Genetic analysis of barrel field size in the first somatosensory area (SI) in inbred and recombinant inbred strains of mice

We measured the combined area of posterior medial barrel subfield (PMBSF) and anterior lateral barrel subfield (ALBSF) areas in four common inbred strains (C3H/HeJ, A /J, C57BL /6J, DBA/2J), B6D2F1, and ten recombinant inbred (RI) strains generated from C57BL/6J and DBA/2J progenitors (BXD) as an initial attempt to examine the genetic influences underlying natural variation in barrel field size in adult mice. These two subfields are associated with the representation of the whisker pad and sinus hairs on the contralateral face. Using cytochrome oxidase labeling to visualize the barrel field, we measured the size of the combined subfields in each mouse strain. We also measured body weight and brain weight in each strain. We report that DBA/2J mice have a larger combined PMBSF/ALBSF area (6.15 +/- 0.10 mm(2), n = 7) than C57BL /6J (5.48 +/- 0.13 mm(2), n = 10), C3H/HeJ (5.37 +/- 0.16 mm(2), n = 10), and A/J mice (5.04 +/- 0.09 mm(2), n = 15), despite the fact that DBA/2J mice have smaller average brain and body sizes. This finding may reflect dissociation between systems that control brain size with those that regulate barrel field area. In addition, BXD strains (average n = 4) and parental strains showed considerable and continuous variation in PMBSF/ALBSF area, suggesting that this trait is polygenic. Furthermore, brain, body, and cortex weights have heritable differences between inbred strains and among BXD strains. PMBSF/ALBSF pattern appears similar among inbred and BXD strains, suggesting that somatosensory patterning reflects a common plan of organization. This data is an important first step in the quantitative genetic analysis of the parcellation of neocortex into diverse cytoarchitectonic zones that vary widely within and between species, and in identifying the genetic factors underlying barrel field size using quantitative trait locus (QTL) analyses.     

High‐throughput behavioral phenotyping in the expanded panel of BXD recombinant inbred strains

Genetic reference populations, particularly the BXD recombinant inbred (BXD RI) strains derived from C57BL/6J and DBA/2J mice, are a valuable resource for the discovery of the bio‐molecular substrates and genetic drivers responsible for trait variation and covariation. This approach can be profitably applied in the analysis of susceptibility and mechanisms of drug and alcohol use disorders for which many predisposing behaviors may predict the occurrence and manifestation of increased preference for these substances. Many of these traits are modeled by common mouse behavioral assays, facilitating the detection of patterns and sources of genetic coregulation of predisposing phenotypes and substance consumption. Members of the Tennessee Mouse Genome Consortium (TMGC) have obtained phenotype data from over 250 measures related to multiple behavioral assays across several batteries: response to, and withdrawal from cocaine, 3,4‐methylenedioxymethamphetamine; “ecstasy” (MDMA), morphine and alcohol; novelty seeking; behavioral despair and related neurological phenomena; pain sensitivity; stress sensitivity; anxiety; hyperactivity and sleep/wake cycles. All traits have been measured in both sexes in approximately 70 strains of the recently expanded panel of BXD RI strains. Sex differences and heritability estimates were obtained for each trait, and a comparison of early (N = 32) and recent (N = 37) BXD RI lines was performed. Primary data are publicly available for heritability, sex difference and genetic analyses using the MouseTrack database, and are also available in GeneNetwork.org for quantitative trait locus (QTL) detection and genetic analysis of gene expression. Together with the results of related studies, these data form a public resource for integrative systems genetic analysis of neurobehavioral traits.     

Identifying human disease genes through cross-species gene mapping of evolutionary conserved processes

Background

Understanding complex networks that modulate development in humans is hampered by genetic and phenotypic heterogeneity within and between populations. Here we present a method that exploits natural variation in highly diverse mouse genetic reference panels in which genetic and environmental factors can be tightly controlled. The aim of our study is to test a cross-species genetic mapping strategy, which compares data of gene mapping in human patients with functional data obtained by QTL mapping in recombinant inbred mouse strains in order to prioritize human disease candidate genes.

Methodology

We exploit evolutionary conservation of developmental phenotypes to discover gene variants that influence brain development in humans. We studied corpus callosum volume in a recombinant inbred mouse panel (C57BL/6J×DBA/2J, BXD strains) using high-field strength MRI technology. We aligned mouse mapping results for this neuro-anatomical phenotype with genetic data from patients with abnormal corpus callosum (ACC) development.

