Multifunctional requirements for ERK1/2 signaling in the development of ganglionic eminence derived glia and cortical inhibitory neurons

Sara J. Knowles
Michael C. Holter
Guohui Li
George R. Bjorklund
Katherina P. Rees
Johan S. Martinez-Fuentes
Kenji J. Nishimura
Ariana E. Afshari
Noah Fry
April M Stafford
Daniel Vogt
Marco Mangone
Trent Anderson
Jason M. Newbern author has email address

School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA
College of Medicine, University of Arizona, Phoenix, AZ 85004, USA
Department of Pediatrics and Human Development, Michigan State University, Grand Rapids, MI 49503, USA
Virginia G. Piper Center for Personalized Diagnostics, The Biodesign Institute at Arizona State University, 1001 S McAllister Ave, Tempe, AZ 85281, USA

https://doi.org/10.7554/eLife.88313.1

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Figures and data

ERK1/2 signaling regulates MGE-derived oligodendrocyte number.
(A) Anatomical image of a P14 Erk1^-/-; Erk2^fl/wt; Nkx2.1^Cre; Ai9^+/-coronal section showing the expected neuroanatomical pattern of Nkx2.1^Cre-mediated recombination. Note the presence of Nkx2.1^Cre-derived oligodendrocytes in the anterior commissure at this stage. (B-H) Representative confocal images of the anterior commissure at P14 display a significant decrease in the density of OLIG2⁺/RFP⁺ co-labeled cells in Erk1^-/-; Erk2^fl/fl; Nkx2.1^Cre; Ai9 mutants (C, F-G) compared to HET controls (B, D-E) (quantification in H; N=3, mean ± SEM, *student’s t-test, p=.0002) (scale bars 100µm (B-C), 50µm (D-G)). (I-O) Confocal images of the anterior commissure at P21 revealed a significant increase in the number of OLIG2⁺/RFP⁺ cells in Mek1^S217/221E; Nkx2.1^Cre; Ai9 mutants (J, M-N) relative to wildtype (I, K-L) (scale bar = 100 µm I-J, 50 µm K-N) (Quantification in O; N=3-4, mean ± SEM, *student’s t-test, p=.004). (P-T) We observed ectopic expression of OLIG2⁺/RFP⁺ cells in the fimbria of Mek1^S217/221E; Nkx2.1^Cre; Ai9 mutants (scale bar = 50 µm) (quantification in T; N=3-4, mean ± SEM, *student’s t-test, p=.005).

Erk1/2 deletion does not alter CIN number.
(A-E) Representative images of P14 somatosensory cortex in HET control (A-B) and Erk1^-/-; Erk2^fl/fl; Nkx2.1^Cre; Ai9 (C-D) mutant animals, showing similar density of Nkx2.1^Cre-positive cells (quantification in E, N=4, mean +/- SEM, student’s t-test, p=0.476) (scale bar = 50µm). (F-J) At P5, heterozygote control (F-G) and Erk1^-/-; Erk2^fl/fl; VGAT^Cre; Ai9 mutant animals (H-I) showed no significant difference in the density of VGAT^Cre-positive cells in the somatosensory cortex (quantification in J, N=4, mean +/- SEM, student’s t-test p=.955) (scale bar = 50µm).

Loss of ERK1/2 does not alter the initial expression of generic regulators of GABAergic function.
(A) HET IP vs MUTANT IP volcano plot of expressed protein-coding genes in Ribotag, CIN-enriched samples illustrates the significant reduction in ERK1/2 pathway target genes in Erk1/2 inactivated (Erk1^-/-; Erk2^fl/fl; VGAT^Cre; Rpl22^HA) immunoprecipitated fractions (magenta - Spry4, Etv5, Egr2) (N=3, p<.05). A significant difference was not detected in the expression of master regulators of CIN specification (green - Lhx6, Dlx2/5/6) and key GABA processing genes (green - Gad1/2, Slc32a1). However, the expression CIN-subtype enriched genes (Crhbp, Sst, Mafb, Grin2d, Sp9) and canonical developmental signals (Notch1, Smo, Fgf18, Bdnf) were significantly altered in mutant CINs. (B-J) Immunolabeling analysis of CIN subtype markers revealed a significant reduction in SST⁺/CALB^- cell density (yellow arrowheads) in mutants, while SST⁺/CALB⁺ co-labeled cell density was unchanged (quantification in B, N=4, mean ± SEM, *student’s t-test p=0.017). Representative images in (D-F, H-J) are from layer 5 primary somatosensory cortex (scale bar = 25µm).

