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Human Molecular Genetics Advance Access originally published online on March 5, 2007
Human Molecular Genetics 2007 16(7):808-819; doi:10.1093/hmg/ddm025
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Assessment and disease comparisons of hybrid developmental defects

Amanda R. Duselis and Paul B. Vrana*

Department of Biological Chemistry, School of Medicine, University of California Irvine, Irvine, CA 92799-1700, USA

* To whom correspondence should be addressed at: Department of Biological Chemistry, 312 Sprague Hall, University of California Irvine, Irvine, CA 92799-1700, USA. Tel: +1 9498249592; Fax: +1 9498242688; Email: pvrana{at}uci.edu

Received November 21, 2006; Revised January 22, 2007; Accepted February 9, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
There are few mammalian systems in which one can assess the effects of natural genetic variants on growth, development and behavior. The rodent genus Peromyscus are commonly referred to as deer mice, white-footed mice or field mice. Peromyscus constitutes arguably the most abundant group of endogenous North American mammals (1), and diverged ~25 million years from house mice (Mus) and rats (Rattus) (2,3). The Peromyscus maniculatus species complex consists of a widely distributed series of partially isolated populations exhibiting diverse ecological adaptations, behaviors and morphologies (1). The best characterized example of hybrid dysgenesis occurs between the prairie deer mouse (P. maniculatus bairdii), a Midwestern subspecies, and populations of the oldfield mouse, P. polionotus, which is confined to the southeastern United States.

While asymmetric post-fertilization hybrid effects had been noted earlier (4,5), Dawson (6) first quantified parent-of-origin effects in these crosses. He demonstrated that P. m. bairdii females crossed with P. polionotus produce growth-retarded offspring, which remain smaller than either parental species throughout life. The growth retardation is apparent by late gestation, and placental weight is approximately half that of the parental strains. Litter sizes of this hybrid cross are smaller than that of either parental strain, indicating some prenatal lethality (6–9). However, the growth-retarded survivors do not exhibit significant shortening of lifespan, are fertile and the sexes are equally represented (10). The reciprocal cross, P. polionotus females mated to P. maniculatus males, has been shown to result in prenatal somatic and placental overgrowth. This cross is also characterized by high degrees of pre-natal lethality as well as maternal death due to inability to pass the hybrid offspring through the birth canal. Rare post-natal survivors of this cross have been female and fertile (11).

These and subsequent studies were facilitated by establishment of laboratory strains of both species at the University of Michigan. The P. m. bairdii stock was begun with 40 individuals from a local population in Washtenaw county MI in 1948 and hence termed BW. The P. polionotus stock PO stock was derived 4 years later from 21 individuals from the Ocala National Forest in central Florida (http://stkctr.biol.sc.edu/peroavail.htm). These two stocks differ from commonly used inbred house mouse strains in several respects. Most obvious of these is that the Peromyscus stocks have been randomly bred to preserve the degree of heterozygosity present in the founders. Perhaps less appreciated is that the common inbred house mouse strains were derived from widely separated geographic locales (12). That is, the allelic combinations found in these strains do not exist in wild populations, rendering attempts to understand co-adapted gene complexes difficult. Elucidation of such interactions will also likely be essential for our understanding of how common human variants may underlie disease (13). Further, while the common approach of completely ablating gene function via transgenic technology often reveals processes requiring gene function and downstream targets, it does not predict the effects of coding-region or regulatory variants. Therefore, the P. maniculatus species complex has a unique potential among mammalian systems to yield insights into how such neutral or adaptive genetic variants can interact to yield deleterious phenotypes.

The parent-of-origin effects observed in the P. maniculatus (BW)–P. polionotus (PO) crosses suggested the involvement of loci subject to genomic imprinting, maternal effect loci, sex chromosome effects or mitochondrial (mt)DNA. Genomic imprinting refers to the biased allelic expression dependent on their parental origin (14). Many imprinted gene products have been shown to be key growth/nutrient regulators, and have been implicated in the etiology of a number of human growth disorders including Beckwith–Wiedemann, Silver–Russell and Prader–Willi syndromes as well as various cases of intrauterine growth retardation (15,16). The placenta is a key site of imprinted gene expression (17).

