Deletion of DLK in postmitotic glutamatergic neurons does not alter gross morphology of hippocampus.

(A) Western blot of DLK and β-actin in protein extracts of hippocampal tissue of Vglut1Cre/+;DLK(cKO)fl/fl and DLK(cKO)fl/fllittermate controls (age P60, each lane representing individual mice, N=3 mice/genotype). (B) Quantification of DLK protein level normalized to β-actin. Statistics: Unpaired t-test, *** P<0.001. (C) Confocal z-stack (max projection) images of NeuN immunostaining of coronal sections of the dorsal hippocampus in P15 and P60 mice of genotype indicated, respectively. Dashed boxes in CA1 pyramidal layers are enlarged below. Scale bar, 1,000 μm in hippocampi; 100 μm in CA1 layer. (D) Quantification of CA1 pyramidal layer thickness. Each dot represents averaged thickness from 3 sections per mouse; N≥4 mice/genotype per timepoint. Statistics: Two-way ANOVA with Holm-Sidak multiple comparison test; ns, not significant. (E) Confocal z-stack (max projection) images of Tuj1 immunostaining of hippocampus CA1, CA3, and DG regions in control and Vglut1Cre/+;DLK(cKO)fl/fl mice (age P60). Dashed outlines mark ROI (region of interest) for fluorescence intensity quantification. Scale bar, 100μm. (F,G,H) Tuj1 mean fluorescence intensity (MFI) after thresholding signals in dendritic regions in each hippocampal area. Each dot represents averaged intensity from 3 sections per mouse; N=4 control,

5 Vglut1Cre/+;DLK(cKO)fl/fl. Statistics: Unpaired t-test. ns, not significant.

Induced DLK overexpression in hippocampal glutamatergic neurons causes degeneration of CA1 neurons.

(A) Confocal z-stack (max projection) images of NeuN immunostaining of coronal sections from dorsal hippocampus in P10, P15, and P60 mice of genotype indicated. Dashed boxes mark CA1 pyramidal layers enlarged below. P60 images shown under different settings compared to P10 and P15 due to older staining. Scale bar, 1,000μm in hippocampi; 100μm in CA1 layer. (B) Quantification of CA1 pyramidal layer thickness. Data points represent averaged measurement from 3 sections per mouse, N≥4 mice/genotype at each timepoint. Statistics: Two-way ANOVA with Holm-Sidak multiple comparison test. ns, not significant; *** P<0.001; **** P<0.0001.

Differentially expressed genes revealed by RiboTag analysis of hippocampal glutamatergic neurons in DLK(cKO) and DLK(iOE) mice.

(A) Volcano plot showing RiboTag analysis in Vglut1Cre/+;H11-DLKiOE/+;Rpl22HA/+vs Vglut1Cre/+;Rpl22HA/+ (age P15). 260 genes (red) show differential expression with adjusted p-values < 0.05 in Vglut1Cre/+;H11-DLKiOE/+, compared to control; names of genes with p<1E-10 are labeled. (B) Volcano plot showing RiboTag analysis in Vglut1Cre/+;DLK(cKO)fl/fl;Rpl22HA/+vs Vglut1Cre/+;Rpl22HA/+ (age P15). 36 genes (blue) show differential expression with adjusted p-values < 0.05; names of genes with p<1E-10 are labeled. (C) Rank-rank hypergeometric overlap (RRHO) comparison of gene expression in DLK(cKO) and DLK(iOE) RiboTag datasets shows enrichment of similar genes when DLK is low or high, respectively. Color represents the -log transformed hypergeometric p-values (blue for weaker p-value, red for stronger p-value). (D,E) Gene ontology (GO) analysis of significantly up- or down-regulated genes in hippocampal glutamatergic neurons of DLK(iOE) mice compared to the control. Colors correspond to P-values; circle size represents fold enrichment for the GO term; X position shows # of genes significantly enriched in the GO term. (F) SynGO sunburst plot shows enrichment of 42 differentially expressed genes from hippocampal glutamatergic neurons of DLK(iOE) mice, with color corresponding to significance. (G,H) Pie charts show distribution of the 42 synaptic genes up- or down-regulated in DLK(iOE), respectively, in CA1, CA3, and DG in dorsal hippocampus, based on in situ data (P56) in the Allen Mouse Brain Atlas.

