Akanksha Jain’s research while affiliated with ETH Zurich and other places

Brain organoids enable the mechanistic study of human brain development and provide opportunities to explore self-organization in unconstrained developmental systems1, 2–3. Here we establish long-term, live light-sheet microscopy on unguided brain organoids generated from fluorescently labelled human induced pluripotent stem cells, which enables tracking of tissue morphology, cell behaviours and subcellular features over weeks of organoid development⁴. We provide a novel dual-channel, multi-mosaic and multi-protein labelling strategy combined with a computational demultiplexing approach to enable simultaneous quantification of distinct subcellular features during organoid development. We track actin, tubulin, plasma membrane, nucleus and nuclear envelope dynamics, and quantify cell morphometric and alignment changes during tissue-state transitions including neuroepithelial induction, maturation, lumenization and brain regionalization. On the basis of imaging and single-cell transcriptome modalities, we find that lumenal expansion and cell morphotype composition within the developing neuroepithelium are associated with modulation of gene expression programs involving extracellular matrix pathway regulators and mechanosensing. We show that an extrinsically provided matrix enhances lumen expansion as well as telencephalon formation, and unguided organoids grown in the absence of an extrinsic matrix have altered morphologies with increased neural crest and caudalized tissue identity. Matrix-induced regional guidance and lumen morphogenesis are linked to the WNT and Hippo (YAP1) signalling pathways, including spatially restricted induction of the WNT ligand secretion mediator (WLS) that marks the earliest emergence of non-telencephalic brain regions. Together, our work provides an inroad into studying human brain morphodynamics and supports a view that matrix-linked mechanosensing dynamics have a central role during brain regionalization.

Morphogens, secreted signaling molecules that direct cell fate and tissue development, are used to instruct neuroepithelium to differentiate towards discrete brain region identities. Neural tissues derived from pluripotent stem cells in vitro (neural organoids) provide new models for studying brain region development, however, we lack a comprehensive survey of how the developing human neuroepithelium responds to morphogen cues. Here, we produce a detailed map of morphogen-induced effects on the axial and regional specification of human neural organoids using a multiplexed single-cell transcriptomics screen. We find that the timing, concentration, and combination of morphogens strongly influence organoid cell type and regional composition, and that cell line and neural induction method strongly impact the response to a given morphogen condition. We apply concentration gradients in microfluidic chips or a range of static concentrations in multi-well plates to explore how human neuroepithelium interprets morphogen concentrations and observe similar dose-dependent induction of patterned domains in both scenarios. Altogether, we provide a detailed resource that supports the development of new regionalized neural organoid protocols and enhances our understanding of human central nervous system patterning.

Brain organoids enable the mechanistic study of human brain development, and provide opportunities to explore self-organization in unconstrained developmental systems. Here, we establish long-term, live light sheet microscopy on unguided brain organoids generated from fluorescently labeled human induced pluripotent stem cells, which enables tracking of tissue morphology, cell behaviors, and subcellular features over weeks of organoid development. We provide a novel dual-channel, multi-mosaic and multi-protein labeling strategy combined with a computational demultiplexing approach to enable simultaneous quantification of distinct subcellular features during organoid development. We track Actin, Tubulin, plasma membrane, nucleus, and nuclear envelope dynamics, and quantify cell morphometric and alignment changes during tissue state transitions including neuroepithelial induction, maturation, lumenization, and brain regionalization. Based on imaging and single-cell transcriptome modalities, we find that lumenal expansion and cell morphotype composition within the developing neuroepithelium are associated with modulation of gene expression programs involving extracellular matrix (ECM) pathway regulators and mechanosensing. We show that an extrinsically provided matrix enhances lumen expansion as well as telencephalon formation, and unguided organoids grown in the absence of an extrinsic matrix have altered morphologies with increased neural crest and caudalized tissue identity. Matrix-induced regional guidance and lumen morphogenesis are linked to the WNT and Hippo (YAP1) signaling pathways, including spatially restricted induction of the Wnt Ligand Secretion Mediator (WLS) that marks the earliest emergence of non-telencephalic brain regions. Altogether, our work provides a new inroad into studying human brain morphodynamics, and supports a view that matrix-linked mechanosensing dynamics play a central role during brain regionalization.

Self-organizing neural organoids grown from pluripotent stem cells1–3 combined with single-cell genomic technologies provide opportunities to examine gene regulatory networks underlying human brain development. Here we acquire single-cell transcriptome and accessible chromatin data over a dense time course in human organoids covering neuroepithelial formation, patterning, brain regionalization and neurogenesis, and identify temporally dynamic and brain-region-specific regulatory regions. We developed Pando—a flexible framework that incorporates multi-omic data and predictions of transcription-factor-binding sites to infer a global gene regulatory network describing organoid development. We use pooled genetic perturbation with single-cell transcriptome readout to assess transcription factor requirement for cell fate and state regulation in organoids. We find that certain factors regulate the abundance of cell fates, whereas other factors affect neuronal cell states after differentiation. We show that the transcription factor GLI3 is required for cortical fate establishment in humans, recapitulating previous research performed in mammalian model systems. We measure transcriptome and chromatin accessibility in normal or GLI3-perturbed cells and identify two distinct GLI3 regulomes that are central to telencephalic fate decisions: one regulating dorsoventral patterning with HES4/5 as direct GLI3 targets, and one controlling ganglionic eminence diversification later in development. Together, we provide a framework for how human model systems and single-cell technologies can be leveraged to reconstruct human developmental biology. A multi-omic atlas of brain organoid development facilitates the inference of an underlying gene regulatory network using the newly developed Pando framework and shows—in conjunction with perturbation experiments—that GLI3 controls forebrain fate establishment through interaction with HES4/5 regulomes.

