Evolution of androgen-regulated mRNA expression in mouse kidney

To gain information on the evolution of mammalian gene expression patterns, we studied the androgen-inducible expression of three kidney mRNAs in several mouse species (genus Mus). The RP2, ornithine decarboxylase, and beta-glucuronidase mRNAs have each evolved independently, in that the pattern of variation among species is unique for each. This suggests a role for gene-specific, cis-acting genetic elements. Relationships between the regulatory phenotypes and the species phylogeny suggest that the variations in hormone-inducible mRNA expression were generated by a series of independent mutations that occurred in specific lineages, resulting in modifications of the progenitor phenotype. Alternatively, the variations may have preexisted within the progenitor population as polymorphisms that were fixed during establishment of individual lineages. Thus, significant alterations in the androgen-regulated mRNA phenotype have occurred either prior to or during speciation within the Mus genus. These alterations are presumed to be in regulatory sequences that control the expression of the corresponding genes and their response to testosterone; as such, they should be useful in further studying the genetic determinants of gene expression and its evolution.      

EVOLUTION OF β-GLUCURONIDASE REGULATION IN THE GENUS MUS

Despite the central role suggested for regulatory mutations in many evolutionary scenarios, there is relatively little information available about the type and extent of regulatory differences between species, or to what extent differences between species are independent of variation within species. To address this issue we have studied the regulatory system of β-glucuronidase, a gene implicated in a murine androgen-inducible pheromone-signalling system. We examined the changes in β-glucuronidase hormonal regulation which have occurred during the radiation of a group of 12 closely related species of mice by assaying β-glucuronidase activity in six different tissues after treatment with estrogen, and with androgen alone and in combination with either estrogen or growth hormone. We also examined in some detail the extent of variation in regulatory responses within species. We found extensive variation in regulatory phenotypes both within and among the species surveyed, suggesting that many of the species examined are currently polymorphic for various regulatory factors that affect inducibility of β-glucuronidase. The variation we observed reflects changes in the ability of the β-glucuronidase gene to respond to hormonal influences, rather than changes in aspects of the hormonal signalling system exterior to the gene. The marked differences among species in the renal and uterine responses to hormonal induction of β-glucuronidase are not easily related to the phylogeny of the genus Mus. If hormonal induction of the gene for β-glucuronidase is subject to natural selection, it appears to be subject to widely fluctuating selective forces. We review evidence that the apparently disorderly evolution of the hormonal responsiveness of β-glucuronidase does not appear to be a unique property of this regulatory system. In contrast to the evolution of many protein sequences, which are tightly correlated with phylogeny and proceed at a relatively constant rate, some, perhaps many, regulatory phenotypes are in rapid evolutionary flux, providing an extensive range of phenotypes upon which selection can act.     

Loss-of-imprinting of Peg1 in mouse interspecies hybrids is correlated with altered growth

Previous studies have shown that loss-of-imprinting (LOI) is a regular occurrence in interspecies hybrids of the genus Peromyscus. Furthermore, evidence was presented that indicated that LOI is involved in a placental hybrid dysgenesis effect resulting in abnormal placental growth and thus possibly in speciation. We show here that LOI of the strictly paternally expressed gene Peg1 (also called Mest) occurs in F1 hybrids between Mus musculus (MMU) and M. spretus (MSP). Peg1 LOI is correlated with increased body weight and increased weight of two of the organs tested, kidney and spleen. X-gal staining of tissues derived from Peg1(+/-) x MSP F1 mice, carrying a maternal LacZ knock-in allele of Peg1, demonstrates that LOI is stochastic in that it affects different tissues to variable extents and that, even within one tissue, not all cells are similarly affected. Furthermore, this expression from the maternal allele does not necessarily follow the endogenous paternal Peg1 expression pattern. Our results indicate that LOI occurs in interspecies hybrids in the genus Mus and that altered growth is a frequent outcome of LOI.     