Principal Findings

From the 61 syndromes which involve an ACC, 51 human candidate genes have been identified. Through interval mapping, we identified a single significant QTL on mouse chromosome 7 for corpus callosum volume with a QTL peak located between 25.5 and 26.7 Mb. Comparing the genes in this mouse QTL region with those associated with human syndromes (involving ACC) and those covered by copy number variations (CNV) yielded a single overlap, namely HNRPU in humans and Hnrpul1 in mice. Further analysis of corpus callosum volume in BXD strains revealed that the corpus callosum was significantly larger in BXD mice with a B genotype at the Hnrpul1 locus than in BXD mice with a D genotype at Hnrpul1 (F = 22.48, p<9.87*10⁻⁵).

Conclusion

This approach that exploits highly diverse mouse strains provides an efficient and effective translational bridge to study the etiology of human developmental disorders, such as autism and schizophrenia.     

Genomewide association analysis in diverse inbred mice: power and population structure

The discovery of quantitative trait loci (QTL) in model organisms has relied heavily on the ability to perform controlled breeding to generate genotypic and phenotypic diversity. Recently, we and others have demonstrated the use of an existing set of diverse inbred mice (referred to here as the mouse diversity panel, MDP) as a QTL mapping population. The use of the MDP population has many advantages relative to traditional F(2) mapping populations, including increased phenotypic diversity, a higher recombination frequency, and the ability to collect genotype and phenotype data in community databases. However, these methods are complicated by population structure inherent in the MDP and the lack of an analytical framework to assess statistical power. To address these issues, we measured gene expression levels in hypothalamus across the MDP. We then mapped these phenotypes as quantitative traits with our association algorithm, resulting in a large set of expression QTL (eQTL). We utilized these eQTL, and specifically cis-eQTL, to develop a novel nonparametric method for association analysis in structured populations like the MDP. These eQTL data confirmed that the MDP is a suitable mapping population for QTL discovery and that eQTL results can serve as a gold standard for relative measures of statistical power.     

PTPN21 exerts pro-neuronal survival and neuritic elongation via ErbB4/NRG3 signaling

Although expression quantitative trait locus, eQTL, serves as an explicit indicator of gene–gene associations, challenges remain to disentangle the mechanisms by which genetic variations alter gene expression. Here we combined eQTL and molecular analyses to identify an association between two seemingly non-associated genes in brain expression data from BXD inbred mice, namely Ptpn21 and Nrg3. Using biotinylated receptor tracking and immunoprecipitation analyses, we determined that PTPN21 de-phosphorylates the upstream receptor tyrosine kinase ErbB4 leading to the up-regulation of its downstream signaling. Conversely, kinase-dead ErbB4 (K751R) or phosphatase-dead PTPN21 (C1108S) mutants impede PTPN21-dependent signaling. Furthermore, PTPN21 also induced Elk-1 activation in embryonic cortical neurons and a novel Elk-1 binding motif was identified in a region located 1919 bp upstream of the NRG3 initiation codon. This enables PTPN21 to promote NRG3 expression through Elk-1, which provides a biochemical mechanism for the PTPN21–NRG3 association identified by eQTL. Biologically, PTPN21 positively influences cortical neuronal survival and, similar to Elk-1, it also enhances neuritic length. Our combined approaches show for the first time, a link between NRG3 and PTPN21 within a signaling cascade. This may explain why these two seemingly unrelated genes have previously been identified as risk genes for schizophrenia.     

Spontaneous mutations in recombinant inbred mice: mutant toll-like receptor 4 (Tlr4) in BXD29 mice

Recombinant inbred (RI) mice are frequently used to identify QTL that underlie differences in measurable phenotypes between two inbred strains of mice. Here we show that one RI strain, C57BL/6J x DBA/2J (BXD29), does not develop an inflammatory response following inhalation of LPS. Approximately 25% of F2 mice [F1(BXD29 x DBA/2J) x F1] are also unresponsive to inhaled LPS, suggesting the presence of a recessive mutation in the BXD29 strain. A genomic scan of these F2 mice revealed that unresponsive animals, but not responsive animals, are homozygous for C57BL/6J DNA at a single locus on chromosome 4 close to the genomic location of Tlr4. All progeny between BXD29 and gene-targeted Tlr4-deficient mice are unresponsive to inhaled LPS, suggesting that the mutation in the BXD29 strain is allelic with Tlr4. Moreover, the intact Tlr4 receptor is not displayed on the cell surface of BXD29 macrophages. Finally, a molecular analysis of the Tlr4 gene in BXD29 mice revealed that it is interrupted by a large insertion of repetitive DNA. These findings explain the unresponsiveness of BXD29 mice to LPS and suggest that data from BXD29 mice should not be included when using BXD mice to study phenotypes affected by Tlr4 function. Our results also suggest that the frequency of such unidentified, spontaneously occurring mutations is an issue that should be considered when RI strains are used to identify QTL.     

