Phenotypic landscape of intestinal organoid development

Intestinal organoids are an ex vivo culture system of the intestinal epithelium that recapitulate many key aspects of the parent tissue: cell type diversity, spatial organization, but also the ability to regenerate and return to homeostatic conditions following damage. Intestinal organoids can develop from single cells, forming an emergent and self-organizing system undergoing temporally controlled cell fate transitions and spatial rearrangements. This complicated process is orchestrated by crosstalk of many pathways that together form a complex network of functional interactions. Although some components of this densely interconnected network have been resolved and described in detail, a systematic approach to mapping all of the involved players and their interdependence has not been attempted.
To address this complex question, I developed an image-based screen in intestinal organoids cultured from single cells using an annotated compound library. I designed a novel approach generating multivariate feature profiles for hundreds of thousands of individual organoids to quantitatively describe phenotypes observed in each of the screened conditions. These were used to identify stable phenotypic outcomes of intestinal organoid development in a data-driven manner. The relative abundance of each of the detected phenotypes produced a unique phenotypic fingerprint for every screened condition, quantitatively describing the phenotypic landscape of organoid development. I used the generated phenotypic fingerprints to find conditions with significant and reproducible effects, identifying 230 target genes. Subsequently, I used this multivariate dataset to infer the functional genetic interactions of identified genes generating the first map of interactions that govern intestinal organoid formation. This allowed me to discover modules of genes that regulate cell identity transitions and maintain the balance between regeneration and homeostasis. With network analysis I confirmed known players involved in key steps of organoid development but also revealed several novel potential upstream regulators.
In the second part of this study, I focused on conditions identified by the screen to enrich for a regenerative phenotype characterized by absence of differentiated cells of both absorptive and secretory lineage. Abundance of this phenotype marked conditions that potentially improved the regeneration potential of the intestinal epithelium. Among these I discovered two key components of the retinoic acid signaling pathway: RXR and RAR. Follow-up studies allowed me to describe novel roles for nuclear retinoic acid receptors and retinol metabolism in intestinal damage response and homeostasis. By combining quantitative imaging with RNA sequencing I confirmed the role of endogenous retinoic acid signaling and metabolism for initiating transcriptional programs that guide intestinal epithelium cell fate transitions. I also observed that RXR inhibition not only suppressed differentiation, but induced a regenerative fetal-like transcriptional identity. To validate the physiological relevance of this finding, together with our collaborators we designed an in vivo study using a mouse model of cycling cell ablation to induce acute damage in the intestine, treating mice with the compound over the course of recovery. The mouse assay corroborated the findings from the organoid studies, showing that a small molecule inhibitor of RXR identified in the organoid screen improved intestinal regeneration in vivo.
Taken together, this study presents a novel approach for data-driven phenotypic discovery suitable for large image-based screens. This approach can be used on any other arrayed screen with single object resolution offering means to robustly identify phenotypic effects also in a complicated landscape characterized by pleiotropic phenotypes. Furthermore, it establishes a novel paradigm in genetic interaction screening applied to an emergent self-organized system that was ultimately instrumental to identify a small molecule that improved regeneration of the intestinal epithelium in vivo.