Denesa Oberbeck

Genetic diversity and striatal gene networks: focus on the heterogeneous stock-collaborative cross (HS-CC) mouse

BackgroundThe current study focused on the extent genetic diversity within a species (Mus musculus) affects gene co-expression network structure. To examine this issue, we have created a new mouse resource, a heterogeneous stock (HS) formed from the same eight inbred strains that have been used to create the collaborative cross (CC). The eight inbred strains capture > 90% of the genetic diversity available within the species. For contrast with the HS-CC, a C57BL/6J (B6) × DBA/2J (D2) F2 intercross and the HS4, derived from crossing the B6, D2, BALB/cJ and LP/J strains, were used. Brain (striatum) gene expression data were obtained using the Illumina Mouse WG 6.1 array, and the data sets were interrogated using a weighted gene co-expression network analysis (WGCNA).ResultsGenes reliably detected as expressed were similar in all three data sets as was the variability of expression. As measured by the WGCNA, the modular structure of the transcriptome networks was also preserved both on the basis of module assignment and from the perspective of the topological overlap maps. Details of the HS-CC gene modules are provided; essentially identical results were obtained for the HS4 and F2 modules. Gene ontology annotation of the modules revealed a significant overrepresentation in some modules for neuronal processes, e.g., central nervous system development. Integration with known protein-protein interactions data indicated significant enrichment among co-expressed genes. We also noted significant overlap with markers of central nervous system cell types (neurons, oligodendrocytes and astrocytes). Using the Allen Brain Atlas, we found evidence of spatial co-localization within the striatum for several modules. Finally, for some modules it was possible to detect an enrichment of transcription binding sites. The binding site for Wt1, which is associated with neurodegeneration, was the most significantly overrepresented.ConclusionsDespite the marked differences in genetic diversity, the transcriptome structure was remarkably similar for the F2, HS4 and HS-CC. These data suggest that it should be possible to integrate network data from simple and complex crosses. A careful examination of the HS-CC transcriptome revealed the expected structure for striatal gene expression. Importantly, we demonstrate the integration of anatomical and network expression data.

Selection for drinking in the dark alters brain gene coexpression networks.

BACKGROUND Heterogeneous stock (HS/NPT) mice have been used to create lines selectively bred in replicate for elevated drinking in the dark (DID). Both selected lines routinely reach a blood ethanol (EtOH) concentration (BEC) of 1.00 mg/ml or greater at the end of the 4-hour period of access in Day 2. The mechanisms through which genetic differences influence DID are currently unclear. Therefore, the current study examines the transcriptome, the first stage at which genetic variability affects neurobiology. Rather than focusing solely on differential expression (DE), we also examine changes in the ways that gene transcripts collectively interact with each other, as revealed by changes in coexpression patterns. METHODS Naïve mice (N = 48/group) were genotyped using the Mouse Universal Genotyping Array, which provided 3,683 informative markers. Quantitative trait locus (QTL) analysis used a marker-by-marker strategy with the threshold for a significant logarithm of odds (LOD) set at 10.6. Gene expression in the ventral striatum was measured using the Illumina Mouse 8.2 array. Differential gene expression and the weighted gene coexpression network analysis (WGCNA) were implemented largely as described elsewhere. RESULTS Significant QTLs for elevated BECs after DID were detected on chromosomes 4, 14, and 16; the latter 2 were associated with gene-poor regions. None of the QTLs overlapped with known QTLs for EtOH preference drinking. Ninety-four transcripts were detected as being differentially expressed in both selected lines versus HS controls; there was no overlap with known preference genes. The WGCNA revealed 2 modules as showing significant effects of both selections on intramodular connectivity. A number of genes known to be associated with EtOH phenotypes (e.g., Gabrg1, Glra2, Grik1, Npy2r, and Nts) showed significant changes in connectivity. CONCLUSIONS We found marked and consistent effects of selection on coexpression patterns; DE changes were more modest and less concordant. The QTLs and differentially expressed genes detected here are distinct from the preference phenotype. This is consistent with behavioral data and suggests that the DID and preference phenotypes are markedly different genetically.

