Shared gene expression changes in maturing layer 4 and 6 cortical pyramidal neurons

A) Experimental Workflow: Rorb-GFP+/− (L4) or 56L (L6) mice were sacrificed at P2 or P30 and GFP+ neurons were isolated by FACS for RNA-seq or ATAC-seq (left). IGV tracks of Gene Expression (RNA-seq) and Chromatin Accessibility (ATAC-seq) of neonatally expressed Doublecortin (Dcx, top right) and adult-expressed CaMKIIa (bottom right). B) RNA-Seq of FACS- sorted Rorb+ Layer 4 neurons and Bmp3+ Layer 6 neurons at P2 and P30 (n=4 mice per group). B1: Heatmap of All DEGs identified in either layer between P2 and P30 identifies k=6 unique clusters (DEGs defined as |log2FoldChange|>1; p.adj<0.01 by Wald’s Test with BH correction; min TPM>10). Data plotted as group averages of z-scored relative log gene expression. B2 (right): Top Gene Ontology terms enriched in shared upregulated genes relative to all genes eligible for Differential Expression (Fisher Exact tests with BH adjustment). B2 (left): Transcription Factor Motif Enrichment in ATAC-seq peaks overlapping the Promoter (TSS −1000/+200 bp) of shared upregulated genes relative to all Promoter peaks using the JASPAR 2022 CORE Motif set (Fisher Exact tests with BH adjustment). B3: Same as B2 but for shared downregulated genes.

Activating (Klf6 and Klf7) and repressive (Klf9 and Klf13) members of the KLF family display opposing patterns of gene expression throughout the developing cortex

A) Developmental dynamics of Klf7 and Klf9 in Layer 2/3 and Layer 6 neurons between E18 and P48. Plotted as mean FPKM from n=2 mice (data replotted from Yuan et al., 2022). B) Expression of KLF6, KLF7, KLF9, and KLF13 mRNA across the human lifespan in 4 sensory cortical regions taken from the human BrainSpan Atlas (Kang et al., 2011). Data are plotted as smoothed loess fits +/− SE. C) RNAScope of Klf7 and Klf9 mRNA P2, P7, and P30 C57 mouse brains demonstrates Klf7 mRNA is more abundant than Klf9 mRNA at P2 (C1) while Klf9 mRNA is more abundant than Klf7 mRNA at P30, where laminar distribution is more uniform (C3. Scale bar: 100 µm (Insert: 25µm). Data quantified in C2 (n=3-4 mice per age, plotted as puncta normalized by nuclei density with 95% confidence intervals; **:p<0.001, Mann-Whitney U Test)

A CRISPR Interference strategy for studying cell-autonomous Transcription Factor knockdown in excitatory cortical neurons.

A) Schematic of experimental approach: Mice harboring the dCas9-KRAB transgene are crossed with Emx1-Cre mice and double heterozygous offspring are intracerebroventricularly injected at Postnatal day 0.5 with AAV9 containing guide RNA’s targeting gene(s) of interest and a floxxed fluorophore. After a 10-20 waiting period, infected neurons are sorted based on fluorescence and processed for RT-qPCR or RNA-seq. (Scale bars = 500µm) B) CRISPRi produces consistent and effective knockdown of Klf9 (99.0±1.7%) and Klf13 (98.6±1.7%) individually or in combination (Klf9: 99.4±0.6%; Klf13: 97.9±2.2%) as measured by RT-qPCR (n=6-8 mice/condition; data plotted as average Fold Change relative to Scramble sgRNA Control ± standard deviation, Kruskal-Wallis test with post-hoc pairwise Mann-Whitney U test, * p<0.05 **p<0.01, ***p<0.001) C) Example RNA-scope image of P20 mouse cortex infected at P0.5 with AAV9-Klf9g1,g2-Klf13g1,g2-hSyn-DIO-EGFP and probed for EGFP (Green) and Klf9 (Magenta). (Scale bar = 50µm)

Klf9 and Klf13 show compensatory activity and additive regulation of target genes