Dawson et al. (11) ruled out mtDNA–nuclear DNA interactions as being responsible for the hybrid defects through breeding congenic strains with both inter-specific mtDNA/nuclear DNA combinations. These authors proposed involvement of imprinted domains as an alternative. We have previously demonstrated perturbations of imprinted gene expression, and linkage of the overgrowth phenotypes to an imprinted domain and the X chromosome (18). Among the loci shown to exhibit loss-of-imprinting (LOI, switch from mono-allelic to bi-allelic expression) in the PO x BW hybrids are Pw1/Peg3, Snrpn, Mest/Peg1 and H19. The latter is surprising in that the tightly linked Igf2 gene retains imprinting, and common sequences have been shown to control both H19 and Igf2 imprinting. A species incompatibility involving a maternal effect locus involved in the imprinting process has been implicated as underlying the LOI (19). That is, multiple interacting loci are thought to contribute to form hybrid-specific epigenetic lesions. For example, DNA methylation at the H19 locus is altered specifically in the PO x BW hybrids. In contrast, studies to date have revealed relatively minor placenta-specific LOI in the growth-retarded bw x po hybrids.

The prior phenotypic, genetic and gene expression studies have focused primarily on the placenta or on post-natal growth, and on allelic usage (rather than expression levels) of imprinted loci. Largely unexplored areas include detailed assessments, histological, gene expression and biochemical analyses of the embryonic defects associated with the hybrid dysgenesis. While these defects are likely indirect (i.e. ‘down stream’), effects of the primary genetic incompatibilities, assessing their scope and frequency may suggest affected pathways. Here, we describe and quantitate a number of these phenotypes for the first time. We compare these to human growth syndromes and other diseases which share aspects of the hybrid phenotypes. We also examine the expression of several key loci known to be associated with these phenotypes from either disease or mouse transgenic models. We have focused on the P. polionotus female x P. maniculatus male (PO x BW) conceptuses as the phenotypes associated with this cross are more severe, and the genetics of the overgrowth are better understood.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Assessment of lethality, resorption and molar conceptus frequency
As noted, the PO x BW cross results in pre- or neo-natal lethality. Previous studies and our observations suggest resorptions are also a common outcome of these matings. One major question is when these deaths occur. Post-copulatory plugs in Peromyscus are not conspicuous. Hence, previous developmental studies have relied on morphological staging, artificial insemination, time since last parturition, or combinations of these. Here, we utilize a combination of cage partitions to allow animal acclimation and subsequent vaginal saline wash examination (for presence of sperm) to obtain more precisely timed natural matings.

We therefore tabulated the frequency of embryonic deaths and resorptions from 27 PO x BW pregnancies. Of these 27 pregnancies, 13 (~48%) contained only resorbing conceptuses by mid-gestation (Fig. 1A). Two pregnancies contained only conceptuses with no associated embryo; rather they appear to consist only of placental-like tissues and associated extra-embryonic tissues. This condition is reminiscent of the human disease termed hydatidiform mole (20). While the etiology of the condition in Peromyscus hybrids may be different, we will by analogy refer to these as molar conceptuses.


Figure 1
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  		Figure 1. Overview of PO x BW survival (E16≤). (A) Survival percentages by whole litter (L-R) % of litters where all conceptuses are dead or resorbed, percent of litters where all conceptuses were alive, percent of litters with at least one conceptus alive, percent of litters with the majority of conceptuses alive. Note that the first three categories are mutually exclusive and sum to 100%. Numbers in parentheses below each bar indicate number of litters in that category. (B) Percentages of dead conceptuses in litters containing at least one live conceptus at E10.5 or later. Numbers in parentheses below each bar indicate number of dead conceptuses at that age. Control litters of parental strains (PO, BW) over the same period showed no such effects.

 
Thirty-two percent of the remaining fourteen PO x BW litters examined contained at least one live conceptus, 36% had the majority of conceptuses alive at the time of dissection and 20% contained only live conceptuses. However, many of these live conceptuses were severely dysmorphic and likely non-viable. That is, they displayed defects that made survival unlikely (e.g. severe hemorrhaging, see in what follows).

In accordance with that observation, even litters with conceptuses surviving to late gestation exhibit high death rates (Fig. 1B). However, this assessment excludes those litters in which all members were largely resorbed, as we could not determine the time point at which that process began. These data suggest that while there is variation in the severity of the PO x BW phenotypes, there is no evidence for a significant ‘escapee’ class. In addition, both we and others have observed that PO x BW litters with conceptuses surviving to parturition typically cannot be passed through the PO birth canal.

The high rates of death and resorption observed limited our ability to examine and quantify other dysmorphic features we had observed in PO x BW offspring. We therefore also examined a set of backcross animals which produces similar phenotypes, but are somewhat easier to produce than the PO x BW hybrids. This backcross utilized an advanced intercross line (AIL) in which the growth retarded offspring (bw x po—use of lower case denotes the phenotype) were bred together for multiple generations. Female AIL animals were then bred to BW males. The varied maternal genetic background of the AIL x BW backcross has made it useful in genetic studies assessing maternal effects on genomic imprinting perturbations and mapping an X chromosome locus involved in placental and somatic overgrowth (19,21). These studies have suggested that a subset of AIL females homozygous PO for a subset of loci produce offspring with a similar set of defects when mated to BW males. We therefore assessed the nature and frequency of abnormalities in the embryos of the remaining 15 PO x BW litters (21 embryos) and from 65 AIL x BW litters (189 embryos).

