Darren W. Logan

Identification of protein pheromones that promote aggressive behaviour

Mice use pheromones, compounds emitted and detected by members of the same species, as cues to regulate social behaviours such as pup suckling, aggression and mating. Neurons that detect pheromones are thought to reside in at least two separate organs within the nasal cavity: the vomeronasal organ (VNO) and the main olfactory epithelium (MOE). Each pheromone ligand is thought to activate a dedicated subset of these sensory neurons. However, the nature of the pheromone cues and the identity of the responding neurons that regulate specific social behaviours are largely unknown. Here we show, by direct activation of sensory neurons and analysis of behaviour, that at least two chemically distinct ligands are sufficient to promote male–male aggression and stimulate VNO neurons. We have purified and analysed one of these classes of ligand and found its specific aggression-promoting activity to be dependent on the presence of the protein component of the major urinary protein (MUP) complex, which is known to comprise specialized lipocalin proteins bound to small organic molecules. Using calcium imaging of dissociated vomeronasal neurons (VNs), we have determined that the MUP protein activates a sensory neuron subfamily characterized by the expression of the G-protein Gαo subunit (also known as Gnao) and Vmn2r putative pheromone receptors (V2Rs). Genomic analysis indicates species-specific co-expansions of MUPs and V2Rs, as would be expected among pheromone-signalling components. Finally, we show that the aggressive behaviour induced by the MUPs occurs exclusively through VNO neuronal circuits. Our results substantiate the idea of MUP proteins as pheromone ligands that mediate male–male aggression through the accessory olfactory neural pathway.

Genome-wide Generation and Systematic Phenotyping of Knockout Mice Reveals New Roles for Many Genes

Summary Mutations in whole organisms are powerful ways of interrogating gene function in a realistic context. We describe a program, the Sanger Institute Mouse Genetics Project, that provides a step toward the aim of knocking out all genes and screening each line for a broad range of traits. We found that hitherto unpublished genes were as likely to reveal phenotypes as known genes, suggesting that novel genes represent a rich resource for investigating the molecular basis of disease. We found many unexpected phenotypes detected only because we screened for them, emphasizing the value of screening all mutants for a wide range of traits. Haploinsufficiency and pleiotropy were both surprisingly common. Forty-two percent of genes were essential for viability, and these were less likely to have a paralog and more likely to contribute to a protein complex than other genes. Phenotypic data and more than 900 mutants are openly available for further analysis. PaperClip

The Vomeronasal Organ Mediates Interspecies Defensive Behaviors through Detection of Protein Pheromone Homologs

Potential predators emit uncharacterized chemosignals that warn receiving species of danger. Neurons that sense these stimuli remain unknown. Here we show that detection and processing of fear-evoking odors emitted from cat, rat, and snake require the function of sensory neurons in the vomeronasal organ. To investigate the molecular nature of the sensory cues emitted by predators, we isolated the salient ligands from two species using a combination of innate behavioral assays in naive receiving animals, calcium imaging, and c-Fos induction. Surprisingly, the defensive behavior-promoting activity released by other animals is encoded by species-specific ligands belonging to the major urinary protein (Mup) family, homologs of aggression-promoting mouse pheromones. We show that recombinant Mup proteins are sufficient to activate sensory neurons and initiate defensive behavior similarly to native odors. This co-option of existing sensory mechanisms provides a molecular solution to the difficult problem of evolving a variety of species-specific molecular detectors.

The structure and evolution of the melanocortin and MCH receptors in fish and mammals

Zebrafish are an excellent genetic model system for studying developmental and physiological processes. Pigment patterns in zebrafish are affected by mutations in three types of chromatophores. The behavior of these cells is influenced by alpha-melanocyte-stimulating hormone (alphaMSH) and melanin-concentrating hormone (MCH). Mammals have five alphaMSH receptors (melanocortin receptors) and one or two MCH receptors. We have identified the full complement of melanocortin and MCH receptors in both zebrafish and the pufferfish, Fugu. Zebrafish have six melanocortin receptors, including two MC5R orthologues, while Fugu, lacking MC3R, has only four. We also demonstrate that Fugu and zebrafish have two and three MCHR genes, respectively. MC2R and MC5R are physically linked in all species examined. Unlike other species, we find the Fugu genes contain introns, one of which is in a conserved location and is probably ancestral. We also detail the differential expression of the zebrafish genes throughout development.

