Figure 1. PyN AIS-enriched NF186 is Dispensable for Neocortical ChC/PyN AIS Innervation.
(A1-A3) Experimental strategy to target/manipulate PyN gene expression and label ChCs in the same neocortical layer. (A1) Schematic drawing of E15.5 in utero electroporation (IUE) targeting nascent layer II/III (LII/III) PyNs in embryos from Swiss Webster (SW) females that were bred with Nkx2.1-CreER+/−;Rosa26-loxpSTOPloxp-tdTomato (Ai9)+/+ males. The position of the positive (+) and negative (−) electrodes used to target neocortical progenitors in the ventricular zone (VZ) is depicted. (A2) Tamoxifen (TMX) administration at E18.5 induces Cre activity and excision of a STOP cassette resulting in tdTomato red fluorescent protein (RFP) expression in ChC progenitors. (A3) Representative 200 μm × 200 μm confocal image of a single RFP+ ChC and neighboring electroporated GFP+ PyNs in LII of somatosensory cortex. Scale bar, 20 μm. Enlarged view of the boxed area showing a GFP+ PyN innervated at its AIS by an RFP+ ChC cartridge (arrow) is depicted on the right. Scale bar, 5 μm. AISs are visualized by immunostaining for ankyrin-G (AnkG) (blue).
(B) Representative images of PyNs innervated by ChC cartridges in LII of somatosensory cortex from Nkx2.1-CreER;Ai9 mice electroporated at E15.5 with plasmids expressing EGFP and miR30-based shRNAs against NF186 (miR.NF#1 or miR.NF#2) or Renilla luciferase (miR.Ctrl) and sacrificed at P28. Scale bar, 10 μm.
(C) Quantification of the percentage of GFP+ PyNs innervated by single RFP+ ChCs at P28. Innervation percentages are indicated for each condition (in Fig. 1C and E). 6–8 ChCs and 37–131 GFP+ PyNs per ChC from 3 animals were analyzed for each condition; one-way ANOVA, post hoc Tukey-Kramer test.
(D) Representative images of PyNs innervated by ChC cartridges in LII of somatosensory cortex from Nkx2.1-CreER;Ai9 mice coelectroporated at E15.5 with plasmids expressing EGFP and Cas9 together with an sgRNA against NF186 (crNF186) or LacZ (crLacZ), as a control, and sacrificed at P28. Endogenous NF186 is visualized by immunostaining for NF186. Scale bars, 10 μm.
(E) Quantification of the percentage of GFP+ PyNs innervated by single RFP+ ChCs at P28. Of note, crNF186-electroporated PyNs still expressing NF186 (crNF186 NF186+) were also analyzed and included as an additional control. 8–9 ChCs and 17–73 GFP+ PyNs per ChC from 3 animals were analyzed for each condition; one-way ANOVA, post hoc Tukey-Kramer test.
For all images of ChC/PyN AIS innervation, stars and arrows indicate GFP+ PyNs innervated by RFP+ ChC cartridges and the site of GFP+ PyN AIS innervation, respectively.
n.s. (not significant) indicates p ≥ 0.05. Data are mean ± SEM. See also Fig. S1.