NEW YORK (GenomeWeb) – Independent research teams have come up with single-cell sequencing methods for following organismal development and teasing apart the targets of neurons passing along messages, respectively, in model organisms.
For the first of the studies, published online today in Nature, researchers from the Hubrecht Institute and University Medical Center in the Netherlands introduced a single-cell transcriptome sequencing method called ScarTrace — an approach that includes repaired double-strand breaks introduced by CRISPR-Cas9 gene editing. Using this approach, they demonstrated that they could follow the clonal origins and ultimate cell type fates of barcoded zebrafish cells.
"We developed ScarTrace as a new method to quantify clonal origin and cell type simultaneously at single-cell resolution," senior author Alexander van Oudenaarden, director of the Hubrecht Institute, and his colleagues wrote. "This enabled us to investigate the embryonic origin of clones found in different organs of the adult zebrafish and their cell-type commitment during development and regeneration."
The team initially added in eight tandem copies of a green fluorescent protein (GFP) transgene, making it possible to get several "scars" per cell.
These scars — small insertions or deletions introduced at break repair sites — were identified in the transcriptome by converting transcripts to complementary DNA followed by nested PCR and single-cell transcriptome sequencing using a robot-assisted protocol, the authors noted.
"Scarring starts after injecting the yolk or cell of the [zebrafish] zygote with Cas9 DNA or protein, and a single-guide RNA that targets GFP," they explained.
In the zebrafish, for example, the researchers were able to tease apart the embryonic progenitor origins for adult cells in several tissue types — from kidney marrow to the zebrafish's brain, eyes, and caudal fins.
"We envision that similar approaches will have major applications in other experimental systems, in which the matching of embryonic clonal origin to adult cell type will ultimately allow reconstruction of how the adult body is built from a single cell," authors of that study wrote.
A team from China, the US, and the UK described their own single-cell sequencing method, known as MAP-seq, for another Nature paper. That approach was compared with a more time-consuming fluorescence imaging method for following axon projections from neuronal cells to their synaptic endpoints in the mouse brain.
For that study, also appearing in Nature, the researchers focused on in 591 single cells — mouse primary visual cortex neurons — teasing apart communication networks in this brain region. In the process, they found evidence of half a dozen main projection patterns in the visual cortex.
"Our finding signals a shift away from the rather convenient idea of every neuron projecting to just one cortical area," co-first author Justus Kebschull said in a statement. Kebschull was a graduate student in co-senior author Anthony Zador's Cold Spring Harbor Laboratory lab when the research was done. He is currently a post-doctoral researcher at Stanford University.
The team did MAP-seq on more than 550 neurons barcoded with random RNA sequences, uncovering cortical and subcortical regions impinged upon by the axon projections, but got a look at the diversity of these interactions. A subset of those interactions was verified with an established — but far more time-consuming — single-cell tracing method, which was applied to 31 mouse neurons.
Because both approaches identified multiple target sites for the visual cortex neurons considered, the researchers reasoned that these interactions are likely more complex than previously appreciated.
"Our results indicate that the dominant mode of intra-cortical information transfer is not based on 'one neuron-one target area' mapping," Kebschull, Zador, and their co-authors wrote. "Instead, signals carried by individual cortical neurons are shared across a subset of target areas, and thus concurrently contribute to multiple functional pathways."