Simultaneous profiling of cell types and lineages in neuro- and gliogenesis

The mammalian brain consists of billions of highly specialized cells and trillions of connections between them. This immense complexity arises from a single layer of neuroepithelial progenitor cells. During development each progenitor cell produces many daughter cells which transform, migrate, survive, or die, settle and connect in such a coordinated manner that complicated brain structures such as cortex and hippocampus are formed. In the last decade, great progress in single-cell technologies have led to the identification and description of many of those cells in the central nervous system. However, conventional single-cell transcriptomics does not capture a cell’s location in three-dimensional tissue space as well as its continuous development over time. The developmental and spatial path leading from a neuroepithelial progenitor to a fully differentiated cell remains largely unresolved.

Recently, a plethora of methods have emerged to reconstruct cellular lineages at the single-cell level. Single-cell lineage tracing includes marking founder cells with a type of label that is inherited to all daughter cells with each cell division and that can be read out at a later timepoint using single-cell methods. However, due to its complexity and density, in vivo single-cell lineage tracing in the central nervous system has remained challenging.

In this work, we developed methods to reconstruct lineages in thousands of murine brain cells in vivo to connect progenitors to their differentiated progeny, their spatial location, and lineage-specific gene expression.

In paper I, we establish TREX (= clonal TRacking and gene EXpression profiling using single-cell RNAseq) for in vivo random DNA barcoding of early murine progenitors through lentiviral integration. Integrated barcodes are passed on to the daughter cells and, since they are expressed as mRNA, read out together with the transcriptome of the cell. We combine TREX with spatial transcriptomics, called Space-TREX, to study the spatial distribution of related cells. With the two tools at hand, we uncover lineage relationships between early neuroepithelial and myeloid progenitors and their progeny, respectively.

Taking advantage of these data for paper II, we explore in detail the relationship between embryonic neural progenitors and adult neurogenic cells in the hippocampus. Here, we present supporting data for an early fate bias of neuroepithelial progenitors, directing them either toward neurogenic cells within the hippocampal neurogenic region or a nonneurogenic path, where they contribute to other areas within the hippocampus.

Finally, in paper III and yet unpublished results, we use our mouse brain lineage tracing data to support a finding initially made in T cells that clonally related cells share gene expression patterns and that those transcriptional patterns can be long-term heritable. With that we identify clonally heritable gene expression as a source of diversity and possibly competition among genetically identical, related cells.

List of scientific papers

I. Michael Ratz, Leonie von Berlin, Ludvig Larsson, Marcel Martin, Jakub Orzechowski Westholm, Gioele La Manno, Joakim Lundeberg and Jonas Frisén. Clonal relations in the mouse brain revealed by single-cell and spatial transcriptomics. Nature Neuroscience. 2022; 25(3):285-294.
https://doi.org/10.1038/s41593-022-01011-x

II. Leonie von Berlin, Jakub Orzechowski Westholm, Michael Ratz, Jonas Frisén. Early fate bias in neuroepithelial progenitors of hippocampal neurogenesis. Hippocampus. 2023; 33(4):391-401.
https://doi.org/10.1002/hipo.23482

III. Jeff E. Mold, Martin H. Weissman, Michael Ratz, Michael Hagemann-Jensen, Joanna Hård, Carl-Johan Eriksson, Hosein Toosi, Joseph Berghenstråhle, Leonie von Berlin, Marcel Martin, Kim Blom, Jens Lagergren, Joakim Lundeberg, Rickard Sandberg, Jakob Michaëlsson, and Jonas Frisén. Clonally heritable gene expression imparts a layer of diversity within cell types. [Submitted]