Lara S. Carroll

Discrimination of MHC-derived odors by untrained mice is consistent with divergence in peptide-binding region residues

Genes of the major histocompatibility complex (MHC) play a central role in immune recognition, yet they also influence the odor of individuals. Mice can be trained to distinguish odors mediated by classical MHC loci; however, training can introduce confounding behavioral artifacts. This study demonstrates that mice can distinguish some, but not all, naturally occurring allelic variants at classical MHC loci without prior training. This result suggests that MHC-disassortative mating preferences might operate by means of small MHC-based odor differences, and could therefore contribute to diversifying selection acting on MHC loci. Here we show that odors of two MHC mutant mouse strains (bm1 and bm3) can be distinguished, even after genetic background is controlled by intercrossing strains. These two strains differ by five amino acids, three of which are predicted to chemically contact peptides bound to the peptide-binding region (PBR), the site of antigen presentation for T cell recognition. However, the odors of neither bm1 nor bm3 were distinguished from their parental B6 haplotype after randomizing genomic background, despite discrimination of pure-bred B6 and bm1 strain odors. These combined results suggest that (i) there may be an MHC odor discrimination threshold based on divergence in PBR residues, providing a more logical pattern of MHC-based odor discrimination than found in previous training studies, where discrimination ability was not correlated with PBR divergence; and (ii) additional (non-MHC) mutations that influence odor have accumulated in these strains during the 100 generations of divergence between pure B6 and bm1 strains.

Fitness effects of a selfish gene (the Mus t complex) are revealed in an ecological context.

Abstract In wild house mice, genes linked to the t transmission distortion complex cause meiotic drive by sabotaging wild‐type gametes. The t complex is consequently inherited at frequencies higher than 90%. Yet, for unclear reasons, in wild mouse populations this selfish DNA is found at frequencies much lower than expected. Here, we examine selection on the t complex in 10 seminatural populations of wild mice based on data from 234 founders and nearly 2000 progeny. Eight of the 10 populations decreased in t frequency over one generation, and the overall frequency oft haplotypes across all 10 populations was 48.5% below expectations based on transmission distortion and 34.3% below Mendelian (or Hardy‐Weinberg) expectations. Behavioral and reproductive data were collected for 10 months for each population, and microsatellite genotyping was performed on seven of the populations to determine parentage. These combined data show t‐associated fitness declines in both males and females. This is the first study to show evidence for a reduction in the ability of +/t males to maintain territories. Because females tend to mate with dominant males, impairment of territorial success can explain much of the selection against t observed in our populations. In nature, selection against heterozygote carriers of the t complex helps solve the puzzlingly low t frequencies found in wild populations. This ecological approach for determining fitness consequences of genetic variants has broad application for the discovery of gene function in general.

SIZE AND FLUCTUATING ASYMMETRY OF MORPHOMETRIC CHARACTERS IN MICE: THEIR ASSOCIATIONS WITH INBREEDING AND t‐HAPLOTYPE

Abstract Fluctuating asymmetry (FA), a ubiquitous type of asymmetry of bilateral characters, often has been used as a measure of developmental instability in populations. FA is expected to increase in populations subjected to genetic stressors such as inbreeding or environmental stressors such as toxins or parasites, although results have not always been consistent. We tested whether FA in four skeletal size characters and mandible shape was greater in a population of wild‐derived mice reared in the laboratory and subjected to one generation of inbreeding (F = 0.25) versus that in an outbred group (F= 0.00). FA did not significantly differ between the inbred and outbred groups, despite the fact that these two groups differed dramatically in fitness under seminatural population conditions. As far as we know, this is the first study to evaluate the relationship between FA and inbreeding in wild house mice, and our general conclusion is opposite that of earlier work on laboratory inbred strains of mice and their hybrids. Size for two of the characters was significantly less in inbreds than in outbreds, however, and there was a significant difference between inbreds and outbreds in the signed differences of right and left sides in one character (humerus length). Some of the mice in both groups also were heterozygous or homozygous carriers of the t‐complex. Because mice carrying this chromosome 17 variant are known to have reduced fitness, we also tested whether they had greater FA than mice carrying non‐t‐haplotypes. The overall level of a composite FA index calculated from all four characters was in fact significantly higher in the t‐bearing mice. These combined results suggest that FA is not a generally sensitive proxy measure for fitness, but can be associated with fitness reductions for certain genetic stressors.

