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Research · Completed · May–July 2025

Droplets that build the germline: imaging oskar RNA in fly embryos

IIT Kharagpur · Prof. Mainak Bose

Organism: Drosophila melanogaster

The question

How do RNA–protein ‘droplets’ that carry oskar RNA help set aside the cells that will become the next generation’s eggs and sperm?

oskar RNA is localised to the posterior of the Drosophila oocyte, where it nucleates the germ plasm that specifies future germ cells. oskar RNA and proteins assemble into ribonucleoprotein condensates whose physical state matters for their function. I optimised single-molecule imaging of oskar condensates in embryos and used quantitative image analysis to study their translational activity around the maternal-to-zygotic transition.

The basics: the life of an mRNA

Why ask this?

A fruit fly sets aside its future germ cells very early, at the back (posterior) end of the egg. A special cytoplasm there, the germ plasm, is organised by the RNA of the gene oskar. oskar RNA and its partner proteins gather into membrane-less condensates, and their physical state, more liquid or more solid, matters for whether they work (Bose et al. 2022). Strong evidence A specific RNA–RNA “kissing-loop” contact between oskar mRNAs helps build the granules (Bose et al. 2024). Emerging

Seeing single mRNAs: smFISH

In single-molecule FISH (smFISH), about 30–50 short DNA probes (~20 nucleotides each), each carrying one fluorophore, are designed to tile one mRNA. One probe is too dim to see, but when all of them bind the same molecule, that mRNA shows up as a bright, diffraction-limited spot that can be counted and mapped (Femino et al. 1998; Raj et al. 2008). Established With smFISH, germ plasm mRNAs were seen to gather into clusters of the same mRNA inside germ granules (Little et al. 2015; Trcek et al. 2015). Strong evidence

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Sequential smFISH with proteinase K. Round 1: probe set 1 (magenta) detects oskar mRNA that probes can reach. A brief proteinase K digestion partly removes bound proteins. Round 2: probe set 2 (green), against the same mRNA, now also detects mRNA that was masked inside protein-rich RNP granules. Green-only spots mark mRNA that was protein-bound.

Two probe sets, before and after proteinase K

Inside dense RNP granules, bound proteins can hide (“mask”) an mRNA from probes (Buxbaum et al. 2014). To tell accessible oskar mRNA from protein-bound oskar mRNA, I used sequential smFISH with two probe sets against the same mRNA, in two colours:

  1. Round 1. Probe set 1 hybridises to the oskar mRNA that probes can reach.
  2. Brief proteinase K. A short, mild protease digestion removes part of the bound protein and unmasks the mRNA.
  3. Round 2. Probe set 2 now detects the oskar mRNA that had been protein-bound, as well as the accessible molecules.

Spots with both colours are accessible mRNA; spots seen only with probe set 2 mark mRNA that was protein-bound. The same logic, a second-colour probe after protease digestion, revealed masked β-actin mRNA in neurons (Buxbaum et al. 2014). Strong evidence Repeated rounds of hybridisation are also the basis of multiplexed methods such as seqFISH (Lubeck et al. 2014).

What I did

  • I optimised smFISH, including the two-probe-set, proteinase K approach above, for high-resolution views of oskar condensates inside embryos.
  • I imaged embryos on a confocal microscope.
  • I used quantitative image analysis to describe the condensates and relate them to translation, the reading of RNA into protein.

What I took away

Seeing single molecules inside a living embryo changes how you think about biology. Location, timing and physical state are information too. The basics are in the life of an mRNA and droplets inside cells.

Why it matters

Condensates are now known throughout biology, from gene regulation to stress responses. When they go wrong (harden or misfold), they are linked to diseases including neurodegeneration. Emerging

Methods used

  • Single-molecule FISH (smFISH)
  • Sequential smFISH with proteinase K unmasking
  • Live imaging of embryos
  • Confocal microscopy
  • Quantitative image analysis

What these methods are →

References

  1. Bose M, Lampe M, Mahamid J, Ephrussi A (2022) Liquid-to-solid phase transition of oskar ribonucleoprotein granules is essential for their function in Drosophila embryonic development. Cell 185:1308–1324. doi:10.1016/j.cell.2022.02.022
  2. Vastenhouw NL, Cao WX, Lipshitz HD (2019) The maternal-to-zygotic transition revisited. Development 146:dev161471. doi:10.1242/dev.161471
  3. Bose M, Rankovic B, Mahamid J, Ephrussi A (2024) An architectural role of specific RNA–RNA interactions in oskar granules. Nature Cell Biology 26:1934–1942. doi:10.1038/s41556-024-01519-3
  4. Femino AM, Fay FS, Fogarty K, Singer RH (1998) Visualization of single RNA transcripts in situ. Science 280:585–590. doi:10.1126/science.280.5363.585
  5. Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nature Methods 5:877–879. doi:10.1038/nmeth.1253
  6. Buxbaum AR, Wu B, Singer RH (2014) Single β-actin mRNA detection in neurons reveals a mechanism for regulating its translatability. Science 343:419–422. doi:10.1126/science.1242939
  7. Lubeck E, Coskun AF, Zhiyentayev T, Ahmad M, Cai L (2014) Single-cell in situ RNA profiling by sequential hybridization. Nature Methods 11:360–361. doi:10.1038/nmeth.2892
  8. Little SC, Sinsimer KS, Lee JJ, Wieschaus EF, Gavis ER (2015) Independent and coordinate trafficking of single Drosophila germ plasm mRNAs. Nature Cell Biology 17:558–568. doi:10.1038/ncb3143
  9. Trcek T, Grosch M, York A, Shroff H, Lionnet T, Lehmann R (2015) Drosophila germ granules are structured and contain homotypic mRNA clusters. Nature Communications 6:7962. doi:10.1038/ncomms8962