The Original Pink-Eyed Dilution Mutation (P) Arose in Asiatic Mice: Implications for the H4 Minor Histocompatibility Antigen, Myod1 Regulation and the Origin of Inbred Strains

Allelic variation of the mouse pink-eyed dilution (p) gene in common laboratory strains and wild mice was examined by Southern blot and by polymerase chain reaction. In these assays the original p mutation allele found in strains SJL/J, 129/J, B10.129(21m), P/J and FS/Ei most closely matches an Asian Mus musculus allele, confirming anecdotal accounts of the Asian origin of this mutation. In contrast, the wild-type allele found in other common laboratory strains was apparently derived from Mus domesticus. Analysis of chromosome 7 loci both proximal and distal to the p locus demonstrates that strains SJL/J, 129/J, B10.129(21M), P/J and FS/Ei contain DNA segments of varying length derived from M. musculus. Strains 129/J and B10.129(21M) contain the largest segment of M. musculus-derived DNA (about 5 cM), including the loci Myod1, p, three clustered GABA(A) receptor subunit loci (Gabrg3, Gabra5 and Gabrb3), and Snrpn. The difference in the species origin of genes from this region of chromosome 7 may underlie the basis of the antigenicity of the minor histocompatibility antigen H4, defined by the strain B10.129(21M), and may account for the enhanced Myod1 activity observed in SJL/J mice.     

The mouse pink-eyed dilution allele of the P-gene greatly inhibits eumelanin but not pheomelanin synthesis

The mouse pink-eyed dilution (p) locus is known to control eumelanin synthesis, melanosome morphology, and tyrosinase activity in melanocytes. However, it has not been fully determined whether the mutant allele, p affects pheomelanin synthesis. Effects of the p allele on eumelanin and phemelanin synthesis were investigated by chemical analysis of dorsal hairs of 5-week-old mice obtained from the F(2) generations (black, pink-eyed black, recessive yellow, pink-eyed recessive yellow, agouti, and pink-eyed agouti) between C57BL/10JHir (B10)-congenic pink-eyed black mice (B10-p/p) and recessive yellow (B10-Mc1r(e)/Mc1r(e)) or agouti (B10-A/A) mice. The eumelanin content was dramatically (>20-fold) decreased in pink-eyed black and pink-eyed agouti mice, whereas the pheomelanin content did not decrease in pink-eyed black, pink-eyed recessive yellow, or pink-eyed agouti mice compared to the corresponding P/- mice. These results suggest that the pink-eyed dilution allele greatly inhibits eumelanin synthesis, but not pheomelanin synthesis.     

The effect of genetic diversity on angiogenesis

Angiogenesis is the process by which new blood vessels are formed from existing vessels. Mammalian populations harbor genetic variations that alter angiogenesis. Some of these changes result in Mendelian traits of variable penetrance, with telangiectasia being a common symptom. Other more subtle variations exist, with promoter variations in the VEGF gene being of particular interest. Genetic diversity in angiogenesis-regulating genes has been linked to increased susceptibility to multiple angiogenesis-dependent diseases in humans. These diseases include cancer, arthritis, atherosclerosis, and cardiovascular disease, endometriosis, diabetic retinopathy, retinopathy of prematurity, psoriasis, and sarcoidosis. Also, multiple disturbances in pregnancy including miscarriage, spontaneous preterm delivery, and severe pre-eclampsia have been linked to alterations in angiogenesis-regulating genes. Present efforts to dissect the complexity of the genetic diversity that regulates angiogenesis have used laboratory animals due to the availability of genome sequence for many species and the ability to perform high volume controlled breeding. Ongoing mapping studies have identified multiple loci that control angiogenic responsiveness in several mouse models. Genetic alterations responsible for discrete angiogenic alterations will then be studied in appropriate mouse disease models.     

Genetic heterogeneity of angiogenesis in mice.

Many diseases, including cancer, are dependent on the growth of new blood vessels, a process known as angiogenesis. Differences in an individual's ability to grow new blood vessels may influence the rate of progression of these diseases. Here we show that different strains of inbred mice have an approximately 10-fold range of response to growth factor-stimulated angiogenesis in the corneal micropocket assay. The in vitro migratory activity of endothelial cells from aortic rings of selected strains correlated with the in vivo responsiveness. Further, a differential sensitivity to angiogenesis inhibitors was seen between strains, with one strain demonstrating resistance to both TNP-470 and thalidomide. These results suggest the presence of genetic factors that control individual angiogenic potential.     

