Corrigendum to “A New Technical Approach for Cross-species Examination of Neuronal Wiring and Adult Neuron-glia Functions” [Neuroscience 508 (2023) 40–51]
Year: 2023
Type: erratum
Abstract: The authors regret that the original legends for Figure 3, Supplementary Figure 2 and Supplementary Figure 5 contained errors. The correct legends should read: Figure 3. Cell-type specific rescue of function. (A) Schematic representations of the misPiggy CRISPR/Cas9 plasmid containing the sgRNA targeting exon 2 of Xenopus tropicalis Dcc and the rescue plasmid containing a CRISPR resistant human DCC sequence fused to a hemagglutinin (HA) tag. (B) Schematic representation of the experimental strategy. The eye primordium was co-injected with the misPiggy CRISPR/Cas9 plasmid and the rescue plasmid expressing human DCC. Injections and electroporation were performed at Nieuwkoop and Faber (NF) stage 28; analysis of RGC axon arborization was performed at NF stage 46. RGC soma (magenta) are located in the eye and axons project contralaterally to their target regions in the tectum (black square), where they develop a highly branched terminal arborization. (C–K) Analysis of RGCs at the tectum in NF stage 46 animals, showing axonal branches from a single RGC expressing TdTomato (TdT) (magenta; C, F, I) and HA-tagged DCC (green; D, G, J); colocalized staining is shown in the merged image (white; E, H, K). The tectum is delineated with a white dotted line. (L-N) Quantification of the length of axon branches (L), total branch number (M) and total branch area (N) in control and rescue conditions. **p < 0.005, ***p < 0.001, ****p < 0.0001. Supplementary Figure 2. Cell-type specific loss of function of Dcc using misPiggy CRISPR/Cas9 in Xenopus tropicalis. (A) Schematic illustrating the single guide RNA (sgRNA) targeting exon 2, exon 3 or exon 4 of the Deleted in Colorectal Cancer (Dcc) gene in Xenopus tropicalis. (B) Multiple bicistronic misPiggy plasmids were produced, each carrying a specific sgRNA targeting independent exons of Xenopus tropicalis Dcc, or a non-target sgRNA which was used as a control. In all cases, sgRNA expression was driven by the ubiquitously active U6 promoter: To achieve LoF in RGC neurons, Cas9 expression was driven by the cell-type specific Crest3 enhancer, with simultaneous expression of TdTomato. (C-J) misPiggy CRISPR/Cas9 plasmids were injected in the eye primordium at NF stage 28 followed by electroporation, resulting in labelling of single RGC. Axon arborization was analyzed at NF stage 46 (see Figure 2). Images of single RGC axon branches at the tectum (dotted white lines) of animals injected with misPiggy CRISPR/Cas9 expressing non-targeting sgRNA (C, D), or sgRNAs targeting X. tropicalis Dcc at exon 2 (E, F), exon 3 (G, H), or exon 4 (I, J). Scale bars: 50 μm. Quantifications are shown in Figure 2. (K) misPiggy CRISPR/Cas9 plasmids containing sgRNAs targeting Dcc exon 2, exon 3 or exon 4 were co-injected with additional Cas9 protein at the 1 cell stage. Protein lysates were then prepared at NF stage 25 to test knock-down of DCC using Western blotting. Injected embryos show a decrease of DCC for each of the three gRNAs used, compared to wild type (WT) embryos. (L) Graph of DCC relative intensity normalized to beta-tubulin for each condition in (K). Supplementary Figure 5. Clonal analysis experiments using misPiggy. (Top) Schematic of the misPiggy plasmids used for transposon-based lineage tracing. Plasmids share a common design with use of a GFAP-promoter to drive fluorescent protein expression in astrocytes. Constructs expressed one of the following fluorescent proteins, mCherry, EYFP, EGFP or mCerulean in either a membrane labelling (kmyr-tag) or nuclear labelling (nls-) form. (Bottom) Specific fluorescent fingerprints label populations of clonally related astrocytes. A mixture of misPiggy plasmids encoding various fluorescent proteins was injected into mice and electroporated at E16. Samples were collected at P21. Due to the stochastic nature of plasmid uptake, progenitors acquire a unique combination of plasmids during electroporation, which is stably inherited by their daughter cells. Hence, clonally related astrocytes can be determined by a unique shared spectral fingerprint: (left) merge image showing the complex spectral fingerprints defining small clusters of clonally related astrocytes, (right) individual color channels used to make the merged image, showing differential fluorescent protein expression between individual cells. Scale bar 50 μm (left), 100 μm (right). Data are representative images seen in a minimum of 3 slices from 3 independent animals for each experiment. The authors would like to apologise for any inconvenience caused. A New Technical Approach for Cross-species Examination of Neuronal Wiring and Adult Neuron-glia FunctionsNeuroscienceVol. 508PreviewComparative analysis across model systems has been extremely powerful in elucidating the principles of CNS development and function. For example, studies on the development of the retino-tectal system in frogs and chickens have been instrumental in developing the concept of topographic maps (Cowan et al., 1961; De Long and Coulombre, 1965; Holt and Harris, 1983), and established important concepts regarding chemotactic guidance and the role of complementary gradients of Eph receptors and ephrin ligands across multiple cell types (Cheng et al., 1995; Yates et al., 2001; Mann et al., 2002). Full-Text PDF Open Access
Source: Neuroscience
Institutions: University of Bonn, VIB-KU Leuven Center for Brain & Disease Research, KU Leuven, VIB-KU Leuven Center for Cancer Biology, Kanazawa University, University of Hawaiʻi at Mānoa, University of Hawaii System, Goethe University Frankfurt, University Hospital Frankfurt, Frankfurt Cancer Institute, Universidade do Porto, i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto
Language: English
Subfield: Cellular and Molecular Neuroscience
Field: Neuroscience
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,000
OpenAlex ID: W4380785091