Characterization and frequency of hybrid defects
The PO x BW and AIL x BW crosses produced a similar array of phenotypes (Fig. 2). Most obvious among these were the previously documented somatic and placental overgrowth, a rounded appearance, edema, prominent blood vessels, hemorrhaging, disproportionate overgrowth of the tongue (macroglossia) as well as the molar conceptuses. While all PO x BW conceptuses display abnormalities, only 69 of the 189 (~37%) of those produced by the AIL x BW cross show obvious defects. Therefore all further phenotypic analysis of the AIL x BW cross will refer to those 69 conceptuses.


Figure 2
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  		Figure 2. Representative examples of PO x BW hybrid phenotypes. (A, C, G; left column) Parental strain embryos at equivalent ages to PO x BW in the right column (B, D, F, H); (G) Associated extra-embryonic tissues. (E; left column) bw x po strain embryo at equivalent age to all other embryos. B, edemic embryo, D, prominent blood vessels/ hemorrhaging, F, macroglossia and edema, H, conceptus without embryo.

 
Many of the PO x BW embryos display multiple defects. Figure 2B, D and F show PO x BW embryos with the rounded appearance and varying degrees of somatic overgrowth. In some cases, this rounded appearance is clearly due to extreme edema (Fig. 2B). Figure 2B and D also display the prominent blood vessels, with the latter also exhibiting internal hemorrhaging. The embryo in Figure 2F also displays prominent macroglossia. The latter phenotype is also typically characterized by an open mouth to accommodate the hypertrophic tongue.

Figure 2H displays a PO x BW molar conceptus, while Figure 2G displays an equivalent BW embryo and associated extra-embryonic tissues. This molar phenotype differs from dead or resorbing conceptuses in that the tissue is non-necrotic, and typically approximately the same size as hyperplastic PO x BW placentae. Indeed, these conceptuses are often difficult to distinguish from other PO x BW placentae and associated membranes. With the exception of transient mild edema, none of the illustrated defects were observed in either parental strain or the bw x po hybrid embryos.

We next tabulated the frequency of the edema, prominent blood vessel/hemorrhaging and macroglossia phenotypes at timepoints from E12.5 to E16. The PO x BW and AIL x BW offspring display similar percentages (15–20%) of edematous offspring at E12.5 (Fig. 3A), and both display a mild increase at E13.5. However, at both E14.5 and E16 there were surprisingly large differences in the percentage of edematous offspring between the PO x BW and AIL x BW crosses.


Figure 3
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  		Figure 3. Graphical representation of the occurrence of selected phenotypes in PO/AIL x BW conceptuses. (A, B, C) Percentages of observed defects by age. Numbers directly below each bar indicate number of embryos in category (e.g. 6 E12.5 AIL x BW embryos exhibiting edema). (D) Total percentages of defects observed from E12.5-birth. Numbers in parentheses below each bar indicate number of embryos in category.

 
The hemorrhaging defect also displayed obvious frequency differences between these two crosses (Fig. 3B). The AIL x BW offspring display a fairly constant percentage (15–25%) exhibiting the defect from E12.5–E16. The PO x BW cross displays similar percentages of hemorrhaging embryos at the earliest and latest ages examined, but none at intermediate ages. However, the macroglossia phenotype is much more prevalent in PO x BW offspring (Fig. 3C).

The overall occurrence of selected PO/AIL x BW defects during the second half of gestation is shown in Figure 3D. We have included placental overgrowth (placentomegaly), which we defined as placentae weighing ≥two times that of the parental strains' mean placental weight (PO and BW placentae are equivalent size) (18). Placentomegaly is easily the most common defect, occurring in 70 of 90 (~78%) of the combined PO/AIL x BW litter sets. In contrast, the edema, macroglossia and the hemorrhaging phenotypes were all seen in approximately one-third of the PO/AIL x BW embryos.

We also tabulated the occurrences of multiple phenotypes in the same embryo to assess whether certain defects co-segregate more frequently than others (Table 1). Placentomegaly showed the highest correlation with other defects as a dependent variable, and the lowest as an independent variable. This dichotomy is undoubtedly to the overall high frequency of placental overgrowth. Both combinations of the hemorrhaging and edematic defects displayed the highest correlation among the individual combinations. Similarly, these same two defects exhibited the highest co-occurrence with multiple additional defects. The least correlative embryonic phenotype, both in individual and multiple combinations, was macroglossia.