Genetic maps of mouse chromosome 17 including 12 new anonymous DNA loci and 25 anchor loci.

An interspecific backcross between lab mice and Mus spretus was used to construct a multilocus map of Chromosome 17 consisting of 12 new anonymous loci and 9 anchor loci. In addition, 7 anonymous DNA loci were added to the Chr 17 map for the BXD strains. Although we were able to identify readily the most likely gene order in the interspecific backcross, we found no evidence for an unambiguous gene order using the BXD recombinant inbred strains. Comparison of the interspecific backcross map and the BXD RI strain map revealed evidence in the interspecific backcross for a longer total genetic length, enhanced recombination distal to H-2, a segment showing suppressed recombination, and strong interference.     

Genetic regulation of bone mineral density in mice

Peak bone mass is a major determinant of risk of osteoporotic fracture. Family and twin studies have found a strong genetic component to the determination of bone mineral density (BMD). However, BMD is a complex trait whose expression is confounded by environmental influences and polygenic inheritance. The number, locations and effects of the individual genes contributing to natural variation in this trait are all unknown. The extreme difficulty of dissecting out environmental factors from genetic ones in humans has motivated the investigation of animal models. Genetically distinct animal strains raised under strict environmental control are critical tools for defining genetic regulation. The availability of inbred strains, combined with its relative fecundity, has established the mouse as the best model system for the study of mammalian genetics and physiology. Importantly, genes identified in murine analyses can usually be readily mapped to particular human chromosomal regions because of the high degree of synteny that exists between the mouse and human genomes. We employed quantitative trait locus (QTL) analysis to examine peak BMD in 24 recombinant inbred (RI) mouse strains, derived from a cross between C57BL/6 (B6) and DBA/2 (D2) progenitors (BXD RI). The distribution of BMD values among these strains clearly indicated the presence of strong genetic influences, with an estimated narrow sense heritability of 35%. The differences in peak whole body BMD in the BXD strains were integrated with a large database of genetic markers previously defined in the RI BXD strains to generate chromosome map sites for QTL locations. This QTL analysis provisionally identified a number of chromosomal sites linked to BMD. In the second phase of our BMD QTL mapping efforts, we used three independent mouse populations (all derived from B6 and D2 progenitor strains) to confirm and narrow the genetic locations of 4 QTLs (on chromosomes 1, 2, 4, and 11) that strongly influence the acquisition of peak BMD in mice. Using a novel, fine-mapping approach (recombinant inbred segregation testing), we have succeeded in narrowing two of the BMD-related chromosomal regions and in the process eliminated a number of candidate genes. The homologous regions in the human genome for each of these murine QTLs have been identified in recent human genetic studies. In light of this, we believe that findings in mice should aid in the identification of specific candidate genes for study in humans.     

Convergent analysis of cDNA and short oligomer microarrays, mouse null mutants and bioinformatics resources to study complex traits

Gene expression data sets have recently been exploited to study genetic factors that modulate complex traits. However, it has been challenging to establish a direct link between variation in patterns of gene expression and variation in higher order traits such as neuropharmacological responses and patterns of behavior. Here we illustrate an approach that combines gene expression data with new bioinformatics resources to discover genes that potentially modulate behavior. We have exploited three complementary genetic models to obtain convergent evidence that differential expression of a subset of genes and molecular pathways influences ethanol-induced conditioned taste aversion (CTA). As a first step, cDNA microarrays were used to compare gene expression profiles of two null mutant mouse lines with difference in ethanol-induced aversion. Mice lacking a functional copy of G protein-gated potassium channel subunit 2 (Girk2) show a decrease in the aversive effects of ethanol, whereas preproenkephalin (Penk) null mutant mice show the opposite response. We hypothesize that these behavioral differences are generated in part by alterations in expression downstream of the null alleles. We then exploited the WebQTL databases to examine the genetic covariance between mRNA expression levels and measurements of ethanol-induced CTA in BXD recombinant inbred (RI) strains. Finally, we identified a subset of genes and functional groups associated with ethanol-induced CTA in both null mutant lines and BXD RI strains. Collectively, these approaches highlight the phosphatidylinositol signaling pathway and identify several genes including protein kinase C beta isoform and preproenkephalin in regulation of ethanol- induced conditioned taste aversion. Our results point to the increasing potential of the convergent approach and biological databases to investigate genetic mechanisms of complex traits.     

