28 March

Genetic scars help understand individual cell lineages

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Scientists of the Hubrecht Institute (KNAW) in Utrecht, The Netherlands, have developed a method enabling them to investigate the embryonic origin of individual cells in an adult organism. For this, a scientific team from the research group of Alexander van Oudenaarden used a combination of techniques, based on studying afflicted genetic scars in separate cells. According to the researchers, their results could shed light on the reconstruction of the adult body from a single cell. The article was published today in Nature.

The researchers combined genome editing and single-cell RNA sequencing. For the first time, this novel method allows for the tracking of individual cells in their development towards an adult organism. They studied their method in zebrafish, a popular model system in developmental biology because of its short maturation times and the easy accessibility to developing embryos.

Barcode
One of the goals of developmental biology is understanding how a single fertilized cell gives rise to a full-grown organism. To this end, researchers from the Van Oudenaarden group developed ScarTrace. Using the genetic cutting-and-pasting abilities of CRISPR/Cas9, they introduce or remove small fragments of DNA in or from embryonic cells, creating a hereditary barcode of “scars” that is the same in each afflicted cell. The detection of these scars in adult cells allows the researchers to identify groups of cells with a common origin and reconstruct a lineage tree. Prof. dr. ir. Alexander van Oudenaarden, group leader and director at the Hubrecht Institute ‘Until now we were able to research the lineage of ten different embryonic cells, while an embryo consists of tens of thousands of cells. ScarTrace gives us the possibility to label all of these cells uniquely.’ Simultaneously to scar detection, the researchers use single-cell RNA sequencing: a technique to assess the genetic expression profile of individual cells and determine cell type. This combination of techniques allows the researchers to predict the embryonic origin of different cell types in the adult body.

Figure 1a: embryonal cells get permanent and unique labels that are transmitted to cells in the adult zebrafish. B: a label is a genetic scar that originates by injecting genetic material for the GFP protein in the embryo, along with (the gene for) Cas9. This protein builds the actual label, GFP, in the embryonal genome. (source: Alemany et al.)

Back-calculating
The researchers applied ScarTrace to three organs in the zebrafish: the blood, the brain and the tail fin. They discovered that the origin of all blood cells is based on a relatively small set of embryonic progenitors, while there are many progenitors that produce specific cell types in the eyes, brain and tail fin of the zebrafish. Additionally, they found a subset of immune cells in the tail fin with a different clonal origin than the rest of blood cells. An interesting find, says Van Oudenaarden. ‘With this complementary technique, we can observe features from cells in an adult organism and back-calculate them to the embryonic stage. That gives us more insight in the process of development.’ The high resolution of ScarTrace to detect and amplify the barcodes proved to be essential for the discovery. ‘We can track the scars with an efficiency of 90 percent’, says Anna Alemany, postdoc in the Van Oudenaarden group and co-author of the paper. ‘It’s beautiful, because this is the first time the yield is this high at the single-cell level.’

Questions
The published results raise new questions in developmental biology: is it possible to precisely determine when embryonic progenitors commit to a particular cell-type fate? Similar approaches will also be useful in research fields where understanding cell lineages is relevant, such as clonal selection in cancer research models. Van Oudenaarden: ‘With this method, we can go back in time to the “embryonic phase” of the tumor, so to speak. From there, we can study the tumor’s evolution.’ The first step for this line of research is the translation to a model that is closer to humans – the mouse. And according to Van Oudenaarden, that is a delicate matter. ‘Before we can test a cancer model in mice, we first have to incorporate all ingredients, such as the Cas9 protein, into the genome.’ The use of organoids, miniature organoids cultured from human tissue-specific stem cells, could bypass this challenge and allow researchers to work with human material faster. ‘But that also has a downside: you’re not working with a living organism. That might distort the results.’ In each case, the research of Alemany and her colleagues shows that the applications of ScarTrace are very diverse. ‘The method goes beyond development.’

Notable cooperation
This research emphasizes the multidisciplinary research conducted at the Hubrecht Institute: all four authors cooperated equally from their own expertise. Also, the article was published together with the papers of two other research groups that researched the same subject in zebrafish, but focused their research on different developmental stages. The back-to-back publishing of three articles on the same subject is unique in the scientific community. Van Oudenaarden: ‘To keep us from obstructing each other’s publication, we constantly talked about our progress.’ The groups, working at the Max Delbrück Center for Molecular Medicine in Berlin, Germany and Harvard University, Boston, USA, kept each other informed with skype calls and during congresses. ‘We even delayed the publication of our paper until we knew that the other papers were accepted too’, says Van Oudenaarden. ‘When another research group publishes their similar results a few months earlier, your year-long research could instantly lose its news value.’ He does think, however, that journals have become more willing to publish content that has been scooped. ‘Publishing remains a competitive business, especially at the bigger journals such as Nature and Science. That is why we are very glad that we have been able to coordinate the publication of these articles.’

Alexander van Oudenaarden is director and group leader at the Hubrecht Institute, professor of Quantitative Biology of Gene Regulation at the UMC Utrecht and the Faculty of Science at Utrecht University, and Oncode Investigator.

About the Hubrecht Institute
The Hubrecht Institute is a research institute focused on developmental and stem cell biology. It encompasses 20 research groups that perform fundamental and multidisciplinary research, both in healthy systems and disease models. The Hubrecht Institute is a research institute of the Royal Netherlands Academy of Arts and Sciences (KNAW), situated on the Utrecht Science Park ‘De Uithof’. Since 2008, the institute is affiliated with the University Medical Center Utrecht, advancing the translation of research to the clinic. The Hubrecht Institute has a partnership with the European Molecular Biology Laboratory (EMBL). 

About the Royal Netherlands Academy of Arts and Sciences (KNAW)
The Royal Netherlands Academy of Arts and Sciences is the forum, conscience, and voice of the arts and sciences in the Netherlands. It promotes quality in science and scholarship and strives to ensure that Dutch scholars and scientists contribute to cultural, social and economic progress. As a research organisation, the Academy is responsible for a group of fifteen outstanding national research institutes. 

About University Medical Center Utrecht
University Medical Center Utrecht (UMC Utrecht) belongs to the largest public healthcare institutions in the Netherlands and is an internationally leading healthcare provider, medical school and research institute that is exciting for its people, attractive to talent and embodies a culture of teamwork, innovation, sustainability and a competitive spirit. As a patient-centered organization, its 11,000 employees are dedicated to prevent disease, improve healthcare, develop new treatment methods and refine existing ones, with quality and patient safety as cornerstones. For more information, visit www.umcutrecht.nl. 

About Oncode Institute
Oncode is an independent institute dedicated to understanding cancer and translating research into practice. The best fundamental cancer researchers in the Netherlands come together in Oncode to bring their research discoveries into the clinic faster. Along with performing vital basic research, Oncode will specialize in cooperating with third parties to guide its scientists’ discoveries towards translational and clinical research and novel diagnostics, drugs and treatments. Oncode’s aims are to help more patients survive, to improve quality of life for those afflicted, and ultimately to cure cancer.