Evolution of gene expression across brain regions in behaviourally divergent deer mice.

The evolution of innate behaviours is ultimately due to genetic variation likely acting in the nervous system. Gene regulation may be particularly important because it can evolve in a modular brain-region specific fashion through the concerted action of cis- and trans-regulatory changes. Here, to investigate transcriptional variation and its regulatory basis across the brain, we perform RNA sequencing (RNA-Seq) on ten brain subregions in two sister species of deer mice (Peromyscus maniculatus and P. polionotus)-which differ in a range of innate behaviours, including their social system-and their F1 hybrids. We find that most of the variation in gene expression distinguishes subregions, followed by species. Interspecific differential expression (DE) is pervasive (52-59% of expressed genes), whereas the number of DE genes between sexes is modest overall (~3%). Interestingly, the identity of DE genes varies considerably across brain regions. Much of this modularity is due to cis-regulatory divergence, and while 43% of genes were consistently assigned to the same gene regulatory class across subregions (e.g. conserved, cis- or trans-regulatory divergence), a similar number were assigned to two or more different gene regulatory classes. Together, these results highlight the modularity of gene expression differences and divergence in the brain, which may be key to explain how the evolution of brain gene expression can contribute to the astonishing diversity of animal behaviours.

When Sex Chromosomes Recombine Only in the Heterogametic Sex: Heterochiasmy and Heterogamety in Hyla Tree Frogs.

Sex chromosomes are classically predicted to stop recombining in the heterogametic sex, thereby enforcing linkage between sex-determining (SD) and sex-antagonistic (SA) genes. With the same rationale, a pre-existing sex asymmetry in recombination is expected to affect the evolution of heterogamety, for example, a low rate of male recombination might favor transitions to XY systems, by generating immediate linkage between SD and SA genes. Furthermore, the accumulation of deleterious mutations on nonrecombining Y chromosomes should favor XY-to-XY transitions (which discard the decayed Y), but disfavor XY-to-ZW transitions (which fix the decayed Y as an autosome). Like many anuran amphibians, Hyla tree frogs have been shown to display drastic heterochiasmy (males only recombine at chromosome tips) and are typically XY, which seems to fit the above expectations. Instead, here we demonstrate that two species, H. sarda and H. savignyi, share a common ZW system since at least 11 Ma. Surprisingly, the typical pattern of restricted male recombination has been maintained since then, despite female heterogamety. Hence, sex chromosomes recombine freely in ZW females, not in ZZ males. This suggests that heterochiasmy does not constrain heterogamety (and vice versa), and that the role of SA genes in the evolution of sex chromosomes might have been overemphasized.

Evolution of mosquito preference for humans linked to an odorant receptor.

Female mosquitoes are major vectors of human disease and the most dangerous are those that preferentially bite humans. A 'domestic' form of the mosquito Aedes aegypti has evolved to specialize in biting humans and is the main worldwide vector of dengue, yellow fever, and chikungunya viruses. The domestic form coexists with an ancestral, 'forest' form that prefers to bite non-human animals and is found along the coast of Kenya. We collected the two forms, established laboratory colonies, and document striking divergence in preference for human versus non-human animal odour. We further show that the evolution of preference for human odour in domestic mosquitoes is tightly linked to increases in the expression and ligand-sensitivity of the odorant receptor AaegOr4, which we found recognizes a compound present at high levels in human odour. Our results provide a rare example of a gene contributing to behavioural evolution and provide insight into how disease-vectoring mosquitoes came to specialize on humans.

The genetics of morphological and behavioural island traits in deer mice.

