Frogs, genes, and cellular antennae: Unlocking the biology of autism

CZ Biohub SF Investigator Helen Willsey uses the western clawed frog — or Xenopus — to study brain development. (Courtesy of Helen Willsey)

Over the years, researchers have identified hundreds of genes that contribute to the risk of developing autism, but the sheer number of these genes has made it challenging to parse their biological roles. Willsey’s goal is to understand how such genes affect the inner workings of the brain — and might also influence a range of non-neurological health issues commonly associated with autism — by linking genetic variations to their effect on cells during key periods of development. Recently, her work has demonstrated that once-overlooked cellular components, including tiny hair-like structures called cilia, may play critical and unexpected roles in brain development and the emergence of neurodevelopmental conditions, including autism.

“Genetics is a bedrock for investigating complex biological questions,” Willsey says. “You can come in thinking about an illness or a condition with a lot of different biases, but the genetics are going to tell you what’s actually going on. With profound autism, genetics is now pointing us to cellular components we might never have thought about otherwise.”

Clues in convergence

Early on in her career, Willsey briefly considered applying her expertise in genetics to study congenital heart disease, but instead decided to focus on the lesser-known molecular mechanisms of brain development and function — an interest shaped by her childhood relationship with her grandfather, who was diagnosed with bipolar disorder and frequently hospitalized when he was experiencing hallucinations.

“With genetics, I could see a path forward for understanding these mechanisms, not only to help people and their families, but also to reduce the stigma around these conditions,” she says.

Still, Willsey’s early interest in cardiovascular health primed her to take special notice of cases where psychiatric and neurological conditions showed up alongside problems in other systems of the body. People with profound autism, for example, are much more likely than their neurotypical peers to suffer from congenital heart disease, vision problems, and digestive issues. For Willsey, such overlaps offered important clues that she could probe further with genetic analysis.

After completing an undergraduate degree in biology at Duke University, a Ph.D. in genetics at Yale, and a year-long postdoctoral position at UC Berkeley, Willsey joined Matthew State’s lab at UCSF as a postdoctoral fellow in 2016. For about a decade, at both Yale and UCSF, State and colleagues had been leading the way in identifying spontaneous genetic mutations — mutations not inherited from either parent — linked to autism. Willsey was eager to look at these rare genetic variations to investigate what they had in common and how they could affect brain development. Because any one gene might have different functions at different stages of development, Willsey decided to look for functions that overlapped across many high-risk genes, in hopes of pinpointing key biological anomalies that might lead to autism symptoms.

For these analyses, Willsey turned to Xenopus. In addition to having thousands of genes — approximately 79% of the genome — that closely correspond to human counterparts, Xenopus has a remarkable additional advantage for research in developmental biology. At the very early two-cell stage, Xenopus embryos are already large enough to be seen with the naked eye, and each of those two cells will independently give rise to either the right side of the frog’s body or the left. This means that by introducing gene mutations to just one of those cells and allowing the embryo to develop, Willsey could easily compare a mutated half-brain to its normal, unaltered second half — a built-in experimental control.

Early in development, Xenopus embryos consist of just two large cells, each of which will independently give rise to either the right or left side of the frog’s body. This means Willsey can introduce mutations to just one half of a frog and, as the embryo develops, have a built-in control in the normal, unaltered second half. Here (left), red denotes the altered half of an embryo. In the second image (right) a mutation that affects eye pigmentation is apparent on the right but not left side of a tadpole. (Courtesy of Helen Willsey)

In 2021, Willsey’s work in Xenopus culminated in a major study published in Neuron, which reported how mutations in 10 autism-linked genes could hinder brain development. Previously, these genes were primarily believed to contribute to autism by disrupting how brain cells communicate at synapses. But Willsey’s research identified a new role for the genes in the process of neuron formation, leading to brains that didn’t achieve normal size.

In subsequent work, Willsey continued to show that other autism risk genes long thought to act primarily at the synapse had important roles in other parts of the cell as well — including in cilia.

“What we’re seeing is that our understanding of these genes is inherently incomplete,” Willsey says. “It’s becoming clearer and clearer that they’re involved in a range of processes important in normal development that researchers may not yet realize could be linked to autism.”

An overlooked cellular component

Cilia come in two main forms. Almost every cell in the body sports a single “primary” cilium, which functions as a sort-of cellular antenna, detecting and transmitting signals from the environment to the inside of the cell. “Motile” cilia, on the other hand, occur in large numbers and can facilitate the flow of fluid, such as mucus or cerebrospinal fluid, by moving back-and-forth in a coordinated whip-like motion.

Xenopus, it turns out, is an excellent model for studying cilia because these structures are present in large numbers on the frog’s skin. Like other frogs, Xenopus spend their early days as tadpoles, living in ponds, and are protected from aquatic pathogens by a fine layer of specialized mucus kept in constant motion by an army of motile cilia. Though human skin doesn’t work like this, surfaces of the human respiratory, digestive, and reproductive systems do.

It wasn’t long after Willsey first began work to map the expression of autism-related genes in Xenopus that her results began to draw her attention to cilia — again and again proteins encoded by critical genes seemed to show up in and around the structures.

“This frog’s skin is just like a playground to understand ciliary biology,” Willsey says. “When you’re trying to understand where a gene has its impact and you’re looking at Xenopus, it’s very obvious if it happens to be in cilia.”

Using Xenopus embryos and human cells, Willsey and her colleagues have discovered that a number of autism-linked genes seem to carry the instructions for making proteins that build functional cilia. In a recent preprint that examined the expression and localization patterns of 30 proteins frequently linked to autism, Willsey and her team found that 12 of them could be found in both primary and motile cilia. When her team used genetic engineering to delete one of those genes, called SYNGAP1, in Xenopus embryonic cells, the mutation caused the corresponding SynGAP protein to travel away from the cilia, leading to defects in cilia structure.

Such work is setting the stage to help researchers understand how shifts in cellular biology might contribute to the core symptoms of profound autism, like trouble using language and socializing with others. But given the diverse roles that cilia play across our bodies, it may also help explain why conditions like gastrointestinal issues and cardiac problems so often occur alongside autism as well.

Other evidence supports these connections too. For example, people with Bardet-Biedl and Joubert syndromes — conditions known to be related to cilia defects — have an elevated risk of struggling with social challenges. And another recent Willsey lab preprint examining genes that carry risk for both autism and heart disease showed that a subset of these genes relate to cilia biology.

Cilia are also critical for our ability to sense our environment. They make up the specialized receptors in our eyes, ears, and noses, for example, that let us detect light, sound, and smell, respectively. This too could contribute to a condition like profound autism, in which individuals seem to struggle to navigate their environments.

“Our research is getting to the very heart of what drives autism and related health challenges, and cilia dysfunction looks to be a very important piece of the puzzle,” Willsey says. “This is the crescendo of my lab’s work so far.”