A) Principal component for top 500 most variable genes in all knockdown samples and scramble controls shows graded effect of KLF knockdown along PC1 (n=4 mice per condition). B) De-repression of target genes following Klf9/13 KD. Volcano plot of gene expression changes in Klf9/13 KD relative to Scramble Control mice (DEGs defined as |log2FoldChange|>1; p.adj<0.05 by Wald’s Test with BH correction). C1) Heatmap of all DEGs identified in Klf9/13 KD (212 genes) across all libraries. Data plotted as z-scored, regularized log-transformed counts. Right bar represents KLF/Sp root cluster motif counts identified in ATAC-seq peaks overlapping respective Promoters (TSS −1000/+200bp) by FIMO (qval<0.05, quantified in C3, Fisher’s Exact Test). C2: Line plots of all upregulated Klf9/13 targets across all libraries. Grey lines are z-scored, regularized log-transformed RNA-seq counts of individual genes, blue lines represent average of all genes +/− SEM. (***:p<0.0001, Kruskal-Wallis test with post-hoc pairwise Mann-Whitney U test) D) Additive regulation of selected targets with roles in cytoskeletal or synaptic function (n=4 mice per group, average TPM +/− standard deviation). E) Example IGV traces of RNA-seq coverage at the Dpysl3 locus. Arrows indicate KLF/Sp motif matches (FIMO, q<0.05).

Putative Klf9/13 targets are developmentally downregulated in the maturing cortex

A) Expression of upregulated DEGs in Klf9/13 KD at early, mid, and late postnatal developmental time points. Grey lines are z-scored, regularized log-transformed RNA-seq counts of individual genes from sorted Rorb+ (Layer 4) excitatory neurons, blue lines represent average of all genes +/− SEM (n=4 mice per group, Kruskal-Wallis test with post-hoc pairwise Mann-Whitney U test, ***p<0.001). B) Gene expression changes in Klf9/13 KD relative to Scramble Controls at P18-20 (n=4 mice per group) relative to gene expression changes between P2 and P30 (n=8 mice per group, aggregate of 2 cell types). Points represent in individual genes, and red points are DEGs identified in Klf9/13 KD samples. Line is linear fit +/− 95% CI to Klf9/13 DEGs (red).C) RiP-normalized coverage of average ATAC-seq signal at Promoter peaks around DEGs identified from Klf9/13 KD RNA-seq (Shaded region = 95% CI; n=4 libraries per group, aggregate of 2 cell types). D) Example IGV traces from P2 & P30 ATAC-seq and RNA-seq for Klf9/13 Targets Rac3 (top, Developmental DEG without Promoter DAR) and Tubb2b (bottom, Developmental DEG with Promoter DAR). Arrows indicate putative KLF/Sp binding site identified by FIMO (q<0.05)

Klf6 and Klf7 promote expression of developmentally regulated genes in the perinatal cortex

A) RT-qPCR quantification of Klf6 and Klf7 in mice injected with Klf6 and Klf7-targeting sgRNA’s at P0, collected at early (P10-12) or late (P18-20) postnatal timepoints (n=7-9; data plotted as average fold-change relative to P10 Scramble control +/− standard deviation, Kruskal-Wallis test with post-hoc pairwise Mann-Whitney U test with BH adjustment, **p<0.005, ***p<0.001) B) Volcano plot of DEGs identified in Klf6/7 KD samples relative to Scramble Controls at P10 (B1) and P20 (B2, DEGs defined as |log2FoldChange|>1; p.adj<0.05 by Wald’s Test with BH correction). C1) Heatmap of all DEGs identified in Klf6/7 KD at P10 (374 genes, k=3). Data plotted as z-scored, regularized log-transformed counts. Right bar represents KLF motif counts identified in ATAC-seq peaks overlapping respective Promoters (TSS −1000/+200bp) by FIMO (qval<0.05, quantified in C3, Fisher’s Exact Test). Gene Ontology overrepresentation analysis of downregulated targets shown in C2. D) Developmental regulation of Klf6/7 Targets D1: Expression of downreguated DEGs in Klf6/7 KD at early, mid, and late postnatal developmental time points. Grey lines are z-scored, regularized log-transformed RNA-seq counts of individual genes from sorted Rorb+ (Layer 4) excitatory neurons, blue lines represent average of all genes +/− SEM (n=4 mice per group, Kruskal-Wallis test with post-hoc pairwise Mann-Whitney U test, ***p<0.001). D2: Gene expression changes in Klf6/7 KD relative to Scramble Controls at P10-12 (n=4 mice per group) relative to gene expression changes between P2 and P30 (n=8 mice per group, aggregate of 2 cell types). Points represent in individual genes, and green points are DEGs identified in Klf6/7 KD samples. Lines are linear fits +/− 95% CI to all points (black) or Klf6/7 DEGs (green).

