Amanda R. Duselis

Mapping and identification of candidate loci responsible for Peromyscus hybrid overgrowth

Crosses between two recently diverged rodent species of the genus Peromyscus result in dramatic parent-of-origin effects on growth and development. P. maniculatus females crossed with P. polionotus males yield growth-retarded conceptuses, whereas the reciprocal cross results in overgrowth and lethality. These hybrid effects are particularly pronounced in the placenta. We previously detected linkage to two regions of the genome involved in the overgrowth effects. One locus, termed Peal, is a paternally expressed autosomal locus mapping to a domain whose house mouse equivalent contains several clusters of imprinted genes. The other locus, termed Mexl, maps to a gene-poor region of the X chromosome. Here we use an advanced intercross line to verify and narrow the regions of linkage and identify candidate genes for Mexl and Peal. While we have previously shown that Mexl affects both pre-and postnatal growth, we show here that Peal affects only prenatal growth. Utilizing criteria such as mutant phenotypes and allelic expression, we identify the loci encoding the homeobox protein Esx1 and the zinc-finger protein Pw1/Peg3 as candidates. Both loci exhibit expression changes in the hybrids.

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.

Patterns of hybrid loss of imprinting reveal tissue- and cluster-specific regulation.

Background Crosses between natural populations of two species of deer mice, Peromyscus maniculatus (BW), and P. polionotus (PO), produce parent-of-origin effects on growth and development. BW females mated to PO males (bw×po) produce growth-retarded but otherwise healthy offspring. In contrast, PO females mated to BW males (PO×BW) produce overgrown and severely defective offspring. The hybrid phenotypes are pronounced in the placenta and include PO×BW conceptuses which lack embryonic structures. Evidence to date links variation in control of genomic imprinting with the hybrid defects, particularly in the PO×BW offspring. Establishment of genomic imprinting is typically mediated by gametic DNA methylation at sites known as gDMRs. However, imprinted gene clusters vary in their regulation by gDMR sequences. Methodology/Principal Findings Here we further assess imprinted gene expression and DNA methylation at different cluster types in order to discern patterns. These data reveal PO×BW misexpression at the Kcnq1ot1 and Peg3 clusters, both of which lose ICR methylation in placental tissues. In contrast, some embryonic transcripts (Peg10, Kcnq1ot1) reactivated the silenced allele with little or no loss of DNA methylation. Hybrid brains also display different patterns of imprinting perturbations. Several cluster pairs thought to use analogous regulatory mechanisms are differentially affected in the hybrids. Conclusions/Significance These data reinforce the hypothesis that placental and somatic gene regulation differs significantly, as does that between imprinted gene clusters and between species. That such epigenetic regulatory variation exists in recently diverged species suggests a role in reproductive isolation, and that this variation is likely to be adaptive.

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 biology and methodology of assisted reproduction in deer mice (Peromyscus maniculatus)

Although laboratory-reared species of the genus Peromyscus-including deer mice-are used as model animals in a wide range of research, routine manipulation of Peromyscus embryogenesis and reproduction has been lagging. The objective of the present study was to optimize conditions for oocyte and/or embryo retrieval and for in vitro culturing. On average, 6.4 oocytes per mouse were recovered when two doses of 15 IU of pregnant mare serum gonadotropin (PMSG) were given 24 h apart, followed by 15 IU of hCG 48 h later. Following this hormone priming, females mated overnight with a fertile male yielded an average of 9.1 two-cell stage embryos. Although two-cell stage embryos developed to 8-cell stage in Potassium Simplex Optimized Medium (KSOM; Millipore-Chemicon, Billerica, MA, USA) in vitro, but not further, embryos recovered at the 8- to 16-cell stages developed into fully expanded blastocysts when cultured in M16 media in vitro. These blastocysts had full potential to develop into late stage fetuses and possibly into live pups. As a result of the present work, all stages of Peromyscus preimplantation development are now obtainable in numbers sufficient for molecular or other analyses. These advances provide the opportunity for routine studies involving embryo transfer (e.g., chimeras, transgenics), and preservation of genetic lines by cryopreservation.

Harvesting Sperm and Artificial Insemination of Mice

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals. Peromyscus species are used in a wide range of research including toxicology, epidemiology, ecology, behavioral, and genetic studies. Here they provide a useful model for demonstrations of artificial insemination. Methods similar to those displayed here have previously been used in several deer mouse studies, yet no detailed protocol has been published. Here we demonstrate the basic method of artificial insemination. This method entails extracting the testes from the rodent, then isolating the sperm from the epididymis and vas deferens. The mature sperm, now in a milk mixture, are placed in the females reproductive tract at the time of ovulation. Fertilization is counted as day 0 for timing of embryo development. Embryos can then be retrieved at the desired time-point and manipulated.Artificial insemination can be used in a variety of rodent species where exact embryo timing is crucial or hard to obtain. This technique is vital for species or strains (including most Peromyscus) which may not mate immediately and/or where mating is hard to assess. In addition, artificial insemination provides exact timing for embryo development either in mapping developmental progress and/or transgenic work. Reduced numbers of animals can be used since fertilization is guaranteed. This method has been vital to furthering the Peromyscus system, and will hopefully benefit others as well.

Retrieval of mouse oocytes.

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology. Together with the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system. A primary goal has been to optimize oocyte/early embryo retrieval.

Nonsurgical Artificial Insemination in Mice

Artificial insemination is used to impregnate female mice using a solution containing motile sperm. For nonsurgical artificial insemination, a blunt needle is inserted through the vaginal canal of naturally ovulating females that are in the proestrus/estrous phase. This method requires practice but can be mastered, although initially it may be easier to have two people perform the procedure. Naturally ovulating or superovulated females are used in the absence of natural mating. Artificial insemination is achieved by isolating sperm from the cauda epididymis and vas deferens into a milk solution and introducing it into the reproductive tract of ovulating females. Only females that are known to be in estrus are used.