Genetic Analysis of the Stereotypic Phenotype in Peromyscus maniculatus (deer mice).

Peromyscus maniculatus, including the laboratory stock BW, have been used as a model organism for autism spectrum disorder and obsessive-compulsive disorder because of the high occurrence of stereotypy. Several studies have identified neurological and environmental components of the phenotype; however, the heritability of the phenotype has not been examined. This study characterizes the incidence and heritability of vertical jumping stereotypy (VS) and backflipping (BF) behavior in the BW stock of the Peromyscus Genetic Stock Center, which are indicative of autism spectrum disorders. In addition, interspecies crosses between P. maniculatus and P. polionotus were also performed to further dissect genetically stereotypic behavior. The inheritance pattern of VS suggests that multiple genes result in a quantitative trait with low VS being dominant over high VS. The inheritance pattern of BF suggests that fewer genes are involved, with one allele causing BF in a dominant fashion. An association analysis in BW could reveal the underlying genetic loci associated with stereotypy in P. maniculatus, especially for the BF behavior.

Using genomic resources for linkage analysis in Peromyscus with an application for characterizing Dominant Spot.

BACKGROUND: Peromyscus are the most common mammalian species in North America and are widely used in both laboratory and field studies. The deer mouse, P. maniculatus and the old-field mouse, P. polionotus, are closely related and can generate viable and fertile hybrid offspring. The ability to generate hybrid offspring, coupled with developing genomic resources, enables researchers to conduct linkage analysis studies to identify genomic loci associated with specific traits. RESULTS: We used available genomic data to identify DNA polymorphisms between P. maniculatus and P. polionotus and used the polymorphic data to identify the range of genetic complexity that underlies physiological and behavioral differences between the species, including cholesterol metabolism and genes associated with autism. In addition, we used the polymorphic data to conduct a candidate gene linkage analysis for the Dominant spot trait and determined that Dominant spot is linked to a region of chromosome 20 that contains a strong candidate gene, Sox10. During the linkage analysis, we found that the spot size varied quantitively in affected Peromyscus based on genetic background. CONCLUSIONS: The expanding genomic resources for Peromyscus facilitate their use in linkage analysis studies, enabling the identification of loci associated with specific traits. More specifically, we have linked a coat color spotting phenotype, Dominant spot, with Sox10, a member the neural crest gene regulatory network, and that there are likely two genetic modifiers that interact with Dominant spot. These results establish Peromyscus as a model system for identifying new alleles of the neural crest gene regulatory network.

A high methyl donor diet affects physiology and behavior in Peromyscus polionotus.

Folic acid and other dietary methyl donors are widely supplemented due to their ability to prevent neural tube defects. Dietary methyl donors are also added to other consumables such as energy drinks due to energy-promoting attributes and other perceived benefits. However, there is mounting evidence that indicates developmental exposure to high levels of dietary methyl donors may have deleterious effects. We assessed whether behavior was affected in the social North American rodent species Peromyscus polionotus exposed to a diet enriched with folic acid, Vitamin B12, choline, and betaine/trimethylglycine(TMG). P. polionotus (PO) animals are very social and exhibit little repetitive behavior, particularly compared to their sister species, P. maniculatus. We assayed the effects of dietary methyl-donor supplementation on anxiety-like repetitive and social behaviors by testing young adult animals for novel cage behavior and in social interaction tests. Animals of both sexes exposed to the diet had increased repetitive behaviors and reduced social interactions. Males exposed to the diet became more aggressive compared to their control counterparts. Since methyl-diet animals were larger than control animals, DEXA scans and hormone analyses were performed. Animals exposed to the diet had increased body fat percentages and experienced hormonal changes typically associated with excess fat storage and anxiety-like behavior changes. Therefore, these data suggest the wide use of these dietary supplements makes further investigation imperative.

Consequences of dietary methyl donor supplements: Is more always better?