Genetic Epilepsy Model Derived from Common Inbred Mouse Strains

The recombinant inbred mouse strain, SWXL-4, exhibits tonic-clonic and generalized seizures similar to the commonest epilepsies in humans. In SWXL-4 animals, seizures are observed following routine handling at about 80 days of age and may be induced as early as 55 days by rhythmic gentle tossing. Seizures are accompanied by rapid, bilateral high frequency spike cortical discharges and followed by a quiescent post-ictal phase. Immunohistochemistry of the immediate early gene products c-Fos and c-Jun revealed abnormal activation within cortical and limbic structures. The seizure phenotype of SWXL-4 can be explained and replicated fully by the inheritance of susceptibility alleles from its progenitor strains, SWR/J and C57L/J. Outcrosses of SWXL-4 with most other common inbred strains result in F(1) hybrids that have seizures at least as frequently as SWXL-4 itself. Quantitative trait locus mapping reveals a seizure frequency determinant, Szf1, near the pink-eyed dilution locus on chromosome 7, accounting for up to 32% of the genetic variance in an F(2) intercross between SWXL-4 and the linkage testing strain ABP/Le. These studies demonstrate that common strains of mice such as SWR and C57L contain latent epilepsy susceptibility alleles. Although the inheritance of susceptibility may be complex, these results imply that a number of potentially important and practical, noninvasive models for this disorder can be constructred and studied in crosses between common mouse strains.     

Angiogenesis activators and inhibitors differentially regulate caveolin-1 expression and caveolae formation in vascular endothelial cells. Angiogenesis inhibitors block vascular endothelial growth factor-induced down-regulation of caveolin-1.

Angiogenesis is the process by which new blood vessels are formed via proliferation of vascular endothelial cells. A variety of angiogenesis inhibitors that antagonize the effects of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have recently been identified. However, the mechanism by which these diverse angiogenesis inhibitors exert their common effects remains largely unknown. Caveolin-1 and -2 are known to be highly expressed in vascular endothelial cells both in vitro and in vivo. Here, we examine the potential role of caveolins in the angiogenic response. For this purpose, we used the well established human umbilical vein endothelial cell line, ECV 304. Treatment of ECV 304 cells with known angiogenic growth factors (VEGF, bFGF, or hepatocyte growth factor/scatter factor), resulted in a dramatic reduction in the expression of caveolin-1. This down-regulation event was selective for caveolin-1, as caveolin-2 levels remained constant under these conditions of growth factor stimulation. VEGF-induced down-regulation of caveolin-1 expression also resulted in the morphological loss of cell surface caveolae organelles as seen by transmission electron microscopy. A variety of well characterized angiogenesis inhibitors (including angiostatin, fumagillin, 2-methoxy estradiol, transforming growth factor-beta, and thalidomide) effectively blocked VEGF-induced down-regulation of caveolin-1 as seen by immunoblotting and immunofluorescence microscopy. However, treatment with angiogenesis inhibitors alone did not significantly affect the expression of caveolin-1. PD98059, a specific inhibitor of mitogen-activated protein kinase and a known angiogenesis inhibitor, also blocked the observed VEGF-induced down-regulation of caveolin-1. Furthermore, we show that caveolin-1 can function as a negative regulator of VEGF-R (KDR) signal transduction in vivo. Thus, down-regulation of caveolin-1 may be an important step along the pathway toward endothelial cell proliferation.     

