Self-organization of human dorsal-ventral forebrain structures by light induced SHH

ARTICLE https://doi.org/10.1038/s41467-021-26881-w OPEN Self-organization of human dorsal-ventral forebrain structures by light induced SHH 1234567890():,; Riccardo De Santis1, Fred Etoc2, Edwin A. Rosado-Olivieri1 & Ali H. Brivanlou 1✉ Organizing centers secrete morphogens that specify the emergence of germ layers and the establishment of the body’s axes during embryogenesis. While traditional experimental embryology tools have been instrumental in dissecting the molecular aspects of organizers in model systems, they are impractical in human in-vitro model systems to dissect the relationships between signaling and fate along embryonic coordinates. To systematically study human embryonic organizer centers, we devised a collection of optogenetic ePiggyBac vectors to express a photoactivatable Cre-loxP recombinase, that allows the systematic induction of organizer structures by shining blue-light on human embryonic stem cells (hESCs). We used a light stimulus to geometrically confine SHH expression in neuralizing hESCs. This led to the self-organization of mediolateral neural patterns. scRNA-seq analysis established that these structures represent the dorsal-ventral forebrain, at the end of the first month of development. Here, we show that morphogen light-stimulation is a scalable tool that induces self-organizing centers. 1 Laboratory of Stem Cell Biology and Molecular Embryology, The Rockefeller University, New York, NY, USA. 2 Center for Studies in Physics and Biology, The Rockefeller University, New York, NY, USA. ✉email: NATURE COMMUNICATIONS | (2021)12:6768 | https://doi.org/10.1038/s41467-021-26881-w | www.nature.com/naturecommunications 1 ARTICLE D NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26881-w uring embryogenesis, the central nervous system (CNS) is derived from an epithelial sheet of cells, the embryonic neural plate, that is induced in a polarized non-cell autonomous manner by a small group of cells called the Spemann organizer1. Neural induction activity of the organizer occurs by a default mechanism that is exerted through the secretion of soluble inhibitors that block both branches (SMAD1/5/8 and SMAD2/3) of the TGFb signaling pathway2–6. Dual SMADs inhibition directly converts pluripotent embryonic stem cells to the anterior neural tissue of the dorso-anterior forebrain7. Downstream of these primary neural inducing signals, highly localized and dynamic organizing centers provide multiple morphogen sources that pattern the CNS8–10. The embryonic neural tissue undergoes antero-posterior (A-P) and medio-lateral (M-L) patterning during neural plate stages11,12. As the neural tube closes, cells in the lateral part of the plate become dorsal (roof plate) and those in the midline become ventral (floor plate). Thus, M-L patterns are converted to dorso-ventral polarity (D-V). The interplay between two signaling pathways, BMP4 and SHH, both acting as morphogens guide the establishment of the M-L and D-V polarity of the embryonic neural tissue. BMP4, is expressed in the lateral edge of the neural plate, and subsequently in the dorsal neural tube, while SHH, is expressed in the ventral midline of the neural plate, and subsequently in the floor plate of the neural tube13. Classical experimental embryology approaches such as ectopic presentation of SHH ligand by grafting coated beads, embryonic explants, or morphogen-secreting cells in mouse, chick or human stem cells demonstrated that SHH activity is sufficient to induce ventral neural fates via its transcriptional effector Gli314–18. While these approaches have been instrumental in shaping our current understanding, they also suffer from technical shortcomings that have hindered a precise mapping of fate acquisition as a function of signaling dynamics in the context of early human development. For example, grafting experiments provide little control over the extent of the inductive field’s spatial limits, control of throughput, and non-specific effects due to wound healing. The development of tools that systematically control these parameters will lead to better dissection of morphogens patterning and will constitute a major step forward in experimental embryology. Optogenetic tools have been recently used to control gene expression with spatiotemporal resolution, taking advantage of different strategies19–25. This carries the potential of creating exogeneous embryonic organizer centers in model tissues for quantitative studying of embryonic induction and for creating in vitro self-organized structures that present the axial organization, that is a key landmark of embryonic development. Light modulation of signaling pathways provides flexibility and high spatial resolution over the morphogenetic stimulus26–29. Here, we have devised a collection of optogenetic ePiggyBac vectors to conditionally express a photoactivatable Cre-loxP recombinase for creating spatially restricted organizing centers that break symmetry in self-organizing hESCs. This collection is an hESCs optimized blue-light inducible split-CRE system based on the Magnets-split-CRE20. This system allows precise spatiotemporal control over the expression of a morphogen under a blue-light input. This experimental setup provides a highly quantitative and simplified method based on blue-light stimulation that can be used to induce organizing centers in in vitro cultures of hESCs. To establish proof of feasibility, we tested our tool for its ability to break symmetry in hESC-derived neural tissue to light-induce M-L polarity by inducing stripes of SHH expression as observed in the midline of the neural plate in embryo. Light induced and polarized expression of SHH during neural induction in absence of exogenous WNT modulation, led to the self-organization of a 2D in vitro human dorsal-ventral 2 forebrain structure, that include a ventral telencephalichypothalamic primordia. Polarization of morphogens using light provides a non-invasive approach to decipher the earliest events that underly symmetry breaking in the embryonic nervous system in stages of human development otherwise inaccessible for scrutiny. Results Engineering a collection of optogenetic ePiggyBac vectors. In order to provide light modulation of gene expression to human developmental studies, we re-engineered our original transposon ePiggyBac vector30,31 to conditionally express a light-inducible Cre-recombinase enzyme that takes advantage of the Magnets dimerization system (Magnet-CRE)20 (Fig. 1A). This allows for a stable integration of a blue-light dependent CRE enzyme in the genome. To avoid culturing cells in the dark, minimize leakage and to gain better control of light-sensitivity, we controlled the light-CRE enzyme using a Dox-inducible promoter and a second T2A peptide to improve the separation of its components (Fig. 1A, left panel). We paired this vector with a receiver ePiggyBac that carries two sequential ORFs (Red and Green modules) to be regulated by LoxP (...truncated)