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Diagrammed above is aveoli sac formation - or sacculation.
Image credit: Barbara Treutlein
Reverse engineering the developing lung
How do embryos form the cells in our lungs, muscles, nerves and other tissues? A new process decodes the genetic instructions that enable the all-purpose cells of the embryo to multiply and transform into the many specialized cell types in the body.
The embryo begins as a glob of identical cells that change shape and function as they multiply to become the cells of our lungs, muscles, nerves and all the other specialized tissues of the body.
Now, in a feat of reverse tissue engineering, Stanford researchers have begun to unravel the complex genetic codes that allows embryonic cells to proliferate and transform into all of the specialized cells of a body that perform a myriad of biological tasks.
Using the new technique of single-cell genomic analysis, a team of interdisciplinary researchers began examining mouse embryo lung cells, choosing samples from different points in the development cycle. Their intent was to record which genes were active in each cell at each point in that cycle - and their technique of study is applicable to any type of cell.
"This lays out a playbook for how to reverse tissue engineer."
Stephen Quake, Lee Otterson Professor, School of Engineering and leader of the research team at Stanford University
Dr. Quake detailed his results in the journal Nature, co-authored along with Mark Krasnow, professor of biochemistry at the Stanford School of Medicine and Tushar Desai, assistant professor of pulmonary and critical care medicine, also at Stanford.
They used their reverse engineering approach to study the cells of the alveoli - the small balloon-like structures at the tips of the lung airways. The alveoli serve as docking stations where blood vessels receive oxygen.
Barbara Treutlein, a postdoctoral scholar in Quake's lab, together with Doug Brownfield, a postdoctoral researcher in the Krasnow lab, isolated 198 lung cells from mouse embryos at three stages of gestation: 14.5 days, 16.5 days and 18.5 days (mice are on average born at 20 days). Lung cells were also taken from fully adult mice.
After using standard enzymatic techniques to dissolve the proteins that hold lung cells together as a tissue, the scientists sorted out specific alveolar cell types to become the focus of their study. Their next step became the heart of their reverse engineering process.
In recent years, biotechnologists have used microfluid devices with such precision that they can suck a single cell out of solution and isolate it in a chamber to study its genes. DNA is in the nucleus of every cell and contains the full genome for that organism even making it possible to build an organism from a single cell. But only some of those genes are active at any given time. That's why lung cells are different than hair cells; each cell has a different set of active genes turning on its functions.
Genes direct cel activity by making or "expressing" a messenger RNA or mRNA. Each mRNA instructs the cell to make a particular protein. Cells are essentially a group of interacting proteins.
Knowing which mRNAs are active in a cell offers a lens into the function of that cell at one point in its lifetime.
Using microfluid processing, the Stanford researchers revealed for the first time precisely which genes regulate the development of lung cells at each step along their way to becomming mature alveoli.
One important finding involved identifying two important cells types at the tip of the alveoli, where the lung meets blood to perform the gas exchange that keeps us alive.
Alveolar type I cells are the flattest cells in the body. Blood cells dock alongside them to deliver oxygen or pick up get rid of carbon dioxide. The thinness of the cell is vital to facilitating this gas transfer.
Alveolar type II cells are compact and cube shaped. They secrete proteins to keep alveoli from collapsing like empty balloons, keeping open the inner space through which oxygen and carbon dioxide move.
Using single cell genomics allowed the researchers to reverse engineer the lung development process and follow how a single progenitor cell gives rise to the different cells of the lung.
The researchers had captured cells in transition from progenitor to a mature cell state, gaining crucial insights into the mechanism of alveolar cell differentiation
Although their study focused on lung cells, the technique of capturing individual cells at different stages of embryonic development and assessing gene activity through mRNA sequencing – can be used to reverse-engineer other tissues.
In addition to studying embryonic development, the technique could be used in clinical settings. For example researchers could study differences between individual cells in a tumor, improving our understanding of the stages of cancers and leading to better, more targeted therapies.
"This technology represents a quantal leap forward in our ability to apprehend the full diversity of cell types in a given population, including rare ones that could have special functions.
"Because a comprehensive molecular characterization of each type is achieved, including the signals they send and receive, a snapshot of the communication between individual cells will also emerge and may suggest attractive therapeutic targets in disease."
Tushar Desai, assistant professor, pulmonary and critical care medicine, Stanford University
The mammalian lung is a highly branched network in which the distal regions of the bronchial tree transform during development into a densely packed honeycomb of alveolar air sacs that mediate gas exchange. Although this transformation has been studied by marker expression analysis and fate-mapping, the mechanisms that control the progression of lung progenitors along distinct lineages into mature alveolar cell types are still incompletely known, in part because of the limited number of lineage markers1, 2, 3 and the effects of ensemble averaging in conventional transcriptome analysis experiments on cell populations1, 2, 3, 4, 5. Here we show that single-cell transcriptome analysis circumvents these problems and enables direct measurement of the various cell types and hierarchies in the developing lung. We used microfluidic single-cell RNA sequencing (RNA-seq) on 198 individual cells at four different stages encompassing alveolar differentiation to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We empirically classified cells into distinct groups by using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or the previous purification of cell populations. The results confirmed the basic outlines of the classical model of epithelial cell-type diversity in the distal lung and led to the discovery of many previously unknown cell-type markers, including transcriptional regulators that discriminate between the different populations. We reconstructed the molecular steps during maturation of bipotential progenitors along both alveolar lineages and elucidated the full life cycle of the alveolar type 2 cell lineage. This single-cell genomics approach is applicable to any developing or mature tissue to robustly delineate molecularly distinct cell types, define progenitors and lineage hierarchies, and identify lineage-specific regulatory factors.
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