TfM mutation and masculinization versus feminization of the mouse central nervous system

The sexual behavior of mice of various genotypes has been studied in our stock in which X-linked genes, Tfm and (O^hv), as well as an autosomal dominant gene, Sxr, are maintained. Since the absence of neonatal imprinting in Tfm (O^hv)/Y^ leads to no sexual behavior, we conclude that neonatal imprinting is the prerequisite for feminization as well as masculinization of the central nervous system. Since individual components of sexual behavior may become feminine or absent instead of being masculine in sex-reversed Tfm (O^hv)/+(O⁺, Sxr/+^♂ with mosaic brains, we conclude that neonatal imprinting directly involves individual neurons, and that different degrees of imprinting by the same agent lead to masculinization or feminization. In accordance with recent views, we believe estradiol to be this imprinting agent.We envisage the role of the Tfm locus in the central nervous system as follows: within individual neurons for sexual behavior, the synthesis of aromatizing enzymes is normally inducible by androgens; these enzymes are therefore noninducible in Tfm (O^hv)/Y^. In normal neonates, exposure to testosterone leads to the induced intracellular conversion of testosterone to estradiol, self imprinting by estradiol causing masculinization. Feminization may normally be due to the direct exposure of these neurons to a low circulating concentration of estradiol. An alternative explanation might be that the estradiol-receptor protein in these neurons also is normally inducible by testosterone. In this case, neonatally testosterone-exposed neurons would become inherently more responsive to estradiol than neonatally estradiol-exposed neurons.     

The analysis of Lyon's hypothesis through preferential X-activation

Lyon's hypothesis of random X-inactivation or activation can be represented as a problem of compound binomials: (p + q)ⁿ, where p = q = 0.5 and n is the number of early cells representing a future embryo, expresses initial clonal compositions of XX-embryos immediately after the random but irreversible decision on the fate of individual X-chromosomes. Clonal compositions of a given somatic cell type in the adult body, on the other hand, can be expressed as (p′ + q′)^n′ where p′ and q′ are now variables determined by the initial clonal composition, while n′ is the number of embryonic cells which serve as the immediate progenitors of that somatic cell type. Since n is considerably larger than 10 even in the mouse, a paradox is created because so long as the initial decision process remains random, experimental tests of the hypothesis also remain impractical.We have determined the frequency with which the entire somatic cell type becomes Tfm + (0^hv) Blo monoclonal on a number of divergent organs of Tfm + (0^hv) Blo/ + + (0⁺) + heterozygotes. The results are compatible with parameters of (0.8 + 0.2)⁶⁰, determining the initial clonal compositions of these heterozygotes. Alleles, such as (0^hv) and (0⁺) of Cattanach's “controlling element” site on the X-chromosome, apparently determine the values which p and q take in the initial decision process.     

Metabolic cooperation between Tfm and wild-type cells in mosaic mice after induction of DNA synthesis

Mosaic mice composed of androgen-insensitive Tfm and androgen-sensitive wild-type cells are constructed by virtue of the natural X inactivation: XX mice heterozygous for X-linked testicular feminization (Tfm) are reverted to males by the sex reversal (Sxr) mutation. After stimulation with testosterone, in the epididymis of the mosaic mice, the incorporation of ³H-thymidine is compared in both cell fractions. The labeling index of Tfm and wild-type cells is in the same order of magnitude. The result indicates that the stimulus for DNA synthesis exerted by testosterone is conveyed from the androgen-sensitive wild-type to the androgen-insensitive Tfm cells by metabolic cooperation on tissue level.On the other hand, the proportion of Tfm cells in the epididymal mosaic is far less than expected from X inactivation. The reason is that in the mosaic, in spite of normal proliferation, the undifferentiated Tfm cells die off steadily. Typical dense bodies are observed as signs of physiological cell death.     