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  		Table 1. Percentages of PO/AIL x BW embryos which display multiple defects

 
Histological assessment of hybrid phenotypes
We next examined stained histological sections of whole embryos to better understand the nature of these three phenotypes. Figure 4 displays sagittal sections illustrating a representative example of PO/AIL x BW macroglossia relative to parental strain and bw x po hybrids embryos. These sections illustrate that the PO x BW tongues are both longer and thicker than those of the parental strains. Less expected was the apparent reduced tongue growth displayed in the bw x po hybrid. Differences in hybrid cranio-facial patterning/growth also seem likely, but have not been examined in detail nor their frequency tabulated.


Figure 4
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  		Figure 4. Representative example of macroglossia in Peromyscus hybrids at E16.5. Embryos were sagittaly sectioned and stained with hematoxylin/eosin (H and E). (A and B) Sections of parental strain animals (PO/BW); (C and D) bw x po sections; (E and F) PO x BW sections. Left column shows upper torso and head; right column are cranio-facial close-ups of the same embryos.

 
The PO x BW sagittal sections also illustrated the edematous phenotype. The ventral side of the embryo in Figure 4E reveals significant space between the skin and the rest of the body. Coronal sections (Fig. 5) show this enlarged space in greater detail. This fluid filled space likely contributes to the rounded appearance often displayed by these embryos. The coronal sections revealed consistently thinner skin in the PO/AIL x BW hybrids. Further, the immediately subcutaneous blood vessels appeared to have thinner walls, larger diameters and fewer surrounding cells than corresponding vessels in the parental strains or bw x po hybrids.


Figure 5
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  		Figure 5. Skin defects in PO x BW embryos illustrated by H and E stained coronal sections. (A and C) Parental strains, (B and D) PO x BW. (C and D) are higher magnification of (A and B). Asterisk denotes hemorrhaging in the PO x BW embryo. Arrows indicate blood vessels. (C and D) display relative lack of cells surrounding the PO x BW blood vessels.

 
These characteristics suggested the basis for the prominent blood vessel and hemorrhaging phenotypes observed in the PO/AIL x BW animals. To better visualize potential defects, we stained sections with an antibody against laminin. This glycoprotein is found in the basement membrane of epithelial tissues, and is prevalent in blood vessels (22). We examined PO/AIL x BW offspring exhibiting the phenotype and found that their vessels have larger diameters, often with proportionally thinner walls. Figure 6 illustrates these differences in the descending aorta and the right subclavian artery.


Figure 6
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  		Figure 6. Laminin antibody staining of coronal sections to illustrate blood vessel phenotypes. (A and C) Parental strain animals, (B and D) PO x BW hybrids. (A and B) show the descending aorta. (C and D) show the right subclavian artery and superior vena cava. Single arrows mark the right subclavian artery and double arrows mark the right superior vena cava.

 
Prior studies had suggested that a number of genes whose products affect extra-cellular matrix (ECM) genes have altered expression levels in PO x BW placentae. The down regulation of the Col1a-2 gene, whose product is a major component of type I collagen, was among the most dramatic examples of the affected ECM loci. To assess whether reduced collagen and ECM might be associated with the embryonic phenotypes, we stained embryo sections with an antibody to type I collagen. Type I collagen production increases greatly during the second half of gestation in house mice, and is highly abundant in the dermal layer of the skin. Figure 7 depicts the reduced levels of type I collagen we observed in PO/AIL x BW epidermis. Accordingly, reduced collagen levels are associated with thinner and defective skin in human diseases such as Ehlers–Danlos syndrome (23). Although we have not observed an obvious skin defect in the bw x po hybrids, their epidermis also displayed reduced amounts of type I collagen. This finding is not entirely unexpected, as bw x po placentae also had much lower levels of Col1a-2 expression than the parental strains.


Figure 7
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  		Figure 7. Hybrid skin phenotypes illustrated by type I collagen antibody staining of sagital sections of E13 embryos. Left column, parental strains; middle column, PO x BW hybrids; right column, bw x po hybrids. (A–C) depict the snout at 10 x magnification, while (D–F) are 40 x magnification of this region. Arrows in (D–F) point to areas of differential staining. (G–I) depict hind limbs at 10 x magnification. (J–L) are higher magnifications of the back. Arrows denote the epidermal layer of the skin. Hair follicles are visible as indentations.