The proportion of SST-expressing CINs decreased following reductions in ERK1/2 signaling.
(A-H) Representative confocal images of the somatosensory cortex at P14. The proportion of PV-expressing CINs was similar between genotypes (C, G, quantification in Q; N=3, mean ± SEM, student’s t-test, p > .05), while the proportion of SST⁺/EYFP⁺ cells was significantly lower in mutants compared to HET controls (D, H, quantification in Q; N=3, mean ± SEM, *student’s t-test, p = .005). (I-P) By P60, no difference in PV-CINs was detected (K, O, quantification in Q; N=3, mean ± SEM, p > .05), whereas a significant decrease in the density of SST⁺/EYFP⁺ cells in mutant cortices was still apparent (L, P, quantification in Q; N=3, mean ± SEM, *student’s t-test p = .03) (scale bars =50 µm).

ERK1/2 is required to induce FOSB expression in CINs after h3MD_q-DREADD mediated stimulation.
(A) Representative traces of firing response to depolarizing current steps from HET control or mutant neurons indicate that recorded neurons had a fast-spiking electrophysiological phenotype. (B) Acute treatment of CINs in acute slices with 10µM CNO led to a depolarizing increase in the holding current in both HET control and mutant neurons (n=8 neurons/genotype, mean +/- SEM, paired t-test, p <.05). (C) HET control and mutant neurons were recorded under current clamp and the response to a depolarizing current step (200pA, 1s) was measured. CNO significantly increased the firing frequency induced by the applied current step in both HET control and mutant neurons when compared to baseline (VEH) (n= 8 neurons/genotype, mean +/- SEM, paired t-test, p<.05). (D) Diagram of the in vivo experimental design. Animals were treated with 2 mg/kg CNO or saline vehicle by IP injection for 7 days beginning at P8. (E) One week of chemogenetic stimulation increased FOSB expression in HET control CINs but not in mutants (mean +/- SEM, N=4-5/group, one-way ANOVA followed by Fisher’s LSD, F_3,14=15.37, *post-hoc p<.001). (F-Q) Representative confocal images of FOSB/PV/HA co-expression in layer 5 somatosensory cortex at P14. White arrowheads indicate HA⁺ cells lacking significant FOSB expression, whereas magenta arrows indicate FOSB⁺/HA⁺ co-labeled cells (scale bar = 25 µm).

Juvenile chemogenetic stimulation increases SST expression in a subset of CINs.
(A) Both CALB1+ and CALB1-SST-expressing cells were reduced in P14 mutant animals when compared to HET controls (mean +/- SEM, N=5, student’s t-test, SST+/CALB- p=.007, SST+/CALB+ p=.014). (B) CNO treatment increased the proportion of CINs expressing SST in ERK1/2 inactivated mutants, though this proportion remained significantly lower than in HET control animals (N=4-5, one-way ANOVA followed by Fisher’s LSD, F_3,14=22.83, *post-hoc p=.005). (C-J) Representative confocal images of CIN subtypes in P14 somatosensory cortex, layer 5 (scale bar = 50µm) (D, F, H, J). Monochrome image highlighting somatostatin immunoreactivity in those cells.

