Developmental exposure of California mice to endocrine disrupting chemicals and potential effects on the microbiome-gut-brain axis at adulthood.

Xenoestrogens are chemicals found in plant products, such as genistein (GEN), and in industrial chemicals, e.g., bisphenol A (BPA), present in plastics and other products that are prevalent in the environment. Early exposure to such endocrine disrupting chemicals (EDC) may affect brain development by directly disrupting neural programming and/or through the microbiome-gut-brain axis. To test this hypothesis, California mice (Peromyscus californicus) offspring were exposed through the maternal diet to GEN (250 mg/kg feed weight) or BPA (5 mg/kg feed weight, low dose- LD or 50 mg/kg, upper dose-UD), and dams were placed on these diets two weeks prior to breeding, throughout gestation, and lactation. Various behaviors, gut microbiota, and fecal metabolome were assessed at 90 days of age. The LD but not UD of BPA exposure resulted in individuals spending more time engaging in repetitive behaviors. GEN exposed individuals were more likely to exhibit such behaviors and showed socio-communicative disturbances. BPA and GEN exposed females had increased number of metabolites involved in carbohydrate metabolism and synthesis. Males exposed to BPA or GEN showed alterations in lysine degradation and phenylalanine and tyrosine metabolism. Current findings indicate cause for concern that developmental exposure to BPA or GEN might affect the microbiome-gut-brain axis.

Embryonic Development of the Deer Mouse, Peromyscus maniculatus.

Deer mice, or Peromyscus maniculatus, are an emerging model system for use in biomedicine. P. maniculatus are similar in appearance to laboratory mice, Mus musculus, but are more closely related to hamsters than to Mus. The laboratory strains of Peromyscus have captured a high degree of the genetic variability observed in wild populations, and are more similar to the genetic variability observed in humans than are laboratory strains of Mus. The Peromyscus Genetic Stock Center at the University of South Carolina maintains several lines of Peromyscus harboring mutations that result in developmental defects. We present here a description of P. maniculatus development from gastrulation to late gestation to serve as a guide for researchers interested in pursuing developmental questions in Peromyscus.

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.

The developmental role of Agouti in color pattern evolution.

Animal color patterns can affect fitness in the wild; however, little is known about the mechanisms that control their formation and subsequent evolution. We took advantage of two locally camouflaged populations of Peromyscus mice to show that the negative regulator of adult pigmentation, Agouti, also plays a key developmental role in color pattern evolution. Genetic and functional analyses showed that ventral-specific embryonic expression of Agouti establishes a prepattern by delaying the terminal differentiation of ventral melanocytes. Moreover, a skin-specific increase in both the level and spatial domain of Agouti expression prevents melanocyte maturation in a regionalized manner, resulting in a novel and adaptive color pattern. Thus, natural selection favors late-acting, tissue-specific changes in embryonic Agouti expression to produce large changes in adult color pattern.

Aberrant growth and pattern formation in Peromyscus hybrid placental development.

Crosses between the North American deer mouse species Peromyscus maniculatus (BW) and P. polionotus (PO) produce dramatic asymmetric developmental effects. BW females mated to PO males (female bw × male po) produce viable growth-retarded offspring. In contrast, PO females mated to BW males (female PO × male BW) produce overgrown but dysmorphic conceptuses. Most female PO × male BW offspring are dead by midgestation; those surviving to later time points display numerous defects reminiscent of several diseases. The hybrid effects are particularly pronounced in the placenta. Here we examine placental morphological defects via histology and in situ hybridization as well as the relationship between growth and mortality in the female PO × male BW cross. These assays indicate altered hybrid fetal:placental ratios by the equivalent of mouse (Mus) Embryonic Day (E) 13 and disorganization and labyrinth defects in female PO × male BW placentas and confirm earlier suggestions of a severely reduced junctional zone in the female bw × male po hybrids. Further, we show that both cellular proliferation and death are abnormal in the hybrids through BrdU incorporation and TUNEL assays, respectively. Together the data indicate that the origin of the effects is prior to the equivalent of Mus E10. Finally, as the majority of these assays had not previously been performed on Peromyscus, these studies provide comparative data on wild-type placentation.

Small mammals collected from a site with elevated selenium concentrations and three reference sites.

Small mammals were trapped in July, August, and September 1999 at Kesterson Reservoir (Merced County, CA), a site with elevated concentrations of selenium (Se), and at three nearby reference sites. Deer mice (Peromyscus maniculatus) were the most frequently trapped species at all of the sites, and western harvest mice (Reithrodontomys megalotis) and house mice (Mus musculus) were also trapped frequently. About half the animals collected from the reference sites were in reproductive condition, compared to less than a quarter of the animals from Kesterson. A lower proportion of the deer mice trapped at Kesterson was male, compared to about half at the reference sites. Deer mice from Kesterson also tended to have a lower condition index (body weight/body length) than those from the reference sites. Mice from Kesterson had enlarged livers, based on a higher liver-to-body-weight ratio (% liver). Male mice from Kesterson tended to have a smaller anogenital distance than male mice from reference sites, suggesting some feminization of Kesterson mice. Mice from Kesterson had higher liver Se concentrations than mice from reference sites, and some mice from reference sites had surprisingly low liver Se concentrations. It is possible that the effects observed were caused by physical stressors at Kesterson such as lower water and food availability but it also suggests that long-term exposure to elevated levels of Se may be, in part, the cause of some of the differences observed in small mammals from Kesterson.

