Progress on a genetic atlas of kidney development shifts to single-cell studies and the tipping points that move cells along their earliest trajectories
by Sarah Stankorb
“There are many cases where if you study populations of cells, chunks of tissue, you don’t really get the complete picture,” says Steven Potter, PhD, a researcher in Cincinnati Children’s Division of Developmental Biology.
Potter likes to get the complete picture. In late 2008, he published the world’s first virtual map of murine kidney development, which he continues to improve upon to this day. Collaborating with Bruce Aronow, PhD, Division of Biomedical Informatics, Potter and a team of scientists defined the activity of nearly the entire kidney genome during normal development. It was a one-of-a-kind atlas, groundbreaking work. But it relied upon the law of averages, pooling data from groups of cells to provide an overview for that cell type. Potter’s recent work is like moving from a standard Google Map to street view, then looking inside houses.
He is observing the smallest living unit of biology — the living cell — as it becomes differentiated.
For the initial atlas, Potter’s lab used laser capture microdissection fluorescence-activated cell sorting and microarrays and RNA sequencing to define gene expression patterns. Put simply, Potter explains, “You have 1,000 cells in a pot, and you study their gene expression patterns. You’re getting an average picture for those thousand cells that you’re looking at.” If you look at those ensemble averages or population pools, says Potter, “you miss the fine resolution differences.”
Seeing The Fine Points
Thanks to an investment of $150,000 in a Fluidigm C1 System, researchers can now isolate and reliably process individual cells for genomic analysis. Potter’s lab uses enzymes to chew apart the connections that bind groups of cells and isolate them in single cell suspensions. From there, a series of biochemical reactions allow researchers to see how each cell is expressing each of its genes. What they have found represents a paradigm shift in the basic model of kidney development.
If you take a snapshot of embryonic kidney development, clusters of epithelial cells begin looking pretty much alike, but within a day’s time, they follow different developmental pathways. “They’re going different directions, and that’s already happening, right here,” says Potter, pointing at a slide of cells. A gene expression study of that collective population of epithelial cells misses the individual story happening within each cell — what directs one cell to become a distal tubule cell and another to become a podocyte of the glomerulus.
There are two competing models of development. One, the blank slate option, imagines a single cell expressing very little, “and then it starts to turn on a few genes that reflect its developmental destiny, where it’s going to go,” Potter describes. This is the model that many accept, but Potter’s lab has found that something else altogether is happening in the kidney.
It is not solely a matter of switching on the right genes.
When Potter’s research team profiled 58 renal vesicle cells, they found that many cells were expressing genes associated with multiple cell types in the nephron. Potter explains that in a manner of speaking, “these cells haven’t made up their minds yet. And not only have they not made up their minds—but they are seriously thinking about doing more than one thing.” This is known as “multilineage priming.”
As development progresses and a cell becomes a podocyte cell, for example, more genes associated with podocyte development are turned on, and genes associated with other cell types are repressed. It’s a matter of switching on the right genes and dialing back others.
“We have very strong evidence from our single cell data that multilineage priming is the model that drives the progression of stem cells into adult differentiated cells,” says Potter.
Getting To The Cause
It remains unknown what triggers repression and activation of various gene expressions. Potter speculates that cells could be experiencing different growth factor environments, different cues from their environments, or signals that are directing them down different pathways. The challenge remains for working out the details of how that differentiation is accomplished for even one cell type, much less all the cell types within the kidney.
According to the National Kidney Foundation, some abnormality occurs in the development of the kidney or urinary tract in about one in 500 live births. But with so many development-related diseases of the kidney — ranging from renal aplasia to horseshoe kidneys — establishing how cells develop is crucial. “As we learn more about the genetic circuitry of how you make a kidney, we’ll better understand the underpinnings of those diseases,” says Potter. This knowledge, combined with the revolution in DNA sequencing technology, he adds, “will also help us better diagnose those diseases.”
Potter’s kidney research is part of a National Institute of Child Health and Human Development–funded consortium, the GenitoUrinary Development Molecular Anatomy Project (GUDMAP). His original kidney atlas was part of this broader investigation, which continues creating a genetic map of the kidneys, bladder and reproductive organs.
Potter is also collaborating on two earlier-stage studies, including the NIH-funded FaceBase, a scientific database that is compiling biological instructions to construct the middle region of the human face. He aims to use single-cell studies to define the genetics underlying normal and abnormal craniofacial development. For the similarly NIH-funded LungMAP, Potter is working with Jeffrey Whitsett, MD, in Neonatology, Perinatal and Pulmonary Biology to begin developing a single-cell molecular anatomy for the developing lung.
The work has broad implications. Potter notes that in single-cell studies, “once you get it, you can just apply it to anything… heart, lung, kidney, spleen, pancreas.” As his kidney investigation deepens and his team digs into studies of the gut, lungs and face, Potter’s lab seeks to trace the lineage of cells from the moment they differentiate and observe these basic units of life as they transform.