Cartana In Situ sequencing (CARTANA ISS) is a next generation cell sequencing technology that provides unique information about spatial interactions through the simultaneous sequencing of hundreds of genes, directly in tissue samples and with single cell resolution.
In a recent study of the cell fate decision progress of neural crest (NC) cells, CARTANA ISS proved to be a crucial complement to the standard methods of single cell sequencing. For the first time, the molecular logic of cell fate decisions involved in these rapidly changing cells was revealed – by the dimensions of time and space.
The ongoing mapping of complete genome sequences for an increasing number of organisms, as well as the compilation of new protein and RNA expression atlases, are continuously giving us new tools for the understanding of life on a molecular level. Although biological tissue is complex with many different cell types interacting in a three-dimensional environment, in situ sequencing (ISS) permits us to study its molecular components in situ to learn more about the entire biological system. By comparing the molecular content of perturbed and unperturbed biological systems during different stages of development, molecular components can be directly linked to their biological function. Still, the investigation of complex cell processes such as cell fate decision mechanisms can be challenging and require leading-edge tools and expertise.
In an international collaboration study, recently published in Science by Soldatov et al. (2019), the molecular mechanisms of cell fate decisions of rapidly changing NC cells were studied in embryonic tissue samples at different developmental stages by employing a combination of state-of-the-art methods:
– Single-cell-RNA sequencing2 and Unbiased clustering3
– RNA velocity analysis4,
– CARTANA ISS5
For the first time , the molecular logic of cell fate decisions involved in NC cell development was
Single-cell RNA and cluster analysis – major cluster of neural crest and neural tube development
Single-cell RNA sequencing and subsequent unbiased cluster analysis showed that NC cells can be transcriptionally separated.
RNA velocity analysis – major directions of cell progression
RNA velocity analysis further exposed that these subdivisions are “bridged” by NC delamination processes and sensory neurogenesis programs. Moreover, the major NC cell clusters appeared to form a continuum of cell states during development.
Neural crest cells
NC cells are multipotent progenitor cells that are unique to vertebrates. They develop into a variety of cell types such as pigmented cells, cartilage, bone, smooth muscle cells, peripheral glia and sensory and autonomic neurons. Understanding the cell decision progress of these versatile cells will give us a better understanding of the development of complex life forms. NC development consists of fast molecular changes with complex mechanisms during migration, making them difficult to study in detail on the single cell level. A challenge that was now possible to overcome by the latest advances in single-cell and in situ RNA sequencing.
International collaboration study
The study was an international collaboration between
• Igor Adameyko and his lab at Karolinska Institute / Medical University of Vienna
• Peter V. Kharchenko and his lab at Harvard Medical School/ Harvard Stem Cell Institute
CARTANA In Situ Sequencing – spatial segregation of distinct neural crest states
CARTANA ISS was applied in order to validate and visualize spatio-temporal aspects of NC cell development. By mapping 32 marker genes on multiple sections along the rostro-caudal axis of developing embryos simultaneously, the major cell states and migratory processes of NC development could be successfully identified.
What has not been possible to resolve by scRNAseq, was revealed by ISS: the NC sub-populations are captured in different regions in the tissue revealing their unique migration patterns.
Through these results, Soldatov et al. could, for the first time, explain the molecular logic of cell fate decision characteristic for the transitions between early and late NC and subsequent cell fates arising from NC. The resulting gene clusters will be a fundamental resource for other in-depth studies of NC biology, as well as a gate-opener for further investigations of other NC-related processes or even NC-derived pathologies.
1. Soldatov, R. et al. (2019). Spatiotemporal structure of cell fate decisions in murine neural crest. Science. 364, eaas9536.
2. Picelli, S. et al. (2014). Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc. 9, 171-181.
3. Fan, J. et al. (2016). Characterizing transcriptional heterogeneity through pathway and gene set overdispersion analysis. Nat Methods. 13, 241-244.
4. La Manno, G. et al. (2018). RNA velocity of single cells. Nature. 560, 494-498.
5. Library preparation was performed at CARTANA. In Situ Sequencing and imaging were performed in collaboration with Science for Life Laboratory in Stockholm, Sweden.