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. 2022 May 12;50(10):5772–5792. doi: 10.1093/nar/gkac337

Figure 4.

Figure 4.

Khsrp mRNA is transported into PNS axons. (A) Representative confocal images for smFISH/IF for Khsrp mRNA (grey) and NF (magenta) in dissociated DRG cultures are shown as indicated. There is a clear signal for Khsrp mRNA in the cell body (left and middle columns) and distal axons (right column) of DRGs from Khsrp+/+ mice. Axons of Khsrp–/– DRGs show Khsrp mRNA signals comparable to the scrambled probe; however, there was faint Khsrp signal in the soma of the Khsrp–/– cultures that was consistently above the scrambled probe signal [scale bar = 10 μm].(B, C)Quantification smFISH signals for Khsrp mRNA in soma (B) and axons (C) is shown as mean ± SEM for scramble (Khsrp+/+) and Khsrp mRNA (Khsrp+/+ and Khsrp–/–) probes; in each case, scramble probe was hybridized to Khsrp+/+ DRG cultures as in panel A (N ≥ 16 neurons over three separate cultures; *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001 by one way ANOVA with Tukey's post-hoc analysis). (D) Representative confocal images for smFISH/IF for Khsrp mRNA (grey) and NF (magenta) in uninjured sciatic nerve are shown as indicated. Left column shows XYZ projections from eight optical planes taken at 0.2 μm Z step intervals; right column shows ‘axon only’ Khsrp mRNA signals generated by extracting FISH signals overlapping with NF in individual Z sections and projecting those as an ‘Axonal Khsrp mRNA’ XYZ image [scale bar = 5 μm]. (E) Quantification of axonal Khsrp mRNA signals from (D) are shown as mean ± SEM (N = 6 animals per genotype; ** P ≤ 0.01 by Student's t-test). (F) Representative matched exposure smFISH/IF images for Khsrp mRNA, NF, GFAP and DAPI in naïve versus 7 days post-crush injured sciatic nerves as indicated. Upper row shows merged signals as single XY planes; lower two rows show XYZ projections for Khsrp mRNA colocalizing with NF (middle row) and GFAP (lower row). Representative matched exposure images for scramble probe are shown in Supplemental Figure S3E [scale bar = 5 μm]. (G) Quantification of axonal smFISH signals for Khsrp mRNA in naive and 7 days regenerating sciatic nerve axons (N = 6 animals; no significant differences detected by Student's t-test).