ERK1/2 is required for activity-dependent responses in adults.
(A) Animals aged 6-10 months were administered 2 mg/kg CNO or saline vehicle via daily IP injection for one week. (B-E) High magnification images from layer 5 of the adult somatosensory cortex showing FOSB expression within CINs following 7 days of daily CNO treatment (scale bar = 10µm). One week of CNO treatment increased FOSB expression in HET control CINs but not Erk1^-/-; Erk2^fl/fl; Nkx2.1^Cre; G_q-DREADD-HA mutant CINs (quantification in J). (F-I) Confocal images of the adult somatosensory cortex layers 2-5, showing the distribution of FOSB+ cells (scale bar = 100µm). The density of FOSB⁺/Nkx2.1^Cre-negative cells was unchanged in response to Erk1/2 deletion or chemogenetic stimulation. (J) Quantification of Nkx2.1^Cre-positive cells expressing FOSB (N=3, one-way ANOVA followed by Fisher’s LSD, F_3,8 =31.87, *post-hoc p<.001). (K) Quantification of Nkx2.1^Cre-negative cells expressing FOSB (N=3, one-way ANOVA, F_3,8 = 0.16, p>.05).

One week of chemogenetic stimulation in adulthood induces a partial rescue of SST expression.
(A) Diagram of experimental design. (B) In adult mice, the proportion of CINs expressing SST is lower in vehicle treated mutants when compared to vehicle treated HET controls. One week of CNO treatment in adult mutants increased SST-CINs relative to vehicle treated mutants (N=4, one-way ANOVA followed by Fisher’s LSD, F_3,12 =126.54, *post-hoc p=.024 MUTVEH/MUTCNO). (C-J) Confocal images showing immunolabeling for HA, PV, and SST in HET control and Erk1^-/-; Erk2^fl/fl; Nkx2.1^Cre; G_q-DREADD-HA mutant mice. Yellow arrowheads indicate SST⁺/HA⁺ co-labeled cells in layers 4-5 of the primary somatosensory cortex (scale bar =25µm).

Cre-targeted modulation of ERK1/2 signaling.
(A) Coronal hemisection of an E13.5 Nkx2.1^Cre; Ai9 brain showing the expected pattern of Nkx2.1^Cre-mediated recombination. (B-J) High-resolution confocal images of neurons demonstrating reduced ERK2 immunoreactivity in RFP-expressing cells in P14 Erk1^-/-; Erk2^fl/fl; Nkx2.1^Cre; Ai9 mice (H-J) compared with wildtype (B-D) or heterozygote controls (E-G) (scale bar = 5µm). (K-P) High-resolution confocal images demonstrating an increase in MEK1 expression in RFP-expressing neurons in Mek1^S217/221E; Nkx2.1^Cre; Ai9 (N-P) animals compared to wildtype (K-M) (scale bar = 5µm).

Reduced ERK1/2 signaling regulates MGE-derived GFAP⁺ astrocyte number.
(A) Extended data on Nkx2.1^Cre-derived OLIG2⁺ cells in the anterior commissure. (B-G) High-resolution confocal images demonstrating reduced ERK2 immunoreactivity in RFP-expressing cells in the anterior commissure of P14 Erk1^-/-; Erk2^fl/fl; Nkx2.1^Cre; Ai9 mice (E-G) compared with heterozygote controls (B-D) (scale bar = 5µm). (H-J) Images demonstrating reduced density of RFP⁺/GFAP⁺ co-labeled cells in the anterior commissure of Erk1^-/-; Erk2^fl/fl; Nkx2.1^Cre; Ai9 mice compared with heterozygous controls. (Quantification in J, N=3, mean ± SEM, *student’s t-test, p<.05) (scale bar = 50µm). (K-P) High-resolution confocal images demonstrating an increase in MEK1 protein expression in RFP-expressing neurons in Mek1^S217/221E; Nkx2.1^Cre; Ai9 animals (N-P) compared to wildtype controls (K-M) (scale bar = 5µm). (Q-S) Images of RFP+/GFAP+ cells in the anterior commissure of wildtype and Mek1^S217/221E; Nkx2.1^Cre; Ai9 mice compared with heterozygous controls. (Quantification in S, N=3-4, mean ± SEM, *student’s t-test, p>.05) (scale bar = 50µm).