 
We did, however, uncover a hitherto undescribed hybrid defect in assessing the hematoxylin and eosin stained histological sections for the blood vessel and hemorrhaging phenotypes. We observed that late gestation PO x BW embryos contain a high proportion of nucleated red blood cells (RBC) during late stage development. While erythrocytes derived from the yolksac early in development retain their nucleus (24), mature RBCs derived from bone marrow are enucleated. Thus the ratio of nucleated:non-nucleated RBCs declines dramatically during late gestation. In house mice, ~90% of erythrocytes lack a nucleus by E16 (25). Examination of both PO and BW strain embryos suggest this is also true in Peromyscus—we examined RBCs in multiple places including the liver at E13.5 and E16. At E13.5, a majority of RBCs are nucleated in both parental strains and both hybrids (Fig. 8). In contrast, both parental strains and the bw x po hybrids displayed almost no nucleated RBCs (~2%) at E16. However, ~30% of E16 PO x BW erythrocytes are nucleated. This abnormal hematopoesis phenotype is reminiscent of targeted gene ‘knockouts’ of the retinoblastoma (Rb1) tumor suppressor locus in house mice (26). We therefore undertook gene expression assays of this and other loci associated with phenotypes similar to those we observed.


Figure 8
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  		Figure 8. Comparison of red blood cell nucleation status between Peromyscus parental strains and reciprocal hybrids. Blood vessel pockets within the liver of H and E stained sagittal sections are depicted. A–C E13.5 embryos. D–E and G–I E16 embryos; the latter is a higher magnification view of the region above. Genotypes depicted above each column. Note the presence of nucleated RBCs in the PO x BW E16 embryo. All pictures taken at 40 x magnification. (G–I) are close inspections of 40 x magnification pictures.

 
Expression assays of loci associated with similar phenotypes
We assayed expression of the insulin-like growth factor 2 (Igf2), glypican-3 (Gpc3), growth factor receptor binding protein 10 (Grb10) and cyclin-dependent kinase inhibitor 1 C (Cdkn1c/p57kip2) and Rb1 genes. Igf2 and Cdkn1c are both imprinted loci associated with Beckwith–Wiedemann syndrome (BWS) in humans (27,28). Similarly, a mouse model combining Igf2 over-expression and a Cdkn1c null mutant, recapitulated many of the BWS defects as well as somatic and placental overgrowth (29,30). Further, Cdkn1c down-regulation is also seen in hydatidiform moles (31). While Igf2 retains imprinting in both hybrid types, we had previously only assessed expression levels very late in gestation (~E18). Finally, Phlda2 (= Tssc3, Ipl) is an imprinted gene, elevated levels of which are associated with intra-uterine growth retardation (32,33). In contrast, reduced Phlda2 levels are associated with complete (versus partial) hydatidiform moles (34). Similarly, the mouse Phlda2 knockout resulted in placental hypertrophy (35).

Null mutations in the X-linked Gpc3 gene are associated with Simpson Golobai-Behmel syndrome (SGBS) (36). Both BWS and SGBS patients exhibit frequent macroglossia as well as somatic overgrowth and placentomegaly (27,36). The Grb10 gene is also imprinted, and has been implicated in Silver–Russell syndrome, which is characterized by intra-uterine and post-natal growth retardation (37). Both hybrid classes exhibited some bi-allelic expression of the Grb10 gene. As noted, Rb1 was investigated due to the effect of the mouse targeted mutation on hematopoiesis (26).

We assessed expression of these loci via quantitative or semi-quantitative PCR assays after obtaining Peromyscus sequence for each gene. Data were gathered at three time points: E10.5, E13.5 and E16. Figure 9 displays the results of these assays grouped by developmental timepoint. Both Igf2 and Gpc3 display are under-expressed in the growth-retarded bw x po embryos at E10.5 (Fig. 9A). Down-regulation of the potent mitogen Igf2 is associated with some cases of Silver–Russell syndrome as well as other examples of intra-uterine growth retardation (IUGR) (33,38). As the complete loss of Gpc3 is associated with overgrowth, the lower expression levels observed in the bw x po embryos are counter-intuitive. Gpc3 expression in the placenta was not detectable with our assay at any of the ages surveyed. Embryonic Gpc3 expression was not significantly different between groups at E13.5 (Fig. 9C) or at E16 (Fig. 9E).


Figure 9
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  		Figure 9. Expression assays of selected loci associated with defects similar to those observed in Peromyscus hybrid embryos. (A, C, E) Relative whole embryo gene expression values at ages E10.5, E13.5 and E16, respectively. (B, D, F) display relative placental gene expression values at the same three ages. (G) depicts Grb10 gene expression data for E13.5 and is shown separately due to the high levels of expression in BW embryos relative to other groups (PO, PO x BW and bw x po), and other genes at this age. Each gene/tissue/timepoint combination represents the average value from 3 or more individuals. Stars denote results where error bars (1 standard deviation) did not overlap with any other class.