Animals on islands often exhibit dramatic differences in morphology and behaviour compared with mainland individuals, a phenomenon known as the 'island syndrome'. These differences are thought to be adaptations to island environments, but the extent to which they have a genetic basis or instead represent plastic responses to environmental extremes is often unknown. Here, we revisit a classic case of island syndrome in deer mice (Peromyscus maniculatus) from British Columbia. We first show that Saturna Island mice and those from neighbouring islands are approximately 35% (approx. 5 g) heavier than mainland mice and diverged approximately 10 000 years ago. We then establish laboratory colonies and find that Saturna Island mice are heavier both because they are longer and have disproportionately more lean mass. These trait differences are maintained in second-generation captive-born mice raised in a common environment. In addition, island-mainland hybrids reveal a maternal genetic effect on body weight. Using behavioural testing in the laboratory, we also find that wild-caught island mice are less aggressive than mainland mice; however, laboratory-raised mice born to these founders do not differ in aggression. Together, our results reveal that these mice have different responses to the environmental conditions on islands-a heritable change in a morphological trait and a plastic response in a behavioural trait.

Pre-Quaternary divergence and subsequent radiation explain longitudinal patterns of genetic and morphological variation in the striped skink, Heremites vittatus.

BACKGROUND: Many animal and plant species in the Middle East and northern Africa have a predominantly longitudinal distribution, extending from Iran and Turkey along the eastern Mediterranean coast into northern Africa. These species are potentially characterized by longitudinal patterns of biological diversity, but little is known about the underlying biogeographic mechanisms and evolutionary timescales. We examined these questions in the striped skink, Heremites vittatus, one such species with a roughly longitudinal distribution across the Middle East and northern Africa, by analyzing range-wide patterns of mitochondrial DNA (mtDNA) sequence and multi-trait morphological variation. RESULTS: The striped skink exhibits a basic longitudinal organization of mtDNA diversity, with three major mitochondrial lineages inhabiting northern Africa, the eastern Mediterranean coast, and Turkey/Iran. Remarkably, these lineages are of pre-Quaternary origin, and are characterized by p-distances of 9-10%. In addition, within each of these lineages a more recent Quaternary genetic diversification was observed, as evidenced by deep subclades and high haplotype diversity especially in the Turkish/Iranian and eastern Mediterranean lineages. Consistent with the genetic variation, our morphological analysis revealed that the majority of morphological traits show significant mean differences between specimens from northern Africa, the eastern Mediterranean coast, and Turkey/Iran, suggesting lineage-specific trait evolution. In addition, a subset of traits exhibits clinal variation along the eastern Mediterranean coast, potentially indicating selection gradients at the geographic transition from northern Africa to Anatolia. The existence of allopatric, morphologically and genetically divergent lineages suggests that Heremites vittatus might represent a complex with several taxa. CONCLUSIONS: Our work demonstrates that early divergence events in the Pliocene, likely driven by both climatic and geological factors, established the longitudinal patterns and distribution of Heremites vittatus. Subsequent radiation during the Pleistocene generated the genetic and morphological diversity observed today. Our study provides further evidence that longitudinal diversity patterns and species distributions in the Middle East and northern Africa were shaped by complex evolutionary processes, involving the region's intricate geological history, climatic oscillations, and the presence of the Sahara.

The neural basis of defensive behaviour evolution in Peromyscus mice.

Evading imminent predator threat is critical for survival. Effective defensive strategies can vary, even between closely related species. However, the neural basis of such species-specific behaviours is still poorly understood. Here we find that two sister species of deer mice (genus Peromyscus) show different responses to the same looming stimulus: P. maniculatus, which occupy densely vegetated habitats, predominantly dart to escape, while the open field specialist, P. polionotus, pause their movement. This difference arises from species-specific escape thresholds, is largely context-independent, and can be triggered by both visual and auditory threat stimuli. Using immunohistochemistry and electrophysiological recordings, we find that although visual threat activates the superior colliculus in both species, the role of the dorsal periaqueductal gray (dPAG) in driving behaviour differs. While dPAG activity scales with running speed and involves both excitatory and inhibitory neurons in P. maniculatus, the dPAG is largely silent in P. polionotus, even when darting is triggered. Moreover, optogenetic activation of excitatory dPAG neurons reliably elicits darting behaviour in P. maniculatus but not P. polionotus. Together, we trace the evolution of species-specific escape thresholds to a central circuit node, downstream of peripheral sensory neurons, localizing an ecologically relevant behavioural difference to a specific region of the complex mammalian brain.

