Defensive responses: behaviour, the brain and the body.

Most animals live under constant threat from predators, and predation has been a major selective force in shaping animal behaviour. Nevertheless, defence responses against predatory threats need to be balanced against other adaptive behaviours such as foraging, mating and recovering from infection. This behavioural balance in ethologically relevant contexts requires adequate integration of internal and external signals in a complex interplay between the brain and the body. Despite this complexity, research has often considered defensive behaviour as entirely mediated by the brain processing threat-related information obtained via perception of the external environment. However, accumulating evidence suggests that the endocrine, immune, gastrointestinal and reproductive systems have important roles in modulating behavioural responses to threat. In this Review, we focus on how predatory threat defence responses are shaped by threat imminence and review the circuitry between subcortical brain regions involved in mediating defensive behaviours. Then, we discuss the intersection of peripheral systems involved in internal states related to infection, hunger and mating with the neurocircuits that underlie defence responses against predatory threat. Through this process, we aim to elucidate the interconnections between the brain and body as an integrated network that facilitates appropriate defensive responses to threat and to discuss the implications for future behavioural research.

A missense mutation in SNRPE linked to non-syndromal microcephaly interferes with U snRNP assembly and pre-mRNA splicing.

Malfunction of pre-mRNA processing factors are linked to several human diseases including cancer and neurodegeneration. Here we report the identification of a de novo heterozygous missense mutation in the SNRPE gene (c.65T>C (p.Phe22Ser)) in a patient with non-syndromal primary (congenital) microcephaly and intellectual disability. SNRPE encodes SmE, a basal component of pre-mRNA processing U snRNPs. We show that the microcephaly-linked SmE variant is unable to interact with the SMN complex and as a consequence fails to assemble into U snRNPs. This results in widespread mRNA splicing alterations in fibroblast cells derived from this patient. Similar alterations were observed in HEK293 cells upon SmE depletion that could be rescued by the expression of wild type but not mutant SmE. Importantly, the depletion of SmE in zebrafish causes aberrant mRNA splicing alterations and reduced brain size, reminiscent of the patient microcephaly phenotype. We identify the EMX2 mRNA, which encodes a protein required for proper brain development, as a major mis-spliced down stream target. Together, our study links defects in the SNRPE gene to microcephaly and suggests that alterations of cellular splicing of specific mRNAs such as EMX2 results in the neurological phenotype of the disease.

Pan-tissue analysis of allelic alternative polyadenylation suggests widespread functional regulation.

Alternative polyadenylation (APA) is a major layer of gene regulation. However, it has recently been argued that most APA represents molecular noise. To clarify their functional relevance and evolution, we quantified allele-specific APA patterns in multiple tissues from an F1 hybrid mouse. We found a clearly negative correlation between gene expression and APA diversity for the 2,866 genes (24.9%) with a dominant polyadenylation site (PAS) usage above or equal to 90%, suggesting that their other PASs represent molecular errors. Among the remaining genes with multiple PASs, 3,971 genes (34.5%) express two or more isoforms with potentially functional importance. Interestingly, the genes with potentially functional minor PASs specific to neuronal tissues often express two APA isoforms with distinct subcellular localizations. Furthermore, our analysis of cis-APA divergence shows its pattern across tissues is distinct from that of gene expression. Finally, we demonstrate that the relative usage of alternative PASs is not only affected by their cis-regulatory elements, but also by potential coupling between transcriptional and APA regulation as well as competition kinetics between alternative sites.

Inheritance of gene expression level and selective constraints on trans- and cis-regulatory changes in yeast.

Gene expression evolution can be caused by changes in cis- or trans-regulatory elements or both. As cis and trans regulation operate through different molecular mechanisms, cis and trans mutations may show different inheritance patterns and may be subjected to different selective constraints. To investigate these issues, we obtained and analyzed gene expression data from two Saccharomyces cerevisiae strains and their hybrid, using high-throughput sequencing. Our data indicate that compared with other types of genes, those with antagonistic cis-trans interactions are more likely to exhibit over- or underdominant inheritance of expression level. Moreover, in accordance with previous studies, genes with trans variants tend to have a dominant inheritance pattern, whereas cis variants are enriched for additive inheritance. In addition, cis regulatory differences contribute more to expression differences between species than within species, whereas trans regulatory differences show a stronger association between divergence and polymorphism. Our data indicate that in the trans component of gene expression differences genes subjected to weaker selective constraints tend to have an excess of polymorphism over divergence compared with those subjected to stronger selective constraints. In contrast, in the cis component, this difference between genes under stronger and weaker selective constraint is mostly absent. To explain these observations, we propose that purifying selection more strongly shapes trans changes than cis changes and that positive selection may have significantly contributed to cis regulatory divergence.

Systematic evaluation of a predator stress model of depression in mice using a hierarchical 3D-motion learning framework.