Expression Quantitative Trait Loci Are Highly Sensitive to Cellular Differentiation State

Genetical genomics is a strategy for mapping gene expression variation to expression quantitative trait loci (eQTLs). We performed a genetical genomics experiment in four functionally distinct but developmentally closely related hematopoietic cell populations isolated from the BXD panel of recombinant inbred mouse strains. This analysis allowed us to analyze eQTL robustness/sensitivity across different cellular differentiation states. Although we identified a large number (365) of “static” eQTLs that were consistently active in all four cell types, we found a much larger number (1,283) of “dynamic” eQTLs showing cell-type–dependence. Of these, 140, 45, 531, and 295 were preferentially active in stem, progenitor, erythroid, and myeloid cells, respectively. A detailed investigation of those dynamic eQTLs showed that in many cases the eQTL specificity was associated with expression changes in the target gene. We found no evidence for target genes that were regulated by distinct eQTLs in different cell types, suggesting that large-scale changes within functional regulatory networks are uncommon. Our results demonstrate that heritable differences in gene expression are highly sensitive to the developmental stage of the cell population under study. Therefore, future genetical genomics studies should aim at studying multiple well-defined and highly purified cell types in order to construct as comprehensive a picture of the changing functional regulatory relationships as possible.     

Random sequence oligonucleotide primers detect polymorphic DNA products which segregate in inbred strains of mice

The random amplification of polymorphic DNA (RAPD) using primers of arbitrary nucleotide sequence has been extremely valuable in identifying heritable markers in a variety of systems. The present studies examined whether the RAPD technique can identify large numbers of polymorphisms that can be used to construct genetic maps in inbred strains of mice. By screening the inbred mouse strains C57BL/6J and DBA/2J with 481 random 10-mer oligonucleotide primers, we identified 95 polymorphisms and mapped 76 of these by use of the BXD series of recombinant inbred (RI) strains. The results clearly demonstrate that the RAPD technique allows for the identification of large numbers of DNA-based polymorphisms that distinguish these two inbred strains of mice,and that such markers can readily be used to construct molecular genetic linkage maps.     

The genetic rescue of two bottlenecked South Island robin populations using translocations of inbred donors

Populations forced through bottlenecks typically lose genetic variation and exhibit inbreeding depression. ‘Genetic rescue’ techniques that introduce individuals from outbred populations can be highly effective in reversing the deleterious effects of inbreeding, but have limited application for the majority of endangered species, which survive only in a few bottlenecked populations. We tested the effectiveness of using highly inbred populations as donors to rescue two isolated and bottlenecked populations of the South Island robin (Petroica australis). Reciprocal translocations significantly increased heterozygosity and allelic diversity. Increased genetic diversity was accompanied by increased juvenile survival and recruitment, sperm quality, and immunocompetence of hybrid individuals (crosses between the two populations) compared with inbred control individuals (crosses within each population). Our results confirm that the implementation of ‘genetic rescue’ using bottlenecked populations as donors provides a way of preserving endangered species and restoring their viability when outbred donor populations no longer exist.     

Genetic dissection of testicular weight in the mouse with the BXD recombinant inbred strains

Testicular weights were studied in the mouse BXD recombinant inbred (RI) strains. These strains were derived from DBA/2J and C57BL/6J progenitors that differ significantly in their testicular weights (0.224 g ± 0.015 vs. 0.161 g ± 0.03, P < 0.0001). The heritability of testicular weights was calculated to be 0.53, and the minimum number of responsible effective factors was estimated to be 5.7. The total genome scanning of the BXD RI strains with over 1000 markers revealed a quantitative trait locus (QTL) on mouse Chromosome (Chr) 13 near the D13Mit3 marker (LOD score 6.9). This QTL region was designated Twq1 and associated with over 75% of genetic variability. Received: 23 January 1998 / Accepted: 16 March 1998     

CellRegMap: a statistical framework for mapping context‐specific regulatory variants using scRNA‐seq

