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Diapause is a state of developmental and metabolic dormancy that precedes exposure to environmental stresses. Yet, diapausing embryos are typically stress-tolerant. Evidence suggests that diapausing embryos “prepare” for stress as part of a gene expression program as they enter dormancy. Here, we investigate if diapause II embryos of the annual killifish Austrofundulus limnaeus, which can survive for hundreds of days of anoxia, can mount a transcriptomic response to anoxic insult. Bulk RNAseq was used to characterize the transcriptomes of diapause II embryos exposed to normoxia, 4 h and 24 h anoxia, and 2 h and 24 h normoxic recovery from anoxia. Differential expression and gene ontology analyses were used to probe for pathways that may mitigate survival. Transcriptional factor analysis was used to predict potential mediators of this response. Diapausing embryos exhibited a robust transcriptomic response to anoxia and recovery that returns to near baseline conditions after 24 h. Anoxia induced an upregulation of genes involved in the integrated stress response, lipid metabolism, p38mapk kinase signaling, and apoptosis. Developmental and mitochondrial genes decreased. We conclude that diapause II embryos mount a robust transcriptomic stress response when faced with anoxic insult. This response is consistent with mediating expected challenges to cellular homeostasis in anoxia. 

In recent years a diversity of small noncoding RNAs have been identified that originate from the mitochondrial genome. These mitosRNAs are often dominated by tRNA-derived small RNAs (mito-tDRs). Differential expression of mito-tDRs is associated with responses to stress. They also appear to be expressed differentially during development and their expression may be enriched in stress-tolerant animals. Very little is currently known about roles or modes of action of these sequences, although they are implicated in a diversity of processes such as cell cycle regulation, mRNA stability, regulation of ROS production, and import of proteins into the mitochondrion. To better understand the various roles these sequences may play, it is critical that we understand their diversity, cellular location, and the context for their expression. This protocol outlines the methodologies used to detect mitosRNAs, including mito-tDRs, in embryos and cells of the annual killifish Austrofundulus limnaeus. We highlight critical steps in the isolation of RNA, creation of sequencing libraries, bioinformatics processing of sequence data, and methods for validation of expression that support a robust discovery pipeline for mitosRNAs even from species with incomplete reference genome sequences. 

Abstract We are entering an era in which we will be able to detect and characterize hundreds of dwarf galaxies within the Local Volume. It is already known that a strong dichotomy exists in the gas content and star formation properties of field dwarf galaxies versus satellite dwarfs of larger galaxies. In this work, we study the more subtle differences that may be detectable in galaxies as a function of distance from a massive galaxy, such as the Milky Way. We compare smoothed particle hydrodynamic simulations of dwarf galaxies formed in a Local Volume-like environment (several megaparsecs away from a massive galaxy) to those formed nearer to Milky Way–mass halos. We find that the impact of environment on dwarf galaxies extends even beyond the immediate region surrounding Milky Way–mass halos. Even before being accreted as satellites, dwarf galaxies near a Milky Way–mass halo tend to have higher stellar masses for their halo mass than more isolated galaxies. Dwarf galaxies in high-density environments also tend to grow faster and form their stars earlier. We show observational predictions that demonstrate how these trends manifest in lower quenching rates, higher Hifractions, and bluer colors for more isolated dwarf galaxies.