LIM protein Ajuba promotes liver cell proliferation through its involvement in DNA replication and DNA damage control.

The LIM-domain protein Ajuba is associated with cell proliferation, a fundamental process of tissue regeneration and cancer. We report that in the liver, Ajuba expression is increased during regeneration and in tumour cells and tissues. Knockout of Ajuba using CRISPR/Cas9 is embryonic lethal in mice. shRNA targeting of Ajuba reduces cell proliferation, delays cell entry into S-phase, reduces cell survival and tumour growth in vivo and increases expression of the DNA damage marker γH2AX. Ajuba binding partners include proteins involved in DNA replication and damage, such as SKP2, MCM2, MCM7 and RPA70. Taken together, our data support that Ajuba promotes liver cell proliferation associated with development, regeneration and tumour growth and is involved in DNA replication and damage repair.

Interspecific variation in cooperative burrowing behavior by Peromyscus mice.

Animals often adjust their behavior according to social context, but the capacity for such behavioral flexibility can vary among species. Here, we test for interspecific variation in behavioral flexibility by comparing burrowing behavior across three species of deer mice (genus Peromyscus) with divergent social systems, ranging from promiscuous (Peromyscus leucopus and Peromyscus maniculatus) to monogamous (Peromyscus polionotus). First, we compared the burrows built by individual mice to those built by pairs of mice in all three species. Although burrow length did not differ in P. leucopus or P. maniculatus, we found that P. polionotus pairs cooperatively constructed burrows that were nearly twice as long as those built by individuals and that opposite-sex pairs dug longer burrows than same-sex pairs. Second, to directly observe cooperative digging behavior in P. polionotus, we designed a burrowing assay in which we could video-record active digging in narrow, transparent enclosures. Using this novel assay, we found, unexpectedly, that neither males nor females spent more time digging with an opposite-sex partner. Rather, we demonstrate that opposite-sex pairs are more socially cohesive and thus more efficient digging partners than same-sex pairs. Together, our study demonstrates how social context can modulate innate behavior and offers insight into how differences in behavioral flexibility may evolve among closely related species.

Loss of Claudin-3 Impairs Hepatic Metabolism, Biliary Barrier Function, and Cell Proliferation in the Murine Liver.

BACKGROUND & AIMS: Tight junctions in the liver are essential to maintain the blood-biliary barrier, however, the functional contribution of individual tight junction proteins to barrier and metabolic homeostasis remains largely unexplored. Here, we describe the cell type-specific expression of tight junction genes in the murine liver, and explore the regulation and functional importance of the transmembrane protein claudin-3 in liver metabolism, barrier function, and cell proliferation. METHODS: The cell type-specific expression of hepatic tight junction genes is described using our mouse liver single-cell sequencing data set. Differential gene expression in Cldn3-/- and Cldn3+/+ livers was assessed in young and aged mice by RNA sequencing (RNA-seq), and hepatic tissue was analyzed for lipid content and bile acid composition. A surgical model of partial hepatectomy was used to induce liver cell proliferation. RESULTS: Claudin-3 is a highly expressed tight junction protein found in the liver and is expressed predominantly in hepatocytes and cholangiocytes. The histology of Cldn3-/- livers showed no overt phenotype, and the canalicular tight junctions appeared intact. Nevertheless, by RNA-seq we detected a down-regulation of metabolic pathways in the livers of Cldn3-/- young and aged mice, as well as a decrease in lipid content and a weakened biliary barrier for primary bile acids, such as taurocholic acid, taurochenodeoxycholic acid, and taurine-conjugated muricholic acid. Coinciding with defects in the biliary barrier and lower lipid metabolism, there was a diminished hepatocyte proliferative response in Cldn3-/- mice after partial hepatectomy. CONCLUSIONS: Our data show that, in the liver, claudin-3 is necessary to maintain metabolic homeostasis, retention of bile acids, and optimal hepatocyte proliferation during liver regeneration. The RNA-seq data set can be accessed at: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE159914.