Position Effects and Variegation Enhancers in an Autosomal Region of DROSOPHILA MELANOGASTER

A dominant eye color mutation was found associated with a third chromosome inversion broken distally at or near the karmoisin (kar) locus in 87C and proximally within centric heterochromatin. Suppressibility of the mutant phenotype by an extra Y chromosome indicated that this was an example of dominant position-effect variegation. When heterozygous with deficiencies uncovering the kar locus, this inversion chromosome was found to be lethal unless a region in 87EF was also deleted. Extra Y chromosomes rescued inversion/deletion heterozygotes, while removal of the Y chromosome from heterozygous males deficient for the region in 87EF was lethal. Thus, a variegating lethal lies near the breakpoint in 87C, and a wild-type gene that enhances its variegation lies in 87EF. Furthermore, deletion of the region in 87EF was found to strongly suppress white-mottled-4 (w^m4) variegation, while deletion of another region in 87BC suppressed less strongly. These results indicate that essential genes on autosomes are sensitive to position effects, and loci that enhance variegation, as defined by deficiency mapping, are very common.     

Testosterone-“Regulon” in the Mouse Kidney

Proximal tubule cells of the mouse kidney (metanephros) are normally extremely responsive to testosterone and its intracellular metabolites. The X-linked Tfm mutation recovered by Lyon and Hawkes seems to represent an i^s (repressor noninducible) mutation of the regulatory locus.     

Nuclear DHT-Receptor in <Emphasis Type="Italic">Tfm</Emphasis>/<Emphasis Type="Italic">Y</Emphasis> Kidney Cell

OUR previous studies on the X-linked testicular feminization (Tfm) mutation¹ of the mouse^2–4 showed that the so-called cytosol and nuclear 5αx-dihydrotestosterone (DHT) receptor protein^5–7 might be a regulatory protein specified by the Tfm locus. The dual role of being a translational repressor in the cytoplasma and a mediator of hypertrophy in the nucleus was envisaged⁸. We found, however, another class of androgen-receptor in the polysome fraction of kidney proximal tubule cells which seems better qualified to be a translational regulator. Since a single gene locus specifies only one kind of polypeptide chain, we re-examined whether the cytosol and nuclear DHT-receptor protein underwent a true mutational change in Tfm/Y individuals.     

Androgen Spares Androgen-Insensitive Motoneurons from Apoptosis in the Spinal Nucleus of the Bulbocavernosus in Rats

The spinal nucleus of the bulbocavernosus (SNB) is a sexually dimorphic motor nucleus in the rat lumbar spinal cord. The sex difference arises through the androgenic sparing of the motoneurons and their target muscles from ontogenetic cell death. Indirect evidence suggests that androgen acts on the target muscles rather than directly on SNB motoneurons to spare them from death. The testicular feminization mutation (Tfm), a defect in the androgen receptor (AR), blocks androgenic sparing of SNB motoneurons and their targets. The pattern of AR immunocytochemistry was previously found to be different in adultTfmand wild-type rats: immunostaining was nuclear in most SNB cells of wild-type rats, but very few SNB cells display nuclear AR immunostaining in affectedTfmrats. Because theTfmmutation is carried on the X chromosome, random X inactivation during development makes female carriers ofTfm(+/Tfm) genetic mosaics for androgen sensitivity.Tfmcarriers, their wild-type sisters, and affectedTfmmales were treated with perinatal testosterone and immunocytochemistry was used to detect androgen receptor in the SNB when the rats reached adulthood. Mosaic females could be distinguished from their wild-type sisters by external morphology. In such perinatally androgenized mosaics, adult SNB cells were equally divided between wild-type andTfmgenotype, as indicated by AR immunocytochemistry. In contrast, the pattern of AR immunocytochemistry in target muscles of mosaics appeared similar to that of wild-type females. These results indicate that early androgen spared both androgen-sensitive and -insensitive motoneurons from cell death, confirming a site of androgen action other than the motoneurons themselves.     

