Build a better brain with optogenetics
Summary by Katherine Rogers: De Santis, R., Etoc, F., Rosado-Olivieri, E. A. & Brivanlou, A. H. Self-organization of human dorsal-ventral forebrain structures by light induced SHH. Nat Commun 12, 6768, doi:10.1038/s41467-021-26881-w (2021).
Image credit: Gaetan Lee, Wikimedia Commons
During human embryogenesis, a fertilized oocyte gives rise to multiple organ systems, including the central nervous system (CNS). Cells are instructed to acquire specific fates by a combination of signaling molecules and antagonists produced in a localized region of the embryo dubbed an “organizer”2. The patterning processes coordinated by organizers are difficult to study directly because human fetuses develop inside the uterus over months. Therefore, researchers have created accessible model culture systems in which a layer of human embryonic stem cells (hESCs) is coaxed into mimicking embryonic development3. Although these models recapitulate features of normal development, CNS models fail to produce some cell types such as hypothalamus precursors. The Brivanlou lab speculated that introducing an artificial organizing center by activating a signaling molecule called “Sonic Hedgehog” (SHH) in a localized region might lead to a model that better reflects normal development.
To create an artificial organizer, the authors designed a method to activate expression of the SHH gene “optogenetically” with light using a modified “CRE/Lox” system. CRE is an enzyme that interacts with a DNA sequence called LoxP. If two LoxP sequences flank a gene, CRE will cut out the gene and stitch the remaining DNA together. The Brivanlou lab flanked a DNA element that inhibits downstream gene expression with LoxP sites, and placed this upstream of the SHH gene. Wherever CRE is present, the inhibitory element is removed, activating SHH expression. To confer light-mediated SHH activation, they used a modified CRE called “Magnet-CRE”4. CRE was split into two halves—which are not functional alone—and blue light-dimerizing proteins called Magnets5 were attached to each half. Blue light exposure leads to Magnet dimerization, combining the two non-functional CRE halves into a functional whole.
This optogenetic system was introduced into hESCs, which were then exposed to culture conditions leading toward CNS fates. To create an artificial organizer, a 1x1 mm subset of cells was exposed to blue light, activating SHH expression locally, similar to normal embryos. This led to activation of SHH-regulated genes that specify CNS fates in and around the light-exposed region. Importantly, different genes were activated in distinct regions that mimic the spatial organization seen in embryos, suggesting that the artificial organizer recapitulates organizer-mediated CNS patterning. Fourteen cell types that are known to occur in the developing CNS were identified, including hypothalamus precursor cells not found in previous iterations of this model. Strikingly, they identified an unknown cell type that simultaneously expresses two genes formerly thought to be mutually exclusive. This might represent a novel “hybrid” state that eventually resolves into two different cell types.
In contrast to many traditional approaches, optogenetics can offer spatially localized signaling activation that may better mimic natural organizers. Future models could employ temporal and combinatorial manipulation of several signaling molecules to more accurately recapitulate normal embryogenesis, or study the developmental consequences of different signaling inputs. In addition, artificial organizers may have applications in other fields including tissue engineering and regeneration.
References
1. De Santis, R., Etoc, F., Rosado-Olivieri, E. A. & Brivanlou, A. H. Self-organization of human dorsal-ventral forebrain structures by light induced SHH. Nat Commun 12, 6768, doi:10.1038/s41467-021-26881-w (2021).
2. Thisse, B. & Thisse, C. Formation of the vertebrate embryo: Moving beyond the Spemann organizer. Semin Cell Dev Biol 42, 94-102, doi:10.1016/j.semcdb.2015.05.007 (2015).
3. Warmflash, A., Sorre, B., Etoc, F., Siggia, E. D. & Brivanlou, A. H. A method to recapitulate early embryonic spatial patterning in human embryonic stem cells. Nat Methods 11, 847-854, doi:10.1038/nmeth.3016 (2014).
4. Kawano, F., Okazaki, R., Yazawa, M. & Sato, M. A photoactivatable Cre-loxP recombination system for optogenetic genome engineering. Nat Chem Biol 12, 1059-1064, doi:10.1038/nchembio.2205 (2016).
5. Kawano, F., Suzuki, H., Furuya, A. & Sato, M. Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins. Nat Commun 6, 6256, doi:10.1038/ncomms7256 (2015).