PLEIOTROPIC EFFECTS OF INDIVIDUAL GENE LOCI ON MANDIBULAR MORPHOLOGY

The genotypic basis of morphological variation is largely unknown. In this study we examine patterns of pleiotropic effects on mandibular morphology at individual gene loci to determine whether the pleiotropic effects of individual genes are restricted to functionally and developmentally related traits. Mandibular measurements were obtained from 480 mice from the F₂ generation of an intercross between the LG/J and SM/J mouse strains. DNA was also extracted from these animals, and 76 microsatellite loci covering the autosomes were scored. Interval mapping was used to detect chromosomal locations with significant effects on various mandibular measurements. Sets of traits mapping to a common chromosomal region were considered as being affected by a single quantitative trait locus (QTL) for mandibular morphology. Thirty-seven such chromosomal regions were identified spread throughout the autosomes. Gene effects were small to moderate with the allele derived from the LG/J strain typically leading to larger size. When dominance was present, the LG/J allele was typically dominant to the SM/J allele. Most loci affected restricted functional and developmental regions of the mandible. Of the 26 chromosomal regions affecting more than two traits, 50% affect the muscular processes of the ascending ramus, 27% affect the alveolar processes carrying the teeth, and 23% affect the whole mandible. Four additional locations affecting two traits had effects significantly associated with alveolar regions. Pleiotropic effects are typically restricted to morphologically integrated complexes.     

A genetic locus responsible for salmonella susceptibility in BSVS mice is not responsible for the limited T-dependent immune responsiveness of BSVS mice

BSVS mice are known to be highly susceptible to salmonella infection. We have shown that the bulk of the difference in susceptibility between BSVS and salmonella-resistant A/J mice is the result of a genetic difference at a single locus not closely linked to H-2, Igh-C, or Hbb, and not X-linked. We have backcrossed the A/J allele at this locus into BSVS mice for 8 successive generations and have demonstrated that the salmonella resistance afforded by this allele is not the result of a restoration of the generalized poor T-dependent responsiveness of BSVS mice. The salmonella resistance locus we have examined with these 2 strains is probably the same as the Ity locus described by others.     

Genetic modifiers of cardiovascular phenotype caused by elastin haploinsufficiency act by extrinsic noncomplementation

Elastin haploinsufficiency causes the cardiovascular complications associated with Williams-Beuren syndrome and isolated supravalvular aortic stenosis. Significant variability exists in the vascular pathology in these individuals. Using the Eln(+/-) mouse, we sought to identify the source of this variability. Following outcrossing of C57Bl/6J Eln(+/-), two backgrounds were identified whose cardiovascular parameters deviated significantly from the parental strain. F1 progeny of the C57Bl/6J; Eln(+/-)x129X1/SvJ were more hypertensive and their arteries less compliant. In contrast, Eln(+/-) animals crossed to DBA/2J were protected from the pathologic changes associated with elastin insufficiency. Among the crosses, aortic elastin and collagen content did not correlate with quantitative vasculopathy traits. Quantitative trait locus analysis performed on F2 C57; Eln(+/-)x129 intercrosses identified highly significant peaks on chromosome 1 (LOD 9.7) for systolic blood pressure and on chromosome 9 (LOD 8.7) for aortic diameter. Additional peaks were identified that affect only Eln(+/-), including a region upstream of Eln on chromosome 5 (LOD 4.5). Bioinformatic analysis of the quantitative trait locus peaks revealed several interesting candidates, including Ren1, Ncf1, and Nos1; genes whose functions are unrelated to elastic fiber assembly, but whose effects may synergize with elastin insufficiency to predispose to hypertension and stiffer blood vessels. Real time RT-PCR studies show background-specific increased expression of Ncf1 (a subunit of the NOX2 NAPDH oxidase) that parallel the presence of increased oxidative stress in Eln(+/-) aortas. This finding raises the possibility that polymorphisms in genes affecting the generation of reactive oxygen species alter cardiovascular function in individuals with elastin haploinsufficiency through extrinsic noncomplementation.     

Genetic heterogeneity of skin microvasculature

Angiogenesis, the formation of new blood vessels from existing vasculature, is a complex process that is essential for normal embryonic development. Current models for experimental evaluation of angiogenesis often use tissue from large vessels like the aorta and umbilical vein, which are phenotypically distinct from microvasculature. We demonstrate that the utilization of skin to measure microvascular angiogenesis in embryonic and adult tissues is an efficient way to quantify microvasculature angiogenesis. We validate this approach and demonstrate its added value by showing significant differences in angiogenesis in monogenic and polygenic mouse models. We discovered that the pattern of angiogenic response among inbred mouse strains in this ex vivo assay differs from the strain distributions of previous in vivo angiogenesis assays. The difference between the ex vivo and in vivo assays may be related to systemic factors present in whole animals. Expression analysis of cultured skin biopsies from strains of mice with opposing angiogenic response was performed to identify pathways that contribute to differential angiogenic response. Increased expression of negative regulators of angiogenesis in C57Bl/6J mice was associated with lower growth rates.     