Modularity of the rodent mandible: integrating bones, muscles, and teeth.

Summary Several models explain how a complex integrated system like the rodent mandible can arise from multiple developmental modules. The models propose various integrating mechanisms, including epigenetic effects of muscles on bones. We test five for their ability to predict correlations found in the individual (symmetric) and fluctuating asymmetric (FA) components of shape variation. We also use exploratory methods to discern patterns unanticipated by any model. Two models fit observed correlation matrices from both components: (1) parts originating in same mesenchymal condensation are integrated, (2) parts developmentally dependent on the same muscle form an integrated complex as do those dependent on teeth. Another fits the correlations observed in FA: each muscle insertion site is an integrated unit. However, no model fits well, and none predicts the complex structure found in the exploratory analyses, best described as a reticulated network. Furthermore, no model predicts the correlation between proximal parts of the condyloid and coronoid, which can exceed the correlations between proximal and distal parts of the same process. Additionally, no model predicts the correlation between molar alveolus and ramus and/or angular process, one of the highest correlations found in the FA component. That correlation contradicts the basic premise of all five developmental models, yet it should be anticipated from the epigenetic effects of mastication, possibly the primary morphogenetic process integrating the jaw coupling forces generated by muscle contraction with those experienced at teeth.

Heavy metal exposure, reproductive activity, and demographic patterns in white-footed mice (Peromyscus leucopus) inhabiting a contaminated floodplain wetland.

We examined the concentrations of selected metals and selenium (Se) in the tissues of white-footed mice (Peromyscus leucopus) collected at a constructed wetland originally created as a retention basin for sediments dredged from Lake DePue, Illinois. These sediments were contaminated with high concentrations of cadmium (Cd), zinc (Zn), and other elements as a result of nearby smelting operations. White-footed mice inhabiting the former retention basin experienced greater exposure to Cd, Pb, and Se than those from nearby reference sites. Concentrations of Cu and Zn in livers of mice from the contaminated wetland and adjacent floodplain reference site were greater than in mice from the more-distant reference sites. Judging by concentrations in their kidneys, white-footed mice inhabiting the floodplain adjacent to the contaminated wetland had greater exposure to Cd than those from the two more-distant reference sites. Concentrations of Hg in tissues of mice did not vary appreciably among sites. Concentrations of Cd and Se in the tissues of some white-footed mice from the contaminated wetland exceeded critical concentrations observed in experimental studies of laboratory mice and rats; with few exceptions tissue Pb concentrations were below published effects levels. However, we did not detect changes in abundance, demographics, or reproductive activity that might suggest population-level effects of contaminant exposure. Mean weight of embryos expressed as a function of crown-rump length did not differ among locations sampled, and no gross lesions indicative of exposure to heavy metals were observed. Kidney and liver weight, corrected for body weight, were nominally, though not significantly, lowest in both male and female mice from areas of increased Cd and Pb exposure. Metals dredged from Lake DePue were still bioavailable 25 years after deposition. However, small mammal populations are resilient to environmental stressors and we did not detect differences in population parameters suggesting that the population of white-footed mice inhabiting the contaminated wetland was at risk from increased exposure to these contaminants.

Assessment and disease comparisons of hybrid developmental defects.

Rodents of the genus Peromyscus are among the most common North American mammals. Crosses between natural populations of two of these species, P. maniculatus (BW) and P. polionotus (PO), produce parent-of-origin effects on growth and development. BW females mated to PO males produce growth-retarded offspring. In contrast, PO females mated to BW males produce overgrown but dysmorphic conceptuses. Variation in imprinted loci and control of genomic imprinting appear to underlie the hybrid effects. Prior morphological and genetic analyses have focused on placental and post-natal growth. Here, we assess the frequency and scope of embryonic defects. The most frequent outcome of the PO x BW cross is death prior to embryonic day 13. Conceptuses lacking an embryo proper are also observed as in gestational trophoblast disease. Among the common embryonic phenotypes described and tabulated are edema, blood vessel enlargement/hemorrhaging, macroglossia, retention of nucleated erythrocytes, placentomegaly. We investigate expression of loci known to be mis-regulated in human growth/placental disorders and/or mouse knockouts with similar phenotypes. These loci are Igf2, Cdkn1c, Grb10, Gpc3, Phlda2 and Rb1. All exhibited significant differences in either placental or embryonic expression levels at one or more of the three timepoints examined. The data underscore the importance of placental gene expression on embryonic defects. We suggest that the hybrid defects offer a novel system to understand how natural allelic combinations interact to produce disease phenotypes. We propose that such interactions and their resulting epimutations may similarly underlie the phenotypic and causal heterogeneity seen in many human diseases.