Stmn4 and microtubule homeostasis show dependency on the expression levels of DLK.

(A) Confocal single-slice image of RNAscope analysis of Stmn4 and Vglut1 mRNAs in hippocampal neurons. Dashed circle outlines single nuclei. Scale bar, 10μm. (B,C) Quantification of the ratio of Stmn4 to Vglut1 RNAscope puncta in same nuclei of CA1 and CA3 neurons, respectively. N= 6,3,3 mice of respective genotypes, quantified from >50 cells per genotype from 4 sections per mouse. Statistics: One way ANOVA with Dunnett’s multiple comparison test, ns, not significant; ** P<0.01; **** P<0.0001. (D-F) Confocal z-stack (max projection) images of CA1 immunostained for Tuj1, tyrosinated tubulin, and acetylated tubulin, respectively, in control and Vglut1Cre/+;H11-DLKiOE/+ mice of P15. SR: stratum radiatum. (G-H) Normalized mean fluorescence intensity (MFI) of tyrosinated and acetylated tubulin, respectively, after thresholding signals in SR in CA1 (dashed outlines on images in E-F). N=9, 6 mice, 3 sections averaged per mouse in (G); N=6, 4 mice, 3 sections averaged per mouse in (H). (I-K) Confocal z-stack (max projection) images of immunostained CA1 sections for Tuj1, tyrosinated tubulin, and acetylated tubulin, respectively, in control and Vglut1Cre/+;H11-DLKiOE/+ mice of P60. (L-M) Normalized MFI of tyrosinated and acetylated tubulin, respectively, after thresholding signal in SR in CA1 (dashed outlines on images in J-K). N=9, 9 mice, 3 sections averaged per mouse in L; N=6, 6 mice, 3 sections averaged per mouse in M. (N-P) Confocal z-stack (max projection) images of immunostained CA1 sections for Tuj1, tyrosinated tubulin, and acetylated tubulin, respectively, in control and Vglut1Cre/+;DLK(cKO)fl/flmice of P60. (Q-R) Normalized MFI for tyrosinated and acetylated tubulin, respectively, after thresholding signal in SR in CA1 (dashed outlines on images in O-P). N=5, 7 mice, 3 sections averaged per mouse in Q; N=6, 7 mice, 3 sections averaged per mouse in R. All tubulin images shown as maximum projection of z-stack. Scale bar, 10μm. In I-J, arrows point to apical dendrites with elevated immunostaining signal; arrowheads point to thin neurites with elevated signal. Statistics in (G,H,L,M,Q,R): Unpaired t-test. ns, not significant; * P<0.05.

Hippocampal dorsal CA1 glutamatergic neurons show altered synapses following increased DLK expression.

(A) Confocal single-slice images of Bassoon and Homer1 immunostaining in CA1 stratum radiatum (SR) of control and Vglut1Cre/+;DLK(cKO)fl/flmice of P60. (B-C) Quantification of Bassoon and Homer1 puncta density, respectively. (D) Quantification of co-localization of Bassoon and Homer1. (E-F) Quantification of Bassoon and Homer1 puncta size. Data points represent average values per mouse from 3 sections. N=7 control, and 8 Vglut1Cre/+;DLK(cKO)fl/flmice. Statistics: unpaired t-test or Mann-Whitney U test if not passing normality. ns, not significant. (G) Confocal single-slice images of Bassoon and Homer1 immunostaining in CA1 SR of control and Vglut1Cre/+;H11-DLKiOE/+ mice of P15. (H-I) Quantification of Bassoon and Homer1 puncta density, respectively. (J) Quantification of co-localization of Bassoon and Homer1. (K-L) Quantification of Bassoon and Homer1 puncta size. Data points represent average values per mouse from 3 sections, N=9 control, and 6 Vglut1Cre/+;H11-DLKiOE/+ mice. Statistics: unpaired t-test or Mann-Whitney U test if not passing normality. ns, not significant; ** P<0.01. Scale bars, 5μm in panel images, and 1μm in enlarged images.

DLK promotes short neurite formation in primary cultured hippocampal neurons.

