Abstract
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.
Highlights
Organizing centers secrete morphogens that specify the emergence of germ layers and the establishment of the body’s axes during embryogenesis
The efficiency of light conversion by single-cell fluorescence measurement shows that 24 h of blue-light stimulation converts 78.3% of the total cells, while controls kept in the dark, or in absence of Dox, show less than 1% of Green positive cells (Fig. 1D)
Light induced gene expression modulation was not confined to a single human embryonic stem cells (hESCs) line as another of our hESC line (RUES1, genetic background, male XY, NIHhESC09-0012) responded in the same manner (Supplementary Fig. 1G)
Summary
Organizing centers secrete morphogens that specify the emergence of germ layers and the establishment of the body’s axes during embryogenesis. 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 Gli[314–18] While these approaches have been instrumental in shaping our current understanding, they 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. 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. 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
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