Species Specificity in Major Urinary Proteins by Parallel Evolution

Species-specific chemosignals, pheromones, regulate social behaviors such as aggression, mating, pup-suckling, territory establishment, and dominance. The identity of these cues remains mostly undetermined and few mammalian pheromones have been identified. Genetically-encoded pheromones are expected to exhibit several different mechanisms for coding 1) diversity, to enable the signaling of multiple behaviors, 2) dynamic regulation, to indicate age and dominance, and 3) species-specificity. Recently, the major urinary proteins (Mups) have been shown to function themselves as genetically-encoded pheromones to regulate species-specific behavior. Mups are multiple highly related proteins expressed in combinatorial patterns that differ between individuals, gender, and age; which are sufficient to fulfill the first two criteria. We have now characterized and fully annotated the mouse Mup gene content in detail. This has enabled us to further analyze the extent of Mup coding diversity and determine their potential to encode species-specific cues. Our results show that the mouse Mup gene cluster is composed of two subgroups: an older, more divergent class of genes and pseudogenes, and a second class with high sequence identity formed by recent sequential duplications of a single gene/pseudogene pair. Previous work suggests that truncated Mup pseudogenes may encode a family of functional hexapeptides with the potential for pheromone activity. Sequence comparison, however, reveals that they have limited coding potential. Similar analyses of nine other completed genomes find Mup gene expansions in divergent lineages, including those of rat, horse and grey mouse lemur, occurring independently from a single ancestral Mup present in other placental mammals. Our findings illustrate that increasing genomic complexity of the Mup gene family is not evolutionarily isolated, but is instead a recurring mechanism of generating coding diversity consistent with a species-specific function in mammals.

Adipocyte Accumulation in the Bone Marrow during Obesity and Aging Impairs Stem Cell-Based Hematopoietic and Bone Regeneration

Summary Aging and obesity induce ectopic adipocyte accumulation in bone marrow cavities. This process is thought to impair osteogenic and hematopoietic regeneration. Here we specify the cellular identities of the adipogenic and osteogenic lineages of the bone. While aging impairs the osteogenic lineage, high-fat diet feeding activates expansion of the adipogenic lineage, an effect that is significantly enhanced in aged animals. We further describe a mesenchymal sub-population with stem cell-like characteristics that gives rise to both lineages and, at the same time, acts as a principal component of the hematopoietic niche by promoting competitive repopulation following lethal irradiation. Conversely, bone-resident cells committed to the adipocytic lineage inhibit hematopoiesis and bone healing, potentially by producing excessive amounts of Dipeptidyl peptidase-4, a protease that is a target of diabetes therapies. These studies delineate the molecular identity of the bone-resident adipocytic lineage, and they establish its involvement in age-dependent dysfunction of bone and hematopoietic regeneration.

Restriction and Recruitment—Gene Duplication and the Origin and Evolution of Snake Venom Toxins

Snake venom has been hypothesized to have originated and diversified through a process that involves duplication of genes encoding body proteins with subsequent recruitment of the copy to the venom gland, where natural selection acts to develop or increase toxicity. However, gene duplication is known to be a rare event in vertebrate genomes, and the recruitment of duplicated genes to a novel expression domain (neofunctionalization) is an even rarer process that requires the evolution of novel combinations of transcription factor binding sites in upstream regulatory regions. Therefore, although this hypothesis concerning the evolution of snake venom is very unlikely and should be regarded with caution, it is nonetheless often assumed to be established fact, hindering research into the true origins of snake venom toxins. To critically evaluate this hypothesis, we have generated transcriptomic data for body tissues and salivary and venom glands from five species of venomous and nonvenomous reptiles. Our comparative transcriptomic analysis of these data reveals that snake venom does not evolve through the hypothesized process of duplication and recruitment of genes encoding body proteins. Indeed, our results show that many proposed venom toxins are in fact expressed in a wide variety of body tissues, including the salivary gland of nonvenomous reptiles and that these genes have therefore been restricted to the venom gland following duplication, not recruited. Thus, snake venom evolves through the duplication and subfunctionalization of genes encoding existing salivary proteins. These results highlight the danger of the elegant and intuitive “just-so story” in evolutionary biology.