In multicellular organisms, the specification, coordination, and compartmentalization of cell types enable the formation of complex body plans. However, some eukaryotic protists such as slime molds generate diverse and complex structures while remaining in a multinucleate syncytial state. It is unknown if different regions of these giant syncytial cells have distinct transcriptional responses to environmental encounters and if nuclei within the cell diversify into heterogeneous states. Here, we performed spatial transcriptome analysis of the slime mold Physarum polycephalum in the plasmodium state under different environmental conditions and used single-nucleus RNA-sequencing to dissect gene expression heterogeneity among nuclei. Our data identifies transcriptome regionality in the organism that associates with proliferation, syncytial substructures, and localized environmental conditions. Further, we find that nuclei are heterogenous in their transcriptional profile and may process local signals within the plasmodium to coordinate cell growth, metabolism, and reproduction. To understand how nuclei variation within the syncytium compares to heterogeneity in single-nucleus cells, we analyzed states in single Physarum amoebal cells. We observed amoebal cell states at different stages of mitosis and meiosis, and identified cytokinetic features that are specific to nuclei divisions within the syncytium. Notably, we do not find evidence for predefined transcriptomic states in the amoebae that are observed in the syncytium. Our data shows that a single-celled slime mold can control its gene expression in a region-specific manner while lacking cellular compartmentalization and suggests that nuclei are mobile processors facilitating local specialized functions. More broadly, slime molds offer the extraordinary opportunity to explore how organisms can evolve regulatory mechanisms to divide labor, specialize, balance competition with cooperation, and perform other foundational principles that govern the logic of life.

Induced pluripotent stem cell (iPSC)-derived organoids provide models to study human organ development. Single-cell transcriptomics enable highly resolved descriptions of cell states within these systems; however, approaches are needed to directly measure lineage relationships. Here we establish iTracer, a lineage recorder that combines reporter barcodes with inducible CRISPR–Cas9 scarring and is compatible with single-cell and spatial transcriptomics. We apply iTracer to explore clonality and lineage dynamics during cerebral organoid development and identify a time window of fate restriction as well as variation in neurogenic dynamics between progenitor neuron families. We also establish long-term four-dimensional light-sheet microscopy for spatial lineage recording in cerebral organoids and confirm regional clonality in the developing neuroepithelium. We incorporate gene perturbation (iTracer-perturb) and assess the effect of mosaic TSC2 mutations on cerebral organoid development. Our data shed light on how lineages and fates are established during cerebral organoid formation. More broadly, our techniques can be adapted in any iPSC-derived culture system to dissect lineage alterations during normal or perturbed development.

In multicellular organisms, the specification, coordination, and compartmentalization of cell types enable the formation of complex body plans. However, some eukaryotic protists such as slime molds generate diverse and complex structures while remaining in a multinucleated syncytial state. It is unknown if different regions of these giant syncytial cells have distinct transcriptional responses to environmental encounters, and if nuclei within the cell diversify into heterogeneous states. Here we performed spatial transcriptome analysis of the slime mold Physarum polycephalum in the plasmodium state under different environmental conditions, and used single-nucleus RNA-sequencing to dissect gene expression heterogeneity among nuclei. Our data identifies transcriptome regionality in the organism that associates with proliferation, syncytial substructures, and localized environmental conditions. Further, we find that nuclei are heterogenous in their transcriptional profile, and may process local signals within the plasmodium to coordinate cell growth, metabolism, and reproduction. To understand how nuclei variation within the syncytium compares to heterogeneity in single-nucleated cells, we analyzed states in single Physarum amoebal cells. We observed amoebal cell states at different stages of mitosis and meiosis, and identified cytokinetic features that are specific to nuclei divisions within the syncytium. Notably, we do not find evidence for predefined transcriptomic states in the amoebae that are observed in the syncytium. Our data shows that a single-celled slime mold can control its gene expression in a region-specific manner while lacking cellular compartmentalization, and suggests that nuclei are mobile processors facilitating local specialized functions. More broadly, slime molds offer the extraordinary opportunity to explore how organisms can evolve regulatory mechanisms to divide labor, specialize, balance competition with cooperation, and perform other foundational principles that govern the logic of life.

Diverse regions develop within cerebral organoids generated from human induced pluripotent stem cells (iPSCs), however it has been a challenge to understand the lineage dynamics associated with brain regionalization. Here we establish an inducible lineage recording system that couples reporter barcodes, inducible CRISPR/Cas9 scarring, and single-cell transcriptomics to analyze lineage relationships during cerebral organoid development. We infer fate-mapped whole organoid phylogenies over a scaring time course, and reconstruct progenitor-neuron lineage trees within microdissected cerebral organoid regions. We observe increased fate restriction over time, and find that iPSC clones used to initiate organoids tend to accumulate in distinct brain regions. We use lineage-coupled spatial transcriptomics to resolve lineage locations as well as confirm clonal enrichment in distinctly patterned brain regions. Using long term 4-D light sheet microscopy to temporally track nuclei in developing cerebral organoids, we link brain region clone enrichment to positions in the neuroectoderm, followed by local proliferation with limited migration during neuroepithelial formation. Our data sheds light on how lineages are established during brain organoid regionalization, and our techniques can be adapted in any iPSC-derived cell culture system to dissect lineage alterations during perturbation or in patient-specific models of disease.