Figure 1. Clstn1, Clstn2, and Clstn3 are co-expressed in overlapping neuronal populations in the mouse hippocampus, but are largely expressed in separate neuronal populations in the mouse cerebellum.
(A) Single-molecule in situ fluorescent hybridization (RNAscope) reveals that Clstn1, Clstn2 and Clstn3 exhibit largely overlapping expression patterns in the dorsal hippocampus. Representative images show sections from a mouse at P30 labeled with probes to Clstn1 (green), Clstn2 (red), and Clstn3 (magenta) and with DAPI (blue) as indicated (DG, dentate gyrus; CA1 and CA3, CA1- and CA3-regions of the hippocampus proper; mHb, medial habenula). (B & C) Different from the hippocampus, Clstn1 (green), Clstn2 (red), and Clstn3 (magenta) exhibit largely distinct, non-overlapping expression patterns in the cerebellum as visualized by single-molecule in situ hybridization (B, overview; C, expanded views of the area boxed in B; Mb, midbrain; ML, molecular layer; PCL, Purkinje cell layer; GCL, granule cell layer). Scale bars apply to all images in a set. (D) Single-cell qRT-PCR demonstrates that Purkinje cells uniquely express high levels of Clstn3 mRNAs. The cytosol of singe Purkinje, granule and basket cells in acute cerebellar slices from wild-type mice (n = 4 mice; number of cells are indicated in the graph) was aspirated via a glass pipette and subjected to qRT-PCR with primers validated in Figure 1—figure supplement 1B. mRNA levels were normalized to those of Gapdh using threshold cycle values (Ct). (E) Analyses of ribosome-associated mRNAs isolated from Purkinje cells confirms enrichment of Clstn3 expression in Purkinje cells. Ribosome-associated mRNAs were immunoprecipitated using HA-antibodies from the cerebellum of RiboTag mice that had been crossed with Pcp2-Cre mice. mRNA levels of Clstn1, Clstn2, Clstn3, Calbindin, and Pcp (Purkinje cell protein-2) were measured using qRT-PCR and normalized to the internal control of β-actin using threshold cycle values (Ct). Samples were from 4 mice. Data in D and E are means ± SEM. Statistical analyses were performed using one-way ANOVA and post-hoc Tukey tests for multiple comparisons. For D, F(8, 138) = 8.786, p < 0.000. ***denotes p < 0.001. For E, F(4,15)=33.065, p < 0.000. *denotes p < 0.05, **denotes p < 0.01, ***denotes p < 0.001.
Figure 1—figure supplement 1. Unbiased single-cell RNAseq analyses demonstrate that most Clstn3 in the cerebellum is expressed in Purkinje cells, which in turn express little Clstn1 or Clstn2, whereas other cells in cerebellum primarily express other calsyntenins (A), and validation of the qRT-PCR primers using standard curves to ensure that the qRT-PCR measurements are reliable (B-E, Clstn1, Clstn2, Clstn3, and Gapdh).
