Deep-learning analysis of micropattern-based organoids enables high-throughput drug screening of Huntington's disease models

Organoids are carrying the promise of modeling complex disease phenotypes and serving as a powerful basis for unbiased drug screens, potentially offering a more efficient drug-discovery route. However, unsolved technical bottlenecks of reproducibility and scalability have prevented the use of current organoids for high-throughput screening. Here, we present a method that overcomes these limitations by using deep-learning-driven analysis for phenotypic drug screens based on highly standardized micropattern-based neural organoids. This allows us to distinguish between disease and wild-type phenotypes in complex tissues with extremely high accuracy as well as quantify two predictors of drug success: efficacy and adverse effects. We applied our approach to Huntington's disease (HD) and discovered that bromodomain inhibitors revert complex phenotypes induced by the HD mutation. This work demonstrates the power of combining machine learning with phenotypic drug screening and its successful application to reveal a potentially new druggable target for HD

WNT signaling memory is required for ACTIVIN to function as a morphogen in human gastruloids

Self-organization of discrete fates in human gastruloids is mediated by a hierarchy of signaling pathways. How these pathways are integrated in time, and whether cells maintain a memory of their signaling history remains obscure. Here, we dissect the temporal integration of two key pathways, WNT and ACTIVIN, which along with BMP control gastrulation. CRISPR/Cas9-engineered live reporters of SMAD1, 2 and 4 demonstrate that in contrast to the stable signaling by SMAD1, signaling and transcriptional response by SMAD2 is transient, and while necessary for pluripotency, it is insufficient for differentiation. Pre-exposure to WNT, however, endows cells with the competence to respond to graded levels of ACTIVIN, which induces differentiation without changing SMAD2 dynamics. This cellular memory of WNT signaling is necessary for ACTIVIN morphogen activity. A re-evaluation of the evidence gathered over decades in model systems, re-enforces our conclusions and points to an evolutionarily conserved mechanism

Huntingtin CAG expansion impairs germ layer patterning in synthetic human 2D gastruloids through polarity defects

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expansion of the CAG repeats in the huntingtin gene (HTT). Although HD has been shown to have a developmental component, how early during human embryogenesis the HTT-CAG expansion can cause embryonic defects remains unknown. Here, we demonstrate a specific and highly reproducible CAG length-dependent phenotypic signature in a synthetic model for human gastrulation derived from human embryonic stem cells (hESCs). Specifically, we observed a reduction in the extension of the ectodermal compartment that is associated with enhanced activin signaling. Surprisingly, rather than a cell-autonomous effect, tracking the dynamics of TGFβ signaling demonstrated that HTT-CAG expansion perturbs the spatial restriction of activin response. This is due to defects in the apicobasal polarization in the context of the polarized epithelium of the 2D gastruloid, leading to ectopic subcellular localization of TGFβ receptors. This work refines the earliest developmental window for the prodromal phase of HD to the first 2 weeks of human development, as modeled by our 2D gastruloids

The RNA-binding protein, Vg1RBP, is required for pancreatic fate specification

Signaling mechanisms underlying the induction of the pre-pancreatic tissue within the endoderm remain poorly understood. Through an expression cloning strategy, we have identified a previously uncharacterized pancreatic factor that we named Shirin. Interestingly, the non-coding RNA regulatory sequence (3 UTR) of Shirin is sufficient to induce insulin expression in Xenopus embryos. Biochemical studies demonstrate that this RNA sequence is able to bind directly to a trans-acting factor, Vg1RBP, which was previously shown to be involved in the localization of endodermal determinant factors. Loss-of-function analysis indicates that Vg1RBP is required for establishment of pancreatic fate within the endoderm, suggesting a synergism between Vg1RBP and Shirin in the embryo. This study argues for a central role of post-transcriptional mechanisms in establishing pancreatic fate, where a 3 UTR may recruit factors necessary for pancreatic development, and highlights an unknown embryological activity of Vg1RBP