Single‐cell RNA sequencing (scRNA‐seq) enables characterizing the cellular heterogeneity in human tissues. Recent technological advances have enabled the first population‐scale scRNA‐seq studies in hundreds of individuals, allowing to assay genetic effects with single‐cell resolution. However, existing strategies to analyze these data remain based on principles established for the genetic analysis of bulk RNA‐seq. In particular, current methods depend on a priori definitions of discrete cell types, and hence cannot assess allelic effects across subtle cell types and cell states. To address this, we propose the Cell Regulatory Map (CellRegMap), a statistical framework to test for and quantify genetic effects on gene expression in individual cells. CellRegMap provides a principled approach to identify and characterize genotype–context interactions of known eQTL variants using scRNA‐seq data. This model‐based approach resolves allelic effects across cellular contexts of different granularity, including genetic effects specific to cell subtypes and continuous cell transitions. We validate CellRegMap using simulated data and apply it to previously identified eQTL from two recent studies of differentiating iPSCs, where we uncover hundreds of eQTL displaying heterogeneity of genetic effects across cellular contexts. Finally, we identify fine‐grained genetic regulation in neuronal subtypes for eQTL that are colocalized with human disease variants.     

Genotyping new BXD recombinant inbred mouse strains and comparison of BXD and consensus maps

Nine additional BXD recombinant inbred (RI) strains have been developed from the F₂ cross of C57BL/6J and DBA/2J mouse strains. A tenth line stopped breeding in the F₁₂ generation. F₂₀ generation breeding pairs from the nine surviving strains and an F₁₂ pair from the extinct line were genotyped at 319 genetic markers (primarily microsatellites) spanning most of the genome. Where typing data were lacking, the established set of 26 BXD strains also were genotyped at these same loci. The availability of these additional nine strains enhances the value of the BXD RI set for analysis of complex phenotypic traits. The proportion of loci still segregating at the F₂₀ generation was found to closely approximate expectation, suggesting that selection favoring the retention of heterozygosity is not a strong factor. However, the number of crossovers between adjacent markers was frequently less than predicted from consensus map distances. A significant deficiency of recombinants was observed on Chrs 3, 4, 14, and X. On Chr 14, the estimated cumulative BXD map distance between the most proximal and distal markers was only 30.2 cM, compared with a distance of 60.0 cM in the consensus map. On the X Chr, the estimated and predicted cumulative distances were 38.8 and 69.5 cM, respectively. Over all chromosomes, the BXD RI map is 14.5% shorter than predicted from the consensus map. It is suggested that distances in some of the consensus maps are inflated. Alternatively, recombinant genotypes could be selected against during inbreeding owing to allelic interactions affecting fitness. The latter interpretation implies that relatively strong intrachromosomal epistasis is common. Received: 2 October 1998 / Accepted: 15 December 1998     

Genetic analysis of barrel field size in the first somatosensory area (SI) in inbred and recombinant inbred strains of mice

We measured the combined area of posterior medial barrel subfield (PMBSF) and anterior lateral barrel subfield (ALBSF) areas in four common inbred strains (C3H/HeJ, A?/J, C57BL?/6J, DBA/2J), B6D2F1, and ten recombinant inbred (RI) strains generated from C57BL/6J and DBA/2J progenitors (BXD) as an initial attempt to examine the genetic influences underlying natural variation in barrel field size in adult mice. These two subfields are associated with the representation of the whisker pad and sinus hairs on the contralateral face. Using cytochrome oxidase labeling to visualize the barrel field, we measured the size of the combined subfields in each mouse strain. We also measured body weight and brain weight in each strain. We report that DBA/2J mice have a larger combined PMBSF/ALBSF area (6.15?±?0.10?mm²,?n?=?7) than C57BL?/6J (5.48?±?0.13?mm²,?n?=?10), C3H/HeJ (5.37?±?0.16?mm²,?n?=?10), and A/J mice (5.04?±?0.09?mm²,?n?=?15), despite the fact that DBA/2J mice have smaller average brain and body sizes. This finding may reflect dissociation between systems that control brain size with those that regulate barrel field area. In addition, BXD strains (average n?=?4) and parental strains showed considerable and continuous variation in PMBSF/ALBSF area, suggesting that this trait is polygenic. Furthermore, brain, body, and cortex weights have heritable differences between inbred strains and among BXD strains. PMBSF/ALBSF pattern appears similar among inbred and BXD strains, suggesting that somatosensory patterning reflects a common plan of organization. This data is an important first step in the quantitative genetic analysis of the parcellation of neocortex into diverse cytoarchitectonic zones that vary widely within and between species, and in identifying the genetic factors underlying barrel field size using quantitative trait locus (QTL) analyses.