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

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 atlas-

es, are continuously giving us new tools for the under-

standing 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 scientists to

study its molecular components in situ to learn more

about the entire biological system. By comparing the

molecular content of perturbed and unperturbed bio-

logical 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.

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 sen-

sory 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 over-

come by the latest advances in single-cell and in situ

sequencing.

International collaboration studyThe study was an international collaboration led by

- 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

C A RTA N A

the single

CARTANA

1

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 sequencing2 and Unbiased clustering3

- RNA velocity analysis4,

- In situ sequencing by Cartana .

-

cisions involved in neural crest cell development was

Results

Single-cell RNA sequencing and subsequent unbiased

cluster analysis showed that neural crest cells and

neural tube cells in different developmental stage can

be transcriptionally separated.

RNA velocity analysis – major directions of cell progressionRNA 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.

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

What has not been possible to resolve by scRNAseq,

was revealed by ISS: the NC sub-populations are

captured and mapped in different regions in the tissue

revealing their unique migration patterns.

C A RTA N A

Fig. 1 Transcriptional aspects: Cluster analysis of cells from E9.5 embryos showed twelve major subdivisions of neural crest and neural tube development.

Pre-EMT neural crest

Delaminating neural crest

Migratory progenitors

Autonomic neurons

Sensory neurons

Mesodermally-derived

mesenchyme

In an international collaboration study - recently

Fig. 2 Temporal aspects: RNA velocity analysis showed the major directions (arrows)of cell progression.

Spatio-temporal aspects: Spatial mapping of gene expression patterns by in situ se-quencing (ISS) and visualization of NC subpopulations revealed the segregated anatomicallocations of different neural crest cell states.

Single-cell RNA and cluster analysis - major clusters of neural crest and neural tube development

RNA velocity

Embryonic day 9.5

Single-cell RNA sequencing and unbiased analysis

Neural crest cell migration

Cartana in situ sequencing and cell type mapping

5

Gene expression patterns

C A RTA N A

ConclusionThrough 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

More Information

Interested to know more about Cartana in situ sequencing?

xiaoyan@cartana.se

Contact information

www.cartana.se

CARTANA AB

Nobels väg 16

SE-17165 Solna

Sweden

2. S. Picelli et al., Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9, 171-181 (2014)

3. J. Fan et al., Characterizing transcriptional heterogeneity through pathway and gene set overdispersion analysis. Nat

Methods 13, 241-244 (2016)

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

5. Sample preparation at CARTANA, sequencing and imaging in collaboration with Science for Life Laboratory, Stockholm

1. R. Soldatov et al., Spatiotemporal structure of cell fate decisions in murine neural crest. Science 364, eaas9536 (2019)