Biology subjects

Ronshaugen, M.

Publications and source records attributed to Ronshaugen, M..

4 recordsLinked to original sources

bioRxiv2021

miR-9a regulates levels of both rhomboid mRNA and protein in the early Drosophila melanogaster embryo

MicroRNAs have subtle and combinatorial effects on the expression levels of their targets. Studying the consequences of a single microRNA knockout often proves difficult as many such knockouts exhibit phenotypes only under stress conditions. This has led to the hypothesis that microRNAs frequently act as buffers of noise in gene expression. Observing and understanding buffering effects requires quantitative analysis of microRNA and target expression in single cells. To this end, we have employed single molecule fluorescence in situ hybridization, immunofluorescence, and high-resolution confocal microscopy to investigate the effects of miR-9a loss on the expression of the serine-protease rhomboid in Drosophila melanogaster early embryos. Our single-cell quantitative approach shows that rhomboid mRNA exhibits the same spatial expression pattern in WT and miR-9a knockout embryos, although the number of mRNA molecules per cell is higher when miR-9a is absent. However, the level of rhomboid protein shows a much more dramatic increase in the miR-9a knockout. Specifically, we see accumulation of rhomboid protein in miR-9a mutants by stage 5, much earlier than in WT. The data therefore show that miR-9a functions in the regulation of rhomboid activity by both inducing mRNA degradation and inhibiting translation in the blastoderm embryo. Temporal regulation of neural proliferation and differentiation in vertebrates by miR-9 is well-established. We suggest that miR-9 family microRNAs are conserved regulators of timing in neurogenic processes. This work shows the power of single-cell quantification as an experimental tool to study phenotypic consequences of microRNA mis-regulation.

developmental biology↗

bioRxiv2021

The developmental transcriptome of Parhyale hawaiensis: microRNAs and mRNAs show different expression dynamics during the maternal-zygotic transition

Parhyale hawaiensis has emerged as the crustacean model of choice due to its tractability, ease of imaging, sequenced genome, and development of CRISPR/Cas9 genome editing tools. However, transcriptomic datasets spanning embryonic development are lacking, and there is almost no annotation of non-protein-coding RNAs, including microRNAs. We have sequenced microRNAs, together with mRNAs and long non-coding RNAs, in Parhyale using paired size-selected RNA-seq libraries at seven time-points covering important transitions in embryonic development. Focussing on microRNAs, we annotate 175 loci in Parhyale, 85 of which have no known homologs. We use these data to annotate the microRNome of 37 crustacean genomes, and suggest a core crustacean microRNA set of around 61 sequence families. We examine the dynamic expression of microRNAs and mRNAs during the maternal-zygotic transition. Our data suggest that zygotic genome activation occurs in two waves in Parhyale with microRNAs transcribed almost exclusively in the second wave. Contrary to findings in other arthropods, we do not predict a general role for microRNAs in clearing maternal transcripts. These data significantly expand the available transcriptomics resources for Parhyale, and facilitate its use as a model organism for the study of small RNAs in processes ranging from embryonic development to regeneration.

developmental biology↗

bioRxiv2020

smiFISH and embryo segmentation for single-cell multi-gene RNA quantification in arthropods

Recently, advances in fluorescent in-situ hybridization techniques and in imaging technology have enabled visualisation and counting of individual RNA molecules in single cells. This has greatly enhanced the resolution in our understanding of transcriptional processes. Here, we adapt a recently published smiFISH protocol (single-molecule inexpensive fluorescent in-situ hybridization) to whole embryos across a range of arthropod model species, and also to non-embryonic tissues. Using multiple fluorophores with distinct spectra and white light laser confocal imaging, we simultaneously detect and separate single RNAs from up to eight different genes in a whole embryo. We also combine smiFISH with cell membrane immunofluorescence, and present an imaging and analysis pipeline for 3D cell segmentation and single-cell RNA counting in whole blastoderm embryos. Finally, using whole embryo single-cell RNA count data, we propose two alternative single-cell variability measures to the commonly used Fano factor, and compare the capacity of these three measures to address different aspects of single-cell expression variability.

developmental biology↗

bioRxiv2017

The house spider genome reveals an ancient whole-genome duplication during arachnid evolution

The duplication of genes can occur through various mechanisms and is thought to make a major contribution to the evolutionary diversification of organisms. There is increasing evidence for a large-scale duplication of genes in some chelicerate lineages including two rounds of whole genome duplication (WGD) in horseshoe crabs. To investigate this further we sequenced and analyzed the genome of the common house spider Parasteatoda tepidariorum. We found pervasive duplication of both coding and non-coding genes in this spider, including two clusters of Hox genes. Analysis of synteny conservation across the P. tepidariorum genome suggests that there has been an ancient WGD in spiders. Comparison with the genomes of other chelicerates, including that of the newly sequenced bark scorpion Centruroides sculpturatus, suggests that this event occurred in the common ancestor of spiders and scorpions and is probably independent of the WGDs in horseshoe crabs. Furthermore, characterization of the sequence and expression of the Hox paralogs in P. tepidariorum suggests that many have been subject to neofunctionalization and/or subfunctionalization since their duplication, and therefore may have contributed to the diversification of spiders and other pulmonate arachnids.

genomics↗