Complex living things are made up of collections of individual cells all working together. Understanding how these assemblages of cells communicate with each other and grow over time is critical to fixing these systems when something goes wrong—for example, in the case of cancer, when cells begin to proliferate uncontrollably and create tumors.
To achieve this goal, scientists have devised a number of tools to trace the history of cells, including their lineage relationships, in various biological systems. Early versions of these techniques worked by introducing pieces of DNA into the cells that acted as molecular "ID cards" and uniquely identified a cell and all its progeny. More sophisticated techniques were made possible with the advent of genome editing technologies, such as CRISPR/Cas9, which allowed researchers to program cells to mutate their DNA barcodes to mark a particular event in the cells' lives, track expression of a specific gene, or reflect the lineage relationships among the cells.
Most previous techniques have used DNA sequencing to detect barcodes. In these methods, a tissue sample first must be broken down for the DNA from individual cells to be extracted and sequenced. Caltech researchers have now developed a new technique that keeps tissues intact. It uses imaging technology to read out the histories of many cells within a tissue sample while the cells remain in their original locations, thus preserving the tissue's spatial organization. This is especially useful when the position of cells reveals information about their function, such as in the central nervous system and in solid tumors.
The research was a collaboration with the laboratories of Michael Elowitz, Professor of Biology and Bioengineering, Howard Hughes Medical Institute Investigator, and executive officer for biological engineering; Long Cai, Professor of Biology and Biological Engineering; and Carlos Lois, Research Professor of Biology. The work was performed as part of the Allen Discovery Center for Cell Lineage Tracing, and also supported by the National Institutes of Health. A paper describing the research appeared in the journal Nature Biotechnology on November 18.
"The behavior of any cell depends on where it is within a tissue and on its own individual history," says Elowitz. "This technology helps us reconstruct that history by using the genome as a memory storage system in which cells record their own molecular histories in a format we can visualize and interpret. This work will allow researchers to better understand the events that occur in all sorts of developmental processes. The work was a fruitful collaboration with the laboratories of Carlos Lois and Long Cai, who are experts and pioneers in neural development and biological imaging."
"To understand a biological system, it is usually not enough to analyze the cells it is made of in isolation. The spatial organization of cells, interactions between them, and their morphology are often essential for the normal function of the tissue and need to be considered in the study of disease," says Amjad Askary, postdoctoral scholar and the first author on the paper. "Using this method, we can look at the cells in their native context and read out information about their past history as well as their current state."
The key innovation of the method is to locally amplify the barcodes directly within the cells, at the endpoint of the experiment, using an enzyme produced by bacterial viruses called RNA polymerase. Because of this amplification, short barcodes that would otherwise be difficult to detect can be transformed into bright "dots" that can be easily and accurately visualized. Furthermore, the amplification method is sensitive enough to not only detect a barcode but also to distinguish it from very similar barcodes that differ at only a single position in their DNA sequence.
What is more, in this technique, the barcodes are "silent" until the scientists choose to read them out—meaning that they will not interfere with the cells' normal functions and their detection will not depend on expression by the cells' own molecular machinery. This work should now allow researchers to uniquely label cells in diverse contexts, enabling a more direct view of cellular relationships in both normal development and disease contexts.
Further, in conjunction with advances in controlled mutation of the genome, or DNA editing, the work will allow researchers to use barcodes as memory storage systems, genetically programming cells to actively record information within barcodes. This work will thus advance a new paradigm for analyzing biological systems by reading out not only the present state of each cell, but also its history, through imaging.
A paper describing the research is titled "In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription." In addition to Askary, Elowitz, Cai, and Lois, co-authors include postdoctoral scholar Luis Sánchez-Guardado, research scientist James Linton, graduate student Duncan Chadly, and senior postdoctoral scholar Mark Budde. Cai and Lois are affiliated faculty members with the Tianqiao and Chrissy Chen Institute for Neuroscience at Caltech.