Genetic compensation triggered by mutant mRNA degradation.

Genetic robustness, or the ability of an organism to maintain fitness in the presence of harmful mutations, can be achieved via protein feedback loops. Previous work has suggested that organisms may also respond to mutations by transcriptional adaptation, a process by which related gene(s) are upregulated independently of protein feedback loops. However, the prevalence of transcriptional adaptation and its underlying molecular mechanisms are unknown. Here, by analysing several models of transcriptional adaptation in zebrafish and mouse, we uncover a requirement for mutant mRNA degradation. Alleles that fail to transcribe the mutated gene do not exhibit transcriptional adaptation, and these alleles give rise to more severe phenotypes than alleles displaying mutant mRNA decay. Transcriptome analysis in alleles displaying mutant mRNA decay reveals the upregulation of a substantial proportion of the genes that exhibit sequence similarity with the mutated gene's mRNA, suggesting a sequence-dependent mechanism. These findings have implications for our understanding of disease-causing mutations, and will help in the design of mutant alleles with minimal transcriptional adaptation-derived compensation.

Functional equivalence of germ plasm organizers.

The proteins Oskar (Osk) in Drosophila and Bucky ball (Buc) in zebrafish act as germ plasm organizers. Both proteins recapitulate germ plasm activities but seem to be unique to their animal groups. Here, we discover that Osk and Buc show similar activities during germ cell specification. Drosophila Osk induces additional PGCs in zebrafish. Surprisingly, Osk and Buc do not show homologous protein motifs that would explain their related function. Nonetheless, we detect that both proteins contain stretches of intrinsically disordered regions (IDRs), which seem to be involved in protein aggregation. IDRs are known to rapidly change their sequence during evolution, which might obscure biochemical interaction motifs. Indeed, we show that Buc binds to the known Oskar interactors Vasa protein and nanos mRNA indicating conserved biochemical activities. These data provide a molecular framework for two proteins with unrelated sequence but with equivalent function to assemble a conserved core-complex nucleating germ plasm.

Induction of interferon-stimulated genes and cellular stress pathways by morpholinos in zebrafish.

The phenotypes caused by morpholino-mediated interference of gene function in zebrafish are often not observed in the corresponding mutant(s). We took advantage of the availability of a relatively large collection of transcriptomic datasets to identify common signatures that characterize morpholino-injected animals (morphants). In addition to the previously reported activation of tp53 expression, we observed increased expression of the interferon-stimulated genes (ISGs), isg15 and isg20, the cell death pathway gene casp8, and other cellular stress response genes including phlda3, mdm2 and gadd45aa. Studies involving segmentation stage embryos were more likely to show upregulation of these genes. We also found that the expression of these genes could be upregulated by increasing doses of an egfl7 morpholino, or even high doses of the standard control morpholino. Thus, these data show that morpholinos can induce the expression of ISGs in zebrafish embryos and further our understanding of morpholino effects.

Genetic compensation: A phenomenon in search of mechanisms.

Several recent studies in a number of model systems including zebrafish, Arabidopsis, and mouse have revealed phenotypic differences between knockouts (i.e., mutants) and knockdowns (e.g., antisense-treated animals). These differences have been attributed to a number of reasons including off-target effects of the antisense reagents. An alternative explanation was recently proposed based on a zebrafish study reporting that genetic compensation was observed in egfl7 mutant but not knockdown animals. Dosage compensation was first reported in Drosophila in 1932, and genetic compensation in response to a gene knockout was first reported in yeast in 1969. Since then, genetic compensation has been documented many times in a number of model organisms; however, our understanding of the underlying molecular mechanisms remains limited. In this review, we revisit studies reporting genetic compensation in higher eukaryotes and outline possible molecular mechanisms, which may include both transcriptional and posttranscriptional processes.

Multi-species genome-wide CRISPR screens identify conserved suppressors of cold-induced cell death.

Cells must adapt to environmental changes to maintain homeostasis. One of the most striking environmental adaptations is entry into hibernation during which core body temperature can decrease from 37°C to as low at 4°C. How mammalian cells, which evolved to optimally function within a narrow range of temperatures, adapt to this profound decrease in temperature remains poorly understood. In this study, we conducted the first genome-scale CRISPR-Cas9 screen in cells derived from Syrian hamster, a facultative hibernator, as well as human cells to investigate the genetic basis of cold tolerance in a hibernator and a non-hibernator in an unbiased manner. Both screens independently revealed glutathione peroxidase 4 (GPX4), a selenium-containing enzyme, and associated proteins as critical for cold tolerance. We utilized genetic and pharmacological approaches to demonstrate that GPX4 is active in the cold and its catalytic activity is required for cold tolerance. Furthermore, we show that the role of GPX4 as a suppressor of cold-induced cell death extends across hibernating species, including 13-lined ground squirrels and greater horseshoe bats, highlighting the evolutionary conservation of this mechanism of cold tolerance. This study identifies GPX4 as a central modulator of mammalian cold tolerance and advances our understanding of the evolved mechanisms by which cells mitigate cold-associated damage-one of the most common challenges faced by cells and organisms in nature.