The transition from KLF activators to repressors constitutes a developmental switch at shared targets

A) Principal component analysis of all RNA-seq libraries shows that Age and KLF valence account for >75% of variance (n=4 mice each). B) GSEA of genes ranked by the bidirectional effect of KLF KD (defined as absolute loadings of PC2 from A) C Heatmap of all DEGs shared between Klf9/13 KD and Klf6/7 KD (|log2FoldChange|>1; p.adj<0.05) shows bidirectional regulation by the KLF family and developmental regulation of all shared targets. Data plotted as z-scored, regularized log-transformed counts. D) Gene expression changes in Klf6/7 KD relative to Scramble Controls at P10 are significantly negatively correlated with gene expression changes in Klf9/13 KD relative to Scramble controls at P20, especially at shared DEGs (p.adj<0.05 by Wald’s Test with BH correction). E) Bidirectional regulation of novel (Dpysl3, Tubb2b), and established (Gap43, Stmn2) targets Klf7. (Data plotted as TPM +/− standard deviation, n=4 mice) F) Representative IGV tracks of RNA-seq data at targets of Klf7 Rac3 (top) and L1cam (bottom) across treatment groups. Arrows indicate the presence of a KLF/Sp motif identified by FIMO (q<0.05).

FISH validation of bidirectional regulation of Tubb2b and Dpyls3 by KLF activators and repressors

A) Maximum intensity projections of slices taken from P20 mice receiving P0.5 i.c.v. injections of Scramble sgRNA or Klf9/13-targeting sgRNA AAV9 probed for Klf9 (A1,A4) and Tubb2b (A2,A5). Infected cells receiving sgRNA are EGFP-positive (A3,A6). Scale bars: 50µm. B) Quantification of results in A. Data plotted as average number of RNA puncta per cell scaled by cell pixel area ± s.e.m. (n=4 mice per condition, n=5 for ScrGFP Dpysl3 and Klf9; ***: p<0.001, **: p<0.005 independent samples t-test). C) Maximum intensity projections of slices taken from P10 mice receiving i.c.v. injections of Scramble sgRNA or Klf6/7-targeting sgRNA AAV9 at P0.5 probed for Tubb2b (C1,C3). Infected cells receiving sgRNA are EGFP-positive (C2,C4). Scale bars: 50µm. D) Quantification of results in C. (n=3 mice per condition, n=4 for Klf6/7 Dpysl3; **: p<0.01 independent samples t-test). Shaded shapes represent individual animals.

A model for the postnatal loss of axon growth capacity through competition between KLF paralogs

During the first week of postnatal development when Klf6 and Klf7 expression are high, the KLF family functions to activate transcription of genes with roles in axon growth including (but not limited to) Tubb2b, Dplys3, Rac3, and Gap43 by binding GC-rich regions in target gene promoters (Top). During the second and third week of postnatal development, the expression of transcriptional repressors Klf9 and Klf13 increases while Klf6 and Klf7 expression decreases, leading to displacement of KLF activators at target promoters and repression of pro-growth transcripts (bottom)

sgRNA Golden Gate Primers

sgRNA Sequences

qPCR Primers

Shared gene expression and chromatin accessibility changes in maturing layer 4 and layer 6 pyramidal neurons

A) Principal Component Analysis of RNA-Seq (A1, n=4 per condition) and ATAC-seq (A2, n=2 per condition) samples used in this analysis B) Distributions of ATAC-seq Peak Annotations for All Peaks, All DARs, Layer 4 DARs, Layer 6 DARs, and DARs identified in both cell types. C) Transcription Factor Motif Enrichment in ATAC-seq peaks overlapping the Promoter (TSS −1000/+200 bp) of shared up- (left) or down-regulated (right) genes relative to all Promoter peaks using the JASPAR 2022 Cluster Root Motifs (Fisher Exact tests with BH adjustment). Cluster 18 includes AP-1 family Transcription Factors. Cluster 27 contains all KLF/Sp Transcription Factors. Consensus motif and representative members are indicated on the right for each gene set.