Epigenetic mechanisms are now recognized to play roles in disease etiology. Several diseases increasing in frequency are associated with altered DNA methylation. DNA methylation is accomplished through metabolism of methyl donors such as folate, vitamin B12, methionine, betaine (trimethylglycine), and choline. Increased intake of these compounds correlates with decreased neural tube defects, although this mechanism is not well understood. Consumption of these methyl donor pathway components has increased in recent years due to fortification of grains and high supplemental levels of these compounds (e.g. vitamins, energy drinks). Additionally, people with mutations in one of the enzymes that assists in the methyl donor pathway (5-MTHFR) are directed to consume higher amounts of methyl donors to compensate. Recent evidence suggests that high levels of methyl donor intake may also have detrimental effects. Individualized medicine may be necessary to determine the appropriate amounts of methyl donors to be consumed, particularly in women of child bearing age.

Hematologic and serum biochemical values of 4 species of Peromyscus mice and their hybrids.

Deer mice (Peromyscus maniculatus) and congeneric species are used in a wide variety of research applications, particularly studies of developmental, physiologic, and behavioral characteristics associated with habitat adaptation and speciation. Because peromyscine mice readily adapt to colony conditions, animals with traits of interest in the field are moved easily into the laboratory where they can be studied under controlled conditions. The purpose of this study was to determine the serum chemistry and hematologic parameters of 4 frequently used species from the Peromyscus Genetic Stock Center species (P. californicus, P. leucopus, P. maniculatus, and P. polionotus) and to determine quantitative differences in these parameters among species and between sexes. Triglyceride values were substantially higher in female compared with male mice in all 4 species. Similar cross-species differences in MCH were present. Overall there was considerable interspecific variation for most blood parameters, with little evidence for covariation of any 2 or more parameters. Because crosses of P. maniculatus and P. polionotus produce fertile offspring, segregation analyses can be applied to determine the genetic basis of any traits that differ between them, such as their 3.8- and 2.1-fold interspecific differences in cholesterol and triglyceride levels, respectively. The current data provide a set of baseline values useful for subsequent comparative studies of species experiencing different circumstances, whether due to natural variation or anthropogenic environmental degradation. To enable such comparisons, the raw data are downloadable from a site maintained by the Stock Center (http://ww2.biol.sc.edu/∼peromyscus).

Pleiotropic effects of a methyl donor diet in a novel animal model.

Folate and other methyl-donor pathway components are widely supplemented due to their ability to prevent prenatal neural tube defects. Several lines of evidence suggest that these supplements act through epigenetic mechanisms (e.g. altering DNA methylation). Primary among these are the experiments on the mouse viable yellow allele of the agouti locus (A(vy)). In the Avy allele, an Intracisternal A-particle retroelement has inserted into the genome adjacent to the agouti gene and is preferentially methylated. To further test these effects, we tested the same diet used in the Avy studies on wild-derived Peromyscus maniculatus, a native North American rodent. We collected tissues from neonatal offspring whose parents were fed the high-methyl donor diet as well as controls. In addition, we assayed coat-color of a natural variant (wide-band agouti = A(Nb)) that overexpresses agouti as a phenotypic biomarker. Our data indicate that these dietary components affected agouti protein production, despite the lack of a retroelement at this locus. Surprisingly, the methyl-donor diet was associated with defects (e.g. ovarian cysts, cataracts) and increased mortality. We also assessed the effects of the diet on behavior: We scored animals in open field and social interaction tests. We observed significant increases in female repetitive behaviors. Thus these data add to a growing number of studies that suggest that these ubiquitously added nutrients may be a human health concern.

Peromyscus (deer mice) as developmental models.

