What does it take to make a worm?
MicroRNAs are important regulators of development, but exactly what different miRNAs do and at which embryonic stages has yet to be determined. Scientists at the Research Institute of Molecular Pathology (IMP) in Vienna have now shown that just two microRNAs are essential for embryogenesis in the worm C. elegans. Their system provides a blueprint for further studies in this dynamic research field.
For decades, biologists assumed that higher organisms were equipped with a large number of genes to account for their complexity. When sequencing whole genomes became feasible, this correlation turned out to be flawed. Instead, other factors were identified that could multiply the information content inscribed in DNA, either by fabricating a number of different products from one gene or by fine-tuning the expression of genes.
Among these regulating factors are so-called microRNAs whose widespread function in animals was revealed as recently as 2000 by studying C. elegans worms. MiRNAs are tiny RNA-molecules that can silence genes with complementary sequences, acting as post-transcriptional repressors of gene expression. They are found universally in plants and animals and play important roles in development and physiology. Unlike protein-coding genes, their numbers do correspond to the complexity of an organism.
In this video abstract, Philipp Dexheimer and Luisa Cochella highlight the key findings of the study.
MicroRNAs are essential for development
The biogenesis of miRNAs is an elaborate process: many enzymes and other factors are required for a series of reactions that take place both in the nucleus and the cytoplasm. Most importantly, a complex called the microprocessor trims a longer RNA to eventually produce the short functional miRNA. In every animal studied so far, loss of the microprocessor is lethal in early embryos, but the reason for this is largely unknown. It implies, however, that at least some miRNAs are essential for development.
But how many and which miRNAs are responsible for the lethal phenotype? It might be just a single essential miRNA that is produced by the microprocessor. Alternatively, embryogenesis could be impaired by the additive effects of losing many miRNAs, each of which may not be individually essential. In a new study published in the journal Current Biology, researchers from the IMP can now show that two microRNAs - out of around 150 that are known to occur in the worm - can revert the lethality of microprocessor defects.
Philipp Dexheimer, a doctoral student in the lab of Luisa Cochella, took a bottom-up approach to identify the minimal set of miRNAs needed for development. Rather than taking away piece after piece and monitoring the effects of removing individual parts, his strategy was to deplete worm-embryos of miRNAs altogether and then adding them back one at a time. To get rid of all miRNAs, he had to completely shut down the function of the microprocessor. This turned out to be the hard part, and it took two years to master the trick. The technology involved fitting the microprocessor with a genetic switch. To this end the respective genes were tagged with a piece of plant-DNA using CRISPR/Cas9. The plant hormone auxin was then used as a signal to turn the switch on or off, allowing the researchers to produce embryos lacking miRNAs.
Two miRNAs are sufficient for a normal phenotype
In the absence of all miRNAs, worm embryos stopped developing at a very early stage. A mere lump of cells, they did not show any defined structures nor the hint of a body plan. But the addition of just two miRNAs, one member of the miR-35 family and one from the miR-51 family, resulted in the development of morphologically normal larvae. These two miRNAs were not chosen randomly. Both are known to be abundant in developing C. elegans worms and are evolutionary very old. In fact, miR-51 is the most ancient animal miRNA that is known.
To reintroduce miR-35 and miR-51, Philipp Dexheimer had to trick the cells into producing them without a functioning microprocessor. He achieved this by constructing an elegant transgenic system that bypassed the microprocessor-mediated cleavage. While only two miRNAs are essential for early embryonic development, worms have more than 100 other miRNAs that seem to play a role in the correct function of specialised cells that develop later during embryogenesis.
Apart from showing that two miRNAs are sufficient for morphogenesis and organogenesis in C. elegans, the publication also provides a useful technological basis, as Philipp Dexheimer explains: “We developed a system to make embryos without miRNAs and designed microprocessor-independent miRNAs. This provides a blueprint for future microprocessor-bypass experiments also in other contexts.”
Luisa Cochella adds: “We believe our work has a major impact on the current understanding of the contribution of miRNAs to animal development, and will guide future work in the fields of miRNAs - and small RNAs in general -, embryogenesis, and gene regulation.”
And finally, the study is a reminder that the tiny nematode C. elegans has been tremendously useful in answering important biological questions - and will probably continue its career as a model organism in the labs around the world.
Philipp J. Dexheimer, Jingkui Wang and Luisa Cochella. Two microRNAs are sufficient for embryonic patterning in C. elegans. Current Biology, 29 October 2020.
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More: Mini Lecture
For more information on miRNAs in development, watch Luisa Cochella’s "Mini Lecture" below.