Striking Balance in Throughput and Resolution
Christophe Lancrin, PhD, leads a Research Group at the European Molecular Biology Laboratory (EMBL) in Rome, Italy. He recently spoke with Fluidigm about his 2018 eLife paper, “Single-cell transcriptomics reveals a new dynamical function of transcription factors during embryonic hematopoiesis,” describing how he used C1™ and Biomark™ HD to identify an intermediate cell population between endothelial and blood cells during endothelial to hematopoietic transition (EHT). In the publication, he also discusses using C1 for full-length mRNA sequencing to identify a new gene regulatory network involving seven key transcription factors.
Christophe Lancrin on revealing new transcription factors using C1 and Biomark
His research group at EMBL explores cell fate decisions and how endothelial cells grow blood progenitors and stem cells during embryonic development. Understanding EHT depends on identifying essential genetic regulatory mechanisms involved in the biological process. For this study, he and first author Dr. Isabelle Bergiers, PhD, set out to reveal the gene regulatory network responsible for switching off the endothelial cell fate and switching on the blood program and generating blood stem cells.
Recognizing that ultrahigh-throughput 3ʹ end RNA sequencing may be insufficient for detecting low abundant transcripts such as those coding for transcription factors, Dr. Lancrin launched his study with a hypothesis: Using targeted gene expression as a starting point, genomicists can apply highly sensitive single-cell real-time PCR technology to gain valuable insights for investigations into health and disease research.
Starting with 95 target genes associated with hematopoietic, endothelial and vascular smooth muscle cells, the team isolated embryonic endothelial cells for targeted gene expression using the Biomark HD system and assessed the expression patterns by single-cell real-time PCR to understand the process of EHT. Dr. Lancrin’s group discovered a population with endothelial and hematopoietic characteristics co-expressing seven essential transcription factors at the single-cell level.
In the eLife paper, the authors make a strong case for hypothesis-driven low-throughput single-cell sequencing employing highly sensitive full-length transcriptome analysis with the C1 platform, noting that sequencing 192 single cells was enough to reveal a new gene regulatory network involving the Runx1, Gata2, Tal1, Fli1, Lyl1, Erg and Lmo2 transcription factors. This finding did not require ultrahigh-throughput RNA sequencing analysis.
Single-cell analysis is powerful. When you work in bulk, you miss information. Working with single cells is definitely a big plus but you need to use the right technology for the right question. ”
– Christophe Lancrin, PhD, EMBL
A summary of the study
The investigators produced data suggesting that even though the proto-oncogene-encoded protein Fli1 (transcription factor Friend leukemia integration 1) initially supports the endothelial cell fate, it acquires a prohematopoietic role when co-expressed with protein Runx1 (runt-related transcription factor 1).
Bergiers et al. concluded that “this work demonstrates the power of single-cell RNA sequencing for characterizing complex transcription factor dynamics.” The eLife paper notes that “although bulk transcriptomics can reveal crucial overall gene correlations between semi-stable cellular states, it cannot resolve subtler gene interactions occurring in complex transitional states. In addition, using a bulk approach makes it difficult to infer the direct consequences on the transcriptional landscape upon which these TFs are acting. These limitations can be overcome by the use of single-cell approaches.”
While stem cells are responsible for an organism’s development of specialized cell types, understanding how they form is necessary for assimilating cellular development and regenerative medicine applications. A vital function of many species, EHT is the embryonic process wherein vascular endothelial cells develop into blood stem cells.
When Dr. Lancrin and the researchers used Biomark with the classical endothelial genes and hematopoietic genes, they realized there were three different populations in the embryonic endothelium: endothelial, hematopoietic and one in between the two. Finding dual identity in both endothelial and hematopoietic genes, they identified a cell type in transition between the two stages. Looking closely, they discovered the seven transcription factors co-expressing both endothelial and hematopoietic genes were the only population expressing them all at single-cell resolution.
To clarify the findings, the team asked whether the genetic expression was a consequence or a cause of this dual identity. Using the embryonic stem cell (ESC) differentiation model into blood cells, they created an ESC line where all these factors could be over-expressed simultaneously at the single-cell level. Using this tool, they discovered that the co-expression of these transcription factors was in fact responsible for the dual endothelial and hematopoietic identity.
After proving their hypothesis, the EMBL scientists used C1 to identify genes linking to those transcription factors, to find certain targets for them. They discovered GPR-56 to be one of the target genes. There was an increase in the expression of this gene, which happens to be involved in hematopoiesis. The Lancrin group was surprised to learn that GPR-56 expression could not explain the dual endothelial and hematopoietic identity.
“Strikingly, we saw there were two transition factors linked to the same genes but in opposite relationships,” he said. “This suggested that some transcription factors were working against each other. We proved that this was not just correlation; it was actually the cause of the dual endothelial and hematopoietic identity.”
By modifying the relative quantity of these transcription factors, the Lancrin group eventually demonstrated that these factors were in a competition, which was responsible for the dual identity. “The competition is basically between two groups of transcription factors,” he said, “one supporting the blood cell fate and the other the endothelial identity.”
For this study, the investigators needed the C1 full-length mRNA sequencing technology because of its sensitivity. Dr. Lancrin explained, “We not only needed to detect transcription factors, we had to detect a range of gene expression levels. We required higher-quality sequencing data to perform these challenging analyses.”
Potential personalized medicine implications
The EMBL group realized that the Fli1 transcription factor supporting the endothelial cell fate essentially switches activity at some point during the EHT transition. Fli1 initially supports the endothelial cell fate but following expression of Runx1—master regulator of blood cell development—Fli1 starts to support the formation of blood cells. a football analogy, Dr. Lancrin likened it to two equal teams competing against each other. If suddenly a number of players switch sides, then one team must win, and it will be the team with more players. That study was the first to provide clear insights for him. “What we found could potentially help researchers to produce blood cells more efficiently, potentially by using small molecules affecting the interaction between Fli1 and Runx1.”
Dr. Lancrin plans to continue doing single-cell transcription analysis for more investigations, such as identifying what other aspects of transcription factor activity can be regulated. The eLife paper makes it clear that despite the small number of cells, his research was impactful because of how the scientists set up and modeled the study. “Single-cell analysis is powerful,” he said. “When you work in bulk, you miss information. Working with single cells is definitely a big plus but you need to use the right technology for the right question.”
Generating embryonic stem cell-like (ESC-like) induced pluripotent stem cells (iPSCs) from fully-differentiated cell types such as skin fibroblasts was a breakthrough in regenerative medicine. Important work remains to be done to efficiently differentiate iPSC or ESC for specific blood cell progenitors like hematopoietic stem cells (HSCs), so Dr. Lancrin is focusing his research on revealing the mechanisms underlying HSC formation from endothelial cells. “Combining single-cell transcriptomics, computational biology, time-lapse microscopy and loss and gain of function experiments in vitro and in vivo, we plan to identify signaling pathways and transcriptional regulators involved in generating hematopoietic stem and progenitor cells during embryonic life,” he said. If all goes as planned, his research will lead to the development of new strategies to improve methods of blood cell generation from ESC or iPSC for regenerative medicine.
Dr. Lancrin believes his findings could potentially help researchers design more efficient approaches to generate blood cells from ESCs or iPSCs by influencing key transcription factor activity. Looking beyond hematopoiesis, investigators could apply the single-cell transcriptomics approaches from this study to gain insights about different cellular transitions such as the epithelial to mesenchymal transition occurring during development and in disease states such as cancer metastasis.
“Our computational approach for the study of transcription factor interaction will help to understand the formation of HSCs from endothelial cells,” he said.