RiboTag in Erk1/2 VGAT^Cre mice enriches for CIN mRNA.
(A) Read coverage maps for Erk1/Mapk3 and Erk2/Mapk1 in a representative immunoprecipitated RNA sample from a wildtype (grey), heterozygous control (blue), and mutant Erk1 Erk2 VGAT^Cre Rpl22^HA (red) cortex. As expected, Erk1 exons 1-6 were not detected in heterozygous (N=3) and mutant (N=3) fractions relative to wildtype (N=2) samples (DEXSeq: p<0.005). Analysis of Erk2/Mapk1 exon 2 (inset), which is loxp-flanked in conditional knockouts, revealed a statistically significant reduction in immunoprecipitated mutant samples when compared to heterozygote controls (DEXSeq: 2.5-fold reduction, p<0.005, N=3). (B) Heatmap of DeSeq2-derived normalized gene expression values for select protein coding genes with known specificity for CINs, excitatory neurons, pan-glia, astrocytes, and oligodendrocytes in all samples. Values were unit-variance scaled for each gene within a row for visualization purposes; purple values indicate relative enrichment while green values indicate relative depletion. (C) Volcano plots including >11,000 expressed protein coding genes in IP vs INPUT fractions within each of the three test conditions. Grey dots indicate genes not significantly changed, yellow-green dots are significantly enriched genes in INPUT fractions, while blue dots are significantly enriched genes in IP fractions. Select genes known to be expressed in distinct cell types noted in (B) are highlighted.

Complete loss of ERK1/2 signaling is required to alter the number of SST-expressing cells.
(A) Quantification of total PV-expressing and SST-expressing cell density in the P60 somatosensory cortex of Cre-negative controls (Erk1^-/-), HET controls (Erk1^-/-; Erk2^fl/wt; Nkx2.1^Cre) and mutant (Erk1^-/-; Erk2^fl/fl; Nkx2.1^Cre) mice (N=3, mean +/- SEM, one-way ANOVA followed by Fisher’s LSD; PV-F_2,6=0.621, p>.05; SST-F_2,6=16.206, p=.004). (B-F) Representative images showing CIN subtypes in the P30 primary somatosensory cortex. Nkx2.1^Cremediated deletion of Erk2 did not alter total Cre-expressing CIN number or the density of SST-or PV-expressing CINs (quantification in F, N=3, mean +/- SEM, students t-test p >.05) (scale bar = 50 µm). (G-L) Hyperactivation of ERK1/2 in Mek1^S217/221E; Nkx2.1^Cre mice reduced total Cre-expressing CIN density (quantification in K; N=3, mean +/- SEM, students t-test p <.05) but did not alter SST⁺/RFP⁺-CIN density (quantification in L; N=3, mean +/- SEM, students t-test p >.05) (scale bar = 50 µm).

Intrinsic excitability of FS-CINs is not changed following ERK1/2 modulation.
(A) Key FS-CIN intrinsic properties appear to be unaffected by ERK1/2 deletion, as there was no difference in resting membrane potential (p=0.73), membrane resistance (p= 0.74), or capacitance (p=0.85) (n=8 neurons/genotype, student’s t-test). (B) HA⁺ cell density was unchanged by 7 days of CNO treatment (N=4-5, one-way ANOVA, F_3,24=0.62, p>.05). (C) CNO did not alter the intensity of SST immunoreactivity across genotypes (N=3 animals, minimum 12 cells/animal, one-way ANOVA, F_3,8=3.446, p=.072). (D-F) Confocal images showing low expression of FOSB in P21 wildtype CINs under basal conditions (D). FOSB expression was relatively unchanged in Mek1^S217/221E; Nkx2.1^Cre CINs (E) (Quantification in F, mean +/- SEM, N=3, student’s t-test, p>.05) (scale bar = 25 µm).

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