Controlling intestinal organoid regeneration with retinoic acid and RXR inhibition

da | Giu 14, 2023 | Biologia Molecolare, Intestine, Organoids, Regeneration

Fig. 1 – RXR is a homing mechanism to exit regenerative state and its inhibition leads to regenerative phenotype; retinoic acid provides tissue-specificity [Created with BioRender.com]


Abstract

The intestine is the place where nutrients are absorbed. Problems of the intestine could lead to weight problems or physical problems such as inflammatory bowel syndrome. In addition, invasive therapy medicine such as bone marrow transplant or chemotherapy can affect intestinal stem cells. The development of intestinal organoids in vitro recapitulates the regenerative capacity of the intestinal epithelium. In this paper, the authors studied the development of mice intestinal organoids, classified them using an image screening method, and studied the gene interactions that modified the final phenotype. From this study, they understood the fundamental role of retinoic acid in maintaining proper homeostasis between differentiated cells and regenerative phenotype. These results are essential in order to better comprehend how some compounds could be used to obtain new experimental models in vitro or to help patient recovery in some situations.

Discussion

The intestinal epithelium is a single layer of polarized cells, arranged into regular protruding units, called villi, interspaced by crypts composed of stem cells [1]. Given the multiple pathologies associated with the loss of intestinal barrier and nutrient malabsorption, it is important to understand the mechanisms involved in the regeneration of the intestinal epithelium. Intestinal organoids represent a powerful tool to study these mechanisms in vitro.

An entire organoid develops from a single cell, forming a self-organized structure. Initially, single cells enter a regenerative state that is dependent on the transcriptional regulator YAP, and forms a symmetric cyst [2]. Subsequently, symmetry is broken by the emergence of Paneth cells that define and maintain the crypt, followed by the differentiation of absorptive enterocytes distal from the crypt [3]. Organoids thus recapitulate the regeneration of the epithelium and subsequent re-establishment of homeostasis [4]. In this work [5], the authors established an image-based screening platform to characterize the phenotypic landscape of organoid development from a single screen, generating the first map of functional genetic interactions that govern intestinal organoid development and self-organization.

Organoids development and screening

Intestinal organoids were generated from a single cell during 4 days cultured in contact with 2789 compounds. The cells were then stained highlighting DNA content, enterocytes, and Paneth cells. Approximately 450.000 organoids were profiled using an image screening technique, and 7 major classes of organoids were defined based on the observed phenotype: wild-type, enterocyte-reduced, Paneth cell hyperplasia, increased self-renewal, progenitor-reduced, regenerative and enterocyst. The authors found 301 compounds, targeting 201 unique genes, that produced strong and reproducible phenotypes. Through the use of the hierarchical interaction score (HIS) algorithm, a statistical index previously developed by the same group [6], they were able to create a map of the functional genetic interactions that underlie intestinal organoid development. Remarkably, the authors found signaling pathways such as WNT and MAPK among the crucial players of the reconstructed genetic network.

RXR inhibition leads to regenerative phenotype

Several target genes such as Casr, Akt and RXR were modified in order to validate the study.

Next, the authors focused on the regenerative phenotype. During this phase, they noticed that the use of the RXR antagonist Cpd2170 (RXRi) [7] led to an increase in the regenerative phenotype. In particular, the authors measured the abundance of enterocytes through aldolaseB staining and noticed that the antagonist of RXR induces a near-complete absence of enterocytes. In contrast, they observed that an RXR agonist, all-trans retinoic acid (atRA), led to the opposite effect. In fact, it increased enterocyte differentiation despite failing to rescue the RXRi-induced differentiation defect, hinting at a retinoic acid-independent role of RXR.

To investigate the lack of Paneth cells that was also observed upon RXR inhibition, the authors turned to Notch signaling and YAP1 localization. In RXRi-treated organoids they observed an absence of cells expressing the Notch ligand DLL1 and a strong homogenous nuclear retention of YAP1, which led to retaining a regenerative state [2] and an active cell cycle. By contrast, in atRA-treated organoids YAP1 was invariably localized to the cytoplasm and cells underwent differentiation to enterocytes. However, both RXRi-and atRA-treated organoids showed a defect in symmetry breaking. Interestingly, RXRi-treatment after YAP1 activation did not result in nuclear translocation of YAP1, suggesting that RXR does not activate YAP1 but rather controls the nuclear export.

Moreover, gene expression profiling by RNA sequencing revealed that in the RXRi condition, YAP targets and genes associated with a fetal-like regenerative state were strongly upregulated, as compared to treatment with atRA. In fact, day 4 organoids with RXRi were similar to day 1 control organoids. In addition, the expression of ANXA genes indicates that RXR inhibition leads to a regenerative phenotype in all gastroenteric cells, not just the intestinal ones.

All-trans retinoic acid role

Next, the author found that genes involved in retinoic acid metabolism were specifically expressed in enterocytes, such as the retinaldehyde dehydrogenase (ALDH1A1) gene, which converts retinol in atRA [8]. Retinoic acid also activates the RAR gene leading to the production of retinoic acid response elements (RARE), and their higher abundance is exclusive to enterocytes.

ALDH1A1 knock-out of this gene or vitamin A depletion led to fewer enterocytes and more cycling cells, suggesting that retinoic acid metabolism is necessary for differentiation of enterocytes but not of Paneth cells.

In vivo mice experiment

Finally, the authors performed an in vivo experiment. A colitis condition was induced in mice with gamma radiation, thus leading to cell cycle loss, shorter villi and improper nutrient absorption paired with weight loss.

By treating mice with RXRi they observed improved barrier function and increasingly significant villi length recovery as days passed but never reached initial normal size.

In addition, the treatment improved crypt morphology by accumulating proliferative cells at the bottom. These results confirmed the regenerative phenotype also in vivo, suggesting RXRi treatment as a possible useful therapy to improve intestine regeneration.

Conclusions

Summarizing the results, the authors found that YAP1 is activated by external stimulus and RXRi keeps it active by ensuring that it does not leave the nucleus, so the cells take on a regenerative phenotype. While RXR is crucial for the retinoic acid signalling pathway and enterocyte differentiation. Furthermore, the authors pointed out that retinoic acid metabolism is necessary for the differentiation of enterocytes but not of Paneth cells.

The interaction between RXRi and retinoic acid could be used to obtain new experimental models in vitro by manipulating the intestinal gene pool. In the future, this regeneration potential could be explored by combining inhibitors with different diets. In addition, inhibition could be used to mitigate problems that may arise with cancer therapies that damage the intestinal barrier by affecting stem cells

References

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Lucia Etzi

Master Industrial Biotechnology student

Cosmin George Mitachi

Master Industrial Biotechnology student

 

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