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Intestinal organoids are complex three-dimensional structures that mimic the cell-type composition and tissue organization of the intestine by recapitulating the self-organizing ability of cell populations derived from a single intestinal stem cell. Crucial in this process is a first symmetry-breaking event, in which only a fraction of identical cells in a symmetrical sphere differentiate into Paneth cells, which generate the stem-cell niche and lead to asymmetric structures such as the crypts and villi. Here we combine single-cell quantitative genomic and imaging approaches to characterize the development of intestinal organoids from single cells. We show that their development follows a regeneration process that is driven by transient activation of the transcriptional regulator YAP1. Cell-to-cell variability in YAP1, emerging in symmetrical spheres, initiates Notch and DLL1 activation, and drives the symmetry-breaking event and formation of the first Paneth cell. Our findings reveal how single cells exposed to a uniform growth-promoting environment have the intrinsic ability to generate emergent, self-organized behaviour that results in the formation of complex multicellular asymmetric structures. Single-cell-based imaging and sequencing approaches are used to characterize organoid development and the intestinal regeneration process, which is driven by transient activation of YAP1.

Organoids provide an accessible in vitro system to mimic the dynamics of tissue regeneration and development. However, long-term live-imaging of organoids remains challenging. Here we present an experimental and image-processing framework capable of turning long-term light-sheet imaging of intestinal organoids into digital organoids. The framework combines specific imaging optimization combined with data processing via deep learning techniques to segment single organoids, their lumen, cells and nuclei in 3D over long periods of time. By linking lineage trees with corresponding 3D segmentation meshes for each organoid, the extracted information is visualized using a web-based “Digital Organoid Viewer” tool allowing combined understanding of the multivariate and multiscale data. We also show backtracking of cells of interest, providing detailed information about their history within entire organoid contexts. Furthermore, we show cytokinesis failure of regenerative cells and that these cells never reside in the intestinal crypt, hinting at a tissue scale control on cellular fidelity. Live imaging of organoid growth remains a challenge: it requires long-term imaging of several samples simultaneously and dedicated analysis pipelines. Here the authors report an experimental and image processing framework to turn long-term light-sheet imaging of intestinal organoids into digital organoids.

Rhythmic and sequential segmentation of the growing vertebrate body relies on the segmentation clock, a multi-cellular oscillating genetic network. The clock is visible as tissue-level kinematic waves of gene expression that travel through the presomitic mesoderm (PSM) and arrest at the position of each forming segment. Here, we test how this hallmark wave pattern is driven by culturing single maturing PSM cells. We compare their cell-autonomous oscillatory and arrest dynamics to those we observe in the embryo at cellular resolution, finding similarity in the relative slowing of oscillations and arrest in concert with differentiation. This shows that cell-extrinsic signals are not required by the cells to instruct the developmental program underlying the wave pattern. We show that a cell-autonomous timing activity initiates during cell exit from the tailbud, then runs down in the anterior-ward cell flow in the PSM, thereby using elapsed time to provide positional information to the clock. Exogenous FGF lengthens the duration of the cell-intrinsic timer, indicating extrinsic factors in the embryo may regulate the segmentation clock via the timer. In sum, our work suggests that a noisy cell-autonomous, intrinsic timer drives the slowing and arrest of oscillations underlying the wave pattern, while extrinsic factors in the embryo tune this timer’s duration and precision. This is a new insight into the balance of cell-intrinsic and -extrinsic mechanisms driving tissue patterning in development.

Rhythmic and sequential segmentation of the growing vertebrate body relies on the segmentation clock, a multi-cellular oscillating genetic network. The clock is visible as tissue-level kinematic waves of gene expression that travel through the presomitic mesoderm (PSM) and arrest at the position of each forming segment. Here, we test how this hallmark wave pattern is driven by culturing single maturing PSM cells. We compare their cell-autonomous oscillatory and arrest dynamics to those we observe in the embryo at cellular resolution, finding similarity in the relative slowing of oscillations and arrest in concert with differentiation. This shows that cell-extrinsic signals are not required by the cells to instruct the developmental program underlying the wave pattern. We show that a cell-autonomous timing activity initiates during cell exit from the tailbud, then runs down in the anterior-ward cell flow in the PSM, thereby using elapsed time to provide positional information to the clock. Exogenous FGF lengthens the duration of the cell-intrinsic timer, indicating extrinsic factors in the embryo may regulate the segmentation clock via the timer. In sum, our work suggests that a noisy cell-autonomous, intrinsic timer drives the slowing and arrest of oscillations underlying the wave pattern, while extrinsic factors in the embryo tune this timer’s duration and precision. This is a new insight into the balance of cell-intrinsic and -extrinsic mechanisms driving tissue patterning in development.

Multicellular systems grow over the course of weeks from single cells to tissues or even full organisms, making live imaging challenging. To bridge spatiotemporal scales, we present an open-top dual-view and dual-illumination light-sheet microscope dedicated to live imaging of large specimens at single-cell resolution. The configuration of objectives together with a customizable multiwell mounting system combines dual view with high-throughput multiposition imaging. We use this microscope to image a wide variety of samples and highlight its capabilities to gain quantitative single-cell information in large specimens such as mature intestinal organoids and gastruloids. This work presents a highly versatile open-top, dual-view and dual-illumination light-sheet microscope for live imaging of large specimens.

An inverted light-sheet microscope enables imaging of mouse embryos from zygote to blastocyst with minimal photodamage and high resolution for automatic lineage tree reconstruction, allowing new insight into cell fate specification. Despite its importance for understanding human infertility and congenital diseases, early mammalian development has remained inaccessible to in toto imaging. We developed an inverted light-sheet microscope that enabled us to image mouse embryos from zygote to blastocyst, computationally track all cells and reconstruct a complete lineage tree of mouse pre-implantation development. We used this unique data set to show that the first cell fate specification occurs at the 16-cell stage.

