
a, Expression levels of marker genes among fs-LM isolated single PLM neurons (n = 123 PLM neurons). b, Expression levels of marker genes among dissociation-FACS isolated TRNs (n = 23 single TRNs). c, Expression levels of marker genes among dissociation-FACS isolated bulk samples (n = 3 bulk samples each with >5,000 TRNs).
Extended Data Fig. 7 Examples of successful fs-LM isolation of single neurons from different locations in multiple C. elegans strains.
a, Isolation of one of the PLM neurons from the tail region in CZ10175 animal (we resected n = 14 PLM neurons out of 15 attempts over 6 independent batches). Scale bar, 50 μm. b, Isolation of VD7 neuron from the mid-body region (close to the vulva muscle and residing on the ventral cord region) in the CZ1200 animal (resected n = 3 VD7 neurons over 3 independent batches). c, Isolation of one of the CEP neurons from the head region (in the head ganglion) in the BY200 animal (resected n = 24 CEP neurons out of 30 attempts over 10 independent batches). d, Isolation of one of the RME neurons from the head region (in the head ganglion) in the CZ1200 animal (We resected n = 8 RME neurons out of 9 attempts over 4 independent batches). The top panel shows the neurons’ schematics. Below are the fluorescence images describing the steps for the axotomy of neuronal processes, neuron dissection, cuticle ablation, neuron extraction, and neuron collection. The lightening arrows indicate the locations of the laser spots for axotomy and fs-LM (light pink, indicating low pulse energies) and cuticle ablation (red, indicating higher pulse energies required to ablate the cuticle). The yellow arrows indicate the isolated neurons outside the animal body and as collected inside the micro-pipette tip. The bottom panel shows 2% agarose gel with PCR amplification products of single-cell lysates and visualized with ethidium bromide. Lanes are marked with the target genes or ladder used in the experiment.
Extended Data Fig. 8 Correlation of gene expression profiles between fs-LM and dissociation-FACS methods.
a, Normalized expression of the selected 36 genes, obtained from CeNGEN feature lists, utilized to visualize and identify cell clusters that were assigned a predicted neuronal category. With this classification analysis, we obtained PLM (n = 6), PVM (n = 5), ALM (n = 8), and AVM (n = 3) subgroups with one remaining unclassified. The color panels show the four neuron categories and the normalized level of expression. b, Principal component analysis (PCA) on gene expression profiles in single PLM neurons isolated by fs-LM (fs-LM (PLM), n = 40) and dissociation-FACS (dissociation-FACS (PLM), n = 6) methods. The six dissociation-FACS isolated neurons were classified using the neuron-specific gene expression data available in CeNGEN data. c, Pearson correlation coefficients as calculated from 50 groups of 6 neurons isolated using the fs-LM method (n = 40), predicted-PLM method (dissociation-FACS, n = 6), and all TRNs (dissociation-FACS, n = 23). Shaded areas represent a 95% confidence interval around the mean. d, Box and whisker plot for the top 10 expressed genes. e, Box and whisker plot for all 1,000 ranked genes. The box extends from the 25th to 75th percentiles, the line is plotted at the median, the whiskers drawn down to the 10th percentile and up to the 90th percentile. Points below and above the whiskers are drawn as individual points. The statistical test used is a two-tailed unpaired Wilcoxon rank sum test. P-value = 0.024 (dissociation FACS and predicted PLM for top 10 genes, *) and P-value < 0.0001 (****).
Extended Data Fig. 9 Axon regeneration of PLM neurons in wild-type and dlk-1 (0) animals following fs-laser axotomy.
a, Regeneration status of axotomized PLM neurons at 24-hour post-axotomy examined over at least 3 independent batches. 1 out of 86 wild-type PLM neurons failed to initiate regrowth. b, Length of axon regrowth at various time points post-axotomy. At 2–4 hours post-axotomy, wild-type PLM neurons started to regrow at an average rate of 6.1 μm/hour. The ectopic regrowth rate of dlk-1 (0) neurons was found to be 1.2 μm/hour. Data presented as mean ± standard deviation (SD). c, d, Microscope images of regrowing wild-type (c) and dlk-1 (0) neurons (d). The regrowth lengths presented in (b) were measured from up to n = 16 wild-type and n = 14 dlk-1(0) animals, collected over at least 3 independent experiments. Asterisk, site of laser axotomy. Arrowhead, the tip of regrowing axon. Scale bar, 10 μm.
Extended Data Fig. 10 Differential gene expression between uninjured and axotomized neurons isolated from wild-type/dlk-1 (0) animals.
a, DEGs between uninjured wild-type (n = 21) and dlk-1 (0) (n = 24) neurons collected from at least 3 independent experiments. Red points in the volcano plot represent significant DEGs with adjusted P-value < 0.05, log2 fold change > 2, and tested using a one-sided Wilcoxon rank test. b, DEGs between uninjured (n = 21 control) and axotomized wild-type (n = 46) neurons collected over at least 3 independent batches. c, DEGs between uninjured (n = 24 control) and axotomized dlk-1 (0) (n = 13) neurons collected over at least 3 independent batches. Boxplots on the right show expression levels of the top 4 DEGs for each set. The box extends from the 25th to 75th percentiles, the line is plotted at the median, the whiskers drawn down to the 10th percentile and up to the 90th percentile. Points below and above the whiskers are drawn as individual points.
Supplementary Notes 1–6 and Figs. 1–5.
Time-lapse video of single-cell isolation and cell-lysate collection from an individual PLM neuron using the fs-LM method.
Time-lapse video of single-cell isolation and cell-lysate collection from an individual CEP neuron using the fs-LM method.
Supplementary Tables 1–9.
Source Data Fig. 1
Statistical source data.
Source Data Fig. 3
Statistical source data.
Source Data Extended Data Fig. 7
Unprocessed gel pictures for Extended Data Fig. 7a,c.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Zhao, P., Mondal, S., Martin, C. et al. Femtosecond laser microdissection for isolation of regenerating C. elegans neurons for single-cell RNA sequencing. Nat Methods 20, 590–599 (2023). https://doi.org/10.1038/s41592-023-01804-3