Physical mapping of the pink-eyed dilution complex in mouse chromosome 7 shows that Atp10c is the only transcript between Gabrb3 and Ube3a.

Phenotypic analyses of a set of homozygous-lethal deletion mutants at the pink-eyed dilution (p) locus has resulted in the identification of p-linked obesity locus 1 (plo 1), distal to the p locus, as a locus involved in the modulation of body fat and/or affecting lipid metabolism in these mice. The plo 1 region maps to mouse chromosome 7 (MMU 7) between two genes, Gabrb3 and Ube3a, which have been used as anchor points to generate an integrated deletion and physical map of plo 1 that encompasses about 1.2-1.3 Mb. A deletion/physical map was constructed and the genomic DNA between the two loci was sequenced to identify genes mapping to this region. Data show that Atp10c, a novel type IV ATPase a putative phospholipid transporter, is the only coding unit in this region of the chromosome.     

Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor or vasculotropin, is a recently characterized endothelial-specific mitogen which is angiogenic in vivo. Here we demonstrate that VEGF is angiogenic in vitro: when added to microvascular endothelial cells grown on the surface of three-dimensional collagen gels, VEGF induces the cells to invade the underlying matrix and to form capillary-like tubules, with an optimal effect at approximately 2.2nM (100ng/ml). When compared to basic fibroblast growth factor (bFGF) at equimolar (0.5nM) concentrations, VEGF was about half as potent. The most striking effect was seen in combination with bFGF: when added simultaneously, VEGF and bFGF induced an in vitro angiogenic response which was far greater than additive, and which occurred with greater rapidity than the response to either cytokine alone. These results demonstrate that like bFGF, VEGF induces an angiogenic response via a direct effect on endothelial cells, and that by acting in concert, these two cytokines have a potent synergistic effect on the induction of angiogenesis in vitro. We suggest that the synergism between VEGF and bFGF plays an important role in the control of angiogenesis in vivo.     

Functional interactions between OCA2 and the protein complexes BLOC-1, BLOC-2, and AP-3 inferred from epistatic analyses of mouse coat pigmentation

The biogenesis of melanosomes is a multistage process that requires the function of cell-type-specific and ubiquitously expressed proteins. OCA2, the product of the gene defective in oculocutaneous albinism type 2, is a melanosomal membrane protein with restricted expression pattern and a potential role in the trafficking of other proteins to melanosomes. The ubiquitous protein complexes AP-3, BLOC-1, and BLOC-2, which contain as subunits the products of genes defective in various types of Hermansky-Pudlak syndrome, have been likewise implicated in trafficking to melanosomes. We have tested for genetic interactions between mutant alleles causing deficiency in OCA2 (pink-eyed dilution unstable), AP-3 (pearl), BLOC-1 (pallid), and BLOC-2 (cocoa) in C57BL/6J mice. The pallid allele was epistatic to pink-eyed dilution, and the latter behaved as a semi-dominant phenotypic enhancer of cocoa and, to a lesser extent, of pearl. These observations suggest functional links between OCA2 and these three protein complexes involved in melanosome biogenesis.     

Constitutive notch signaling in adult transgenic mice inhibits bFGF‐induced angiogenesis and blocks ovarian follicle development

Notch signaling is important in angiogenesis during embryonic development. However, the embryonic lethal phenotypes of knock‐out and transgenic mice have precluded studies of the role of Notch post‐natally. To develop a mouse model that would bypass the embryonic lethal phenotype and investigate the possible role of Notch signaling in adult vessel growth, we developed transgenic mice with Cre‐conditional expression of the constitutively active intracellular domain of Notch1 (IC‐Notch1). Double transgenic IC‐Notch1/Tie2‐Cre embryos with endothelial specific IC‐Notch1 expression died at embryonic day 9.5. They displayed collapsed and leaky blood vessels and defects in angiogenesis development. A tetracycline‐inducible system was used to express Cre recombinase postnatally in endothelial cells. In adult mice, IC‐Notch1 expression inhibited bFGF‐induced neovascularization and female mice lacked mature ovarian follicles, which may reflect the block in bFGF‐induced angiogenesis required for follicle growth. Our results demonstrate that Notch signaling is important for both embryonic and adult angiogenesis and indicate that the Notch signaling pathway may be a useful target for angiogenic therapies. genesis 52:809–816, 2014. © 2014 Wiley Periodicals, Inc.     

