MicroRNA-mediated control of developmental lymphangiogenesis

Background

Lymphatic vessels are crucial components in vertebrate fluid homeostasis. They build a network of vessels and nodes that is important for drainage of interstitial fluid, circulation of immune cells and transport of dietary lipids.

Over the years, studies on the molecular mechanisms that control lymphatic development have revealed the involvement of several signalling pathways and transcriptional regulators. However, information on post-transcriptional regulation of lymphatic development is limited. MicroRNAs (miRNAs) are non-coding, ~20-24 nucleotide sequences that regulate gene expression by silencing the mRNAs of their target genes. Post-transcriptional regulation through specific miRNAs has been documented to be important for normal development of a variety of tissues and as many as 60% of human protein-coding genes are predicted to have miRNA target sites¹. In the context of lymphatic development, some miRNAs have been reported to have a role in lymphatic endothelial cell (LEC) specification, development and inflammation². However, only a few of these studies have utilized in vivo model systems.

In the past decade, zebrafish has been well-established as a model system for lymphatic development^3,4. In this study, Jung et al. combined human endothelial cell culture system and zebrafish embryos to identify how miRNA-204 and its target nfatc1 is required to generate optimal levels of lymphatic structures.

Important results

To identify miRNAs that are enriched in lymphatic endothelial cells (LECs), Jung et al. performed small RNA sequencing on human dermal lymphatic microvascular endothelial cells (HMVEC-dLy, representing LECs) and human umbilical vein endothelial cells (HUVECs, representing blood endothelial cells). Comparative analysis of these datasets revealed 98 differentially expressed miRNAs, among which 30 were highly expressed in LECs. miR-204, with 105 times higher expression in LECs, was singled out for in vivo characterization.

Enrichment of miR-204 was also found in developing LECs of zebrafish embryos, but the zebrafish genome harbors three paralogues of miR-204 (referred as miR-204-1, 204-2 and 204-3), all of which produce a mature miR-204 whose sequence that is 100% identical to its human counterpart. Injection of a pan-miR-204 morpholino, that suppresses the mature miR-204 produced from all the three loci, caused loss of early lymphatic structures in the trunk of the embryos. However, a CRISPR mutant that was designed to disrupt 204-1 showed no phenotype and caused only 20% reduction in the total miR-204 content, indicating that 204-2 and/or 204-3 can compensate for this mutation. Using morpholinos that specifically suppressed miR-204-1, 204-2, 204-3 or a combination of them, the authors could conclude that miR-204-1 and 204-2 are the major contributors towards the total miR-204 and suppressing products from these two loci together caused prominent loss of early lymphatics in the trunk. The role of miR-204 as a positive regulator for lymphatic formation was further strengthened when lymphatic-specific overexpression of miR-204 resulted in faster development of the thoracic duct, the early forming lymphatic vessel in the trunk.

To identify the gene(s) regulated by miR-204, the authors utilized RNA22, a computational tool for miRNA target discovery⁵, and identified nfatc1 as a possible target. The authors showed that miR-204 can indeed supress nfatc1 and it is mediated through miR-204 binding in the 3’UTR region of the nfatc1 transcript. Furthermore, suppression of miR-204 indeed increased endogenous nfatc1 transcript levels both in human LECs and in zebrafish. Loss of Nfatc1 has been previously shown to cause lymphatic hyperplasia in mouse⁶. Similarly, Jung et al. found thoracic duct enlargement upon suppression of nfatc1 transcript or inhibition of Nfatc1 downstream pathway.

Thus, while miR-204 promotes lymphatic growth, Nfatc1 seems to restrict lymphatic growth (since its downregulation causes lymphatic hyperplasia). To establish a functional link between these two, morpholino-mediated co-suppression of miR-204 and nfatc1 was performed. As expected, the downregulation of both ended up giving rise to a somewhat normal lymphatic vasculature.

 

Why I chose this preprint

In this work, Jung et al. have successfully utilized human cells and zebrafish in vivo system to identify an evolutionary conserved post-transcriptional regulatory mechanism for lymphatic development. Apart from presenting a new regulator of lymphatic development, this study also provides strong evidence for lymphatic developmental conservation between mammalian and fish model systems.