Shared chromatin accessibility changes in maturing layer 4 and layer 6 pyramidal neurons

A) Heatmap of all DARs in gene Promoters identified in either layer between P2 and P30, separated into k=2 clusters. (DARs defined as |log2FoldChange|>1; p.adj<0.01 by Wald’s Test with BH correction; min(TMM)>10). Data plotted as group averages of z-scored log2(TMM). B) Transcription Factor Motif Enrichment in Gained (B1) and Lost (B2) ATAC-seq Promoter peaks (TSS −1000/+200 bp) relative to all Promoter peaks using the JASPAR 2022 Cluster Root motif set (Fisher Exact tests with BH adjustment). C) RiP- normalized coverage of average ATAC-seq signal at Promoter peaks +/−1.5kb of shared downregulated (top) or shared upregulated (bottom) DEGs in all P2 or P30 samples (Shaded region = 95% CI; n=4 libraries per group, aggregate of 2 cell types).

Activating (Klf6 and Klf7) and repressive (Klf9 and Klf13) members of the KLF family display opposing patterns of gene expression throughout the developing cortex

A) Expression of all KLF family members in major excitatory neuronal cell types of the cortex obtained by bulk RNA-seq (left, Sugino et al., 2019) and in the principal cell clusters of the mouse cortex and hippocampus obtained by scRNA-seq (right, Yao et al., 2021) B) Expression patterns of developmentally regulated KLFs between P2 and P30 in Layer 4 and Layer 6 neurons (RNA-seq, n=4 mice per group).

Klf9 and Klf13 show compensatory activity and additive regulation of target genes

A) Volcano plots of gene expression changes in Klf9 KD (A1) or Klf13 KD (A2) relative to Scramble Controls (DEGs defined as |log2FoldChange|>1; p.adj<0.05 by Wald’s Test with BH correction) B) Venn Diagrams comparing All DEGs (B1), Upregulated DEGs (B2), and Downregulated DEGs (B3) identified in Klf9, Klf13, and Klf9/13 KD samples relative to scramble controls C) Line plot of all downregulated Klf9/13 targets across all libraries. Grey lines are z-scored, regularized log-transformed RNA-seq counts of individual genes, red lines represent average of all genes +/− SEM. (***:p<0.0001, Kruskal-Wallis test with post-hoc pairwise Mann-Whitney U test) D) Transcription Factor Motif Enrichment in ATAC-seq peaks overlapping the Promoter (TSS −1000/+200 bp) of Klf9/13 targets (upregulated DEGs) relative to Promoter peaks in non-DEGs using the JASPAR 2022 CORE Motif set (Fisher Exact tests with BH adjustment).

Normal intrinsic and synaptic properties of Klf9/13 KD neurons

A) Example biocytin fill of Layer 5 pyramidal neuron (green) infected with Klf9/13-mCherry KD virus (red) at P0.5 (Scale bar = 200µm) B) Intrinsic excitability is unchanged between Klf9/13 KD neurons (N=15) and Scrambled controls (N=10). (p = 0.787; Virus::Current effect from 2-Way ANOVA for Current and Virus) C) Miniature Excitatory Postsynaptic Current (mEPSC) properties of Klf9/13 KD neurons (red, N=12) and Scrambled controls (blue, N=15) are not different (top) (Amplitude: Klf9/13-mCherry: 12.9±0.614 pA, Scr- mCherry: 12.7±0.47 pA, p=0.90, Mann-Whitney U test; Frequency: Klf9/13-mCherry: 5.85±0.76 Hz; Scr-mCherry: 5.4±42 Hz p= 0.83, Mann-Whitney U test)

Putative Klf9/13 Targets are developmentally downregulated in the maturing cortex

