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. 2021 Dec 23;10:e71892. doi: 10.7554/eLife.71892

Figure 1. Combining cell-type-specific protein tagging and manual microdissection allows for precise definition of the CA1 dendritic transcriptome.

(A) Experimental design. Hippocampal slices from Camk2a-Cre-expressing conditionally tagged mouse lines were subject to microdissection in order to separate the CA1 cell bodies and CA1 neuropil layers. These layers contained material from pyramidal neurons (in which proteins of interest contain an affinity tag, green) and contaminating cell types (other colors). Microdissected compartments were subject to affinity purification in order to obtain pyramidal neuron-specific ribosomes or affinity-tagged RNA-binding proteins and bound mRNAs. In order to obtain the dendritic ribosome-bound transcriptome, TRAP-seq was performed from tagged ribosomes in the CA1 neuropil compartment. Compartment-specific cTag-PAPERCLIP was performed in order to determine mRNAs with 3′UTR isoforms that undergo differential localization, and compartment-specific FMRP regulation was determined by cTag FMRP-CLIP of the microdissected compartments. (B) Identification of dendritic mRNAs. Differential gene expression analysis was performed on bulk RNA-seq and TRAP-seq from microdissected CA1 compartments. All CA1-expressed mRNAs as shown. Colors indicate the log2 fold change (LFC)/SE (standard error, stat) from DESeq analysis. mRNAs significantly enriched in CA1 neuropil TRAP over bulk RNA-seq of the CA1 neuropil were defined as ‘dendrite-present.’ mRNAs that were also significantly enriched in CA1 neuropil TRAP when compared to cell bodies TRAP were considered to be ‘dendrite-enriched.’ In addition, only mRNAs that were enriched in CA1 neuropil TRAP in Camk-Cre-expressing RiboTag mice when compared to RiboTag mice not expressing Cre were considered. (C) Localized mRNAs are highly enriched for genes involved in synaptic signaling and synapse organization. GO analysis was performed comparing dendrite-enriched mRNAs to all mRNAs expressed in CA1 neurons. (D–G) Validation of localized mRNAs. Fluorescence in situ hybridization (FISH) was performed using the RNAscope method using probes designed against the entire mRNA of the indicated gene. Left: representative FISH image of RNAscope on the CA1 region of coronal brain sections. mRNA spots are shown in green, and DAPI staining is shown in blue. Scale bars represent 30 µm. Middle: the distance between the mRNA punctae and the cell bodies was quantitated for three representative images. Density is plotted for all collected images (red) and compared to a negative control mRNA (Snca) that was identified as sequestered in the cell bodies (gray). Right: mRNAs more than 10 µm from the cell bodies were considered to be in the neuropil. The proportion of indicated mRNAs that were found in the neuropil is plotted. A cell body-sequestered mRNA (Snca) is used as a negative control (neg). Asterisks indicate results of the Wilcoxon rank-sum test (****p<0.00001).

Figure 1.

Figure 1—figure supplement 1. CA1 compartment-specific TRAP-seq enriches for dendritic mRNAs derived specifically from Camk2a-expressing pyramidal neurons.

Figure 1—figure supplement 1.

(A) Crossing the RiboTag mice with Camk2aIIα-Cre mice allows for expression of HA-tagged Rpl22 ribosomal subunits only in the pyramidal neurons of the hippocampus. Immunostaining was performed on coronal brain sections from 8- to 10-week Camk2aIIα-RiboTag mice. Sections were stained with a pan-neuronal marker (NeuN, left panel) and for the HA-tagged ribosomal subunit RPL22 (right panel). (B) Principal component analysis of microdissected TRAP- and RNA-seq samples. Point shape is decided by sample type (TRAP or bulk RNA-seq), and color is determined by compartment (cell body layer [CB] or CA1 neuropil). (C) Gene coverage in TRAP- and bulk RNA-seq samples. Coverage of sequenced samples, as determined by Picard. (D) Contaminating cell types are not enriched in CA1 neuropil TRAP. Genes were ranked by log2 fold change/standard error (LFC/SE) in TRAP samples (CA1 neuropil TRAP/bulk RNA-seq), and marker gene sets were used for gene set enrichment analysis (GSEA). Significantly enriched (false discovery rate [FDR] < 0.05) markers are shown in red, significantly de-enriched markers are shown in blue, and not enriched cell types are shown in gray. Point size indicates -log10(FDR). (E) CA1 pyramidal markers are enriched in CA1 neuropil TRAP samples. Boxplots compare LFC (CA1 neuropil TRAP/bulk RNA-seq) of various marker genes in CA1 neuropil TRAP. Asterisks indicate significance in Wilcoxon rank-sum test (****p<0.00001).
Figure 1—figure supplement 2. Characteristics of CA1 dendritic mRNAs.

Figure 1—figure supplement 2.

(A) Some dendrite-present mRNAs were found in previous rat CA1 bulk RNA-seq experiments. Overlap of mRNAs found to be dendrite-present in CA1 neurons (as defined by enrichment in CA1 neuropil TRAP vs. bulk RNA-seq, Figure 1B) with mRNAs identified previously (Cajigas et al., 2012). (B) Previously identified localized mRNAs are enriched in CA1 dendrites. Cumulative distribution function (CDF) plots show the level of enrichment in CA1 neuropil TRAP vs. neuropil bulk RNA-seq. Plotted is the log2 fold change/standard error (CA1 neuropil TRAP/bulk RNA-seq, determined by DESeq2) for all genes (black) and the localized genes identified in rats by Cajigas et al., 2012 (cyan). Significance is determined by Kolmogorov–Smirnov test. (C) Comparison of mRNA length of all CA1-expressed mRNAs and the dendrite-enriched mRNAs. The mRNA with the highest expression for each gene in CA1 neurons (determined by TRAP; Sawicka et al., 2019) was used for this analysis. Length of dendrite-enriched and all CA1 mRNAs was compared for the full-length transcript, 5′UTRs, coding sequence (CDS), and 3′UTRs. Significance was determined by Wilcoxon rank-sum test (***p<0.0001, ****p<0.00001).