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Cyp26b1 is required for proper airway epithelial differentiation during lung development

Edward Daniel, Gabrielle I. Sutton, Yadanar Htike, Ondine Cleaver

Preprint posted on June 20, 2019 https://www.biorxiv.org/content/10.1101/678581v1

Endothelial cells fine-tune retinoic acid levels through Cyp261b, enabling the differentiation of lung epithelial cells.

Selected by Julio Sainz de Aja

Why I think this paper is important for the scientific community:

 The effort to achieve differentiated human lungs in vitro out of human induced pluripotent stem cells (hiPSCs) is an ongoing struggle in the fields of stem cell biology and development. It is clear that to correctly and fully differentiate human lung cells, or indeed any organ, it is crucial to understand the developmental process by which those cells are produced.

Nowadays, most of the lung organoid differentiation methods rely on co-culturing stem cells with niche cells (Lee et al., 2017; Leeman et al., 2019). Differentiation from hiPSCs to lung organoids have been achieved in the last years (McCauley et al., 2017), however, cells remain in their progenitor state, being unable to differentiate towards the final differentiation stage of lung development. In order to understand how the in vitro cultures could be improved, it is important to study the interaction in vivo between stromal, endothelial or smooth muscle cells and the lung epithelial cells in the context of differentiation.

In this work, Daniel and colleagues explore the role of endothelial cells in lung differentiation. They observe that levels of Retinoic acid (RA) are crucial for proper lung distal differentiation and that Cyp26b1, a RA-catabolizing enzyme highly expressed in endothelial cells, is crucial for the late differentiation of the lung distal progenitor population. More specifically, AT2 cells (surfactant producing cells that in the alveoli can give rise to AT1 cells) fail to differentiate towards AT1 (alveoli epithelial cells in charge of the oxygen exchange). Since alveoli differentiation from hiPS cells is challenging using current protocols (Jacob et al., 2017), this work could point to a possible solution for this problem in the lung organoid field.

 Background:

 The spatiotemporal regulation of RA is vital for proper specification of the organs during embryonic development and further on (Rhinn and Dollé, 2012). In this regard, RA catabolizing enzymes play an important role in regulating the levels of RA and determining the spatial distribution of this molecule (Abu-Abed et al., 2002). More specifically, in the lung, it is known that enzymes that synthesize RA are necessary for lung bud growth (Ribes et al., 2006). However, the role of the RA-catabolizing Cyp26 enzymes remains unknown. Cyp26a1 is expressed in the lung epithelium in early stages, while Cyp26b1 is expressed in the mesenchyme in later stages of development (Abu-Abed et al., 2002). The question remains as to whether these enzymes have a role in lung distal differentiation, particularly Cyp26b1 given that it is expressed in later stages of lung development.

 Key findings:

Cyp26b1 is highly expressed in endothelial cells of the developing lung. The authors generated two different KO for Cyp26b1 by CRISPR/Cas9, in both distal airway morphogenesis is delayed at E16.5 with the lung’s lumens being small and rarely open (Figure A).  KO mice died right after birth probably due to respiratory distress, as they display air hunger reflexes. More specifically, AT2 cells fail to differentiate into AT1 cells. The authors quantify AT2 cells using Lamp3 and pro-SPC, and AT1 with Aqp5 and Pdpn by IF, western and qPCR (Figure B). Other cell types in the lung such as proximal airways, stroma, endothelia and lymphatics are not affected in the KO. Exogenous RA administered to the pregnant mothers has a similar phenotype as the Cyp26b1 KO, although AT2 cells only increased in the heterozygous embryos treated but not the wildtypes treated with exogenous RA (Figure C). Cyp26b1 full KO treated with exogenous RA died by E18.5. Still, exogenous RA and loss of Cyp26b1 have a different transcriptional response in a number of RA targets by qPCR, suggesting the Cyp26b1 phenotype is not completely RA-dependent.

Figure A: Loss of Cyp26b1 in lung endothelial cells delays the formation of distal airways.

Figure B: Cyp26b1 KO lungs have increased cellular density and reduced AT1 cells presence.

Figure C: Cyp26b1phenotype is at least partially due to the excess of RA.

Questions to the authors:

  1. Would you expect the same results in lung epithelial cells in a conditional KO for endothelial cells?
  2. How do you believe endothelial cells interact with epithelial cells in the RA context? Do they interact physically with each other?
  3. Have you contemplated following this work in adult lung, to see whether Cyp26b1 has an impact in alveoli regeneration upon injury? Is it still expressed in endothelial cells?

