Rett syndrome is a rare disorder caused by mutations in the MECP2 gene, which is characterized by symptoms including severe intellectual disability and impaired social behavior. Cerebral organoid, or minibrain, models of Rett syndrome allow researchers to study the mutation’s impact on early brain development, but imaging these organoids is difficult due to the high scattering properties of the tissue, and many methods require approaches that alter or kill the cells. Researchers from The Picower Institute for Learning and Memory at MIT have now gained new insights into neuronal migration in Rett syndrome models using a label-free, non-destructive and deeply penetrating microscopy method that enabled them to track early brain development with micron-scale resolution.
The researchers used third harmonic generation (THG) three-photon microscopy in order to image the developing organoids over time; this technique uses a low-power laser (less than 5 milliwatts) and does not require chemical labeling, which can pose the risk of damaging the cells or interfering with their functions. The THG technique is also very sensitive to changes in the refractive index of materials, enabling the researchers to resolve boundaries between biological structures such as blood vessels, cell membranes and extracellular spaces. Because neural shapes change during their development, the team was able to clearly see delineation between the ventricular zone and the cortical plate of the minibrains, as well as very easily resolve various ventricles and segment them into distinct regions. The researchers imaged both Rett syndrome cerebral organoid models and otherwise identical cultures without the MECP2 mutations in order to compare their development, imaging the samples every 20 minutes for up to four days.
The imaging revealed several differences between the Rett syndrome models and controls. In the models with the MECP2 mutation, neuronal migration was significantly slower, with the nascent neurons traveling from the ventricle zone at about two-thirds the speed of the newborn neurons from the non-Rett syndrome cultures. The mutated neurons also followed a more “torturous” and less linear path than the non-mutated neurons, covering barely half the distance in the same amount of time. Additionally, the researchers found that the ventricles of the Rett syndrome organoids were larger and more numerous, and that the ventricular zones were thinner. This study represents the first time THG three-photon microscopy has been used to image human cerebral organoids, and the insights gained from the high-resolution method can help further research into the impact of MECP2 mutations in early development. This research was published in eLife.
Video Credit: Sur Lab/MIT Picower Institute
“We know from postmortem brains and brain imaging methods that things go awry during brain development in Rett syndrome, but it has been astonishingly difficult to figure out what and why,” said corresponding author Mriganka Sur, who directs the Simons Center for the Social Brain at MIT. “This method has enabled us to directly visualize a key contributor.”
The researchers hope to continue their research into Rett syndrome by imaging organoids later in development, as well as identify which specific cell types may struggle to migrate more or less than others. The team also plans to continue advancing the THG three-photon microscopy method for fine-grained imaging of living human tissues.