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

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.

Loss of transcriptional heterogeneity in aged human muscle stem cells

“…Moreover, similar cluster markers such as AP-1 transcription factor unit ( JUN, FOS ), COL1A1 (Collagen Type Alpha 1 Chain), SOX8 (Sry-Box Transcription Factor 8), IGFBP7 (Insulin Like Growth Factor Binding Protein 7), MX1, HSPA1A (Heat Shock Protein A, Hsp70)) or CAV1 (Calveolin-1) were found in each age group ( Figure 1-figure supplement 1B ). To compare each age group to another, we used INGEST (De Santis, Etoc, Rosado-Olivieri, & Brivanlou, 2021; Stuart et al, 2019). Unlike BBKNN or CCA (Canonical Correlation Analysis, e.g.…”

Spatio-temporal, optogenetic control of gene expression in organoids

“…Light-inducible activation of the SHH promoter is therefore sufficient to stimulate SHH transcription and exert a detectable biological effect by inducing the expression of some of its known targets. In parallel, we used the PA-Cre/Lox system to generate a stable hiPSC line that overexpresses a NeonGreen-SHH cassette upon light stimulation and doxycycline treatment (SHH-GFP for simplicity, De Santis et al, 2021, suppl. video 1).…”

“…We first developed an experimental setup, for which we provide a general blueprint and the necessary tools, which allows the following: 1) choose a gene or a gene set of interest, 2) perturb it (by activation, knock-down, or both) in a specific area of interest of a 2D or 3D cellular model and 3) measure the impact of the perturbation in the transcriptomic and physical space. To this aim, we implemented and optimized light-inducible CRISPRa-based transcription (Nihongaki et al, 2017), Tet-ON transcriptional control (Yamada et al, 2018) and Cre/Lox recombination (De Santis et al, 2021) and combined these with synthetic gene expression cassettes, including a Cas13-based knock-down module, to provide versatile tools of light-inducible gene perturbations.…”

“…Li et al, 2018) or fusing a SHH-expressing spheroid with organoids (Cederquist et al, 2019). Very recently, De Santis et al (2021) used a light-inducible Cre/Lox system to overexpress SHH in human induced pluripotent stem cells and showed that this approach can induce strong cell-autonomous and non-autonomous responses. We adopted this approach, as well as a CRISPRa-based system to activate the endogenous SHH locus, and combined it with spatial transcriptomics, by establishing a protocol for transferring a cell monolayer to a glass slide suitable for analysis by 10X Visium.…”

“…To address both the need to control organoid patterning as well as the need for temporally controlled and spatially multiplexed (“programmable”) gene expression perturbations, we developed a flexible system that allows light-inducible activation and repression of target genes, by combining optogenetic transcription (Nihongaki et al, 2017, Yamada et al, 2018, De Santis et al, 2021) with CRISPR/Cas13 knock-downs (Abudayyeh et al, 2017, Cox et al, 2017, Konerman et al, 2018). We then engineered laser scanning and digital light projection setups for live-cell photo-stimulation and imaging and were able to “print” complex patterns of gene expression onto single cells and organoids.…”