Deer mice (Peromyscus) are the most common native North American mammals, and exhibit great natural genetic variation. Wild-derived stocks from a number of populations are available from the Peromyscus Genetic Stock Center (PGSC). The PGSC also houses a number of natural variants and mutants (many of which appear to differ from Mus). These include metabolic, coat-color/pattern, neurological, and other morphological variants/mutants. Nearly all these mutants are on a common genetic background, the Peromyscus maniculatus BW stock. Peromyscus are also superior behavior models in areas such as repetitive behavior and pair-bonding effects, as multiple species are monogamous. While Peromyscus development generally resembles that of Mus and Rattus, prenatal stages have not been as thoroughly studied, and there appear to be intriguing differences (e.g., longer time spent at the two-cell stage). Development is greatly perturbed in crosses between P. maniculatus (BW) and Peromyscus polionotus (PO). BW females crossed to PO males produce growth-restricted, but otherwise healthy, fertile offspring which allows for genetic analyses of the many traits that differ between these two species. PO females crossed to BW males produce overgrown but severely dysmorphic conceptuses that rarely survive to late gestation. There are likely many more uses for these animals as developmental models than we have described here. Peromyscus models can now be more fully exploited due to the emerging genetic (full linkage map), genomic (genomes of four stocks have been sequenced) and reproductive resources.

Caring for Peromyscus spp. in research environments.

Peromyscus spp. are the most abundant native North American mammals. They have gained popularity as research animals in the last 20 years, and this trend is expected to continue as new research tools, such as whole genome sequences, baseline physiological data and others, become available. Concurrently, advances have been made in the recommendations for the care of laboratory animals. The authors provide insight into how the Peromyscus Genetic Stock Center successfully breeds and maintains several stocks of deer mice and related species. This information is beneficial to researchers that plan to include Peromyscus spp. in their research programs.

A genetic map of Peromyscus with chromosomal assignment of linkage groups (a Peromyscus genetic map).

The rodent genus Peromyscus is the most numerous and species-rich mammalian group in North America. The naturally occurring diversity within this genus allows opportunities to investigate the genetic basis of adaptation, monogamy, behavioral and physiological phenotypes, growth control, genomic imprinting, and disease processes. Increased genomic resources including a high quality genetic map are needed to capitalize on these opportunities. We produced interspecific hybrids between the prairie deer mouse (P. maniculatus bairdii) and the oldfield mouse (P. polionotus) and scored meiotic recombination events in backcross progeny. A genetic map was constructed by genotyping of backcross progeny at 185 gene-based and 155 microsatellite markers representing all autosomes and the X-chromosome. Comparison of the constructed genetic map with the molecular maps of Mus and Rattus and consideration of previous results from interspecific reciprocal whole chromosome painting allowed most linkage groups to be unambiguously assigned to specific Peromyscus chromosomes. Based on genomic comparisons, this Peromyscus genetic map covers ~83% of the Rattus genome and 79% of the Mus genome. This map supports previous results that the Peromyscus genome is more similar to Rattus than Mus. For example, coverage of the 20 Rattus autosomes and the X-chromosome is accomplished with only 28 segments of the Peromyscus map, but coverage of the 19 Mus autosomes and the X-chromosome requires 40 chromosomal segments of the Peromyscus map. Furthermore, a single Peromyscus linkage group corresponds to about 91% of the rat and only 76% of the mouse X-chromosomes.

Natural genetic variation underlying differences in Peromyscus repetitive and social/aggressive behaviors.

Peromyscus maniculatus (BW) and P. polionotus (PO) are interfertile North American species that differ in many characteristics. For example, PO exhibit monogamy and BW animals are susceptible to repetitive behaviors and thus a model for neurobehavioral disorders such as Autism. We analyzed these two stocks as well as their hybrids, a BW Y(PO) consomic line (previously shown to alter glucose homeostasis) and a natural P. maniculatus agouti variant (A(Nb) = wide band agouti). We show that PO animals engage in far less repetitive behavior than BW animals, that this trait is dominant, and that trait distribution in both species is bi-modal. The A(Nb) allele also reduces such behaviors, particularly in females. PO, F1, and A(Nb) animals all dig significantly more than BW. Increased self-grooming is also a PO dominant trait, and there is a bimodal trait distribution in all groups except BW. The inter-stock differences in self-grooming are greater between males, and the consomic data suggest the Y chromosome plays a role. The monogamous PO animals engage in more social behavior than BW; hybrid animals exhibit intermediate levels. Surprisingly, A(Nb) animals are also more social than BW animals, although A(Nb) interactions led to aggressive interactions at higher levels than any other group. PO animals exhibited the lowest incidence of aggressive behaviors, while the hybrids exhibited BW levels. Thus this group exhibits natural, genetically tractable variation in several biomedically relevant traits.