Specificity of androgen resistance inMus caroli kidney

Androgen controls the expression of beta-glucuronidase and several other proteins in the kidney of the standard laboratory mouse, Mus musculus. Other species within the genus Mus exhibit a variety of response patterns for kidney beta-glucuronidase and other markers of androgen action. We have investigated the mechanism of androgen action in M. caroli, a Mus species that does not produce beta-glucuronidase in response to testosterone. The failure of testosterone to induce beta-glucuronidase in M. caroli females cannot be overcome by treatment with dihydrotestosterone, with pharmacological doses of testosterone propionate or dihydrotestosterone propionate, or with a variety of potent androgen analogues. All of these compounds induce kidney beta-glucuronidase in M. musculus females and kidney ornithine decarboxylase, submandibular gland renin, and submandibular gland epidermal growth factor in both M. caroli and M. musculus females. Furthermore, kidney androgen receptor proteins from M. caroli and M. musculus animals have the same sedimentation characteristics on sucrose density gradients. These data indicate that androgen resistance in M. caroli is not due to deficient 5 alpha-reductase or aberrant hormone metabolism producing suboptimal levels of functional androgen and is not caused by a defective androgen receptor. They suggest that the resistance of beta-glucuronidase in M. caroli kidney to induction by androgen occurs at the level of the beta-glucuronidase gene.     

Long-term balancing selection at the blood group-related gene B4galnt2 in the genus Mus (Rodentia; Muridae)

Recent surveys of the human genome have highlighted the significance of balancing selection in relation to understanding the evolutionary origins of disease-associated variation. Cis-regulatory variation at the blood group-related glycosyltransferase B4galnt2 is associated with a phenotype in mice that closely resembles a common human bleeding disorder, von Willebrand disease. In this study, we have performed a survey of the 5' flanking region of the B4galnt2 gene in several Mus musculus subspecies and Mus spretus. Our results reveal a clear pattern of trans-species polymorphism and indicate that allele classes conferring alternative tissue-specific expression patterns have been maintained for >2.8 My in the genus Mus. Furthermore, analysis of B4galnt2 expression patterns revealed the presence of an additional functional class of alleles, supporting a role for gastrointestinal phenotypes in the long-term maintenance of expression variation at this gene.     

Expression of the alpha 1-proteinase inhibitor gene family during evolution of the genus Mus

alpha 1-Proteinase inhibitors (alpha 1-PIs) are members of the serpin superfamily of proteinase inhibitors, and are important in the maintenance of homeostasis in a wide variety of animal taxa. Previous studies have shown that in mice (genus Mus), evolution of alpha 1-PIs is characterized by gene amplification, region-specific concerted evolution, and rapid accumulation of amino acid substitutions. The latter occurs primarily in the reactive center, which is the region of the alpha 1-PI molecule that determines the inhibitor's specificity for target proteinases. The P1 residue within the reactive center, which is methionine in so-called orthodox alpha 1-PIs and an amino acid other than methionine in unorthodox alpha 1-PIs, is a primary determinant of inhibitor specificity. In the present study, we find that the expression of mRNAs encoding unorthodox alpha 1-PIs is polymorphic within Mus species, i.e., among individuals or inbred strains. This is in striking contrast to mRNAs that encode orthodox alpha 1-PIs, whose concentrations are relatively invariant. The intraspecies variations in mRNA expression represent polymorphisms in the structure of the alpha 1- PI gene family. The results, taken together with previously described aspects of alpha 1-PI evolution, indicate that the dissimilar levels of polymorphism exhibited by orthodox and unorthodox alpha 1-PIs, which likely have distinct physiological functions, may reflect different levels of selective constraint. The significance of this finding to the evolution of gene families is discussed.      

A toxicological basis to derive generic interspecies uncertainty factors for application in human and ecological risk assessment

A new method is proposed to derive the size of the interspecies uncertainty factor (UF) that is toxicologically and statistically based. The method is based on the biological/evolutionary assumption that similarity in susceptibility to toxic substances is a function of phylogenetic relatedness. This assumption is assessed via a large and highly structured aquatic database with over 500 agents tested in specific binary toxicity comparison (i.e., when two species have been tested with the same chemical under identical conditions) for dozens of species of wide phylogenetic relatedness. The methodology takes into account the generic need to estimate a response in any species (not just human) and the need to predict responses for new chemical agents. The method involves quantifying interspecies variation in susceptibility to numerous toxic substances via the use of binary interspecies comparisons that are converted to a 95% UF. This interspecies UF represents an estimate of the upper 95% of the population of 95% prediction intervals (PI) for binary interspecies comparisons within four categories of phylogenetic relatedness (species‐within‐genus, genera‐within‐family, families‐within‐order, orders‐within‐class). The 95% interspecies UFs range from a low of 10 for species‐within‐genus up to 65 for orders‐within‐class. Most mammalian toxicology studies involving mice, rats, cats, dogs, gerbils, and rabbits are orders‐within‐class categories for human risk assessment and would be provided a 65‐fold UF. Larger or smaller interspecies UF values could be selected based on the level of protection desired. The procedures described have application to both human and ecological risk assessment.     