Investigation of the neurobiology of depression in humans depends on animal models that attempt to mimic specific features of the human disorder. However, frequently-used paradigms based on social stress cannot be easily applied to female mice which has led to a large sex bias in preclinical studies of depression. Furthermore, most studies focus on one or only a few behavioral assessments, with time and practical considerations prohibiting a comprehensive evaluation. In this study, we demonstrate that predator stress effectively induced depression-like behaviors in both male and female mice. By comparing predator stress and social defeat models, we observed that the former elicited a higher level of behavioral despair and the latter elicited more robust social avoidance. Furthermore, the use of machine learning (ML)-based spontaneous behavioral classification can distinguish mice subjected to one type of stress from another, and from non-stressed mice. We show that related patterns of spontaneous behaviors correspond to depression status as measured by canonical depression-like behaviors, which illustrates that depression-like symptoms can be predicted by ML-classified behavior patterns. Overall, our study confirms that the predator stress induced phenotype in mice is a good reflection of several important aspects of depression in humans and illustrates that ML-supported analysis can simultaneously evaluate multiple behavioral alterations in different animal models of depression, providing a more unbiased and holistic approach for the study of neuropsychiatric disorders.

The subthalamic corticotropin-releasing hormone neurons mediate adaptive REM-sleep responses to threat.

When an animal faces a threatening situation while asleep, rapid arousal is the essential prerequisite for an adequate response. Here, we find that predator stimuli induce immediate arousal from REM sleep compared with NREM sleep. Using in vivo neural activity recording and cell-type-specific manipulations, we identify neurons in the medial subthalamic nucleus (mSTN) expressing corticotropin-releasing hormone (CRH) that mediate arousal and defensive responses to acute predator threats received through multiple sensory modalities across REM sleep and wakefulness. We observe involvement of the same neurons in the normal regulation of REM sleep and the adaptive increase in REM sleep induced by sustained predator stress. Projections to the lateral globus pallidus (LGP) are the effector pathway for the threat-coping responses and REM-sleep expression. Together, our findings suggest adaptive REM-sleep responses could be protective against threats and uncover a critical component of the neural circuitry at their basis.

Mammalian splicing divergence is shaped by drift, buffering in trans, and a scaling law.

Alternative splicing is ubiquitous, but the mechanisms underlying its pattern of evolutionary divergence across mammalian tissues are still underexplored. Here, we investigated the cis-regulatory divergences and their relationship with tissue-dependent trans-regulation in multiple tissues of an F1 hybrid between two mouse species. Large splicing changes between tissues are highly conserved and likely reflect functional tissue-dependent regulation. In particular, micro-exons frequently exhibit this pattern with high inclusion levels in the brain. Cis-divergence of splicing appears to be largely non-adaptive. Although divergence is in general associated with higher densities of sequence variants in regulatory regions, events with high usage of the dominant isoform apparently tolerate more mutations, explaining why their exon sequences are highly conserved but their intronic splicing site flanking regions are not. Moreover, we demonstrate that non-adaptive mutations are often masked in tissues where accurate splicing likely is more important, and experimentally attribute such buffering effect to trans-regulatory splicing efficiency.

Systematic Analysis of Monoallelic Gene Expression and Chromatin Accessibility Across Multiple Tissues in Hybrid Mice.

In diploid eukaryotic organisms, both alleles of each autosomal gene are usually assumed to be simultaneously expressed at similar levels. However, some genes can be expressed preferentially or strictly from a single allele, a process known as monoallelic expression. Classic monoallelic expression of X-chromosome-linked genes, olfactory receptor genes and developmentally imprinted genes is the result of epigenetic modifications. Genetic-origin-dependent monoallelic expression, however, is caused by cis-regulatory differences between the alleles. There is a paucity of systematic study to investigate these phenomena across multiple tissues, and the mechanisms underlying such monoallelic expression are not yet fully understood. Here we provide a detailed portrait of monoallelic gene expression across multiple tissues/cell lines in a hybrid mouse cross between the Mus musculus strain C57BL/6J and the Mus spretus strain SPRET/EiJ. We observed pervasive tissue-dependent allele-specific gene expression: in total, 1,839 genes exhibited monoallelic expression in at least one tissue, and 410 genes in at least two tissues. Among these 88 are monoallelic genes with different active alleles between tissues, probably representing genetic-origin-dependent monoallelic expression. We also identified six autosomal monoallelic genes with the active allele being identical in all eight tissues, which are likely novel candidates of imprinted genes. To depict the underlying regulatory mechanisms at the chromatin layer, we performed ATAC-seq in two different cell lines derived from the F1 mouse. Consistent with the global expression pattern, cell-type dependent monoallelic peaks were found, and a higher proportion of C57BL/6J-active peaks were observed in both cell types, implying possible species-specific regulation. Finally, only a small part of monoallelic gene expression could be explained by allelic differences in chromatin organization in promoter regions, suggesting that other distal elements may play important roles in shaping the patterns of allelic gene expression across tissues.

