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. 2023 Nov 29;43(48):8104–8125. doi: 10.1523/JNEUROSCI.0431-23.2023

Figure 6.

Figure 6.

Constitutive deletion of the miR-218. a, b, CRISPR-cas9-mediated deletion of miR-218-1 and miR-218-2 by using two gRNAs for each targeted locus. The chromosomic regions containing Slit2 and Slit3 and the location of miR-218-1 and miR-218-2 within these genes are reported in a and b, respectively. Uppercase letters represent the pre-miRNA sequences. Red represents gRNAs. The sequences of mature miRNAs are underlined. Dashes indicate deleted nucleotides. Right panels, The Sanger-sequencing results. Two red lines and a vertical dashed black line indicate sites of nonhomologous end joining recombination. c, Representative images of P0 pups are in d (see also Movie 1). d, e, The expression of miR-218 (d) in the midbrain of E14.5 KO embryos was evaluated by TaqMan assay. Data are normalized to the average of the reference sno-202 and represent the mean ± SEM of 2−ΔCt values from 3 animals. *p < 0.05 (two-way ANOVA followed by Sidak). The expression of Slit2 and Slit3 (e) was evaluated in the midbrain of E14.5 WT and KO models by qPCR. Data are normalized to the average of the reference Hprt and represent the mean ± SEM of 2−ΔCt values from three embryos. WT: miR-218-1+/±; miR-218-2+/±. KO1: miR-218-1–/–; miR-218-2+/±. KO2: miR-218-1+/±; miR-218-2–/–. dKO: miR-218-1–/–; miR-218-2–/–. f, The expression of Th in the midbrain of E14.5 KO embryos was evaluated by qPCR. Data are normalized to the average of the reference Hprt and represent the mean ± SEM of 2−ΔCt values from three independent experiments. g, Boxplot represents the percentage of TH-GFP+ DAn isolated from the midbrain of E14.5 KO embryos. h-k, Immunostaining and quantifications for TH (i), PITX3 (j), and ISL1 (k) in the midbrain of E14.5 WT and double KO embryos. The count was performed from 10 randomly selected fields from at least three different litters for each condition. Images were acquired at 10× magnification. Scale bar, 100 μm. l-n, Functional characterization of iDAn obtained from dKO E14 MEF. Current to voltage relationships of K+ (l) and Na+(m) voltage-dependent currents, recorded in dKO and WT iDAn, elicited by depolarizing voltage steps (–40 to 40 mV, 5 mV increment, 300 ms) from VH = − 60 mV. A significant difference between dKO and WT iDAn is present for both INa+ and IK+ current areas at most stimulus intensities. Data are mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; two-way repeated-measures ANOVA followed by Bonferroni test. n, In current-clamp recordings, dKO iDAn fire a higher number of APs in response to depolarizing current injections (15-105 pA, 10 pA increment, 300 ms) compared with WT iDAn. Data are mean ± SEM. *p < 0.05; ***p < 0.001; two-way repeated-measures ANOVA, followed by Bonferroni test. iDAn were identified as TH-GFP+ cells.