Parental mutations influence wild-type offspring via transcriptional adaptation.

Transgenerational epigenetic inheritance (TEI) is mostly discussed in the context of physiological or environmental factors. Here, we show intergenerational and transgenerational inheritance of transcriptional adaptation (TA), a process whereby mutant messenger RNA (mRNA) degradation affects gene expression, in nematodes and zebrafish. Wild-type offspring of animals heterozygous for mRNA-destabilizing alleles display increased expression of adapting genes. Notably, offspring of animals heterozygous for nontranscribing alleles do not display this response. Germline-specific mutations are sufficient to induce TA in wild-type offspring, indicating that, at least for some genes, mutations in somatic tissues are not necessary for this process. Microinjecting total RNA from germ cells of TA-displaying heterozygous zebrafish can trigger TA in wild-type embryos and in their progeny, suggesting a model whereby mutant mRNAs in the germline trigger a TA response that can be epigenetically inherited. In sum, this previously unidentified mode of TEI reveals a means by which parental mutations can modulate the offspring's transcriptome.

Transcriptional adaptation in Caenorhabditis elegans.

Transcriptional adaptation is a recently described phenomenon by which a mutation in one gene leads to the transcriptional modulation of related genes, termed adapting genes. At the molecular level, it has been proposed that the mutant mRNA, rather than the loss of protein function, activates this response. While several examples of transcriptional adaptation have been reported in zebrafish embryos and in mouse cell lines, it is not known whether this phenomenon is observed across metazoans. Here we report transcriptional adaptation in C. elegans, and find that this process requires factors involved in mutant mRNA decay, as in zebrafish and mouse. We further uncover a requirement for Argonaute proteins and Dicer, factors involved in small RNA maturation and transport into the nucleus. Altogether, these results provide evidence for transcriptional adaptation in C. elegans, a powerful model to further investigate underlying molecular mechanisms.

Metabolic modulation regulates cardiac wall morphogenesis in zebrafish.

During cardiac development, cardiomyocytes form complex inner wall structures called trabeculae. Despite significant investigation into this process, the potential role of metabolism has not been addressed. Using single cell resolution imaging in zebrafish, we find that cardiomyocytes seeding the trabecular layer actively change their shape while compact layer cardiomyocytes remain static. We show that Erbb2 signaling, which is required for trabeculation, activates glycolysis to support changes in cardiomyocyte shape and behavior. Pharmacological inhibition of glycolysis impairs cardiac trabeculation, and cardiomyocyte-specific loss- and gain-of-function manipulations of glycolysis decrease and increase trabeculation, respectively. In addition, loss of the glycolytic enzyme pyruvate kinase M2 impairs trabeculation. Experiments with rat neonatal cardiomyocytes in culture further support these observations. Our findings reveal new roles for glycolysis in regulating cardiomyocyte behavior during cardiac wall morphogenesis.

The flow responsive transcription factor Klf2 is required for myocardial wall integrity by modulating Fgf signaling.

Complex interplay between cardiac tissues is crucial for their integrity. The flow responsive transcription factor KLF2, which is expressed in the endocardium, is vital for cardiovascular development but its exact role remains to be defined. To this end, we mutated both klf2 paralogues in zebrafish, and while single mutants exhibit no obvious phenotype, double mutants display a novel phenotype of cardiomyocyte extrusion towards the abluminal side. This extrusion requires cardiac contractility and correlates with the mislocalization of N-cadherin from the lateral to the apical side of cardiomyocytes. Transgenic rescue data show that klf2 expression in endothelium, but not myocardium, prevents this cardiomyocyte extrusion phenotype. Transcriptome analysis of klf2 mutant hearts reveals that Fgf signaling is affected, and accordingly, we find that inhibition of Fgf signaling in wild-type animals can lead to abluminal cardiomyocyte extrusion. These studies provide new insights into how Klf2 regulates cardiovascular development and specifically myocardial wall integrity.

Loss of pyruvate kinase M2 limits growth and triggers innate immune signaling in endothelial cells.

Despite their inherent proximity to circulating oxygen and nutrients, endothelial cells (ECs) oxidize only a minor fraction of glucose in mitochondria, a metabolic specialization that is poorly understood. Here we show that the glycolytic enzyme pyruvate kinase M2 (PKM2) limits glucose oxidation, and maintains the growth and epigenetic state of ECs. We find that loss of PKM2 alters mitochondrial substrate utilization and impairs EC proliferation and migration in vivo. Mechanistically, we show that the NF-κB transcription factor RELB is responsive to PKM2 loss, limiting EC growth through the regulation of P53. Furthermore, S-adenosylmethionine synthesis is impaired in the absence of PKM2, resulting in DNA hypomethylation, de-repression of endogenous retroviral elements (ERVs) and activation of antiviral innate immune signalling. This work reveals the metabolic and functional consequences of glucose oxidation in the endothelium, highlights the importance of PKM2 for endothelial growth and links metabolic dysfunction with autoimmune activation in ECs.