Selective requirement for polycomb repressor complex 2 in the generation of specific hypothalamic neuronal subtypes

Abstract

The hypothalamus displays staggering cellular diversity, chiefly established during embryogenesis by the interplay of several signalling pathways and a battery of transcription factors. However, the contribution of epigenetic cues to hypothalamus development remains unclear. We mutated the polycomb repressor complex 2 gene Eed in the developing mouse hypothalamus, which resulted in the loss of H3K27me3, a fundamental epigenetic repressor mark. This triggered ectopic expression of posteriorly expressed regulators (e.g. Hox homeotic genes), upregulation of cell cycle inhibitors and reduced proliferation. Surprisingly, despite these effects, single cell transcriptomic analysis revealed that most neuronal subtypes were still generated in Eed mutants. However, we observed an increase in glutamatergic/GABAergic double-positive cells, as well as loss/reduction of dopamine, hypocretin and Tac2-Pax6 neurons. These findings indicate that many aspects of the hypothalamic gene regulatory flow can proceed without the key H3K27me3 epigenetic repressor mark, but points to a unique sensitivity of particular neuronal subtypes to a disrupted epigenomic landscape.

Cytocipherdetermines significantly different populations of cells in single cell RNA-seq data

“…To test the enrichment methods on an extremely difficult test-case, we utilise our previously published mouse E18.5 hypothalamus single cell RNA-seq data 8 , representing 79 different neuronal subtypes with complex gene co-expression between clusters (Figure 3A). Each cluster is annotated with the top combination of marker genes for each cluster, which were taken as the marker genes for gene enrichment analysis using the same four enrichment methods (Figure 3A-B).…”

“…Importantly, the idea of significantly different clusters in single cell data is novel, and current single cell analysis does not consider any kind of significance testing for cluster membership. Indeed, the large-scale single cell atlas efforts currently undertaken will be largely manually annotated, with analysts manually checking gene expression on over-clustered data to ensure that clustering represents distinct gene expression 3,4,8,19 . This process is likely error prone, especially in the case where clusters are distinct due to gene combinatorics (as exemplified in the E18.5 hypothalamus data).…”

“…Single cell count matrices, cell annotations, and UMAP coordinates were obtained from the original study 8 (availability detailed below). Gene expression was normalised using log-counts-per-million (log-cpm) in Scanpy .…”

“…Single cell count matrices, cell annotations, and UMAP coordinates were obtained from the original study 11 . Gene expression was normalised using log1p counts normalised to total cell counts with the median read counts observed across all cells in Scanpy .…”

“…All data analysed are publicly available and were downloaded as detailed in the relevant methods section for each case study. The E18.5 hypothalamus neuronal subtype data is available in the Gene Expression Omnibus (GEO) database at accession GSE154995 with the cell annotations and UMAP coordinates available in the supplemental data of the original paper 8 13 with the scv.datasets.pancreas() function. The human PBMC 3K data are hosted by 10X Genomics, and downloadable via wget http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pmbc3k/pbmc3k_filtered_gene_bc_matricies.tar.gz .…”