There is also something exciting about identifying miRNAs that strongly influence certain biological processes. Since miRNAs are small sequences, they can be easily delivered, sometimes with suitable modifications, for miRNA-based therapeutics⁷. In fact, several miRNAs have been utilized for preclinical studies, and a few are being considered for clinical trials. This is of importance in the context of lymphatics, as lymphatic dysfunction causes a range of debilitating conditions, called lymphedema, which lacks a proper cure. Identification of miRNAs that can have impact on lymphatic development and maintenance offers opportunities for therapeutics, and thus this study, although very preliminary, opens such an opportunity.

Questions to the authors

• The data presented in this manuscript provides support for a model where Nfatc1 is suppressing LEC proliferation, and miR-204, by suppressing the levels of Nfatc1, allows for optimal lymphatic development. However, the presence of Nfatc1 indicates some role for it either in the early lymphatic progenitors or in the PCV cells (that generate lymphatic progenitors). Have the authors attempted to detect Nfatc1 (using in situ hybridization or in the transcriptome of sorted cells) in the zebrafish PCV/early LECs? It would be great if the authors can share their views on the possible role of Nfatc1 in this context.
• While there is a clear evidence of miR-204 suppressing Nfatc1, it is still possible that miR-204 regulates other transcripts required for lymphatic development (as also discussed by the authors). Do the authors think that a Nfatc1 overexpression experiment can resolve this issue based on whether it phenocopies miR-204 suppression? Have the authors also identified, in the miRNA target prediction, any other molecular players in the lymphatic pathway as potential targets of miR-204?
• There are number of reports of involvement of miR-204 in certain cancers, where miR-204 is described as a tumor suppressor. Other unrelated studies have also implicated Nfatc1 in certain cancers and in maintenance of stem cell quiescence⁸. Do the findings from these studies provide any interesting clues that can explain some of the phenotypes described in the present work?
• This work utilizes morpholinos for many of the important experiments. In recent years, morpholino usage in zebrafish has been recommended with caution⁹. I feel it would be great for other zebrafish researchers if the authors share some of the crucial aspects of their experiments that allows for a greater reliability in their morpholino experiments. I was also wondering if the authors plan to use a double mutant for 204-1 and 204-2 for a more precise demonstration of their findings.

 References

1. Gebert, L. F. R. & MacRae, I. J. Regulation of microRNA function in animals. Nature Reviews Molecular Cell Biology (2019). doi:10.1038/s41580-018-0045-7
2. Yee, D., Coles, M. C. & Lagos, D. microRNAs in the lymphatic endothelium: Master regulators of lineage plasticity and inflammation. Frontiers in Immunology (2017). doi:10.3389/fimmu.2017.00104
3. Yaniv, K. et al. Live imaging of lymphatic development in the zebrafish. Nat. Med. 12, 711–716 (2006).
4. Hogan, B. M. & Schulte-Merker, S. How to Plumb a Pisces: Understanding Vascular Development and Disease Using Zebrafish Embryos. Developmental Cell (2017). doi:10.1016/j.devcel.2017.08.015
5. Miranda, K. C. et al. A Pattern-Based Method for the Identification of MicroRNA Binding Sites and Their Corresponding Heteroduplexes. Cell (2006). doi:10.1016/j.cell.2006.07.031
6. Norrmén, C. et al. FOXC2 controls formation and maturation of lymphatic collecting vessels through cooperation with NFATc1. J. Cell Biol. (2009). doi:10.1083/jcb.200901104
7. Rupaimoole, R. & Slack, F. J. MicroRNA therapeutics: Towards a new era for the management of cancer and other diseases. Nature Reviews Drug Discovery (2017). doi:10.1038/nrd.2016.246
8. Horsley, V., Aliprantis, A. O., Polak, L., Glimcher, L. H. & Fuchs, E. NFATc1 Balances Quiescence and Proliferation of Skin Stem Cells. Cell (2008). doi:10.1016/j.cell.2007.11.047
9. Stainier, D. Y. R. et al. Guidelines for morpholino use in zebrafish. PLoS Genet. (2017). doi:10.1371/journal.pgen.1007000

Tags: lymphatics, microrna, zebrafish

doi: https://doi.org/10.1242/prelights.9240

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