Full-length transcriptome reconstruction reveals a large diversity of RNA and protein isoforms in rat hippocampus.

Gene annotation is a critical resource in genomics research. Many computational approaches have been developed to assemble transcriptomes based on high-throughput short-read sequencing, however, only with limited accuracy. Here, we combine next-generation and third-generation sequencing to reconstruct a full-length transcriptome in the rat hippocampus, which is further validated using independent 5´ and 3´-end profiling approaches. In total, we detect 28,268 full-length transcripts (FLTs), covering 6,380 RefSeq genes and 849 unannotated loci. Based on these FLTs, we discover co-occurring alternative RNA processing events. Integrating with polysome profiling and ribosome footprinting data, we predict isoform-specific translational status and reconstruct an open reading frame (ORF)-eome. Notably, a high proportion of the predicted ORFs are validated by mass spectrometry-based proteomics. Moreover, we identify isoforms with subcellular localization pattern in neurons. Collectively, our data advance our knowledge of RNA and protein isoform diversity in the rat brain and provide a rich resource for functional studies.

Pervasive allele-specific regulation on RNA decay in hybrid mice.

Cellular RNA abundance is determined by both RNA transcription and decay. Therefore, change in RNA abundance, which can drive phenotypic diversity between different species, could arise from genetic variants affecting either process. However, previous studies in the evolution of RNA expression have been largely focused on transcription. Here, to globally investigate the effects of cis-regulatory divergence on RNA decay in mammals for the first time, we quantified allele-specific differences in RNA decay rates (ASD) in an F1 hybrid mouse. Out of 8,815 genes with sufficient data, we identified 621 genes exhibiting significant cis-divergence. Systematic analysis of these genes revealed that the genetic variants affecting microRNA binding and RNA secondary structures contribute to the observed divergences. Finally, we demonstrated that although the divergences in RNA abundance were predominantly determined by allelic differences in RNA transcription, most genes with significant ASD did not exhibit significant difference in RNA abundance. For these genes, the apparently compensatory effect between the allelic differences in RNA transcription and ASD suggests that changes in RNA decay could serve as important means to stabilize RNA abundances during mammalian evolution.

The evolution of posttranscriptional regulation.

"DNA makes RNA makes protein." After transcription, mRNAs undergo a series of intertwining processes to be finally translated into functional proteins. The "posttranscriptional" regulation (PTR) provides cells an extended option to fine-tune their proteomes. To meet the demands of complex organism development and the appropriate response to environmental stimuli, every step in these processes needs to be finely regulated. Moreover, changes in these regulatory processes are important driving forces underlying the evolution of phenotypic differences across different species. The major PTR mechanisms discussed in this review include the regulation of splicing, polyadenylation, decay, and translation. For alternative splicing and polyadenylation, we mainly discuss their evolutionary dynamics and the genetic changes underlying the regulatory differences in cis-elements versus trans-factors. For mRNA decay and translation, which, together with transcription, determine the cellular RNA or protein abundance, we focus our discussion on how their divergence coordinates with transcriptional changes to shape the evolution of gene expression. Then to highlight the importance of PTR in the evolution of higher complexity, we focus on their roles in two major phenomena during eukaryotic evolution: the evolution of multicellularity and the division of labor between different cell types and tissues; and the emergence of diverse, often highly specialized individual phenotypes, especially those concerning behavior in eusocial insects. This article is categorized under: RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution Translation > Translation Regulation RNA Processing > Splicing Regulation/Alternative Splicing.

Gains and Losses of Transcription Factor Binding Sites in Saccharomyces cerevisiae and Saccharomyces paradoxus.

Gene expression evolution occurs through changes in cis- or trans-regulatory elements or both. Interactions between transcription factors (TFs) and their binding sites (TFBSs) constitute one of the most important points where these two regulatory components intersect. In this study, we investigated the evolution of TFBSs in the promoter regions of different Saccharomyces strains and species. We divided the promoter of a gene into the proximal region and the distal region, which are defined, respectively, as the 200-bp region upstream of the transcription starting site and as the 200-bp region upstream of the proximal region. We found that the predicted TFBSs in the proximal promoter regions tend to be evolutionarily more conserved than those in the distal promoter regions. Additionally, Saccharomyces cerevisiae strains used in the fermentation of alcoholic drinks have experienced more TFBS losses than gains compared with strains from other environments (wild strains, laboratory strains, and clinical strains). We also showed that differences in TFBSs correlate with the cis component of gene expression evolution between species (comparing S. cerevisiae and its sister species Saccharomyces paradoxus) and within species (comparing two closely related S. cerevisiae strains).