Stephen J. Bush

Alternative splicing and the evolution of phenotypic novelty

Alternative splicing, a mechanism of post-transcriptional RNA processing whereby a single gene can encode multiple distinct transcripts, has been proposed to underlie morphological innovations in multicellular organisms. Genes with developmental functions are enriched for alternative splicing events, suggestive of a contribution of alternative splicing to developmental programmes. The role of alternative splicing as a source of transcript diversification has previously been compared to that of gene duplication, with the relationship between the two extensively explored. Alternative splicing is reduced following gene duplication with the retention of duplicate copies higher for genes which were alternatively spliced prior to duplication. Furthermore, and unlike the case for overall gene number, the proportion of alternatively spliced genes has also increased in line with the evolutionary diversification of cell types, suggesting alternative splicing may contribute to the complexity of developmental programmes. Together these observations suggest a prominent role for alternative splicing as a source of functional innovation. However, it is unknown whether the proliferation of alternative splicing events indeed reflects a functional expansion of the transcriptome or instead results from weaker selection acting on larger species, which tend to have a higher number of cell types and lower population sizes. This article is part of the themed issue ‘Evo-devo in the genomics era, and the origins of morphological diversity’.

A high resolution atlas of gene expression in the domestic sheep (Ovis aries)

Sheep are a key source of meat, milk and fibre for the global livestock sector, and an important biomedical model. Global analysis of gene expression across multiple tissues has aided genome annotation and supported functional annotation of mammalian genes. We present a large-scale RNA-Seq dataset representing all the major organ systems from adult sheep and from several juvenile, neonatal and prenatal developmental time points. The Ovis aries reference genome (Oar v3.1) includes 27,504 genes (20,921 protein coding), of which 25,350 (19,921 protein coding) had detectable expression in at least one tissue in the sheep gene expression atlas dataset. Network-based cluster analysis of this dataset grouped genes according to their expression pattern. The principle of ‘guilt by association’ was used to infer the function of uncharacterised genes from their co-expression with genes of known function. We describe the overall transcriptional signatures present in the sheep gene expression atlas and assign those signatures, where possible, to specific cell populations or pathways. The findings are related to innate immunity by focusing on clusters with an immune signature, and to the advantages of cross-breeding by examining the patterns of genes exhibiting the greatest expression differences between purebred and crossbred animals. This high-resolution gene expression atlas for sheep is, to our knowledge, the largest transcriptomic dataset from any livestock species to date. It provides a resource to improve the annotation of the current reference genome for sheep, presenting a model transcriptome for ruminants and insight into gene, cell and tissue function at multiple developmental stages.

Integration of quantitated expression estimates from polyA-selected and rRNA-depleted RNA-seq libraries

BackgroundThe availability of fast alignment-free algorithms has greatly reduced the computational burden of RNA-seq processing, especially for relatively poorly assembled genomes. Using these approaches, previous RNA-seq datasets could potentially be processed and integrated with newly sequenced libraries. Confounding factors in such integration include sequencing depth and methods of RNA extraction and selection. Different selection methods (typically, either polyA-selection or rRNA-depletion) omit different RNAs, resulting in different fractions of the transcriptome being sequenced. In particular, rRNA-depleted libraries sample a broader fraction of the transcriptome than polyA-selected libraries. This study aimed to develop a systematic means of accounting for library type that allows data from these two methods to be compared.ResultsThe method was developed by comparing two RNA-seq datasets from ovine macrophages, identical except for RNA selection method. Gene-level expression estimates were obtained using a two-part process centred on the high-speed transcript quantification tool Kallisto. Firstly, a set of reference transcripts was defined that constitute a standardised RNA space, with expression from both datasets quantified against it. Secondly, a simple ratio-based correction was applied to the rRNA-depleted estimates. The outcome is an almost perfect correlation between gene expression estimates, independent of library type and across the full range of levels of expression.ConclusionA combination of reference transcriptome filtering and a ratio-based correction can create equivalent expression profiles from both polyA-selected and rRNA-depleted libraries. This approach will allow meta-analysis and integration of existing RNA-seq data into transcriptional atlas projects.