 
Placental Igf2 expression at E10.5 in the bw x po hybrids was slightly lower than that observed in the PO x BW class (Fig. 9B). Surprisingly, BW placental Igf2 expression was much higher than any other class at this age. While the average Igf2 BW levels were higher than those of PO at E16, the E10.5 placenta was the only tissue/time in which this apparent species difference in Igf2 regulation was significant. While the embryonic bw x po Igf2 levels were equivalent to the other classes at later ages, bw x po placental expression was down-regulated at E13.5 and E16 (Fig. 9D and F). A further counter-intuitive finding was that PO x BW placental Igf2 expression was also significantly lower than the parental strains at these ages.

The only significant difference observed in embryonic gene expression at E13.5 was an ~15-fold increase in expression of the Grb10 gene in the BW strain relative to PO and both hybrids (Fig. 9G). While the BW samples displayed large variation, they also had a greater average value than that of PO expression levels. However, these same BW E13.5 samples exhibited neither little variation nor little difference from the PO strain in Igf2 and Gpc3 expression. As the Grb10 product is thought to negatively regulate Igf2 product signaling, the higher Grb10 levels may represent a species-specific adaptation to higher Igf2 levels.

The Cdkn1c product also negatively regulates growth via the cell-cycle as a cyclin-dependent kinase inhibitor. Embryonic Cdkn1c levels were equivalent in all classes at the three timepoints assessed. Consistent with our prediction, however, placental Cdkn1c expression was lower in the PO x BW hybrids at E13.5 and E16.5. This relaxation of cell-control may then negate the observed lower levels of Igf2 in the overgrown hybrids. The final gene encoding a negative growth regulator is Phlda2, whose expression is largely confined to the placenta. Again PO x BW Phlda2 expression is lower than other classes at E13.5 and E16.5.

We examined the expression of the Rb1 gene in both whole embryos and placentas due to the demonstrated involvement of the latter tissue in affecting erythrocyte differentiation. Whole embryo expression assays of Rb1 did not reveal any discernable pattern at any age (data not shown). Patterns of placental Rb1 expression, however, were consistent with the observed hematopoetic defect. At E10 and E13, the majority of erythrocytes are nucleated in all the genetic backgrounds, and all express equivalent levels of placental Rb1. At E16, placental Rb1 levels rise approximately three-fold in the parental strains and bw x po hybrids, which coincides with the majority of RBCs being enucleated. In contrast, E16 PO x BW hybrids maintain the same relatively low levels of Rb1 seen at earlier stages, and the same high proportion of nucleated RBCs.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Here we have described and quantified a suite of the Peromyscus hybrid phenotypes for the first time. Further, we have shown hybrid perturbations and intra-specific variation in a number of known growth-regulating and disease-associated genes. Our hope is that other research groups will also use this broad analysis as the basis for future detailed investigations and disease modeling. Comparisons of the Peromyscus hybrids with selected human diseases are shown in Table 2.


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  		Table 2. Comparison of Peromyscus hybrids with human diseases

 
Among the phenotypes that had either not been previously described or had not been quantitated are macroglossia, edema, blood vessel hypertrophy, epidermal collagen deficiency, improper erythrocyte differentiation, molar conceptuses and early embryonic death/resorption. All phenotypes co-occurred frequently; two combinations of defects exhibited the lowest co-occurrence frequency of 23%. In contrast, four combinations of defects co-occurred in greater than 50% of the PO/AIL x BW embryos.

The thinner skin associated with the overgrowth phenotypes, lower levels of type I collagen and down-regulation of ECM-related genes suggests that epithelial and other tissues for which ECM is particularly critical will be affected in the PO x BW embryos. Some forms of Ehlers–Danlos syndrome are characterized by abnormalities of blood vessel walls as well as of the skin (23). Prominent blood vessels and hemorrhaging were another frequently observed defect in the overgrowth crosses. The prominent appearance is due to the enlarged diameter of the vessels rather than abnormal patterning. We suggest that the increased diameter and hemorrhaging are due to ECM defects which result in weakened vessel walls. Leplace's law states that wall tension increases with vessel radius (http://hyperphysics.phy-astr.gsu.edu/hbase/hph.html). Weaker blood vessels would then dilate to the point of rupturing. An alternative but not mutually exclusive hypothesis is that the PO x BW embryos have increased blood pressure. Several human overgrowth syndromes also manifest weakened vasculature (39, http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM).

We also observed phenotypes which we have not yet formally described or quantitated. For example, we have frequently observed that PO x BW embryos are found with the abdominal wall open and protrusion of intestines. This condition is known as omphalocele, and is a common feature of BWS and other human overgrowth syndromes (39). As we have not yet determined the exact timing of closure of the gut wall in Peromyscus embryos, we have taken the conservative approach of not claiming a frequency of this phenotype.