Site selective adsorption of the spin crossover complex Fe(phen)2(NCS) on Au(111).

The iron(II) spin crossover complex Fe(1,10-phenanthroline)2(NCS)2, dubbed Fe-phen, has been studied with scanning tunneling microscopy, after adsorption on the 'herringbone' reconstructed surface of Au(111) for sub-monolayer coverages. The Fe-phen molecules attach, through their NCS-groups, to the Au atoms of the fcc domains of the reconstructed surface only, thereby lifting the herringbone reconstruction. The molecules stack to form 1D chains, which run along the Au[110] directions. Neighboring Fe-phen molecules are separated by approximately 2.65 nm, corresponding to 9 atomic spacings in this direction. The molecular axis, defined by the two phenanthroline groups, is aligned perpendicular to the chain axis, along the Au [Formula: see text] direction, thereby bridging over 5 atomic spacings, in this direction. Experimental evidence suggests that the molecular spins are locked in a mixed state in the sub-monolayer regime at temperatures between 100 K and 300 K.

The LIM Protein Ajuba Augments Tumor Metastasis in Colon Cancer.

Colorectal cancer, along with its high potential for recurrence and metastasis, is a major health burden. Uncovering proteins and pathways required for tumor cell growth is necessary for the development of novel targeted therapies. Ajuba is a member of the LIM domain family of proteins whose expression is positively associated with numerous cancers. Our data shows that Ajuba is highly expressed in human colon cancer tissue and cell lines. Publicly available data from The Cancer Genome Atlas shows a negative correlation between survival and Ajuba expression in patients with colon cancer. To investigate its function, we transduced SW480 human colon cancer cells, with lentiviral constructs to knockdown or overexpress Ajuba protein. The transcriptome of the modified cell lines was analyzed by RNA sequencing. Among the pathways enriched in the differentially expressed genes, were cell proliferation, migration and differentiation. We confirmed our sequencing data with biological assays; cells depleted of Ajuba were less proliferative, more sensitive to irradiation, migrated less and were less efficient in colony formation. In addition, loss of Ajuba expression decreased the tumor burden in a murine model of colorectal metastasis to the liver. Taken together, our data supports that Ajuba promotes colon cancer growth, migration and metastasis and therefore is a potential candidate for targeted therapy.

Claudin-3-deficient C57BL/6J mice display intact brain barriers.

The tight junction protein claudin-3 has been identified as a transcriptional target of the Wnt/ß-catenin signaling pathway regulating blood-brain barrier (BBB) maturation. In neurological disorders loss of claudin-3 immunostaining is observed at the compromised BBB and blood-cerebrospinal fluid barrier (BCSFB). Although these observations support a central role of claudin-3 in regulating brain barriers' tight junction integrity, expression of claudin-3 at the brain barriers has remained a matter of debate. This prompted us to establish claudin-3-/- C57BL/6J mice to study the role of claudin-3 in brain barrier integrity in health and neuroinflammation. Bulk and single cell RNA sequencing and direct comparative qRT-PCR analysis of brain microvascular samples from WT and claudin-3-/- mice show beyond doubt that brain endothelial cells do not express claudin-3 mRNA. Detection of claudin-3 protein at the BBB in vivo and in vitro is rather due to junctional reactivity of anti-claudin-3 antibodies to an unknown antigen still detected in claudin-3-/- brain endothelium. We confirm expression and junctional localization of claudin-3 at the BCSFB of the choroid plexus. Our study clarifies that claudin-3 is not expressed at the BBB and shows that absence of claudin-3 does not impair brain barrier function during health and neuroinflammation in C57BL/6J mice.

Publisher Correction: Claudin-3-deficient C57BL/6J mice display intact brain barriers.

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Impaired liver regeneration in aged mice can be rescued by silencing Hippo core kinases MST1 and MST2.