The genomic architecture of mastitis resistance in dairy sheep

BackgroundMastitis is the most prevalent disease in dairy sheep with major economic, hygienic and welfare implications. The disease persists in all dairy sheep production systems despite the implementation of improved management practises. Selective breeding for enhanced mastitis resistance may provide the means to further control the disease. In the present study, we investigated the genetic architecture of four mastitis traits in dairy sheep. Individual animal records for clinical mastitis occurrence and three mastitis indicator traits (milk somatic cell count, total viable bacterial count in milk and the California mastitis test) were collected monthly throughout lactation for 609 ewes of the Greek Chios breed. All animals were genotyped with a custom-made 960-single nucleotide polymorphism (SNP) DNA array based on markers located in quantitative trait loci (QTL) regions for mastitis resistance previously detected in three other distinct dairy sheep populations.ResultsHeritable variation and strong positive genetic correlations were estimated for clinical mastitis occurrence and the three mastitis indicator traits. SNP markers significantly associated with these mastitis traits were confirmed on chromosomes 2, 3, 5, 16 and 19. We identified pathways, molecular interaction networks and functional gene clusters for mastitis resistance. Candidate genes within the detected regions were identified based upon analysis of an ovine transcriptional atlas and transcriptome data derived from milk somatic cells. Relevant candidate genes implicated in innate immunity included SOCS2, CTLA4, C6, C7, C9, PTGER4, DAB2, CARD6, OSMR, PLXNC1, IDH1, ICOS, FYB, and LYFR.ConclusionsThe results confirmed the presence of animal genetic variability in mastitis resistance and identified genomic regions associated with specific mastitis traits in the Chios sheep. The conserved genetic architecture of mastitis resistance between distinct dairy sheep breeds suggests that across-breed selection programmes would be feasible.

Species-Specific Transcriptional Regulation of Genes Involved in Nitric Oxide Production and Arginine Metabolism in Macrophages

Activated mouse macrophages metabolize arginine via NO synthase (NOS2) to produce NO as an antimicrobial effector. Published gene expression datasets provide little support for the activation of this pathway in human macrophages. Generation of NO requires the coordinated regulation of multiple genes. We have generated RNA-sequencing data from bone marrow–derived macrophages from representative rodent (rat), monogastric (pig and horse), and ruminant (sheep, goat, cattle, and water buffalo) species, and analyzed the expression of genes involved in arginine metabolism in response to stimulation with LPS. In rats, as in mice, LPS strongly induced Nos2, the arginine transporter Slc7a2, arginase 1 (Arg1), GTP cyclohydrolase (Gch1), and argininosuccinate synthase (Ass1). None of these responses was conserved across species. Only cattle and water buffalo showed substantial NOS2 induction. The species studied also differed in expression and regulation of arginase (ARG2, rather than ARG1), and amino acid transporters. Variation between species was associated with rapid promoter evolution. Differential induction of NOS2 and ARG2 between the ruminant species was associated with insertions of the Bov-A2 retrotransposon in the promoter region. Bov-A2 was shown to possess LPS-inducible enhancer activity in transfected RAW264.7 macrophages. Consistent with a function in innate immunity, NO production and arginine metabolism vary greatly between species and differences may contribute to pathogen host restriction.

Characterisation of subpopulations of chicken mononuclear phagocytes that express TIM4 and the macrophage colony-stimulating factor receptor (CSF1R)

The phosphatidylserine receptor, TIM4, encoded by TIMD4, mediates the phagocytic uptake of apoptotic cells. We applied anti-chicken TIM4 monoclonal antibodies, in combination with CSF1R reporter transgenes to dissect the function of TIM4 in chick (Gallus gallus). During development in ovo, TIM4 was present on the large majority of macrophages but expression became more heterogeneous post-hatch. Blood monocytes expressed KUL01, class II MHC and CSF1R-mApple uniformly. Around 50% of monocytes were positive for surface TIM4. They also expressed many other monocyte-specific transcripts at a higher level than TIM4− monocytes. In liver, highly-phagocytic TIM4hi cells shared many transcripts with mammalian Kupffer cells and were associated with uptake of apoptotic cells. Although they expressed CSF1R mRNA, Kupffer cells did not express the CSF1R-mApple transgene, suggesting that additional CSF1R transcriptional regulatory elements are required by these cells. By contrast, CSF1R-mApple was detected in liver TIM4lo and TIM4− cells which were not phagocytic and were more abundant than Kupffer cells. These cells expressed CSF1R, alongside high levels of FLT3, MHCII, XCR1 and other markers associated with conventional dendritic cells (cDC) in mice. In bursa, TIM4 was present on the cell surface of two populations. Like Kupffer cells, bursal TIM4hi phagocytes co-expressed many receptors involved in apoptotic cell recognition. TIM4lo cells appear to be a sub-population of bursal B cells. In overview, TIM4 is associated with phagocytes that eliminate apoptotic cells in the chick. In the liver, TIM4 and CSF1R reporters distinguished Kupffer cells from an abundant population of DC-like cells.

Characterisation of the UK honey bee (Apis mellifera) metagenome.