Triplication of whole autosomes or large autosomal segments is detrimental to the development of a mammalian embryo. The trisomy of human chromosome (Chr) 21, known as Down's syndrome, is regularly associated with mental retardation and a variable set of other developmental anomalies. Several mouse models of Down's syndrome, triplicating 33-104 genes of Chr16, were designed in an attempt to analyze the contribution of specific orthologous genes to particular developmental features. However, a recent study challenged the concept of dosage-sensitive genes as a primary cause of an abnormal phenotype. To distinguish between the specific effects of dosage-sensitive genes and nonspecific effects of a large number of arbitrary genes, we revisited the mouse Ts43H/Ph segmental trisomy. It encompasses >310 known genes triplicated within the proximal 30 megabases (Mb) of Chr17. We refined the distal border of the trisomic segment to the interval bounded by bacterial artificial chromosomes RP23-277B13 (location 29.0 Mb) and Cbs gene (location 30.2 Mb). The Ts43H mice, viable on a mixed genetic background, exhibited spatial learning deficits analogous to those observed in Ts65Dn mice with unrelated trisomy. Quantitative analysis of the brain expression of 20 genes inside the trisomic interval and 12 genes lying outside on Chr17 revealed 1.2-fold average increase of mRNA steady-state levels of triplicated genes and 0.9-fold average down-regulation of genes beyond the border of trisomy. We propose that systemic comparisons of unrelated segmental trisomies, such as Ts65Dn and Ts43H, will elucidate the pathways leading from the triplicated sequences to the complex developmental traits.

Rhythmic and sequential segmentation of the growing vertebrate body relies on the segmentation clock, a multi-cellular oscillating genetic network. The clock is visible as tissue-level kinematic waves of gene expression that travel through the pre-somitic mesoderm (PSM) and arrest at the position of each forming segment. Here we test how this hallmark wave pattern is driven by culturing single maturing PSM cells. We compare their cell-autonomous oscillatory and arrest dynamics to those we observe in the embryo at cellular resolution, finding similarity in the relative slowing of oscillations and arrest in concert with differentiation. This shows that cell-extrinsic signals are not required by the cells to instruct the developmental program underlying the wave pattern. We show that a cell-autonomous timing activity initiates during cell exit from the tailbud, then runs down in the anterior-ward cell flow in the PSM, thereby using elapsed time to provide positional information to the clock. Exogenous FGF lengthens the duration of the cell-intrinsic timer, indicating extrinsic factors in the embryo may regulate the segmentation clock via the timer. In sum, our work suggests that a noisy cell-autonomous, intrinsic timer drives the slowing and arrest of oscillations underlying the wave pattern, while extrinsic factors in the embryo tune this timer’s duration and precision. This is a new insight into the balance of cell-intrinsic and -extrinsic mechanisms driving tissue patterning in development.

We present an experimental and image processing framework capable of turning long term light-sheet imaging of intestinal organoids into digital organoids. The framework takes advantage of specific imaging optimization on the experimental side together with data processing using a combination of existing deep learning techniques to faithfully segment single organoids, their lumen, cells and nuclei in 3D and over long periods of time. In parallel, large lineage trees for each organoid are predicted and corrected to iteratively improve the tracking and segmentation performances over time. To visualize all the extracted information, we developed a web-based Digital Organoid Viewer that allows a unique understanding of the multivariate and multiscale data by linking lineage trees with the corresponding 3D segmentation meshes. We also backtracked single cells of interest after fixation obtaining detailed information about their history within the entire organoid context. Furthermore, we show nuclei merging events that arise from cytokinesis failure and that these polyploid never reside in the intestinal crypt, hinting at a tissue scale control and feedback on cellular fidelity. Molecularly, these cytokinesis failures may depend on a regenerative state of the organoids and are regulated by Lats1 and we propose a model of tissue integrity by multi-scale check points. This discovery helps us understanding further the robustness of a regenerative YAP cellular state, questioning the role of polyploidy in intestinal regeneration. Competing Interest Statement A.B. and P.S. are co-founders of Viventis Microscopy Sarl that commercializes the light-sheet microscope used in this study. Footnotes * We have updated the work and added: 1) comparison of the LSTree prediction methods with existing networks (Stardist, Elephant) 2) added information on the role of Lats1 and Limk1 on the influence of binucleated cell appearance 3) added extra information on backtracking of stained cells in Supplementary 3) improved readability of the text and added detailed documentation in our Supplementary and in the LSTree repository with example data 4) cross-checked text and corrected typos, added complementary information, etc. * https://github.com/fmi-basel/LSTree * https://zenodo.org/record/6828906 * https://zenodo.org/record/6826915

Abstract Sequential segmentation of the body axis is fundamental to vertebrate embryonic patterning. This relies on the segmentation clock, a multi-cellular oscillating genetic-network, which mainifests as tissue-level kinematic waves of gene expression that arrest at the position of each new segment. How this hallmark wave pattern is generated is an open question. We compare cellular-resolution oscillatory patterns in the embryo to those generated cell-autonomously in culture without extrinsic signals. We find striking similarity, albeit with greater variability in the timing of clock arrest in culture. Our simple physical description of a clock controlled by a noisy cell-intrinsic timer captures these dynamics. We propose the segmentation clock integrates an intrinsic, timer-driven oscillatory program, which underlies the waves and arrest, with extrinsic cues regulating the intrinsic timer’s duration and precision. One-sentence Summary Segmentation clock and wavefront activities underlying tissue-level wave patterns are cell-autonomous properties in the PSM. Competing Interest Statement The authors have declared no competing interest.