Electrolytic lesions of the medial nucleus accumbens shell selectively decrease ethanol consumption without altering preference in a limited access procedure in C57BL/6J mice ☆

The central extended amygdala (cExtA) is a limbic region proposed to play a key role in drug and alcohol addiction and to contain the medial nucleus accumbens shell (MNAc shell). The aim of this study was to examine the involvement of the MNAc shell in ethanol and sucrose consumption in a limited and free access procedure in the C57BL/6J (B6) mouse. Separate groups of mice received bilateral electrolytic lesions of the MNAc shell or sham surgery, and following recovery from surgery, were allowed to voluntarily consume ethanol (15% v/v) in a 2 h limited access 2-bottle-choice procedure. Following 1 week of limited access ethanol consumption, mice were given 1 week of limited access sucrose consumption. A separate group of lesioned and sham mice were given free access (24 h) to ethanol in a 2-bottle choice procedure and were run in parallel to the mice receiving limited access consumption. Electrolytic lesions of the MNAc shell decreased ethanol (but not sucrose) consumption in a limited access procedure, but did not alter free access ethanol consumption. These results suggest that the MNAc shell is a component of the underlying neural circuitry contributing to limited access alcohol consumption in the B6 mouse.

Differential Network Analysis Reveals Genetic Effects on Catalepsy Modules

We performed short-term bi-directional selective breeding for haloperidol-induced catalepsy, starting from three mouse populations of increasingly complex genetic structure: an F2 intercross, a heterogeneous stock (HS) formed by crossing four inbred strains (HS4) and a heterogeneous stock (HS-CC) formed from the inbred strain founders of the Collaborative Cross (CC). All three selections were successful, with large differences in haloperidol response emerging within three generations. Using a custom differential network analysis procedure, we found that gene coexpression patterns changed significantly; importantly, a number of these changes were concordant across genetic backgrounds. In contrast, absolute gene-expression changes were modest and not concordant across genetic backgrounds, in spite of the large and similar phenotypic differences. By inferring strain contributions from the parental lines, we are able to identify significant differences in allelic content between the selected lines concurrent with large changes in transcript connectivity. Importantly, this observation implies that genetic polymorphisms can affect transcript and module connectivity without large changes in absolute expression levels. We conclude that, in this case, selective breeding acts at the subnetwork level, with the same modules but not the same transcripts affected across the three selections.

Cosplicing network analysis of mammalian brain RNA-Seq data utilizing WGCNA and Mantel correlations.

Across species and tissues and especially in the mammalian brain, production of gene isoforms is widespread. While gene expression coordination has been previously described as a scale-free coexpression network, the properties of transcriptome-wide isoform production coordination have been less studied. Here we evaluate the system-level properties of cosplicing in mouse, macaque, and human brain gene expression data using a novel network inference procedure. Genes are represented as vectors/lists of exon counts and distance measures sensitive to exon inclusion rates quantifies differences across samples. For all gene pairs, distance matrices are correlated across samples, resulting in cosplicing or cotranscriptional network matrices. We show that networks including cosplicing information are scale-free and distinct from coexpression. In the networks capturing cosplicing we find a set of novel hubs with unique characteristics distinguishing them from coexpression hubs: heavy representation in neurobiological functional pathways, strong overlap with markers of neurons and neuroglia, long coding lengths, and high number of both exons and annotated transcripts. Further, the cosplicing hubs are enriched in genes associated with autism spectrum disorders. Cosplicing hub homologs across eukaryotes show dramatically increasing intronic lengths but stable coding region lengths. Shared transcription factor binding sites increase coexpression but not cosplicing; the reverse is true for splicing-factor binding sites. Genes with protein-protein interactions have strong coexpression and cosplicing. Additional factors affecting the networks include shared microRNA binding sites, spatial colocalization within the striatum, and sharing a chromosomal folding domain. Cosplicing network patterns remain relatively stable across species.

Diversity Outbred Mice at 21: Maintaining Allelic Variation in the Face of Selection.

Multi-parent populations (MPPs) capture and maintain the genetic diversity from multiple inbred founder strains to provide a resource for high-resolution genetic mapping through the accumulation of recombination events over many generations. Breeding designs that maintain a large effective population size with randomized assignment of breeders at each generation can minimize the impact of selection, inbreeding, and genetic drift on allele frequencies. Small deviations from expected allele frequencies will have little effect on the power and precision of genetic analysis, but a major distortion could result in reduced power and loss of important functional alleles. We detected strong transmission ratio distortion in the Diversity Outbred (DO) mouse population on chromosome 2, caused by meiotic drive favoring transmission of the WSB/EiJ allele at the R2d2 locus. The distorted region harbors thousands of polymorphisms derived from the seven non-WSB founder strains and many of these would be lost if the sweep was allowed to continue. To ensure the utility of the DO population to study genetic variation on chromosome 2, we performed an artificial selection against WSB/EiJ alleles at the R2d2 locus. Here, we report that we have purged the WSB/EiJ allele from the drive locus while preserving WSB/EiJ alleles in the flanking regions. We observed minimal disruption to allele frequencies across the rest of the autosomal genome. However, there was a shift in haplotype frequencies of the mitochondrial genome and an increase in the rate of an unusual sex chromosome aneuploidy. The DO population has been restored to genome-wide utility for genetic analysis, but our experience underscores that vigilant monitoring of similar genetic resource populations is needed to ensure their long-term utility.

