Purkinje Cell Patterning-Insights from Single-Cell Sequencing

Abstract

Despite their homogeneous appearance, Purkinje cells are remarkably diverse with respect to their molecular phenotypes, physiological properties, afferent and efferent connectivity, as well as their vulnerability to insults. Heterogeneity in Purkinje cells arises early in development, with molecularly distinct embryonic cell clusters present soon after Purkinje cell specification. Traditional methods have characterized cerebellar development and cell types, including Purkinje cell subtypes, based on knowledge of selected markers. However, recent single-cell RNA sequencing studies provide vastly increased resolution of the whole cerebellar transcriptome. Here we draw together the results of multiple single-cell transcriptomic studies in developing and adult cerebellum in both mouse and human. We describe how this detailed transcriptomic data has increased our understanding of the intricate development and function of Purkinje cells and provides first clues into features specific to human cerebellar development.

Keywords: Purkinje cell; RNA sequencing; Zebrin; cerebellum; development; stripes; transcriptomics.

Figure 1

Schematic of mouse cerebellar development. Mid-sagittal sections show the stages of mouse cerebellar development over time from E10.5 to adult. (A) At E10.5, two germinal zones are established: the VZ and the RL, which are identified by expression of PTF1A and ATOH1, respectively. (B) SKOR2+ post-mitotic Purkinje cells are generated from PTF1A+ OLIG2+ progenitors in the VZ. The VZ also gives rise to PAX2+ interneurons from PTF1A+ GSX1+ progenitors. (C) From E14.5, Purkinje cells migrate outwards from the VZ to form the PCP. The PCP is initially several layers of cells thick and lies underneath the appearing EGL. (D) In early postnatal stages, the PCP spreads to form the characteristic monolayer of Purkinje cells that exists in the adult. (E) The adult cerebellum consists of ten lobules (I–X) from anterior to posterior that are grouped into four transverse zones. The cerebellar cortex has three layers, from the outside: molecular layer, Purkinje cell layer, granule cell layer. AZ, anterior zone; CP, choroid plexus; CZ, central zone; DCN, deep cerebellar nuclei; E, embryonic day; EGL, external granule layer; NZ, nodular zone; P, postnatal day; PCP, Purkinje cell plate; PZ, posterior zone; RL, rhombic lip; VZ, ventricular zone.

Figure 2

Single-cell transcriptomics of the developing human cerebellum identifies cell type markers and highlights changes in cell populations over time. Expression of Purkinje cell markers is consistent across two transcriptomic datasets. Expression of the top 10 common Purkinje cell markers (Table 2) are shown across cell types in (A) Human [26] and (B) Mouse [25] developing cerebellar datasets. (C) The proportion of different cerebellar cell types changes over the course of human cerebellar development [33]. Only the six cell types with highest frequencies are shown for clarity. (D) The developing human cerebellum has an approximately two-fold higher peak percentage of Purkinje cells compared to mouse. This observation is consistent across human (blue circle [26], green square [33]) and mouse (orange triangle [25], pink cross [33]) RNA-seq datasets. BS, brain stem; CS Carnegie stage; DCN, deep cerebellar nuclei; E, embryonic day; eCN, excitatory cerebellar interneurons; Epend, Ependymal; iCN, inhibitory cerebellar nuclei; IN, interneuron; MLI, molecular layer interneurons; OPC, oligodendrocyte progenitor cells; P, postnatal day; pcw, post conception week; PIP, PAX2+ interneuron progenitors; UBC, unipolar brush cell; VZ, ventricular zone.

Figure 3

Purkinje cell embryonic clusters defined by imaging and birth dating studies in mice. (A) Zebrin stripes define Purkinje cell populations in the adult cerebellum. Stripes are parasagittal with most of the Z+ cells located in the posterior cerebellum. A complete description of the zebrin map is provided in [41] (B) E15.5 clusters can be defined by lineage tracing of different Purkinje cell birthdates E10.5 (red), E11.5 (orange), E12.5 (yellow) or (C) by expression of different combinations of molecular markers. Colors reflect different molecular clusters identified by [42]. Only one hemisphere of the cerebellum is shown in coronal diagrams. (D) Distribution of Purkinje cell markers at E15.5: Foxp2 is broadly expressed in most Purkinje cells. Epha4, Plcb4, Pcdh10, and Itpr1 are differentially expressed and often used as molecular markers to define distinct clusters. Foxp1 is included as an additional marker showing spatially specific expression in the anterior and lateral developing cerebellum. Images of sagittal and coronal sections from Allen Developing Mouse Brain Atlas (https://developingmouse.brain-map.org/ accessed on 7 July 2022) are shown across lateral-medial and anterior-posterior axis. Coronal sections were not available for Pcdh10 and Itpr1. A, anterior; AZ, anterior zone; CZ, central zone; E, embryonic day; L, lateral; M, medial; P, posterior; PC, Purkinje cell; PZ, posterior zone; NZ, nodular zone.

Figure 4

Mouse embryonic Purkinje cell clusters defined by single-cell transcriptomics. (A) Sepp et al. define four Purkinje cell subtypes based on transcriptomic differences and labelled by differentially expressed markers Chd9, Etv1, Foxp1, and Rorb [33]. All subtypes show broad expression of Purkinje cell markers (Foxp2, Skor2, Rora) but variable expression of genes previously used in imaging studies to define clusters (Epha4, Pcdh10, Itpr1, Plcb4) [52,53]. The different subtypes can also be defined by the combination of Ebf1 and Ebf2 expression. In addition, subtypes differ with regards to birth date, location, and predicted adult Zebrin phenotype [33]. (B) Representative images of key marker gene expression in E15.5 sagittal cerebellar sections corresponding to Purkinje cell clusters in (A). Cdh9 and Rorb are shown in medial sections and Foxp1 and Etv1 in lateral sections. ISH images taken from the Allen Developing Mouse Brain Atlas (https://developingmouse.brain-map.org/ accessed on July 2022). (C) Correlation between embryonic Purkinje cell subtypes identified by single-cell transcriptomic studies of the developing mouse cerebellum. Wizeman et al. found genes differentially expressed by five Purkinje cell subtypes [30]. The average expression of these genes is shown across the four Purkinje cell subtypes identified by Sepp et al. [33]. Expression of each gene is normalized across samples. (D) Dimension reduction analysis of the Purkinje cell cluster from a third developing mouse cerebellar scRNA-seq dataset [25] suggests similar distinct molecular identities to previously reported [30,33]. Top left: A UMAP projection at resolution 0.2 indicates three clusters within the Purkinje cells. Top center: Foxp2 is expressed across all Purkinje cells. Top right: Across UMAP_2 (y axis) cells are separated by sample date into two embryonic clusters and one postnatal cluster. The embryonic clusters show distinct expression patterns of Foxp1 and Ebf2. The postnatal cells show expression of more mature Purkinje cell markers such as Thy1 and Itpr1. Expression of the top two markers for each cluster are shown.