The liver has an intrinsic capacity to regenerate in response to injury or surgical resection. Nevertheless, circumstances in which hepatocytes are unresponsive to proliferative signals result in impaired regeneration and hepatic failure. As the Hippo pathway has a canonical role in the maintenance of liver size, we investigated whether it could serve as a therapeutic target to support regeneration. Using a standard two-thirds partial hepatectomy (PH) model in young and aged mice, we demonstrate that the Hippo pathway is modulated across the phases of liver regeneration. The activity of the core kinases MST1 and LATS1 increased during the early hypertrophic phase and returned to steady state levels in the proliferative phase, coinciding with activation of YAP1 target genes and hepatocyte proliferation. Moreover, following PH in aged mice, we demonstrate that Hippo signaling is anomalous in non-regenerating livers. We provide pre-clinical evidence that silencing the Hippo core kinases MST1 and MST2 with siRNA provokes hepatocyte proliferation in quiescent livers and rescues liver regeneration in aged mice following PH. Our data suggest that targeting the Hippo core kinases MST1/2 has therapeutic potential to improve regeneration in non-regenerative disorders.

Preconditioning boosts regenerative programmes in the adult zebrafish heart.

During preconditioning, exposure to a non-lethal harmful stimulus triggers a body-wide increase of survival and pro-regenerative programmes that enable the organism to better withstand the deleterious effects of subsequent injuries. This phenomenon has first been described in the mammalian heart, where it leads to a reduction of infarct size and limits the dysfunction of the injured organ. Despite its important clinical outcome, the actual mechanisms underlying preconditioning-induced cardioprotection remain unclear. Here, we describe two independent models of cardiac preconditioning in the adult zebrafish. As noxious stimuli, we used either a thoracotomy procedure or an induction of sterile inflammation by intraperitoneal injection of immunogenic particles. Similar to mammalian preconditioning, the zebrafish heart displayed increased expression of cardioprotective genes in response to these stimuli. As zebrafish cardiomyocytes have an endogenous proliferative capacity, preconditioning further elevated the re-entry into the cell cycle in the intact heart. This enhanced cycling activity led to a long-term modification of the myocardium architecture. Importantly, the protected phenotype brought beneficial effects for heart regeneration within one week after cryoinjury, such as a more effective cell-cycle reentry, enhanced reactivation of embryonic gene expression at the injury border, and improved cell survival shortly after injury. This study reveals that exposure to antecedent stimuli induces adaptive responses that render the fish more efficient in the activation of the regenerative programmes following heart damage. Our results open a new field of research by providing the adult zebrafish as a model system to study remote cardiac preconditioning.

Distinct effects of inflammation on preconditioning and regeneration of the adult zebrafish heart.

The adult heart is able to activate cardioprotective programmes and modifies its architecture in response to physiological or pathological changes. While mammalian cardiac remodelling often involves hypertrophic expansion, the adult zebrafish heart exploits hyperplastic growth. This capacity depends on the responsiveness of zebrafish cardiomyocytes to mitogenic signals throughout their entire life. Here, we have examined the role of inflammation on the stimulation of cell cycle activity in the context of heart preconditioning and regeneration. We used thoracotomy as a cardiac preconditioning model and cryoinjury as a model of cardiac infarction in the adult zebrafish. First, we performed a spatio-temporal characterization of leucocytes and cycling cardiac cells after thoracotomy. This analysis revealed a concomitance between the infiltration of inflammatory cells and the stimulation of the mitotic activity. However, decreasing the immune response using clodronate liposome injection, PLX3397 treatment or anti-inflammatory drugs surprisingly had no effect on the re-entry of cardiac cells into the cell cycle. In contrast, reducing inflammation using the same strategies after cryoinjury strongly impaired cardiac cell mitotic activity and the regenerative process. Taken together, our results show that, while the immune response is not necessary to induce cell-cycle activity in intact preconditioned hearts, inflammation is required for the regeneration of injured hearts in zebrafish.