The Gata5 target, TGIF2, defines the pancreatic region by modulating BMP signals within the endoderm

Mechanisms underlying regional specification of distinct organ precursors within the endoderm, including the liver and pancreas, are still poorly understood. This is particularly true for stages between endoderm formation and the initiation of organogenesis. In this report, we have investigated these intermediate steps downstream of the early endodermal factor Gata5, which progressively lead to the induction of pancreatic fate. We have identified TGIF2 as a novel Gata5 target and demonstrate its function in the establishment of the pancreatic region within dorsal endoderm in Xenopus. TGIF2 acts primarily by restricting BMP signaling in the endoderm to allow pancreatic formation. Consistently, we found that blocking BMP signaling by independent means also perturbs the establishment of pancreatic identity in the endoderm. Previous findings demonstrated a crucial role for BMP signaling in determining dorsal/ventral fates in ectoderm and mesoderm. Our results now extend this trend to the endoderm and identify TGIF2 as the molecular link between dorsoventral patterning of the endoderm and pancreatic specification

Synthetic embryology: controlling geometry to model early mammalian development

Differentiation of embryonic stem cells in vitro is an important tool in dissecting and understanding the mechanisms that govern early embryologic development. In recent years, there has been considerable progress in creating organoids that model gastrulation, neurulation or organogenesis. However, one of the key challenges is reproducibility. Geometrically confining stem cell colonies considerably improves reproducibility and provides quantitative control over differentiation and tissue shape. Here, we review recent advances in controlling the two-dimensional or three-dimensional organization of cells and the effect on differentiation phenotypes. Improved methods of geometrical control will allow for an even more detailed understanding of the mechanisms underlying embryologic development and will eventually pave the way for the highly reproducible generation of specific tissue types

Genomics and embryology in amphibians

A report on the Eighth Biannual Xenopus Conference, Estes Park, Colorado, August 16-20, 2000

Self-organizing neuruloids model developmental aspects of Huntington's disease in the ectodermal compartment

Harnessing the potential of human embryonic stem cells to mimic normal and aberrant development with standardized models is a pressing challenge. Here we use micropattern technology to recapitulate early human neurulation in large numbers of nearly identical structures called neuruloids. Dual-SMAD inhibition followed by bone morphogenic protein 4 stimulation induced self-organization of neuruloids harboring neural progenitors, neural crest, sensory placode and epidermis. Single-cell transcriptomics unveiled the precise identities and timing of fate specification. Investigation of the molecular mechanism of neuruloid self-organization revealed a pulse of pSMAD1 at the edge that induced epidermis, whose juxtaposition to central neural fates specifies neural crest and placodes, modulated by fibroblast growth factor and Wnt. Neuruloids provide a unique opportunity to study the developmental aspects of human diseases. Using isogenic Huntington's disease human embryonic stem cells and deep neural network analysis, we show how specific phenotypic signatures arise in our model of early human development as a consequence of mutant huntingtin protein, outlining an approach for phenotypic drug screening

A balance between secreted inhibitors and edge sensing controls gastruloid self-organization

The earliest aspects of human embryogenesis remain mysterious. To model patterning events in the human embryo, we used colonies of human embryonic stem cells (hESCs) grown on micropatterned substrate and differentiated with BMP4. These gastruloids recapitulate the embryonic arrangement of the mammalian germ layers and provide an assay to assess the structural and signaling mechanisms patterning the human gastrula. Structurally, high-density hESCs localize their receptors to transforming growth factor β at their lateral side in the center of the colony while maintaining apical localization of receptors at the edge. This relocalization insulates cells at the center from apically applied ligands while maintaining response to basally presented ones. In addition, BMP4 directly induces the expression of its own inhibitor, NOGGIN, generating a reaction-diffusion mechanism that underlies patterning. We develop a quantitative model that integrates edge sensing and inhibitors to predict human fate positioning in gastruloids and, potentially, the human embryo