Intravitreal AAV2.COMP-Ang1 Prevents Neurovascular Degeneration in a Murine Model of Diabetic Retinopathy

Diabetic retinopathy (DR) is the leading cause of blindness in the working-age population in the U.S. The vision-threatening processes of neuroglial and vascular dysfunction in DR occur in concert, driven by hyperglycemia and propelled by a pathway of inflammation, ischemia, vasodegeneration, and breakdown of the blood retinal barrier. Currently, no therapies exist for normalizing the vasculature in DR. Here, we show that a single intravitreal dose of adeno-associated virus serotype 2 encoding a more stable, soluble, and potent form of angiopoietin 1 (AAV2.COMP-Ang1) can ameliorate the structural and functional hallmarks of DR in Ins2Akita mice, with sustained effects observed through six months. In early DR, AAV2.COMP-Ang1 restored leukocyte-endothelial interaction, retinal oxygenation, vascular density, vascular marker expression, vessel permeability, retinal thickness, inner retinal cellularity, and retinal neurophysiological response to levels comparable with nondiabetic controls. In late DR, AAV2.COMP-Ang1 enhanced the therapeutic benefit of intravitreally delivered endothelial colony-forming cells by promoting their integration into the vasculature and thereby stemming further visual decline. AAV2.COMP-Ang1 single-dose gene therapy can prevent neurovascular pathology, support vascular regeneration, and stabilize vision in DR.

Hormonal induction of thumb pads and the evolution of secondary sexual characteristics of the Southeast Asian fanged frog, Rana blythii

The fanged frogs of Southeast Asia do not express most of the hormone-dependent secondary sexual characteristics such as thumb pads that are common to other ranid frogs. At the same point in the evolutionary history of the group that these androgen-mediated characteristics are lost, male parental care first evolves. This behavior is often correlated with low androgen levels. Prior work indicates that in one of the fanged frogs, Rana blythii, adult males have low androgen levels compared to North Temperate species of Rana. This leads to the question of whether these low androgen levels are related to the unusual male parental care and the lack of expression of the thumb pad and other hormone-dependent secondary sexual characteristics in this species. We tested that hypothesis by examining the effects of exogenous dihydrotestosterone supplements on the expression of thumb pads in Rana blythii. Dihydrotestosterone injections appear to stimulate the expression of the thumb pad in R. blythii. These results support the hypothesis that low androgen levels are involved in the loss of the thumb pad in R. blythii. This work provides an example of how mapping characters on phylogenies can be a powerful approach for gaining insights into proximate physiological mechanisms of selection at the evolutionary level.

Accumulated background variation among H2 mutant congenic strains: elimination through PCR-based genotyping of F2 segregants.

Many commercially and privately available congenic strains of laboratory animals were founded decades ago and are likely to differ from one another by dozens of fixed mutational differences at background loci. This problem is often ignored despite growing evidence that such background variation exists. Eliminating this confounding variation can be largely accomplished by crossing congenic strains to produce F2 segregants that are homozygous (or heterozygous) for relevant genes. Discriminating F2 homozygotes can be difficult when strain differences are minor, as are mutant mouse strains differing at single major histocompatibility loci (H2 mutant congenics). Here, we describe a two-step polymerase chain reaction (PCR) method utilizing heteroduplex analysis and sequence specific primers (SSP-PCR) that efficiently discriminates the F2 progeny of two such H2 mutant congenic mice crosses (bm1xB6 and bm1xbm3). A third H2 mutant cross cannot be resolved by heteroduplexing, but is discriminated (albeit less efficiently) with SSP-PCR alone. This sensitive application can be extended to any congenic mutant strains.

Hoxc8 initiates an ectopic mammary program by regulating Fgf10 and Tbx3 expression and Wnt/β-catenin signaling

The role of Hox genes in the formation of cutaneous accessory organs such as hair follicles and mammary glands has proved elusive, a likely consequence of overlapping function and expression among various homeobox factors. Lineage and immunohistochemical analysis of Hoxc8 in mice revealed that this midthoracic Hox gene has transient but strong regional expression in ventrolateral surface ectoderm at E10.5, much earlier than previously reported. Targeted mice were generated to conditionally misexpress Hoxc8 from the Rosa locus using select Cre drivers, which significantly expanded the domain of thoracic identity in mutant embryos. Accompanying this expansion was the induction of paired zones of ectopic mammary development in the cervical region, which generated between three and five pairs of mammary placodes anterior to the first wild-type mammary rudiment. These rudiments expressed the mammary placode markers Wnt10b and Tbx3 and were labeled by antibodies to the mammary mesenchyme markers ERα and androgen receptor. Somitic Fgf10 expression, which is required for normal mammary line formation, was upregulated in mutant cervical somites, and conditional ablation of ectodermal Tbx3 expression eliminated all normally positioned and ectopic mammary placodes. We present evidence that Hoxc8 participates in regulating the initiation stages of mammary placode morphogenesis, and suggest that this and other Hox genes are likely to have important roles during regional specification and initiation of these and other cutaneous accessory organs. Highlighted article: Hoxc8 misexpression in cervical somites and the overlying ectoderm of mice can convert skin into mammary ectoderm, with subsequent placode, bud and sprout development.