Bibliography

 Abu-Abed, S., MacLean, G., Fraulob, V., Chambon, P., Petkovich, M., and Dollé, P. (2002). Differential expression of the retinoic acid-metabolizing enzymes CYP26A1 and CYP26B1 during murine organogenesis. Mech. Dev. 110, 173–177.

Jacob, A., Morley, M., Hawkins, F., McCauley, K.B., Jean, J.C., Heins, H., Na, C.-L., Weaver, T.E., Vedaie, M., Hurley, K., et al. (2017). Differentiation of Human Pluripotent Stem Cells into Functional Lung Alveolar Epithelial Cells. Cell Stem Cell 21, 472-488.e10.

Lee, J.-H., Tammela, T., Hofree, M., Jacks, T., Regev, A., and Kim Correspondence, C.F. (2017). Anatomically and Functionally Distinct Lung Mesenchymal Populations Marked by Lgr5 and Lgr6. Cell 170.

Leeman, K.T., Pessina, P., Lee, J.-H., and Kim, C.F. (2019). Mesenchymal Stem Cells Increase Alveolar Differentiation in Lung Progenitor Organoid Cultures. Sci. Rep. 9, 6479.

McCauley, K.B., Hawkins, F., Serra, M., Thomas, D.C., Jacob, A., and Kotton, D.N. (2017). Efficient Derivation of Functional Human Airway Epithelium from Pluripotent Stem Cells via Temporal Regulation of Wnt Signaling. Cell Stem Cell 20, 844-857.e6.

Rhinn, M., and Dollé, P. (2012). Retinoic acid signalling during development. Development 139, 843–858.

Ribes, V., Wang, Z., Dollé, P., Niederreither, K., and Gossler, A. (2006). Retinaldehyde dehydrogenase 2 (RALDH2)-mediated retinoic acid synthesis regulates early mouse embryonic forebrain development by controlling FGF and sonic hedgehog signaling. Development 133, 351–361.

 

Posted on: 19th August 2019 , updated on: 20th August 2019

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  • Author's response

    Edward Daniel & Ondine Cleaver shared

    1. Would you expect the same results in lung epithelial cells in a conditional KO for endothelial cells?

    Yes. Our data and sequencing data from multiple other groups demonstrate that Cyp26b1 is highly enriched in lung endothelial cells compared to lung non-endothelial cells throughout development and in the adult. This restricted expression suggests it is the endothelial cells lacking Cyp26b1 that drive the phenotype. We are very much interested in following up this work by analyzing endothelial cell conditional KOs for Cyp26b1

     

    1. How do you believe endothelial cells interact with epithelial cells in the RA context? Do they interact physically with each other?

    We believe endothelial cells act as a sink for RA signaling. By expressing Cyp26b1, endothelial cells reduce local RA signaling both within the endothelial cell itself and in the cells immediately adjacent to it (RA is lipid soluble and thus readily goes through plasma membranes). Thus, a gradient forms between the RA-producing cells and the RA-catabolizing endothelial cells. We posit that where an epithelial cell falls in this gradient, in part, determines its fate.

     

    An alternative model is that epithelial cells are indirectly affected by the change in RA signaling. In this model, increased RA exclusively affects endothelial cells and their paracrine signaling, which, in turn, affects epithelial cell differentiation. Although this model is less likely given our work and work done differentiating hiPSCs in vitro, our data cannot exclude this possibility.

     

    1. Have you contemplated following this work in adult lung, to see whether Cyp26b1 has an impact in alveoli regeneration upon injury? Is it still expressed in endothelial cells?

    Adult lung regeneration is a really fascinating topic. The data produced by several groups including seminal work from the Kim and Rafii labs highlight how crucial cell-to-cell communication between endothelial cells, epithelial cells, and stromal cells is to recreate lost or dysfunctional alveoli. RA has been implicated in promoting both proper alveolar formation postnatally and alveolar regeneration. As such, an important follow-up question to our work is to see whether Cyp26b1, which is still highly expressed in endothelial cells in the adult according to sequencing data from other groups, affects adult alveolar regeneration. The hypothesis will be that Cyp26b1, as a negative regulator of RA, inhibits alveolar regeneration. We hope to one day be able to perform these experiments and expand our work to adult models.

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