Peromyscus as a Mammalian epigenetic model.

Deer mice (Peromyscus) offer an opportunity for studying the effects of natural genetic/epigenetic variation with several advantages over other mammalian models. These advantages include the ability to study natural genetic variation and behaviors not present in other models. Moreover, their life histories in diverse habitats are well studied. Peromyscus resources include genome sequencing in progress, a nascent genetic map, and >90,000 ESTs. Here we review epigenetic studies and relevant areas of research involving Peromyscus models. These include differences in epigenetic control between species and substance effects on behavior. We also present new data on the epigenetic effects of diet on coat-color using a Peromyscus model of agouti overexpression. We suggest that in terms of tying natural genetic variants with environmental effects in producing specific epigenetic effects, Peromyscus models have a great potential.

Adh1 and Adh1/4 knockout mice as possible rodent models for presymptomatic Parkinson's disease.

Alcohol dehydrogenases (ADH) catalyze the reversible metabolism of many types of alcohols and aldehydes to prevent the possible toxic accumulation of these compounds. ADHs are of interest in Parkinson's disease (PD) since these compounds can be harmful to dopamine (DA) neurons. Genetic variants in ADH1C and ADH4 have been found to associate with PD and lack of Adh4 gene activity in a mouse model has recently been reported to induce changes in the DA system. Adh1 knockout (Adh1-/-) and Adh1/4 double knockout (Adh1/4-/-) mice were investigated for possible changes in DA system related activity, biochemical parameters and olfactory function compared to wild-type (WT) mice. Locomotor activity was tested at ∼7 (adult) and >15 months of age to mimic the late onset of PD. Adh1-/- and Adh1/4-/- mice displayed a significantly higher spontaneous locomotor activity than WT littermates. Both apomorphine and d-amphetamine increased total distance activity in Adh1-/- mice at both age intervals and in Adh1/4-/- mice at 7 months of age compared to WT mice. No significant changes were found regarding olfactory function, however biochemical data showed decreased 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratios in the olfactory bulb and decreased homovanillic acid (HVA)/DA ratios in the olfactory bulb, frontal cortex and striatum of Adh1/4-/- mice compared to WT mice. Our results suggest that lack of Adh1 alone or Adh1 and Adh4 together lead to changes in DA system related behavior, and that these knockout mice might be possible rodent models to study presymptomatic PD.

Tissue expression pattern of class II and class V genes found in the Adh complex on mouse chromosome 3.

The alcohol dehydrogenase enzymes in mice and humans are encoded by a linked group of genes in the same transcriptional orientation. The enzymes play important roles in alcohol metabolism and retinoid signaling and homeostasis. The expression patterns at the mRNA level of the mouse Adh4 (class II) gene and the recently identified Adh6a and Adh6b genes (class V) are now reported to complete this analysis for the entire family. Adh4 is expressed at high levels in liver and is detectable in small intestine and testes. Adh6b is expressed in liver but Adh6a is not. Adh6a is expressed at high levels in small intestine while Adh6b is not. Adh6a expression is detectable in the female adrenal and not at all in the male adrenal, but Adh6b is expressed at moderate levels in both sexes. Although Adh6a and Adh6b have expression patterns different from each other, neither expresses like any other gene in the complex, suggesting different control mechanisms and possibly different functions.

High and complementary expression patterns of alcohol and aldehyde dehydrogenases in the gastrointestinal tract: implications for Parkinson's disease.

