Each living creature starts its journey from a single cell, differentiating and specializing at a mind-boggling scale. Developmental information in that first cell is carried in the location and movement of maternal molecular messages (mRNA). But the systematic analysis of these fluid and fleeting processes has, until now, been hindered by the lack of methods that can gather information on both the time and location of mRNA.

Using state-of-the-art single-cell transcriptomics, scientists have identified emerging developments patterns by arranging individual cells according to the similarity of the pool of RNA molecules present in individual cells. However, this method cannot reconstruct the earliest stages of embryonic development where the information is embedded in the spatial arrangement of RNA molecules.

A new study combines spatially resolved transcriptomics and single-cell RNA labeling to perform spatio-temporal experiments on the total pool of transcripts (transcriptome) in single-celled zebrafish and frog embryos during the first few hours of development. The analysis shows blueprints for different cell types already exist at the one-cell stage in embryos.

These findings are reported in the article “Spatio-temporal mRNA tracking in the early zebrafish embryo” in the journal Nature Communications.

“We wanted to find out whether the later differences between the various cells are already partly hard-wired into the fertilized egg cell,” says Jan Philipp Junker, PhD, who heads the Quantitative Developmental Biology Lab at the Berlin Institute for Systems Biology (BIMSB) of the Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC).

Researchers had previously succeeded in finding individual genes whose RNA is localized at specific sites within one-cell zebrafish embryos. The current study now shows there are many more such genes.

The researchers measure RNA localization in one-cell stage zebrafish embryos with high spatial resolution by improving upon tomo-seq—a method in which RNA from frozen serial sections is extracted, barcoded, and sequenced.

Using the new approach the authors track the location of individual mRNA molecules and identify a class of mRNAs that are specifically positioned at the vegetal pole of the embryo—the part that gives rise to extra-embryonic structures such as the yolk sac.

The scientists compare the spatial transcriptomes of two species of African clawed frog and zebrafish to indicates that the location of a number of transcripts and sequence motifs at the vegetal pole are evolutionarily conservation in all three species.

The team also establishes a method for high-throughput single-cell RNA labeling in early zebrafish embryos that enables them to follow the fate of individual maternal mRNA until gastrulation—the first large-scale developmental milestone where the embryo separates spatially into the three germinal layers—ectoderm, mesoderm and endoderm.  This novel approach reveals that many localized transcripts are specifically transported to the primordial germ cells—transient embryonic cells that give rise to sperm and ova.

“We have discovered ten times more genes whose RNA is spatially localized in the fertilized egg cell than previously known,” explains Karoline Holler, PhD, lead author of the study. “Many of these RNA molecules are later transported into the primordial germ cells. This means that the program for subsequent cell differentiation is hard-wired into the fertilized egg cell.”

“We labeled the RNA molecules to track them over different developmental stages. This allows us to observe the RNA not only in space but also over time,” says Junker.

This RNA labeling method, called scSLAM-seq, allows the scientists to distinguish the RNA transferred to the embryo by the mother from the RNA produced by the embryo itself. The method was refined in the labs of Markus Landthaler, PhD, and Nikolaus Rajewsky, PhD, at BIMSB, enabling it to be applied in living zebrafish.

The impact of the novel RNA labeling method goes beyond the current study. “We can use it in organoids to investigate how different cell types respond to substances,” Junker says. Although not suitable for visualizing dynamic processes long-term it can be used to see changes in gene expression within five to six hours after treatment and may therefore be applied it drug discovery.

Spatial analysis is also applicable in studying diseases caused by defects in molecular transport, such as cancer or neurodegenerative diseases. “If we understand these transport processes, we may be able to identify risk factors for these diseases,” says Holler.

“There is still much work to be done before the one-cell zebrafish embryo can be used as a model system for studying human neurodegenerative diseases,” says Junker.

The next steps for the team are to focus on mechanisms of RNA localization. They are refining their method so that it can be used in other systems as well.

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