Effects of selection for ethanol preference on gene expression in the nucleus accumbens of HS-CC mice

Previous studies on changes in murine brain gene expression associated with the selection for ethanol preference have used F2 intercross or heterogeneous stock (HS) founders, derived from standard laboratory strains. However, these populations represent only a small proportion of the genetic variance available in Mus musculus. To investigate a wider range of genetic diversity, we selected mice for ethanol preference using an HS derived from the eight strains of the collaborative cross. These HS mice were selectively bred (four generations) for high and low ethanol preference. The nucleus accumbens shell of naive S4 mice was interrogated using RNA sequencing (RNA‐Seq). Gene networks were constructed using the weighted gene coexpression network analysis assessing both coexpression and cosplicing. Selection targeted one of the network coexpression modules (greenyellow) that was significantly enriched in genes associated with receptor signaling activity including Chrna7, Grin2a, Htr2a and Oprd1. Connectivity in the module as measured by changes in the hub nodes was significantly reduced in the low preference line. Of particular interest was the observation that selection had marked effects on a large number of cell adhesion molecules, including cadherins and protocadherins. In addition, the coexpression data showed that selection had marked effects on long non‐coding RNA hub nodes. Analysis of the cosplicing network data showed a significant effect of selection on a large cluster of Ras GTPase‐binding genes including Cdkl5, Cyfip1, Ndrg1, Sod1 and Stxbp5. These data in part support the earlier observation that preference is linked to Ras/Mapk pathways.

On the relationships in rhesus macaques between chronic ethanol consumption and the brain transcriptome

This is the first description of the relationship between chronic ethanol self‐administration and the brain transcriptome in a non‐human primate (rhesus macaque). Thirty‐one male animals self‐administered ethanol on a daily basis for over 12 months. Gene transcription was quantified with RNA‐Seq in the central nucleus of the amygdala (CeA) and cortical Area 32. We constructed coexpression and cosplicing networks, and we identified areas of preservation and areas of differentiation between regions and network types. Correlations between intake and transcription included largely distinct gene sets and annotation categories across brain regions and between expression and splicing; positive and negative correlations were also associated with distinct annotation groups. Membrane, synaptic and splicing annotation categories were over‐represented in the modules (gene clusters) enriched in positive correlations (CeA); our cosplicing analysis further identified the genes affected only at the exon inclusion level. In the CeA coexpression network, we identified Rab6b, Cdk18 and Igsf21 among the intake‐correlated hubs, while in the Area 32, we identified a distinct hub set that included Ppp3r1 and Myeov2. Overall, the data illustrate that excessive ethanol self‐administration is associated with broad expression and splicing mechanisms that involve membrane and synapse genes.

Splicing landscape of the eight collaborative cross founder strains

BackgroundThe Collaborative Cross (CC) is a large panel of genetically diverse recombinant inbred mouse strains specifically designed to provide a systems genetics resource for the study of complex traits. In part, the utility of the CC stems from the extensive genome-wide annotations of founder strain sequence and structural variation. Still missing, however, are transcriptome-specific annotations of the CC founder strains that could further enhance the utility of this resource.ResultsWe provide a comprehensive survey of the splicing landscape of the 8 CC founder strains by leveraging the high level of alternative splicing within the brain. Using deep transcriptome sequencing, we found that a majority of the splicing landscape is conserved among the 8 strains, with ~65% of junctions being shared by at least 2 strains. We, however, found a large number of potential strain-specific splicing events as well, with an average of ~3000 and ~500 with ≥3 and ≥10 sequence read coverage, respectively, within each strain. To better understand strain-specific splicing within the CC founder strains, we defined criteria for and identified high-confidence strain-specific splicing events. These splicing events were defined as exon-exon junctions 1) found within only one strain, 2) with a read coverage ≥10, and 3) defined by a canonical splice site. With these criteria, a total of 1509 high-confidence strain-specific splicing events were identified, with the majority found within two of the wild-derived strains, CAST and PWK. Strikingly, the overwhelming majority, 94%, of these strain-specific splicing events are not yet annotated. Strain-specific splicing was also located within genomic regions recently reported to be over- and under-represented within CC populations.ConclusionsPhenotypic characterization of CC populations is increasing; thus these results will not only aid in further elucidating the transcriptomic architecture of the individual CC founder strains, but they will also help in guiding the utilization of the CC populations in the study of complex traits. This report is also the first to establish guidelines in defining and identifying strain-specific splicing across different mouse strains.