Controlling cilia formation in mammalian embryos
Some might say it looks like a finger. Others might see a worm. Scientists in the field often liken it to an antenna. The technical name is primary cilium. This slender, microscopic appendage juts out from the surface of most cells in the human body — and yet for many years, it was completely missing from textbook illustrations.
Scientists began paying more attention to primary cilia around 2003. Primary cilia play a hugely important role in embryo development: They process signals from a family of proteins called Hedgehog that cue the patterning of the early embryo, including the formation of the neural tube.
Since then, this antenna-like organelle has become a hot topic for research, in part because defects in primary cilia can lead to a whole host of medical problems — everything from hearing loss and cleft palate to having one’s internal organs located on the wrong side of the body. But a major unanswered question is which molecular cues tell cells to make a primary cilium in the first place.
In a new paper published in Science, the researchers report that they have solved part of the mystery: Two transcription factors, SP5 and SP8, act as a kind of on-off switch for the initiation of cilium formation. In cells without a primary cilium, turning these genes on can cause them to build one.
The results have immediate and long-term implications for the understanding and treatment of diseases caused by missing or malfunctioning primary cilia — called ciliopathies — which affect 1 in 2,000 people worldwide.
Why some cell types have primary cilia and others don’t. The authors suspected it was a case of preferential disassembly — that is, cells lacking primary cilia actively promote the disassembly of the structure. In cells that make a primary cilium, perhaps that disassembly process is turned off, so the proteins can build up and form one.
If disassembly isn’t the key, they thought, then maybe the transcription factors that turn specific genes on or off is what determines whether a cell builds a primary cilium.
To get at that question, the authors first used a method called single-cell RNA sequencing (scRNAseq) to identify which of the genes involved in cilia formation and function are turned on in cell types with and without primary cilia. The scientists took advantage of the fact that most cells in the mouse embryo have primary cilia while cells from the mouse extraembryonic yolk sac do not. Using scRNAseq, they identified more than 100 such genes that are more highly expressed in ciliated cells.
Next, they wondered whether any of those genes were so-called transcription factors — proteins whose main function is turning on other genes. To answer that question, they turned to a technique called Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq), which reveals where the genome is “open” and can be “read” or transcribed.
Using that method, the scientists were able to home in on two genes that had telltale signs of being transcription factors. Further analysis revealed that these two genes, Sp5 and Sp8, were very active in cell types with a primary cilium, but inactive in cells lacking one.
The most pleasing results came when they either knocked out these genes in cells with cilia or introduced them into cells lacking cilia. Those results showed that Sp5 and Sp8 were required for primary cilium formation in the early mouse embryo. Moreover, simply overexpressing one gene, Sp8, was enough to initiate primary cilium formation in cells without them.
“If you add SP8 to extraembryonic cells, many of the cells now make cilia,” the author says. “No one’s ever seen that result before.”
The authors reasoned that these two proteins, SP5 and SP8, sit at the top of the cell’s decision tree for making primary cilia. “We see it as a big breakthrough to find the upstream transcription factors that switch the whole thing on,” the author adds.
