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Spatio-Temporal Structure of Cell Fate Decisions in Murine Neural Crest

By June 18, 2020 No Comments
Spatial gene expression of brain tissues

Spatio-Temporal Structure
of Cell Fate Decisions in
Murine Neural Crest

 

Cartana in situ sequencing sheds new
light on the cell fate decision progress
of neural crest cells by mapping the
developmental stages in space

Soldatov, R., Kaucka, M. and Kastriti,
M. E. et al. Science, 2019.

Summary
Cartana in situ sequencing (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 cells, in situ sequencing 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.

Introduction
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 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,
requiring leading-edge tools and expertise.

In an international collaboration study – recently published in Science by Soldatov, Kaucka, Kastriti et al – the molecular mechanisms of cell fate decisions of rapidly changing neural crest cells were studied in embryonic tissue samples at different developmental stages, by employing a combination of state-of-the-art methods:

– Single-cell-RNA sequencing1 and Unbiased clustering2
– RNA velocity analysis3,
– In situ sequencing by Cartana.

For the first time , the molecular logic of cell fate
cisions involved in neural crest cell development was

Results
Single-cell RNA and cluster analysis – major clusters of neural crest and neural tube development
Single-cell RNA sequencing and subsequent unbiased cluster analysis showed that neural crest cells can be transcriptionally separated from neural tube cells by a combination of robust marker genes.

Transcriptional aspects: Cluster analysis of cells from E9.5 embryos showed twelve major subdivisions of neural crest and neural tube development.

RNA velocity analysis – major directions of
cell progression
RNA velocity analysis further exposed that these
subdivisions are “bridged” by neural crest delamination
processes and sensory neurogenesis programs.
Moreover, the major neural crest cell clusters appeared
to form a continuum of cell states during development.

Neural crest cells
Neural crest 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 as well as 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.
Neural crest development consists of fast molecular
changes with complex mechanisms during migration,
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

Temporal aspects: RNA velocity analysis showed the major directions (arrows) of cell progression.

In situ sequencing by Cartana – special
segregation of distinct neural crest states

In order to validate and visualize spatio-temporal
aspects of neural crest cell development, Cartana
in situ sequencing (ISS) was applied. By mapping
32 selected marker genes on multiple sections
along the rostrocaudal axis of developing embryos
simultaneously, the major cell states and migratory
processes of neural crest 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

Spatio-temporal cell type mapping of NC subpopulations by in situ sequencing (ISS) revealed the segregated anatomical local- izations of different neural crest cell states.

Conclusion
Through these results, Soldatov, Kaucka and Kastriti et al. could –
characteristic for the transitions between early and late neural crest and subsequent cell fates arising from neural crest.
The resulting gene clusters will be a fundamental resource for other in-depth studies of neural crest biology, as well as gate-openers for further investigations of other neural crest-related processes or even neural crest-derived pathologies.


References

1. S. Picelli et al., Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9, 171-181 (2014)
2. J. Fan et al., Characterizing transcriptional heterogeneity through pathway and gene set overdispersion analysis. Nat

Methods 13, 241-244 (2016)

3. G. La Manno et al., RNA velocity of single cells. Nature 560, 494-498 (2018).

tissue samples

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