Nonrandom X Chromosome Inactivation Is Influenced by Multiple Regions on the Murine X Chromosome

During the development of female mammals, one of the two X chromosomes is inactivated, serving as a dosage-compensation mechanism to equalize the expression of X-linked genes in females and males. While the choice of which X chromosome to inactivate is normally random, X chromosome inactivation can be skewed in F1 hybrid mice, as determined by alleles at the X chromosome controlling element (Xce), a locus defined genetically by Cattanach over 40 years ago. Four Xce alleles have been defined in inbred mice in order of the tendency of the X chromosome to remain active: Xce^a < Xce^b < Xce^c < Xce^d. While the identity of the Xce locus remains unknown, previous efforts to map sequences responsible for the Xce effect in hybrid mice have localized the Xce to candidate regions that overlap the X chromosome inactivation center (Xic), which includes the Xist and Tsix genes. Here, we have intercrossed 129S1/SvImJ, which carries the Xce^a allele, and Mus musculus castaneus EiJ, which carries the Xce^c allele, to generate recombinant lines with single or double recombinant breakpoints near or within the Xce candidate region. In female progeny of 129S1/SvImJ females mated to recombinant males, we have measured the X chromosome inactivation ratio using allele-specific expression assays of genes on the X chromosome. We have identified regions, both proximal and distal to Xist/Tsix, that contribute to the choice of which X chromosome to inactivate, indicating that multiple elements on the X chromosome contribute to the Xce.     

Third chromosome suppressor of position-effect variegation loci in Drosophila melanogaster

Summary As a result of a genetic analysis of 63 third chromosome suppressor mutations of position-effect variegation 12 different loci showing dominant suppression have been identified and their map positions determined. A compilcation of the genetic data available for each suppressor locus is given. The strong suppressor effects of the mutations have been quantified by measurements of white variegation inw ^m4h /w ^m4h ,w ^m4h /Y andw ^m4h /O flies. Mutant alleles of three loci were found in these studies to dominate over the strong enhancer effect of complete loss of the Y chromosome. Most of the identified loci suppressing position-effect variegation represent essential genetic funtions; only three loci represent nonessential functions. Mutations of two loci display recessive butyrate sensitivity and lethal interaction with the heterochromatic Y chromosome suggesting that these genes affect chromosomal condensation. Studies with deficiencies and triploids revealed that most of the loci represent haplo-abnormal suppressor functions. The use of the isolated mutant material for genetic, developmental and molecular studies of processes connected with gene inactivation in position-effect variegation is discussed.Dedicated to Prof. H.J. Becker on the occasion of his 6th birthday     

Carnitine suppression of position-effect variegation in Drosophila melanogaster

Carnitine is a well-known naturally occurring compound, very similar to butyrate, with an essential role in intermediary metabolism mainly at the mitochondrial level. Since butyrate inhibits the enzyme histone deacetylase and is capable of suppressing position-effect variegation in Drosophila melanogaster, we tested a further possible function of carnitine in the nucleus, using an assay for the suppression of position-effect variegation. We tested three physiological forms of carnitine (l-carnitine, l-propionylcarnitine, l-acetylcarnitine) for the ability to suppress two different chromosomal rearrangements, inducing variegation of the white ⁺ and brown ⁺ genes. The results show that the carnitine derivatives are capable of suppressing the position-effect variegation, albeit with different efficiencies. The carnitine derivatives interact lethally with Su-var(2)1 ⁰¹, a mutation that induces hyperacetylation of histones, whilst hyperacetylated histories accumulated in both the nuclei of HeLa cells and Drosophila polytene chromosomes treated with the same compounds. These results strongly suggest that the carnitine derivatives suppress position-effect variegation by a mechanism similar to that of butyrate. It is suggested that carnitines may have a functional role in the nucleus, probably at the chromatin level.     

