Cell types and clonal relations in the mouse brain revealed by single-cell and spatial transcriptomics

Abstract

The mammalian brain contains a large number of specialized cells that develop from a thin sheet of neuroepithelial progenitor cells. Recently, high throughput single-cell technologies have been used to define the molecular diversity of hundreds of cell types in the nervous system. However, the lineage relationships between mature brain cells and progenitor cells are not well understood, because transcriptomic studies do not allow insights into clonal relationships and classical fate-mapping techniques are not scalable. Here we show in vivo barcoding of early progenitor cells that enables simultaneous profiling of cell phenotypes and clonal relations in the mouse brain using single-cell and spatial transcriptomics. We reconstructed thousands of clones to uncover the existence of fate-restricted progenitor cells in the mouse hippocampal neuroepithelium and show that microglia are derived from few primitive myeloid precursors that massively expand to generate widely dispersed progeny. By coupling spatial transcriptomics with clonal barcoding, we disentangle migration patterns of clonally related cells in densely labelled tissue sections. Compared to classical fate mapping, our approach enables high-throughput dense reconstruction of cell phenotypes and clonal relations at the single-cell and tissue level in individual animals and provides an integrated approach for understanding tissue architecture.

Clonally heritable gene expression imparts a layer of diversity within cell types

“…Ultrasound check was performed at E8.5 to verify the pregnancy. Pregnant females at E9.5 of gestation were anaesthetized with isoflurane, uterine horns were exposed, each embryonic forebrain injected with 0.6µl of lentivirus corresponding to 0.94×10 6 unique cloneIDs ( 31 ) and 4-8 embryos injected per litter. Surgical procedures were limited to 30 min to maximize survival rates.…”

“…For mouse experiments, lentivirus preparations have been used as described previously ( 31 ). Briefly, plasmid libraries encoding a 30N random barcode (“cloneID”) downstream of an H2B-EGFP transgene driven by the human EF1a promoter (EF1a-H2B-EGFP-30N) were generated using Gibson assembly.…”

“…Plasmid cloneID libraries were used for virus particle production by GEG-Tech (Paris, France) and viruses with a titre of 1.27 x 10 9 TU/ml were used for all applications. A typical lentivirus preparation contained about 1.57×10 6 cloneIDs/µl with a largely uniform representation and high sequence diversity ( 31 ).…”

“…We next addressed whether our findings extend beyond human lymphocytes. For this we took advantage of TREX, a genetic barcoding approach developed in our lab that enables simultaneous clonal tracking and gene expression profiling in the mouse brain via single-cell transcriptomics ( 31 ). We delivered a lentiviral barcode library ( Fig.…”

Single cell biology—a Keystone Symposia report

“…Michael Ratz from Jonas Frisén's lab at the Karolinska Institute presented unpublished work on combining in vivo clonal tracking and scRNA-seq to understand neurogenesis in the mouse brain. 122 The approach utilizes ultrasound-guided in utero injections of complex lentivirus barcode libraries into the developing mouse brain to label progenitor cells. Ratz reconstructed thousands of clones to uncover the existence of fate-restricted progenitor cells in the early hippocampal neuroepithelium and showed that microglia are derived from few primitive myeloid precursors that massively expand to generate widely dispersed progeny.…”