Fig 2: Coexpression of nNOS with other interneuron markers. (A) Expression of CB, PV, SST, NPY, CR, or RLN in nNOS type I cells. (B) Quantification of the extent of coexpression between nNOS and the different interneuron markers in type I cells. (C) The number of marker+ nNOS type I cells is presented as a percentage of the total number of marker+ cells. (D) Expression of CB, PV, SST, NPY, CR, or RLN in nNOS type II cells. (E) Quantification of the extent of coexpression between nNOS and the different interneuron markers in type II cells. (F) The number of marker+ nNOS type II cells is presented as a percentage of the total number of marker+ cells. Error bars indicate SEM. Scale bar: 20 µm.

Fig 3: Developmental expression of nNOS in the neocortex. (A–E) nNos mRNA expression at different embryonic and postnatal stages. Black and red arrowheads in (A) and (B) point to expression in the superficial cortex and the ventral MGE, respectively. Arrowheads in (C') and (C”) point to cells expressing high levels of nNOS in the cortex and striatum, respectively. (F–J) nNOS protein expression at different embryonic and postnatal stages. Type I and type II cells are marked by black and red arrows, respectively in (I) and (J). Scale bars: A–J, 500 µm. C, C” and inserts in D, E, I, J, 50 µm. Insert in H, 20 µm.

Fig 4: Foxp1 misexpression rescues the Foxp1-mutant phenotype.Analysis of MN subtypes in the ventral horn of E12.5 Foxp+/+, Foxp−/− and Hb9::Foxp1; Foxp1−/− littermate embryo spinal cords. Scale bar, 50 μm. (a) MN subtypes at cervical levels. Foxp1−/− embryos had reduced numbers of LMCm (Isl1+/Foxp1+) and LMCl (Hb9+/Lhx1+) MNs, while HMC (Hb9+/Isl1+) and MMC-rhomboideus (Isl1+/Lhx3low) MNs were expanded compared with Foxp1+/+ embryos13. Hb9::Foxp1; Foxp1−/− embryos had partially restored LMCm and LMCl MNs that resided at the correct lateral positions, and reduced numbers of ectopic HMC and MMC-Rb MNs, compared with Foxp1−/− embryos. Rb, rhomboideus. (b) MN subtypes at thoracic levels. The loss of PGC (Isl1+/nNOS+) MNs in Foxp1−/− embryos was rescued in Hb9::Foxp1; Foxp1−/− embryos. (c) Quantification of MN percentages at cervical levels (mean±s.e.m.; n=3 sections averaged per embryo, 3 embryos per genotype; two-way analysis of variance (ANOVA) with Bonferroni adjustment). MMC MNs: ***P<0.0001. HMC MNs: ***P<0.0001 and **P=0.001. LMCm MNs: ***P<0.0001 and **P=0.006. LMCl MNs: ***P<0.0001 and *P=0.01. NS, not significant, P>0.5. (d) Quantification of MN percentages at thoracic levels (mean±s.e.m.; n=3 sections averaged per embryo, 3 embryos per genotype; two-way ANOVA with Bonferroni adjustment). HMC MNs: ***P<0.0001. PGC MNs: ***P<0.0001, **P=0.0001 and *P=0.0023. NS, not significant, P>0.5. (e) Quantification of total number of MNs per section (mean±s.e.m.; n=3 sections averaged per embryo, 3 embryos per genotype; two-way ANOVA with Bonferroni adjustment). NS, not significant. P>0.5.

Fig 5: Laminar distribution of nNOS immunoreactive cells originating in different embryonic neuroepithelial regions. The density of type I (A–F) and type II cells (G–L) coexpressing GFP/YFP/Venus/ß-gal (yellow) as well as the total number of nNOS-expressing cells (gray) across cortical bins are shown for each transgenic mouse line. Error bars indicate SEM.