Functional Genomics Requires Ecology

Publisher Summary This chapter focuses on model organisms as yeast, Drosophila, C. elegans, and mice because the tools to generate genetic mutations and gene disruptions to study specific gene function are already available. However, the principles are general and can be applied to similar enterprises in any species. The chapter provides numerous examples where ecological approaches reveal phenotypes that were undetectable in the laboratory. The chapter also demonstrates that even for mutations with known phenotypes, fitness testing is a useful screen for additional unknown phenotypes and to quantify the relative importance of the mutation in the evolutionary currency of fitness. Various lines of reasoning have been invoked to explain the surplus of gene knockouts that yield no phenotype in animals homozygous for the mutation. The era of functional genomics presentsa great opportunity for organismal biologists to collaborate with molecular biologists to truly evaluate how genes function through all levels of biological organization. The enterprise of determining the function of genes is by far the most difficult portion of genome projects. This reflects the sheer complexity of the genome, with genes interacting to influence function (epistasis), genes influencing more than one function (pleiotropy), the involvement of many genes to effect one function (polygenic traits), and countless gene associated phenotypes yet to be discovered. Functional genomics often require an understanding of ecology and behavior to gain a useful understanding of gene function.

Sexual selection constrains the body mass of male but not female mice

Abstract Sexual size dimorphism results when female and male body size is influenced differently by natural and sexual selection. Typically, in polygynous species larger male body size is thought to be favored in competition for mates and constraints on maximal body size are due to countervailing natural selection on either sex; however, it has been postulated that sexual selection itself may result in stabilizing selection at an optimal mass. Here we test this hypothesis by retrospectively assessing the influence of body mass, one metric of body size, on the fitness of 113 wild‐derived house mice (Mus musculus) residing within ten replicate semi‐natural enclosures from previous studies conducted by our laboratory. Enclosures possess similar levels of sexual selection, but relaxed natural selection, relative to natural systems. Heavier females produced more offspring, while males of intermediate mass had the highest fitness. Female results suggest that some aspect of natural selection, absent from enclosures, acts to decrease their body mass, while the upper and lower boundaries of male mass are constrained by sexual selection.

Detection of microvascular retinal changes in type I diabetic mice with optical coherence tomography angiography

ABSTRACT Optical coherence tomography (OCT) angiography is a dye‐free and non‐invasive angiography which allows visualization of retinal and choroid vascular flow, enabling observation of highly permeable and three dimensional vasculature. Although OCT angiography is providing new insights in human retinal and choroidal diseases, a few studies have been reported in experimental mice. In this study, to determine the potential of OCT angiography in experimental mice, we sought to examine whether OCT angiography can detect vascular change in type I diabetic mice. To conduct age dependent analysis, 2 and 6 month old male type 1 diabetic Ins2Akita/+ and age matched C57BL/6J mice were used. OCT angiography was performed by Heidelberg Spectralis OCT Angiography Module with 30° lens + mouse adapter lens. We acquired the OCT angiography image from the peripheral nasal position. For analysis of OCT angiography images, OCT angiography positive area were used for vascular density. We analyzed vascular density from the retinal surface (inner limiting membrane) to 120 &mgr;m depth with 4 &mgr;m steps in order to correlate vascular density vs depth (N = 4 per group). Vascular density of both mouse strains demonstrated three different peaks. By comparing with the OCT image, the first peak (superficial), second peak (intermediate) and third peak (deep) were located in nerve fiber layer/ganglion cell layer, inner plexiform layer/inner nuclear layer and outer plexiform layer/outer nuclear layer, respectively. We calculated vascular density of these peaks separately. In C57BL/6J mice, the vascular density in all three layers do not show significant difference between 2‐ and 6‐month‐old. On the other hand, 6‐month‐old Ins2Akita/+ mice showed a significant decrease of the vascular density in all three layers compared to 2‐month‐old Ins2Akita/+ mice. Also, the vascular density of 6‐month‐old Ins2Akita/+ mice in the deep layer showed a significant decrease compared to 2‐ and 6‐month‐old C57BL/6J mice. Thus, OCT angiography successfully detects retinal vascular difference between type I diabetic mice and control mice, and age‐dependent vasculature change in type I diabetic mice. The diabetic mice demonstrated reduced vascular density due to reduced density of flowing deep vessels. Importantly, we observed this difference without retinal blood leakage, hemorrhage or neovascularization. Our analysis (vascular density vs retinal depth) suggests that OCT angiography is useful for in vivo detection of retinal vasculature alteration in experimental mice. HighlightsOCT angiography enables three dimensional retinal vasculature analysis in mice.Aged diabetic mice show diminished deep retinal vascular layer.Aged diabetic mice demonstrate a similar phenotype to human diabetic retinopathy.