Live cell imaging of X chromosome reactivation during somatic cell reprogramming

Generation of induced pluripotent stem cells (iPSCs) with naive pluripotency is important for their applications in regenerative medicine. In female iPSCs, acquisition of naive pluripotency is coupled to X chromosome reactivation (XCR) during somatic cell reprogramming, and live cell monitoring of XCR is potentially useful for analyzing how iPSCs acquire naive pluripotency. Here we generated female mouse embryonic stem cells (ESCs) that carry the enhanced green fluorescent protein (EGFP) and humanized Kusabira-Orange (hKO) genes inserted into an intergenic site near either the Syap1 or Taf1 gene on both X chromosomes. The ESC clones, which initially expressed both EGFP and hKO, inactivated one of the fluorescent protein genes upon differentiation, indicating that the EGFP and hKO genes are subject to X chromosome inactivation (XCI). When the derived somatic cells carrying the EGFP gene on the inactive X chromosome (Xi) were reprogrammed into iPSCs, the EGFP gene on the Xi was reactivated when pluripotency marker genes were induced. Thus, the fluorescent protein genes inserted into an intergenic locus on both X chromosomes enable live cell monitoring of XCI during ESC differentiation and XCR during reprogramming. This is the first study that succeeded live cell imaging of XCR during reprogramming.     

Nuclei in testicular feminization (Tfm) are not activated by intact testosterone receptor complexes: A morphometric study in striated urethral muscle of mosaic mice

Summary Sex reversed mice heterozygous for the X-linked Tfm mutation are mosaics with respect to the Tfm locus. In the androgen-dependent striated urethral muscle, nuclei coding for the intact testosterone receptor protein and nuclei coding for the defective Tfm receptor protein are incorporated in the same multinucleate muscle fibres. The intact testosterone receptor complex can thus be expected to enter the Tfm nuclei. Our measurements show that the fibre diameters of the mosaic muscle form a homogeneous population, intermediate in size between induced male and non-inducible Tfm phenotypes. By contrast, the nuclear size shows a bimodal distribution, the subpopulations corresponding to Tfm and wild type nuclei. The results indicate that the Tfm nuclei are not activated by the intact testosterone receptor complex.     

Alopecia and male infertility in oligotriche mutant mice are caused by a deletion on distal chromosome 9

The recessive mutation oligotriche (olt) affects the coat and male fertility in the mouse. In homozygous (olt/olt) mutants, the coat is sparse, most notably in the inguinal and medial femoral region. In these regions, almost all hair shafts are bent and distorted in their course through the dermis and rarely penetrate the epidermis because the hair cortex is not fully keratinized. During hair follicle morphogenesis, mutant hair follicles exit from anagen one day before those of normal littermates and show a prolongation of the catagen stage. The oligotriche (olt) locus was mapped to distal chromosome 9 within a 5-Mbp interval distal to D9Mit279. Analysis of candidate gene expression revealed that olt/olt mutant mice do not express functional phospholipase C delta 1 (Plcd1) mRNA. This deficiency is the consequence of a 234-kbp deletion involving not only the Plcd1 locus but also the chromosomal segment harboring the genes Vill (villin-like), Dlec1 (deleted in lung and esophageal cancer 1), Acaa1b (acetyl-Coenzyme A acyltransferase 1B, synonym thiolase B), and parts of the genes Ctdspl (carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase-like) and Slc22a14 (solute carrier family 22 member 14). Offspring of olt/olt females, mated with Plcd1 ^?/? knockout males, exhibit coat defects similar to those observed in homozygous olt/olt mutant mice but the spermiogenesis in male offspring is normal. We conclude that the 234-kbp deletion from chromosome 9 harbors a gene involved in spermiogenesis and we propose that the oligotriche mutant be used as a model for the study of the putative tumor suppressor genes Dlec1, Ctdspl, and Vill. We also suggest that the oligotriche locus be named Del(9Ctdspl-Slc22a14)1Pas.     