Despite only including AIL x BW embryos that displayed defects, the PO x BW and AIL x BW embryos displayed certain phenotypes (edema, macroglossia, enlarged blood vessels) at different frequencies. This discrepancy suggests that PO alleles of other genes affect the PO x BW hybrid phenotypes. Such modifier alleles may be at reduced frequency in the AIL population due to fixation or negative selection. For example, we previously reported evidence suggesting that heterozygous genotypes of the imprinted H19 gene (which is tightly linked to Igf2 and Cdkn1c) were selected against in the advanced intercross line (19). Thus it may be possible to map and characterize variant wild-type alleles which modulate specific phenotypes.

While all the assessed loci displayed significant inter-class variation, they did not all meet prior expectations. For example while Igf2 expression was predictably reduced in the growth-retarded bw x po hybrids, so was the Gpc3 locus. Particularly surprising was the finding that expression levels of the imprinted Grb10 gene, and to a more limited extent the Igf2 gene, vary dramatically between the two parental populations. The Grb10 product has been shown to be a negative regulator of growth and insulin signaling (37). One would expect that the biased expression in favor of the Grb10 maternal allele would lead to higher levels of expression in bw x po than PO x BW. While the data at E16 suggests this may be the case, it is not conclusive. However, observed loss-of-imprinting and/or other upstream effects may significantly affect Grb10 transcript levels. It will be of interest to assess other loci in this pathway for species-specific expression levels.

One explanation for the lack of phenotype/gene expression correlation in the hybrid embryos is that misregulation of placental gene expression underlies these defects. The correlation of placental Rb1 gene expression with the nucleated erthyrocyte phenotype and the placenta-specific reduction in Cdkn1c and Phlda2 expression associated with the overgrowth support such a hypothesis. A more thorough analysis of hybrid placental dysplasias and cell fate is forthcoming.

The etiologies of the Peromyscus hybrid dysgenesis are not yet fully understood, nor are the genetic analyses complete. Our hypothesis is that epigenetic dysregulation in early development leads to improper differentiation and rates of proliferation. The molar pregnancy phenotype is consistent with such a hypothesis. Hydatidiform moles are often characterized by lack of a maternal genome, or an excess of paternal genomes (e.g. due to fertilization by multiple sperm) (20,40). However, a subset of cases have equal contributions from both parental genomes and are termed bi-parental complete hydatidiform moles (BiCHM) (41). While the two classes are phenotypically indistinguishable, BiCHM are characterized by a loss of DNA methylation at discrete parts of the genome including imprinted domains. Genes similarly affected in complete moles and/or the choriocarcinomas that are thought to derive from them and the PO x BW conceptuses are reduced Cdkn1c and Phlda2 levels, increased H19 expression (8) and a counter-intuitive lack of increased Igf2 expression (42). One locus affecting PO x BW placental overgrowth maps near a BiCHM susceptibility locus (21). The latter is thought to be caused by mutations in the NALP7 gene, a locus which better characterized rodents apparently lack. However, a significant number of BiCHM cases associated with genetic susceptibility do not map to NALP7 (43). Similarly, ~15–20% of BWS cases, as well as many cases of Ehlers–Danlos Syndrome and IUGR have not been genetically characterized (15,39,44–46). Indeed, several other overgrowth syndromes including SGBS, Perlman and Sotos syndromes may be difficult to distinguish from BWS. Sotos syndrome is typically caused by mutations in the NSD1 gene, which encodes a histone methyltranserase, and thus may also affect epigenetic gene regulation (47,48).

The heterogeneity of the PO x BW phenotypes could be explained by genetic variation in either the PO or BW strains. Several pieces of evidence suggest this is not the case. First, PO naturally has a high inbreeding coefficient, likely due to the monogamous lifestyle of P. polionotus (http://stkctr.biol.sc.edu/peroavail.htm). Secondly, the bw x po phenotypes are very consistent. Thirdly, another strain of P. polionotus, Pp. leucocephalus (LS), yield indistinguishable results in crosses to BW. Similarly, LS/PO hybrid animals crossed to BW animals also yield equivalent results as PO x BW crosses. Finally, preliminary microsatellite marker studies by the Peromyscus Genetic Stock Center suggest a small number of alleles in BW relative to another stock population (SM2—http://stkctr.biol.sc.edu/peroavail.htm) founded from a similar number of individuals.

