Kozono, Y. | Search bioRxiv

bioRxiv2025

EC-isHCR: a rapid method for in situ hybridization chain reaction in diverse animal samples

The in situ hybridization chain reaction (isHCR) visualizes RNA across multiple spatial scales, from organs to subcellular structures, in diverse samples. We previously proposed a rapid protocol, EC-isHCR, for Drosophila embryos and ovaries. Whether EC-isHCR retains the features of conventional isHCR, including wide-spatial-scale analyses in various samples, however, has remained unclear. Here, we show that EC-isHCR enables robust RNA detection in a broad range of samples, such as whole-mount fruit fly, parasitoid wasp, and aphid preparations; paraffin sections of trout; frozen mouse sections; and human cultured cells. Moreover, EC-isHCR enabled detection of subcellular RNA localization. EC-isHCR also visualized co-localization of RNA with phase-separated condensates in fruit fly embryos and detected the protrusion-enriched mRNA in HeLa cells. To broaden the applicability of EC-isHCR, we developed an automated probe design tool (https://github.com/ShuntaYorimoto/hcrkit). By combining this tool with EC-isHCR, we provide a fast and versatile framework to visualize mRNAs. This framework will help reduce the barrier to using fast isHCR and thereby facilitate research across diverse areas of the life sciences. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/696653v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1852584org.highwire.dtl.DTLVardef@621682org.highwire.dtl.DTLVardef@1b3bf5corg.highwire.dtl.DTLVardef@1d86841_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- EC-isHCR enables rapid acquisition of high-contrast images. - EC-isHCR preserves features of conventional isHCR, including versatile sample compatibility and high-resolution imaging. - An automated probe design tool was developed for EC-isHCR. - EC-isHCR/probe tool framework will help reduce the barrier to using fast isHCR.

bioengineering↗

bioRxiv2025

Nanos downregulates maternal mRNAs in germline during Drosophila early embryogenesis

BackgroundMany maternal mRNAs in Drosophila primordial germ cells (PGCs) are degraded in concert with the synthesis of new transcripts from the zygotic genome during gastrulation and germ band elongation. However, few studies have focused on maternal mRNA destabilization in PGCs at the blastoderm stage that is prior to zygotic genome activation (ZGA). Thus, the stability of maternal mRNAs at this stage and regulation of their degradation remain poorly understood. To address this gap, we examined the role of Nanos, an RNA-binding protein known to promote mRNA degradation, in blastoderm-stage PGCs. ResultsBy combining flow cytometry and RNA-seq analysis of PGCs, we identified the transcripts of 898 genes that were increased in nanos- PGCs. Among them, maternal mRNAs encoded by 298 genes were downregulated by Nanos in PGCs. ConclusionsOur results show that Nanos downregulates maternal mRNA expression in PGCs before ZGA. As Nanos in C. elegans PGCs has also been reported to promote maternal-to-zygotic transition (MZT) via maternal mRNA downregulation during a transcriptionally silent state, our findings highlight the importance of investigating the function of Nanos for understanding the MZT in PGCs across various animal species.

developmental biology↗