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18 | JULY 2016 | GENengnews.com | Genetic Engineering & Biotechnology News At present, RNA-seq is helping researchers take a fresh look at biological phenomena such as development, malignant transformation, and the behavior of microbial populations. Already, the new views are yielding conceptual advances. "Over the years, we have become increasingly aware that what we once treated as homogeneous populations of cells are, in fact, heterogeneous," says Alex K. Shalek, Ph.D., a core member of the Institute for Medical Engineering and Science at MIT. "In parallel, we developed a greater appre- ciation for the different cellular players that are involved in shaping systems-level phenotypes." One of the fundamental problems in dissecting the link between genotype and phenotype is that biological samples are usually complex mixtures of cells. In certain cases, such as with blood, there is a relatively good agreement as to the identity of the major cellular components. "In other cases, such as with tumors, there are unknown mixtures of differ- ent cell types and states that drive the ensemble behaviors we observe," adds Dr. Shalek. One of the promises of single-cell genomics is the possibil- ity of performing transcriptome-wide analyses of the genes expressed by each cell. By uncovering patterns in gene ex- pression and co-variation, researchers can identify what cell types are present and which pathways are active or silent. Survey the Tumor Microenvironment In a recent analysis, Dr. Shalek and colleagues used single- cell RNA-seq to profle 430 cells from fve primary glioblasto- mas. This approach unveiled considerable intratumor variabil- ity in transcriptional programs related to processes fundamen- tal for cancer biology, such as oncogene signaling, hypoxia, and proliferation. "By initially focusing on just tumor cells, we missed a lot of essential information," notes Dr. Shalek. "Non- malignant cells, such as immune infltrate or stroma, can make up a large fraction of a tumor," explains Dr. Shalek. Subsequently, Dr. Shalek's team participated in a study that was focused on characterizing the cellular microenvi- ronment of melanoma. Other study participants included Levi Garraway, M.D., Ph.D., an associate professor of medi- cine at the Dana Farber Cancer Institute, and Aviv Regev, Ph.D., a computational biologist at the Broad Institute of MIT and Harvard. "This was our frst foray toward understanding the di- versity of the cells that are implicated in a tumor," relates Dr. Shalek. During this work, Dr. Shalek and colleagues profled immune, stromal, and endothelial cells in addition to ma- lignant ones. Looking at both cancerous and noncancerous cell states provided opportunities to understand the interplay among cellular phenotypes in the tumor microenvironment. "Going forward," states Dr. Shalek, "we hope to leverage this deeper understanding to guide more effective therapies." Get to the Heart of Development "During our studies on mesoderm patterning in the early embryo, we became particularly interested in the specifcation of the cells that form the second heart feld," says Michael Kyba, Ph.D., the Carrie Ramey/CCRF Endowed Professor in Pediatric Cancer Research at the University of Minnesota. One of the features of the mammalian heart is its multi- chamber organization, an evolutionary innovation that al- lows much more effcient circulation. Compared with the single-chambered heart, which is found in less-developed species, the multichambered heart evolutionarily developed to facilitate predation. Paralleling the evolution of the mul- tichambered heart was the coordinated development of new muscle types to help meet the requirement for predatory bit- ing and for a new type of food intake. In the four-chambered human heart, the left ventricle is the ancestral part of the heart, while the atria and the right ventricle are the more evolved part and originate from the second heart feld. "The facial muscles, used for eating, also originate from those new founder cells that produce the sec- ond heart feld," elaborates Dr. Kyba. "The evolutionary in- novation that enabled predatory feeding involved specifying a new common progenitor population that gives rise to facial muscles and to the new parts of the heart." The mesoderm-promoting transcription factor MESP1 is expressed in these cells and in much of the early mesoderm. Previously, MESP1 was thought to be a heart-specifc master regulator. "Several years ago," notes Dr. Kyba, "we showed that MESP1 promotes the development of not only the heart, but also of other lineages, such as skeletal muscle and blood, If "Overall" Underwhelms, Try RNA-Seq See RNA-Seq on page 20 A t-distributed stochastic neighbor embedding (t-SNE) plot showing the clustering of diverse cell types. Each dot represents a cell and is colored according to the cell's density clustering assignment. This plot, which summarizes RNA-seq data obtained from cells within a clinical isolate, was generated at the MIT laboratory of Alex Shalek, Ph.D. OMICS Richard A. Stein, M.D., Ph.D. The contribution of individual cells to aggregate phenotypes is of increasing interest in biology, as is evident in the rise of technologies that can resolve cell-level details from the blur of population-level generalities. Among these technologies is RNA sequencing (RNA-seq), which can take snapshots of the transcriptome, capturing feeting expression profles, zooming in on single cells, and exposing cell-cell variations.