We suggest that stochastic or micro-environmentally induced variation in the epigenetic dysregulation accounts for the PO x BW variation. These effects may be similar to variable epigenetic mutations and defects seen in ‘cloned’ animals produced by somatic cell nuclear transfer clones (49). We do not suggest that the hybrid phenotypes or etiology perfectly model any one human disease. Rather, the breadth of the hybrid phenotypes induced by natural allelic combinations offer a novel system to study genetic interactions. We propose that such interactions and their resulting epimutations may similarly underlie the phenotypic and causal heterogeneity seen in human diseases such as those discussed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Timed pregnancies
We purchased PO and BW animals from the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu/). Animals were housed with food and water ad libitum on a 16:8 h light:dark cycle. We bred PO females with BW males and BW females with PO males to obtain reciprocal hybrids. Males and females were placed in the same cage with a separation cage top to allow the estrous cycle of the female to be induced by the presence of a male without contact. After 3 days, a normal cage top was used so females and males could mate at will. Vaginal smears were performed two times a day (morning and evening) and visually assessed for sperm. We designated day 0 of development when sperm was found in vagina of cycling females.

Peromyscus pre-implantation development is 4 days longer than that of Mus. To facilitate comparisons, however, we have presented the data without inclusion of these 4 days such that the developmental stages of the two genera coincide; hence, all age references in the text refer to M. musculus equivalents. Breeding of advanced intercross line females to BW males (AIL x BW) was described by Duselis et al. (19).

Histological analysis
Placentas were fixed in 4% paraformaldehyde. Fixed samples were dehydrated through ascending concentrations of ethanol and then sent to Histoserv Inc. (www.histoservinc.com) for sectioning and haematoxylin/eosin staining. Sections used for type I collagen staining using primary antibodies were obtained from Abcam, and for laminins 1 and 2. Vectostain ABC elite kit with horseradish peroxidase was used to visualize the antibody selection.

Gene expression
We first obtained Peromyscus sequence for all genes assayed by previously described strategies (19). Quantitative real-time PCR assays were used for Cdkn1c, Phlda2, Rb1 expression and gene expression. Gpc3, Grb10, Igf2 and RNA expression levels were assessed using semi-quantitative low cycle radioactive PCR. The ribosomal protein encoding gene Rpl-32 was used as a control gene in all experiments.

We utilized three or more samples from each timepoint/genetic background combination.

Total RNA was obtained from whole placental tissue either through lithium chloride-urea treatment (Auffray and Rougeon 1980) or by RNeasy Micro kit or Mini kit purchased from Qiagen. In all cases, the RNA samples were DNased prior to reverse transcription using the Qiagen On-Column DNase digestion. Reverse transcription was made using Invitrogen SuperScript First Strand Synthesis for RT-PCR with oligo (dT).

For real-time PCR assays, Peromyscus specific Taqman assays were developed using Assays-by-design technology (Applied Biosystems). The Taqman protocol was used according to Applied Biosystems (ABI) recommendations. Reactions were done in triplicate in 96 well plates and cycled on an ABI 7900 machine. Quantitative analysis was conducted according to Critical Factors for Successful Real-Time PCR from Qiagen (www.Qiagen.com).

Semi-quantitative PCR assays were conducted with 32P labeled deoxy-cytosine. Samples were assayed with primer sets for both experimental (Igf2, Grb10 and Gpc3) and control (Rpl-32) genes in the same reactions. Reactions were subjected to low (18–20) numbers of cycles to ensure that they remained in the linear range of amplification. Samples were electrophoresed on 7.5% polyacrylamide gels, and then dehydrated. Quantitation and visualization was conducted on an Amersham Bioscience Typhoon 9400 according to the manufacturer's instructions. Samples were normalized to their level of Rpl-32 expression.

Primer sets for development of PCR assays are as follows: Rb1 F: GTC TTC CCA TGG ATT CTG A, R: GGG ATT CCA TGA TTC GAT GT; Igf2 F: CAA TGG GGR TCM CRR YRG GRA A, R: CAG GGG ACG RTG RCK YTT GGC CTC YCT; Grb10 F: GTC AAA GTC TTT AGT GAA GAT GG, R: TCT GAA SGC WGT CAT CCA GCA; Gpc3 F: GAA CAA CTA CCC CAG CCT GA, R: GCC AAA TAC CCT TCA GGT CA; Cdkn1c F: AGA GAA CTG CGC AGG AGA AC, R: GCT TTA CAC CTT GGG ACC AG; Phlda2 F: GCG GAA GTC GAT CTC CTT AT, R: AAA TGG CTT CGA AAA TCG TG.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We thank Xing Dai, Tamara Caspary and Marisa Bartolomei as well as two anonymous reviewers for helpful discussions on the manuscript. This work was supported by grants from the American Cancer Society (RSG-03–070-01-MGO) and National Science Foundation (MCB-0517754) to P.B.V.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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