A) Gene expression changes in Klf9/13 KD relative to Scramble Controls at P18-20 (n=4 mice per group) relative to chromatin accessibility changes between P2 and P30 (n=4 mice per group, aggregate of 2 cell types). Only DEGs or genes with at least one Differentially Accessible Promoter peak are plotted. Points represent in individual genes, and colors indicate the direction of change in Klf9/13 KD samples. Genes with more than one Promoter peak may appear multiple times. B) Example IGV traces from P2 & P30 ATAC-seq and RNA-seq for Klf9/13 Targets Plppr1 (B1, Developmental DEG without Promoter DAR) and Dpysl3 (B2, Developmental DEG with Promoter DAR). Arrows: putative KLF/Sp binding site identified by FIMO (q<0.05)

Klf6 and Klf7 promote expression of developmentally regulated genes in the perinatal cortex

A) Transcription Factor Motif Enrichment in ATAC-seq peaks overlapping the Promoter (TSS −1000/+200 bp) of Klf6/7 P10 targets (downregulated DEGs in Clusters 1 and 2) relative to all Promoter peaks in non-DEGs using the JASPAR 2022 CORE Motif set (Fisher Exact tests with BH adjustment). B) RiP-normalized coverage of average ATAC-seq signal at Promoter peaks around DEGs identified from Klf6/7 P10 KD RNA-seq (Shaded region = 95% CI; n=4 libraries per group, aggregate of 2 cell types). C) Gene expression changes in Klf6/7 KD relative to Scramble Controls at P10-12 (n=4 mice per group) relative to chromatin accessibility changes between P2 and P30 (n=4 mice per group, aggregate of 2 cell types). Only DEGs or genes with at least one Differentially Accessible Promoter peak are plotted. Points represent in individual genes, and colors indicate the direction of change in Klf6/7 KD samples. Genes with more than one Promoter peak may appear multiple times.

Shared KLF targets are similarly expressed and developmentally regulated in layer 4 and layer 6 neurons

A) Heatmap of scored, log- normalized gene expression of 144 Shared KLF Targets in Layer 4 and Layer 6 neurons at P2 and P30 (n=4 mice per group). Candidate genes with significant axonal or neuronal

The transition from KLF Activators to Repressors constitutes a Developmental switch at shared targets

A) Shared Targets are highly enriched for the KLF/Sp Motif. Transcription Factor Motif Enrichment in ATAC-seq peaks overlapping the Promoter (TSS −1000/+200 bp) of shared targets for the root KLF/Sp motif (top, Fisher Exact Test) and the full JASPAR 2022 CORE Motif set (bottom, Fisher Exact tests with BH adjustment). Enrichment is measured relative to promoter peaks in all non-DEGs. B) Klf6/7 Targets are upregulated by Klf9/13 KD (left) and Klf9/13 Targets are downregulated by Klf6/7 KD (right). Grey lines are z-scored, averaged regularized log-transformed RNA-seq counts of individual genes and dark line represent average of all genes +/− SEM (n=4 mice per group, Kruskal-Wallis test with post-hoc pairwise Mann-Whitney U test with BH adjustment, *:p<0.01; **p<1e-6 ***: p<2e-16). C) Loss of chromatin accessibility around at Shared Targets. RiP-normalized coverage of average ATAC-seq signal at Promoter peaks around Shared Targets and unaffected control genes (left; Shaded region = 95% CI; n=4 libraries per group, aggregate of 2 cell types).

FISH validation of bidirectional regulation of Tubb2b and Dpyls3 by KLF

A) Maximum intensity projections of slices taken from P20 mice receiving i.c.v. injections of Scramble sgRNA or Klf9/13- targeting sgRNA AAV9 at P0.5 probed for Klf9 (A1,A4) and Dpysl3 (A2,A5). Infected cells receiving sgRNA are EGFP- positive (A3,A6). Scale bars: 50µm. B) Maximum intensity projections of slices taken from P10 mice receiving i.c.v. injections of Scramble sgRNA or Klf6/7-targeting sgRNA AAV9 at P0 probed for Dpysl3 (B1,B3). Infected cells receiving sgRNA are EGFP-positive (B2,B4). Scale bars: 50µm.