Further Characterization of <Emphasis Type="Italic">o</Emphasis><Superscript>s</Superscript> Mutation of Mouse β-Glucuronidase Locus

The operator noninducible mutation of the mouse β-glucuronidase locus is associated with a reduced electro-phoretic mobility for the enzyme. This suggests that the translational repressor, specified by the Tfm locus, recognizes a translatable segment of the messenger RNA it controls.     

Genetic dissection of testis weight in mice: quantitative trait locus analysis using F2 intercrosses between strains with extreme testis weight, and association study using Y-consomic strains

In the present study, dissection of genetic bases of testis weight in mice was performed. Autosomes and the X chromosome were searched using traditional quantitative trait locus (QTL) scans, and the Y chromosome was searched by association studies of Y-consomic strains. QTL analysis was performed in ??DDD?×???CBA F₂ mice; the inbred mouse DDD has the heaviest testes, whereas the inbred mouse CBA has the lightest testes. Two significant testis weight QTLs were identified on chromosomes 1 and X. A DDD allele was associated with increased and decreased testis weight at the locus on chromosomes 1 and X, respectively. In the reciprocal cross ??CBA?×???DDD F₂ mice, QTL on chromosome 1, and not on chromosome X, had a significant effect on testis weight. The DDD allele at the X-linked locus could not sustain testis weight in combination with the Y chromosome of the CBA strain. The Y chromosome per se had a significant effect on testis weight, i.e., DH-Chr Y^DDD had significantly heavier testes than DH-Chr Y^CBA. On the basis of the results of Y-chromosome-wide association studies using 17 Y-consomic strains, variations in Uty, Usp9y, and Sry were significantly associated with testis weight. Thus, testis weight is a complex quantitative phenotype controlled by multiple genes on autosomes and sex chromosomes and their interactions.     

Traits That Influence Longevity in Mice

Analysis of genetic interactions in the segregating backcross [(C57BL/6 x DBA/2)F₁ x DBA/2] mice revealed influences of genetic and environmental factors on life span. Using determinants of coat color (brown locus of chromosome 4 and dilute locus of chromosome 9), serologically determined H-2 antigens (chromosome 17 ) and sex as genetic markers, we studied the effects of these genes on longevity. The results suggested that genes in the brown locus (b) segment of chromosome 4, genes in a segment of the sex chromosomes and, to a more limited extent, genes in the segment of chromosome 17 which contains the H-2 haplotype all influenced longevity. The coat color (b locus) segment of chromosome 4 was associated with life span predominantly in females, whereas the chromosome 17 (H-2 haplotype) segment was associated with longer life primarily in males. The dilute locus d segment on chromosome 9 did not affect life span. Longevity appears to be influenced by interactions between genes in the chromosomal segment carrying H-2, those in the b segment, gender and the month of birth. Greater heterozygosity at the loci studied was associated with longer life span. Histopathological findings on mice that died at or after 28 months of age were comparable for all genetic combinations except that there was an increased frequency of lymphoma in females and an increased frequency of amyloidosis in males. Our analysis emphasizes the need for comprehensive studies of aging and longevity that would simultaneously determine the effects of several genetic regions and their interactions with the environment with respect to possible causes of death.     

Meiotic mutations from natural populations ofDrosophila melanogaster

Two meiotic genes from natural populations are described. A female meiotic mutation,mei(1)g13, mapped to 17.4 on the X chromosome, causes nondisjunction of all homologs except for the fourth chromosomes. In addition, it reduces recombination by 10% in the homozygotes and causes 18% increased recombination in the heterozygotes. A male meiotic mutation,mei-1223 ^m144 , is located on the third chromosome. Although this mutation causes nondisjunction of all chromosomes, each chromosome pair exhibits a different nondisjunction frequency. Large variations in the sizes of the premature sperm heads observed in the homozygotes may reflect irregular meiotic pairing and the subsequent abnormal segregation, resulting in aneuploid chromosome complements.     