The European honey bee (Apis mellifera) plays a major role in pollination and food production, but is under threat from emerging pathogens and agro-environmental insults. As with other organisms, honey bee health is a complex product of environment, host genetics and associated microbes (commensal, opportunistic and pathogenic). Improved understanding of bee genetics and their molecular ecology can help manage modern challenges to bee health and production. Sampling bee and cobiont genomes, we characterised the metagenome of 19 honey bee colonies across Britain. Low heterozygosity was observed in bees from many Scottish colonies, sharing high similarity to the native dark bee, A. mellifera mellifera. Apiaries exhibited high diversity in the composition and relative abundance of individual microbiome taxa. Most non-bee sequences derived from known honey bee commensal bacteria or known pathogens, e.g. Lotmaria passim (Trypanosomatidae), and Nosema spp. (Microsporidia). However, DNA was also detected from numerous additional bacterial, plant (food source), protozoan and metazoan organisms. To classify sequences from cobionts lacking genomic information, we developed a novel network analysis approach clustering orphan contigs, allowing the identification of a pathogenic gregarine. Our analyses demonstrate the power of high-throughput, directed metagenomics in agroecosystems identifying potential threats to honey bees present in their microbiota.

Environmentally enriched pigs have transcriptional profiles consistent with neuroprotective effects and microglial depletion

Environmental enrichment (EE) is widely used to study the effects of external factors on brain development, function and health in rodent models, but very little is known of the effects of EE on the brain in a large animal model such as the pig. Twenty-four young pigs (aged 5 weeks at start of study, 1:1 male: female ratio) were housed in environmentally enriched (EE) pens and provided with additional enrichment stimulation (a bag filled with straw) once daily. Litter, weight and sex matched controls n= (24) were housed in barren (B) conditions. Behaviour was recorded on alternate days from study day 10. After 21 days, RNA-sequencing of the frontal cortex of male piglets culled one hour after the enrichment stimulation, but not those at 4 hours after stimulation, showed upregulation of genes involved in neuronal activity and synaptic plasticity in the EE compared to the B condition. This result is mirrored in the behavioural response to the stimulation which showed a peak in activity around the 1 hour time-point. By contrast, EE piglets displayed a signature consistent with a relative decrease in microglial activity compared to those in the B condition. These results confirm those from rodents, suggesting that EE may also confer neuronal health benefits in large mammal models, through a potential relative reduction in neuroinflammatory process and increase in neuroprotection driven by an enrichment-induced increase in behavioural activity.

Pleiotropic Impacts of Macrophage and Microglial Deficiency on Development in Rats with Targeted Mutation of the Csf1r Locus

We have produced Csf1r-deficient rats by homologous recombination in embryonic stem cells. Consistent with the role of Csf1r in macrophage differentiation, there was a loss of peripheral blood monocytes, microglia in the brain, epidermal Langerhans cells, splenic marginal zone macrophages, bone-associated macrophages and osteoclasts, and peritoneal macrophages. Macrophages of splenic red pulp, liver, lung, and gut were less affected. The pleiotropic impacts of the loss of macrophages on development of multiple organ systems in rats were distinct from those reported in mice. Csf1r−/− rats survived well into adulthood with postnatal growth retardation, distinct skeletal and bone marrow abnormalities, infertility, and loss of visceral adipose tissue. Gene expression analysis in spleen revealed selective loss of transcripts associated with the marginal zone and, in brain regions, the loss of known and candidate novel microglia-associated transcripts. Despite the complete absence of microglia, there was little overt phenotype in brain, aside from reduced myelination and increased expression of dopamine receptor-associated transcripts in striatum. The results highlight the redundant and nonredundant functions of CSF1R signaling and of macrophages in development, organogenesis, and homeostasis.

ADGRE1 (EMR1, F4/80) Is a Rapidly-Evolving Gene Expressed in Mammalian Monocyte-Macrophages

The F4/80 antigen, encoded by the Adgre1 locus, has been widely-used as a monocyte-macrophage marker in mice, but its value as a macrophage marker in other species is unclear, and has even been questioned. ADGRE1 is a seven transmembrane G protein-coupled receptor with an extracellular domain containing repeated Epidermal Growth Factor (EGF)-like calcium binding domains. Using a new monoclonal antibody, we demonstrated that ADGRE1 is a myeloid differentiation marker in pigs, absent from progenitors in bone marrow, highly-expressed in mature granulocytes, monocytes, and tissue macrophages and induced by macrophage colony-stimulating factor (CSF1) treatment in vivo. Based upon these observations, we utilized RNA-Seq to assess the expression of ADGRE1 mRNA in bone marrow or monocyte-derived macrophages (MDM) and alveolar macrophages from 8 mammalian species including pig, human, rat, sheep, goat, cow, water buffalo, and horse. ADGRE1 mRNA was expressed by macrophages in each species, with inter-species variation both in expression level and response to lipopolysaccharide (LPS) stimulation. Analysis of the RNA-Seq data also revealed additional exons in several species compared to current Ensembl annotations. The ruminant species and horses appear to encode a complete duplication of the 7 EGF-like domains. In every species, Sashimi plots revealed evidence of exon skipping of the EGF-like domains, which are highly-variable between species and polymorphic in humans. Consistent with these expression patterns, key elements of the promoter and a putative enhancer are also conserved across all species. The rapid evolution of this molecule and related ADGRE family members suggests immune selection and a role in pathogen recognition.