Persistent induction of somatic reversions of the pink-eyed unstable mutation in F1 mice born to fathers irradiated at the spermatozoa stage

Untargeted mutation and delayed mutation are features of radiation-induced genomic instability and have been studied extensively in tissue culture cells. The mouse pink-eyed unstable (p(un)) mutation is due to an intragenic duplication of the pink-eyed dilution locus and frequently reverts back to the wild type in germ cells as well as in somatic cells. The reversion event can be detected in the retinal pigment epithelium as a cluster of pigmented cells (eye spot). We have investigated the reversion p(um) in F1 mice born to irradiated males. Spermatogonia-stage irradiation did not affect the frequency of the reversion in F1 mice. However, 6 Gy irradiation at the spermatozoa stage resulted in an approximately twofold increase in the number of eye spots in the retinal pigment epithelium of F1 mice. Somatic reversion occurred for the paternally derived p(un) alleles. In addition, the reversion also occurred for the maternally derived, unirradiated p(un) alleles at a frequency equal to that for the paternally derived allele. Detailed analyses of the number of pigmented cells per eye spot indicated that the frequency of reversion was persistently elevated during the proliferation cycle of the cells in the retinal pigment epithelium when the male parents were irradiated at the spermatozoa stage. The present study demonstrates the presence of a long-lasting memory of DNA damage and the persistent up-regulation of recombinogenic activity in the retinal pigment epithelium of the developing fetus.     

In Vitro Angiogenic and Proteolytic Properties of Bovine Lymphatic Endothelial Cells

The aim of the present study was to determine whether angiogenic cytokines, which induce neovascularization in the blood vascular system, might also be operative in the lymphatic system. In an assay of spontaneous in vitro angiogenesis, endothelial cells isolated from bovine lymphatic vessels retained their histotypic morphogenetic properties by forming capillary-like tubes. In a second assay, in which endothelial cells could be induced to invade a three-dimensional collagen gel within which they formed tube-like structures, lymphatic endothelial cells responded to basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in a manner similar to what has previously been observed with endothelial cells derived from the blood vascular system. Finally, since angiogenesis is believed to require extracellular proteolytic activity, we investigated the effects of bFGF and VEGF on lymphatic endothelial cell proteolytic properties by focussing on the plasminogen activator (PA) system. bFGF and VEGF increased urokinase, urokinase receptor, and tissue-type PA expression. This was accompanied by an increase in PA inhibitor-l, which is thought to play an important permissive role in angiogenesis by protecting the extracellular matrix against excessive proteolytic degradation. Taken together, these results demonstrate that with respect to in vitro morphogenetic and proteolytic properties, lymphatic endothelial cells respond to the previously described angiogenic factors, bFGF and VEGF, in a manner very similar to what has been described for endothelial cells derived from the blood vascular system.     

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.     

Imaging of angiogenesis: from microscope to clinic

Advances in imaging are transforming our understanding of angiogenesis and the evaluation of drugs that stimulate or inhibit angiogenesis in preclinical models and human disease. Vascular imaging makes it possible to quantify the number and spacing of blood vessels, measure blood flow and vascular permeability, and analyze cellular and molecular abnormalities in blood vessel walls. Microscopic methods ranging from fluorescence, confocal and multiphoton microscopy to electron microscopic imaging are particularly useful for elucidating structural and functional abnormalities of angiogenic blood vessels. Magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), ultrasonography and optical imaging provide noninvasive, functionally relevant images of angiogenesis in animals and humans. An ongoing dilemma is, however, that microscopic methods provide their highest resolution on preserved tissue specimens, whereas clinical methods give images of living tissues deep within the body but at much lower resolution and specificity and generally cannot resolve vessels of the microcirculation. Future challenges include developing new imaging methods that can bridge this resolution gap and specifically identify angiogenic vessels. Another goal is to determine which microscopic techniques are the best benchmarks for interpreting clinical images. The importance of angiogenesis in cancer, chronic inflammatory diseases, age-related macular degeneration and reversal of ischemic heart and limb disease provides incentive for meeting these challenges.