Parkinson's disease (PD) is a heterogeneous movement disorder characterized by progressive degeneration of dopamine neurons in substantia nigra. We have previously presented genetic evidence for the possible involvement of alcohol and aldehyde dehydrogenases (ADH; ALDH) by identifying genetic variants in ADH1C and ADH4 that associate with PD. The absence of the corresponding mRNA species in the brain led us to the hypothesis that one cause of PD could be defects in the defense systems against toxic aldehydes in the gastrointestinal tract. We investigated cellular expression of Adh1, Adh3, Adh4 and Aldh1 mRNA along the rodent GI tract. Using oligonucleotide in situ hybridization probes, we were able to resolve the specific distribution patterns of closely related members of the ADH family. In both mice and rats, Adh4 is transcribed in the epithelium of tongue, esophagus and stomach, whereas Adh1 was active from stomach to rectum in mice, and in duodenum, colon and rectum in rats. Adh1 and Adh4 mRNAs were present in the mouse gastric mucosa in nonoverlapping patterns, with Adh1 in the gastric glands and Adh4 in the gastric pits. Aldh1 was found in epithelial cells from tongue to jejunum in rats and from esophagus to colon in mice. Adh3 hybridization revealed low mRNA levels in all tissues investigated. The distribution and known physiological functions of the investigated ADHs and Aldh1 are compatible with a role in a defense system, protecting against alcohols, aldehydes and formaldehydes as well as being involved in retinoid metabolism.

Distal and proximal cis-linked sequences are needed for the total expression phenotype of the mouse alcohol dehydrogenase 1 (Adh1) gene.

Mouse alcohol dehydrogenase 1 (Adh1) gene expression occurs at high levels in liver and adrenal, moderate levels in kidney and intestine, low levels in a number of other tissues, and is undetectable in thymus, spleen and brain by Northern analysis. In transgenic mice, a minigene construct containing 10 kb of upstream and 1.5 kb of downstream flanking sequence directs expression in kidney, adrenal, lung, epididymis, ovary and skin but promotes ectopic expression in thymus and spleen while failing to control expression in liver, eye, intestine and seminal vesicle. Cosmids containing either 7 kb of upstream and 21 kb of downstream or 12 kb of upstream and 23 kb of downstream sequence flanking genetically marked Adh1 additionally promotes seminal vesicle expression suggesting downstream or intragenic sequence controls expression in this tissue. However, expression in liver, adrenal, or intestine is not promoted. The Adh1(a) allele on the cosmid expresses an enzyme electrophoretically distinct from that of the endogenous Adh1(b) allele, and presence of the heterodimeric enzyme in expressing tissues confirms that transgene activity occurs in the same cell-type as the endogenous gene. Transgene expression levels promoted by cosmids were at physiologically relevant amounts and exhibited greater copy-number dependence than observed with minigenes. Transgene mRNA expression correlated with expression measured at the enzyme level. A bacterial artificial chromosome containing 110 kb of 5'- and 104 kb of 3'-flanking sequence surrounding the Adh1 gene promoted expression in tissues at levels comparable to the endogenous gene most importantly including liver, adrenal and intestinal tissue where high level Adh1 expression occurs. Transgene expression in liver was in the same cell types as promoted by the endogenous gene. Although proximal elements extending 12 kb upstream and 23 kb downstream of the Adh1 gene promote expression at physiologically relevant levels in most tissues, more distal elements are additionally required to promote normal expression levels in liver, adrenal and intestinal tissue where Adh1 is most highly expressed.

Organization of six functional mouse alcohol dehydrogenase genes on two overlapping bacterial artificial chromosomes.

Mammalian alcohol dehydrogenases (ADH) form a complex enzyme system based on amino-acid sequence, functional properties, and gene expression pattern. At least four mouse Adh genes are known to encode different enzyme classes that share less than 60% amino-acid sequence identity. Two ADH-containing and overlapping C57BL/6 bacterial artificial chromosome clones, RP23-393J8 and -463H24, were identified in a library screen, physically mapped, and sequenced. The gene order in the complex and two new mouse genes, Adh5a and Adh5b, and a pseudogene, Adh5ps, were obtained from the physical map and sequence. The mouse genes are all in the same transcriptional orientation in the order Adh4-Adh1-Adh5a-Adh5b-Adh5ps-Adh2-Adh3. A phylogenetic tree analysis shows that adjacent genes are most closely related suggesting a series of duplication events resulted in the gene complex. Although mouse and human ADH gene clusters contain at least one gene for ADH classes I-V, the human cluster contains 3 class I genes while the mouse cluster has two class V genes plus a class V pseudogene.