Genetic evidence for a maternal effect locus controlling genomic imprinting and growth.

Crosses between two species of deer mouse (Peromyscus) yield dramatic parent-of-origin effects. Female P. maniculatus (BW) crossed with male P. polionotus (PO) produce animals smaller than either parent. PO females crossed with BW males yield lethal overgrowth that has been associated with loss-of-imprinting (LOI). Previously, we mapped two loci influencing fetal growth. These two loci, however, do not account for the LOI, nor for the dysmorphic phenotypes. Here we report that maternal genetic background strongly influences the LOI. Analyses of crosses wherein maternal genetic background is varied suggest that this effect is likely due to the action of a small number of loci. We have termed these putative loci Meil. Estimation of Meil loci number was confounded by skewed allelic ratios in the intercross line employed. We show that the Meil loci are not identical to any of the DNA methyltransferases shown to be involved in regulation of genomic imprinting.

Genetic and epigenetic incompatibilities underlie hybrid dysgenesis in Peromyscus.

Crosses between the two North American rodent species Peromyscus polionotus (PO) and Peromyscus maniculatus (BW) yield parent-of-origin effects on both embryonic and placental growth. The two species are approximately the same size, but a female BW crossed with a male PO produces offspring that are smaller than either parent. In the reciprocal cross, the offspring are oversized and typically die before birth. Rare survivors are exclusively female, consistent with Haldane's rule, which states that in instances of hybrid sterility or inviability, the heterogametic sex tends to be more severely affected. To understand these sex- and parent-of-origin-specific patterns of overgrowth, we analysed reciprocal backcrosses. Our studies reveal that hybrid inviability is partially due to a maternally expressed X-linked PO locus and an imprinted paternally expressed autosomal BW locus. In addition, the hybrids display skewing of X-chromosome inactivation in favour of the expression of the BW X chromosome. The most severe overgrowth is accompanied by widespread relaxation of imprinting of mostly paternally expressed genes. Both genetic and epigenetic mechanisms underlie hybrid inviability in Peromyscus and hence have a role in the establishment and maintenance of reproductive isolation barriers in mammals.

Isolation of Bartonella spp. from embryos and neonates of naturally infected rodents.

Embryos and neonatal offspring of wild-captured cotton rats (Sigmodon hispidus) and white-footed mice (Peromyscus leucopus) were tested for the presence of Bartonella spp. Isolates of Bartonella spp. were obtained from 18 of 31 embryos and 7 of 19 neonates from bacteremic dams of the two species; no isolates were obtained from material from non-bacteremic dams. Sequence analysis demonstrated that the isolates from embryos and neonates matched the phylogenetic group of Bartonella spp. isolates obtained from the mother. No antibodies to homologous Bartonella spp. antigens were detected in maternal and neonatal blood or embryonic tissue. These findings suggest the possibility of vertical transmission of Bartonella spp. among natural rodent hosts.

Day length affects immune cell numbers in deer mice: interactions with age, sex, and prenatal photoperiod.

The extent to which day length affects immune function was examined in the present study. Three goals were pursued: 1) to confirm and extend the observation that the immune systems of adult deer mice (Peromyscus maniculatus) are responsive to changes in photoperiod, 2) to examine the development of the photoperiod-associated changes in immune function, and 3) to discover whether photoperiodic information transmitted to the young during gestation influences immune function. In experiment 1, adult mice housed in short days had higher white blood cell and lymphocyte numbers than their long-day cohorts. Red blood cell and differential cell counts did not differ between long- and short-day animals. No sex differences were observed in the pattern of immune responses to photoperiod. The effect of photoperiod on immune cells in prepubertal animals was examined in experiment 2; a similar pattern of results was obtained as that for experiment 1, suggesting that the photoperiodic effect on the immune system is not mediated by sex steroid hormones. Prenatal and postnatal photoperiodic effects on immune cells were examined in experiment 3; pups gestated in one day length were cross-fostered to mothers in the same day length conditions or to mothers maintained in the alternative day length. The results of experiment 3 suggested that photoperiodic information transmitted from the mother to the young in utero subsequently affected immune systems of the pups. Animals gestated in short day lengths displayed higher immune status throughout life than mice gestated in long days. These results are discussed from an adaptive functional perspective.

Medroxyprogesterone, immunosuppression, and conceptus size in Peromyscus.

Effects of exogenous medroxyprogesterone acetate (MPA) on skin transplant retention and conceptus size were studied in the deer mouse, Peromyscus maniculatus, and the oldfield mouse Peromyscus polionotus. Daily injections of 2 mg MPA significantly prolonged survival of allografts in P. maniculatus and transspecific grafts between P. maniculatus and P. polionotus. Allografts were retained significantly longer than transspecific grafts (17.2 vs 12.4 days) on MPA-treated P. maniculatus. Near-term fetal and placental sizes and weights were not detectably influenced by daily 1-mg MPA injections given to the mother from the 5th through the 19th day of pregnancy. The data are discussed relative to the possible immunosuppressive role of progestins in protecting the allogeneic conceptus from maternal immune rejection.