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'Brain on a chip' reveals how the brain folds
Being born with a "tabula rasa" - a clean slate - in the case of the brain is something of a curse. Our brains are wrinkled like walnuts by the time we are born and babies born without these wrinkles - smooth brain syndrome - suffer from severe developmental deficiencies with a markedly reduced life expectancy.
A gene known as LIS1 is crucial for ensuring the proper placement of neurons in the developing brain. When an LIS1 gene is missing, brains fail to develop these characteristic folds. Babies with lissencephaly or 'smooth brain' are born severely mentally retarded. Research by Prof. Orly Reiner of the Institute's Molecular Genetics Department, also showed that having extra LIS1 genes causes problems as well.
Reiner continues investigating LIS1 role in the developing brain as it appears it also regulates the cytoskeleton and molecular motors within a cell. Her latest research on the physical forces causing brain wrinkles to form is reported in Nature Physics. The team describes a method they developed for growing tiny "brains on chips" from human cells, allowing them to better track physical and biological mechanisms underlying the wrinkling process.
Tiny brains grown in the lab from embryonic stem cells were pioneered in the last decade by Profs. Yoshiki Sasai in Japan and Juergen Knoblich in Austria.
Eyal Karzbrun PhD, in the Reiner lab, is a physicist by training and uses physical models to understand the behavior of elastic materials in the formation of brain wrinkles. He developed a new approach to growing organoids by limiting their growth along their vertical axis. The new organoids now have a 'pita'-shape, round and flat with a thin center. This shape enables better observation through the thin center tissue while allowing nutrients to be better supplied to all of the cells. By their second week of development, wrinkles began to appear and deepen in these organoids. The scientists could now observe mechanical instability in two places: (1) as the cytoskeleton (internal skeleton) in the center of the organoid contracts, and (2) in the nuclei of the cells near the surface as they expand. Or, to think of it another way, the outside of the 'pita' grew faster than its inside.
Folds or wrinkles in a surface result from mechanical instability - compression forces applied to some part of the material. For example, if there is uneven expansion in one part of the material, another part might be forced to fold inward to accommodate the increase in pressure.
While this achievement was impressive, Reiner was not convinced that the wrinkles in the new organoids were really modeling the folds of a developing brain. So the group grew newer organoids, this time bearing the same mutations carried by babies with lissencephaly or 'smooth brain' syndrome.
This approach using the mutated LIS1 gene grew organoids at the same proportions as the previously organoids, but developed few folds and the folds they did develop were very different in shape from normal wrinkles. Working on the assumption that differences in physical properties of the cell were responsible for these variations, Reiner's group investigated the organoid's cells using atomic force microscopy, assisted by Dr. Sidney Cohen of the Chemical Research Support Department. By elasticity, the normal cells were about twice as stiff as the mutated ones, which were basically soft. Observed Prof. Reiner: "We discovered a significant difference in the physical properties of cells in the two organoids, but we observed difference in their biological properties as well. For example, the nuclei in the centers of the mutant organoids moved more slowly, and we saw significant differences in gene expression."
The researchers plan to continue developing their approach, which they believe could lead to new insights into other brain disorders including microcephaly, epilepsy and schizophrenia.
Human brain wrinkling has been implicated in neurodevelopmental disorders and yet its origins remain unknown. Polymer gel models suggest that wrinkling emerges spontaneously due to compression forces arising during differential swelling, but these ideas have not been tested in a living system. Here, we report the appearance of surface wrinkles during the in vitro development and self-organization of human brain organoids in a microfabricated compartment that supports in situ imaging over a timescale of weeks. We observe the emergence of convolutions at a critical cell density and maximal nuclear strain, which are indicative of a mechanical instability. We identify two opposing forces contributing to differential growth: cytoskeletal contraction at the organoid core and cell-cycle-dependent nuclear expansion at the organoid perimeter. The wrinkling wavelength exhibits linear scaling with tissue thickness, consistent with balanced bending and stretching energies. Lissencephalic (smooth brain) organoids display reduced convolutions, modified scaling and a reduced elastic modulus. Although the mechanism here does not include the neuronal migration seen in vivo, it models the physics of the folding brain remarkably well. Our on-chip approach offers a means for studying the emergent properties of organoid development, with implications for the embryonic human brain.
Authors: Eyal Karzbrun, Aditya Kshirsagar, Sidney R. Cohen, Jacob H. Hanna & Orly Reiner
Also participating in this research were Prof. Yaqub Hanna, who assisted with growing the embryonic stem cells, and research student Aditya Kshirsagar in Reiner's group.
The Weizmann Institute of Science in Rehovot, Israel, is one of the world's top-ranking multidisciplinary research institutions. Noted for its wide-ranging exploration of the natural and exact sciences, the Institute is home to scientists, students, technicians and supporting staff. Institute research efforts include the search for new ways of fighting disease and hunger, examining leading questions in mathematics and computer science, probing the physics of matter and the universe, creating novel materials and developing new strategies for protecting the environment.
Prof. Orly Reiner's research is supported by the Helen and Martin Kimmel Institute for Stem Cell Research; the Nella and Leon Benoziyo Center for Neurological Diseases; the Kekst Family Institute for Medical Genetics; the Dr. Beth Rom-Rymer Stem Cell Research Fund; the Dears Foundation; Mr. and Mrs. Jack Lowenthal; the estate of David Georges Eskinazi; and the estate of Jacqueline Hodes. Prof. Reiner is the incumbent of the Bernstein-Mason Chair of Neurochemistry.
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Fluorescence images show the development of an organoid over days 3-11, in which the emergence of wrinkles is clearly seen. Image credit: Weizmann Institute.