Developmental expression, cellular localization, and testosterone regulation of alpha 1-antitrypsin in Mus caroli kidney

alpha 1-Antitrypsin (alpha 1-protease inhibitor), an essential plasma protein, is synthesized predominantly in the liver of all mammals. We have previously shown that Mus caroli, a Southeast Asian mouse species is exceptional in that it expresses abundantly alpha 1-antitrypsin mRNA and polypeptide, in the kidney as well as the liver (Berger, F.G., and Baumann, H. (1985) J. Biol. Chem. 260, 1160-1165) providing a unique model for examination of the evolution of genetic determinants of tissue-specific gene expression. In the present paper, we have further characterized alpha 1-antitrypsin expression in M. caroli. The extrahepatic expression of alpha 1-antitrypsin is limited to the kidney, specifically within a subset of the proximal tubule cells. The developmental pattern of alpha 1-antitrypsin mRNA expression in the kidney differs from that in the liver. In the kidney, alpha 1-antitrypsin mRNA is present at only 2-4% adult level at birth and increases very rapidly to adult level during puberty between 26 and 36 days of age. There are no significant changes in liver alpha 1-antitrypsin mRNA levels during this period. Testosterone, while having only modest affects on alpha 1-antitrypsin mRNA accumulation in the adult kidney, causes a 20-fold induction of the mRNA in the pre-pubertal kidney. This suggests that the increase in alpha 1-antitrypsin mRNA expression during puberty is testosterone mediated. Southern blot analyses of Mus domesticus and M. caroli genomic DNA and a cloned M. caroli alpha 1-antitrypsin genomic sequence, indicate that a single alpha 1-antitrypsin gene exists in M. caroli, whereas multiple copies exist in M. domesticus. These data show that the alteration in tissue specificity of alpha 1-antitrypsin mRNA accumulation that has occurred during Mus evolution is associated with distinctive developmental and hormonally regulated expression patterns.     

Genetics and ontogeny of aldehyde dehydrogenase isozymes in the mouse: Evidence for a locus controlling the inducibility of the liver microsomal isozyme

Variation in the inducibility of the liver microsomal isozyme of aldehyde dehydrogenase (designated AHD-Cy) by phenobarbital administration was observed among inbred strains and linkage testing stocks of Mus musculus. The phenotypes were inherited in a normal Mendelian fashion with two alleles showing codominance at a proposed regulatory locus (designated Ahd-3r). Strain variation was also observed for the induction of liver AHD-Cy by 17-β-oestradiol administration to ovarectimized female mice. Moreover, this enzyme was elevated in activity by the administration of high (nonphysiological) levels of progesterone. Development studies showed that the liver and kidney AHD-Cy isozyme exhibited low activities in late-stage fetal and neonatal mice and reached adult levels by approximately 6 weeks of age.     

Genetic control of ornithine transcarbamylase induction in chick kidney

After ornithine transcarbamylase (OTC) induction by egg-yolk feeding, OTC activity increases rapidly in chicks bearing an Ocb gene. This response to an egg yolk diet does not appear in chicks having no Ocb gene (showing low OTC activity). The chicks showing intermediate OTC activity also respond to the diet, but moderately. Crossing experiments revealed that OTC induction by egg yolk-diet feeding is inherited as a simple autosomal dominant trait. Since a chick develops during embryonic life by utilizing egg yolk from the yolk sac, the variation of OTC activity among chicken breeds and within a breed in 2-day-old chicks seems to depend on a genetically controlled difference of inducibility by egg yolk. The Ocb is an autosomal gene which controls the induction of OTC activity, but it is difficult to explain the consistent difference in OTC activity between sexes by involving this gene or this locus alone.