The olfactory transcriptomes of mice.

The olfactory (OR) and vomeronasal receptor (VR) repertoires are collectively encoded by 1700 genes and pseudogenes in the mouse genome. Most OR and VR genes were identified by comparative genomic techniques and therefore, in many of those cases, only their protein coding sequences are defined. Some also lack experimental support, due in part to the similarity between them and their monogenic, cell-specific expression in olfactory tissues. Here we use deep RNA sequencing, expression microarray and quantitative RT-PCR in both the vomeronasal organ and whole olfactory mucosa to quantify their full transcriptomes in multiple male and female mice. We find evidence of expression for all VR, and almost all OR genes that are annotated as functional in the reference genome, and use the data to generate over 1100 new, multi-exonic, significantly extended receptor gene annotations. We find that OR and VR genes are neither equally nor randomly expressed, but have reproducible distributions of abundance in both tissues. The olfactory transcriptomes are only minimally different between males and females, suggesting altered gene expression at the periphery is unlikely to underpin the striking sexual dimorphism in olfactory-mediated behavior. Finally, we present evidence that hundreds of novel, putatively protein-coding genes are expressed in these highly specialized olfactory tissues, and carry out a proof-of-principle validation. Taken together, these data provide a comprehensive, quantitative catalog of the genes that mediate olfactory perception and pheromone-evoked behavior at the periphery.

Olfactory mechanisms of stereotyped behavior: on the scent of specialized circuits

Investigation of how specialized olfactory cues, such as pheromones, are detected has primarily focused on the function of receptor neurons within a subsystem of the nasal cavity, the vomeronasal organ (VNO). Behavioral analyses have long indicated that additional, non-VNO olfactory neurons are similarly necessary for pheromone detection; however, the identity of these neurons has been a mystery. Recent molecular, behavioral, and genomic approaches have led to the identification of multiple atypical sensory circuits that display characteristics suggestive of a specialized function. This review focuses on these non-VNO receptors and neurons, and evaluates their potential for mediating stereotyped olfactory behavior in mammals.

Hierarchical deconstruction of mouse olfactory sensory neurons: from whole mucosa to single-cell RNA-seq

The mouse olfactory mucosa is a complex chemosensory tissue composed of multiple cell types, neuronal and non-neuronal. We have here applied RNA-seq hierarchically, in three steps of decreasing cellular heterogeneity: starting with crude tissue samples dissected from the nose, proceeding to flow-cytometrically sorted pools of mature olfactory sensory neurons (OSNs), and finally arriving at single mature OSNs. We show that 98.9% of intact olfactory receptor (OR) genes are expressed in mature OSNs. We uncover a hitherto unknown bipartition among mature OSNs. We find that 19 of 21 single mature OSNs each express a single intact OR gene abundantly, consistent with the one neuron-one receptor rule. For the 9 single OSNs where the two alleles of the abundantly expressed OR gene exhibit single-nucleotide polymorphisms, we demonstrate that monoallelic expression of the abundantly expressed OR gene is extremely tight. The remaining two single mature OSNs lack OR gene expression but express Trpc2 and Gucy1b2. We establish these two cells as a neuronal cell type that is fundamentally distinct from canonical, OR-expressing OSNs and that is defined by the differential, higher expression of 55 genes. We propose this tiered experimental approach as a paradigm to unravel gene expression in other cellularly heterogeneous systems.