Discovering cell-type dynamics in the nervous system by single-cell transcriptomics

The mammalian nervous system is arguably the most intricate system known to science. At its basis lie highly specialized single cells, specifically interacting to ensure everything from normal functionality to complex behavior and cognition. For over a century, neuroscientists have been fascinated by the diversity of cell types that make up the nervous system, and have sought ever-new strategies to characterize them. With the advance of single-cell transcriptomics, particularly RNA-seq, a new toolbox has become available for molecular cell type classification. In this thesis, I will discuss the development of relevant technologies leading up to cellular taxonomy studies, the concept of cell types on a more generalized level, and focus on cell type characterization in the context of continuous, dynamic processes such as development and maturation. Further, I will present the results of two published papers and two manuscripts, as well as preliminary data from our lab’s biggest effort so far, to build an atlas of cell types across the entire nervous system.

In paper I, we describe previously uncharacterized heterogeneity in the CNS myelinating cell population, the oligodendrocytes (OL). We delineate the continuous maturation process from oligodendrocyte progenitors (OPCs), via a number of distinct stages, to mature OLs. In paper II, we use single-cell RNA-seq to explore neurons in the sympathetic nervous system, describing seven distinct types. Retrograde and developmental tracing directly associated two of the cell types with distinct functions as erector muscle neurons. Paper III describes the development and application of STRT-seq-2i, a 5’ single-cell RNAseq platform adapted to a high-throughput 9600-well plate. We discuss technical aspects, throughput and flexibility, as well as results from cortical samples of fresh mouse cells and human post mortem nuclei. In paper IV, we performed high throughput unbiased sampling of early postnatal and adult mouse dentate gyrus, a region known for postnatal and maintained adult neurogenesis. We describe distinct stages in the developmental trajectory, holding true for the early and adult neurogenesis.

Overall, this thesis aims to shed light on molecular cell-type dynamics in different contexts, as well as discuss key concepts emerging and reevaluated along with the technological advances in the field.

List of scientific papers

I. Marques S, Zeisel A, Codeluppi S, van Bruggen D, Mendanha Falcão A, Xiao L, Li H, Häring M, Hochgerner H, Romanov RA, Gyllborg D, Muñoz- Manchado AB, La Manno G, Lönnerberg P, Floriddia E, Rezayee F, Ernfors P, Arenas E, Hjerling-Leffler J, Harkany T, Richardson WD, Linnarsson S, Castelo-Branco G. Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system. Science. 2016 June, 352(6291), 1326-1369.
https://doi.org/10.1126/science.aaf6463

II. Furlan A, La Manno G, Lübke M, Häring M, Abdo H, Hochgerner H, Kupari J, Usoskin D, Airaksinen MS, Oliver G, Linnarsson S, Ernfors P. Visceral motor neuron diversity delineates a cellular basis for nipple- and pilo-erection muscle control. Nature Neuroscience. 2016 October; 19(10), 1331-1340.
https://doi.org/10.1038/nn.4376

III. Hochgerner H, Lönnerberg P, Hodge R, Mikes J, Heskol A, Hubschle H, Lin P, Picelli S, La Manno G, Ratz M, Dunne J, Husain S, Lein E, Srinivasan M, Zeisel A and Linnarsson S. STRT-seq-2i: dual-index 5ʹ single cell and nucleus RNA-seq on an addressable microwell array. [Submitted]

IV. Hochgerner H, Zeisel A, Lönnerberg P, Linnarsson S. Postnatal neurogenesis is conserved in the adult mouse dentate gyrus. [Submitted]