Genetics of Sex-linked yellow in the Syrian Hamster

Alternating patches of black and yellow pigment are a ubiquitous feature of mammalian color variation that contributes to camouflage, species recognition, and morphologic diversity. X-linked determinants of this pattern—recognized by variegation in females but not in males—have been described in the domestic cat as Orange, and in the Syrian hamster as Sex-linked yellow (Sly), but are curiously absent from other vertebrate species. Using a comparative genomic approach, we develop molecular markers and a linkage map for the euchromatic region of the Syrian hamster X chromosome that places Sly in a region homologous to the centromere-proximal region of human Xp. Comparison to analogous work carried out for Orange in domestic cats indicates, surprisingly, that the cat and hamster mutations lie in nonhomologous regions of the X chromosome. We also identify the molecular cause of recessively inherited black coat color in hamsters (historically referred to as nonagouti) as a Cys115Tyr mutation in the Agouti gene. Animals doubly mutant for Sly and nonagouti exhibit a Sly phenotype. Our results indicate that Sly represents a melanocortin pathway component that acts similarly to, but is genetically distinct from, Mc1r and that has implications for understanding both the evolutionary history and the mutational mechanisms of pigment-type switching.     

Cytogenetic and molecular aspects of position effect variegation in Drosophila melanogaster

A chromosomal region subjected to position effect variegation was analysed for possible DNA under-replication. DNA clones from the vicinity of the euheterochromatin junction and from a distance of hundreds of kilobase pairs were used as probes. Formation of compact blocks of chromatin is regarded as a characteristic feature of position effect variegation. It was shown that in T (1;2) dor ^var7 males undergoing position effect variegation clones representing the DNA nearest to the breakpoint in 2B7 hybridized normally in situ to the compact blocks, providing evidence against DNA underreplication. In females the same clones did not hybridize to the compact blocks. These variations in hybridization may be related to different degrees of compaction of chromosome regions in males and females. A correlation between the degree of underreplication and the level of cell polyteny was shown by Southern-blot hybridization of a DNA probe from the 2B region to DNA from an X/O strain carrying Dp (1;1)pn2b displaying position effect variegation and compaction in 94% of salivary gland cells. Almost complete underreplication of the DNA of this region was found in salivary gland cells (with a maximal degree of polyteny), intermediate underreplication was found in fat body cells (with an intermediate degree of polyteny), and replication was not disturbed in diploid cells of the larval cephalic complex.by W. Beermann     

Histone Gene Multiplicity and Position Effect Variegation in DROSOPHILA MELANOGASTER

The histone genes of wild-type Drosophila melanogaster are reiterated 100–150 times per haploid genome and are located in the segment of chromosome 2 that corresponds to polytene bands 39D2-3 to E1-2. The influence of altered histone gene multiplicity on chromatin structure has been assayed by measuring modification of the gene inactivation associated with position effect variegation in genotypes bearing deletions of the 39D-E segment. The proportion of cells in which a variegating gene is active is increased in genotypes that are heterozygous for a deficiency that removes the histone gene complex. Deletions that remove segments adjacent to the histone gene complex have no effect on the expression of variegating genes. Suppression of position effect variegation associated with reduction of histone gene multiplicity applies to both X-linked and autosomal variegating genes. Position effects exerted by both autosomal and sex-chromosome heterochromatin were suppressible by deletions of the histone gene complex. The suppression was independent of the presence of the Y chromosome. A deficiency that deletes only the distal portion of the histone gene complex also has the ability to suppress position effect variegation. Duplication of the histone gene complex did not enhance position effect variegation. Deletion or duplication of the histone gene complex in the maternal genome had no effect on the extent of variegation in progeny whose histone gene multiplicity was normal. These results are discussed with respect to current knowledge of the organization of the histone gene complex and control of its expression.