(A) Confocal images of DIV2 primary hippocampal glutamatergic neurons immunostained with DLK and STMN4. Neurons with indicated genotypes are labeled by tdTomato from Cre-dependent Rosa26-tdTomato, generated from hippocampi in P1 pups from the following crosses: for control: Vglut1Cre/+ X Rosa26tdT/+; for DLKcKO: Vglut1Cre/+;DLK(cKO)fl/fl X Vglut1Cre/+;DLK(cKO)fl/fl;Rosa26tdT/+; for DLKiOE: H11-DLKiOE/iOE X Vglut1 Cre/+;Rosa26tdT/+. Orange arrows point to some of the thin neurites from neurons overexpressing DLK. Red dashes outline enlarged view of neurites. Scale bar, 10μm neuron, 1μm enlarged view. (B) Graph shows positive correlation between STMN4 immunostaining, measured as integrated density (Area X MFI) in neuronal soma, to integrated density of DLK immunostaining. N≥3 cultures/genotype, ≥60 cells/genotype. Spearman correlation r=0.7454. (C) Quantification of percentage of neurons with no, one, or more than one axon (defined by neurites longer than 90μm) in each genotype. Number of neurons: 47 from 3 Vglut1Cre (control) cultures, 49 from 3 DLK(cKO) cultures, 42 from 4 DLK(iOE) cultures. Statistics: Fisher’s exact test shows significance (P<0.0001) between the three genotypes and number of axons. Axon formation in control vs DLK(cKO): P=0.1857. Formation of multiple axons in control vs DLK(cKO): P>0.9999. Axon formation in control vs DLK(iOE): P=0.0042. Formation of multiple axons in control vs DLK(iOE): P=0.0001. (D) Quantification of number of primary neurites, which include both branches and filopodia, per neuron. Number of neurons: 55 from 4 Vglut1Cre (control) cultures, 70 from 4 DLK(cKO) cultures, 45 from 5 DLK(iOE) cultures. Statistics, Kruskal-Wallis test with Dunn’s multiple comparison test. **** P<0.0001. (E) Confocal z-stack images of tyrosinated tubulin immunostaining from DIV2 cultures of genotypes indicated, showing that filopodia structures (arrows) around the soma and axons of neurons with high expression of DLK have tyrosinated tubulin. (F) Confocal z-stack images of acetylated tubulin immunostaining from DIV2 cultures of genotypes indicated, showing that filopodia structures (arrows) around the soma and axons of neurons with high expression of DLK do not have acetylated tubulin. Asterisks indicate stable branches containing acetylated tubulin. Scale bar in E-F,10μm. Tyrosinated tubulin and acetylated tubulin staining shows saturated appearance to visualize staining in thin neurites.

Increasing DLK expression alters synapse formation in primary cultured hippocampal neurons.

(A) Confocal images of axons of DIV14 neurons of indicated genotype, co-stained with Bassoon and DLK. Neurons with indicated genotypes are labeled by tdTomato from Cre-dependent Rosa26-tdTomato generated from the following crosses: for control: Vglut1Cre/+ X Rosa26tdT/+; for DLKcKO: Vglut1Cre/+;DLK(cKO)fl/fl X Vglut1Cre/+;DLK(cKO)fl/fl;Rosa26tdT/+; for DLKiOE: H11-DLKiOE/iOE X Vglut1 Cre/+;Rosa26tdT/+. (B) Quantification of bassoon puncta density. (C) Quantification of average bassoon puncta size from individual neurons. Number of neurons: 30 from 3 Vglut1-cre (control) cultures, 41 from 3 DLK(cKO) cultures, 46 from 4 DLK(iOE) cultures. Statistics: One way ANOVA with Dunnett’s multiple comparison test. ns, not significant; * P<0.05. (D) Confocal z-stack images of DIV14 neurons of indicated genotype, labeled by Rosa26-tdTomato. Dashed boxes outline dendrites enlarged below for dendritic spines. Asterisks provide some examples of spine types; long thin with purple; thin with blue; mushroom with white; stubby with yellow. Scale bar, 10μm top, 5μm bottom. (E) Quantification of dendritic spine density. (F) Quantification of mushroom spine density. (G) Distribution of spine types. (E-G) Number of neurons: 35 from 3 Vglut1-cre (control) cultures, 31 from 3 DLK(cKO) cultures, 31 from 3 DLK(iOE) cultures. Statistics: One way ANOVA with Dunnett’s multiple comparison test. * P<0.05; ** P<0.01.