Rett syndrome: Researchers used an emerging microscopy technology to see that new neurons struggled to reach their developmental destination while using organoids to model early development.

Rett syndrome
In Rett syndrome lab models, advanced imaging reveals mired neuron migration.

Scientists at MIT’s Picower Institute for Learning and Memory used an innovative microscopy method to observe how newborn neurons struggle to reach their proper places in advanced human brain tissue models of Rett syndrome, providing new insight into how developmental deficits observed in the brains of patients with the devastating disorder may emerge.

Mutations in the gene MECP2 cause Rett syndrome, which is characterized by symptoms such as severe intellectual disability and impaired social behavior. Researchers in the lab of Mriganka Sur, Newton Professor of Neuroscience in MIT’s Department of Brain and Cognitive Sciences, grew 3D cell cultures called cerebral organoids, or mini brains, using cells from people with MECP2 mutations and compared them to otherwise identical cultures without the mutations to gain new insight into how the mutation affects the early stages of human brain development. The team, led by postdoc Murat Yildirim, then used third harmonic generation (THG) three-photon microscopy to examine the development of each type of mini brain.

THG, which Yildirim helped to pioneer in Sur’s lab with MIT mechanical engineering Professor Peter So, enables very high-resolution imaging deep into live, intact tissues without the use of chemicals to label cells. According to Yildirim, the new study, published in eLife, is the first to use THG to image organoids while leaving them virtually undisturbed. Previous organoid imaging studies necessitated the use of technologies that could not imagine all the way through the 3D tissue, or methods that necessitated the cultures being killed: either by slicing them into thin sections or by chemically clearing and labeling them.

Three-photon microscopy uses a laser, but Yildirim and So designed the lab’s microscope to have no more power than a cat toy laser pointer (less than 5 milliwatts).

“You must ensure that you are not changing or affecting neuronal physiology in any way,” Yildirim said. “You should really keep everything intact and avoid bringing anything external that could be harmful. That is why we are so concerned about power (as well as chemical labeling).”

Even at low power, they were able to obtain sufficient signals for label-free, intact imaging of fixed and live organoids. They compared their THG images to images created using more traditional chemical labeling methods to validate this.

They were able to track the migration of newborn neurons as they moved from the rim around open spaces in the minibrains (called ventricles) to the outer edge, which is directly analogous to the brain’s cortex, using the THG system. They discovered that nascent neurons in mini-brains with Rett syndrome moved slowly and in meandering paths, as opposed to faster motion in straighter lines in mini-brains without MECP2 mutation. According to Sur, the consequences of such migration deficits are consistent with what scientists, including those in his lab, have hypothesized is happening in Rett syndrome fetuses.

“We know from postmortem brains and brain imaging methods that things go wrong during brain development in Rett syndrome,” said Sur, who directs the Simons Center for the Social Brain at MIT. “We were able to directly visualize a key contributor using this method.” THG images tissues without labels because it is extremely sensitive to changes in material refractive index, according to Yildirim.

As a result, it is capable of resolving boundaries between biological structures such as blood vessels, cell membranes, and extracellular spaces. Because neural shapes change during development, the researchers were able to distinguish between the ventricular zone (the area around the ventricles where newborn neurons emerge) and the cortical plate (an area that mature neurons settle into). It was also very simple to separate the ventricles and segment them into distinct regions.

The researchers were able to see that the ventricles in Rett syndrome organoids were larger and more numerous, and the ventricular zones (the rims around the ventricles where neurons are born) were thinner. They were able to track some of the neurons making their way toward the cortex in live organoids over a few days, taking a new picture every 20 minutes, just as neurons in developing brains do. They discovered that Rett syndrome neurons were only about two-thirds as fast as non-mutated neurons. The Rett neurons’ paths were also noticeably wackier. The combination of the two differences meant that the Rett cells only got half as far.

“We now want to know how MECP2 affects genes and molecules that affect neuronal migration,” Sur explained. “We have some good guesses from screening Rett syndrome organoids that we are eager to test.” Yildirim, who will begin his own lab as an assistant professor at the Cleveland Clinic’s Lerner Research Institute in September, said the findings have raised new questions for him. He wishes to image later in organoid development in order to track the effects of the sinuous migration. He also wants to learn whether certain cell types migrate more or less, which could affect how cortical circuits work.

Yildirim also expressed his desire to continue developing THG three-photon microscopy, which he believes has the potential for fine-grained imaging in humans. It can be a significant benefit in people, particularly because the imaging method can penetrate deep into living tissue without the use of artificial labels.

The paper’s other authors include Chloe Delepine, Danielle Feldman, Vincent Pham, Stephanie Chou, Jacque Pak Kan Ip, Alexi Nott, Li-Huei Tsai, and Guo-li Ming, in addition to Yildirim, Sur, and So.

The research was funded by the National Institutes of Health, the National Science Foundation, the JPB Foundation, and the Massachusetts Life Sciences Initiative.

Source: Materials provided by Picower Institute at MIT.

Reference: DOI: 10.7554/eLife.78079

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