A Genetic Analysis of Intersex, a Gene Regulating Sexual Differentiation in Drosophila Melanogaster Females

Sex-type in Drosophila melanogaster is controlled by a hierarchically acting set of regulatory genes. At the terminus of this hierarchy lie those regulatory genes responsible for implementing sexual differentiation: genes that control the activity of target loci whose products give rise to sexually dimorphic phenotypes. The genetic analysis of the intersex (ix) gene presented here demonstrates that ix is such a terminally positioned regulatory locus. The ix locus has been localized to the cytogenetic interval between 47E3-6 and 47F11-18. A comparison of the morphological and behavioral phenotypes of homozygotes and hemizygotes for three point mutations at ix indicates that the null phenotype of ix is to transform diplo-X animals into intersexes while leaving haplo-X animals unaffected. Analysis of X-ray induced, mitotic recombination clones lacking ix(+) function in the abdomen of diplo-X individuals indicates that the ix(+) product functions in a cell-autonomous manner and that it is required at least until the termination of cell division in this tissue. Taken together with previous analyses, our results indicate that the ix(+) product is required to function with the female-specific product of doublesex to implement appropriate female sexual differentiation in diplo-X animals.     

Antibody-Mediated Immune Control of a Retrovirus Does Not Require the Microbiota

Commensal microbes are often required to control viral infection by facilitating host immune defenses. However, we found that this does not hold true for retroviral infection. We report that retrovirus-resistant mice control the pathogen with virus-neutralizing antibodies independently of commensal microbiota. This is in contrast to orthomyxoviruses and arenaviruses, where resistance is ablated in animals depleted of microbiota. Clearly, when it comes to antiviral immunity, the role of the microbiota cannot be generalized.     

Essential Genes in Autosomal Heterochromatin of Drosophila Melanogaster

We are taking two approaches to understanding the structure, function and regulation of essential genes within Drosophilaheterochromatin. In the first, we have undertaken a genetic and molecular characterization of essential genes within proximal 3L heterochromatin. The expression of such ‘resident’ genes within a heterochromatic environment is paradoxical and poorly understood, given that the same environment can inactivate euchromatic sequences (position effect variegation, or PEV). A second approach involves the study of the local chromosomal environment of heterochromatic (het) genes, as assayed both biochemically, and via the effects of genetic modifiers of PEV, the latter being putative components important for het gene expression. Our results to date suggest that the three most proximal genes in 3L heterochromatin have key roles in development, and indicate strong effects of combinations of genetic modifiers of PEV on het gene expression. This revised version was published online in July 2006 with corrections to the Cover Date.     

Tissue-Specific Expression Phenotypes of Hawaiian Drosophila Adh Genes in Drosophila Melanogaster Transformants

Interspecific differences in the tissue-specific patterns of expression displayed by the alcohol dehydrogenase (Adh) genes within the Hawaiian picture-winged Drosophila represent a rich source of evolutionary variation in gene regulation. Study of the cis-acting elements responsible for regulatory differences between Adh genes from various species is greatly facilitated by analyzing the behavior of the different Adh genes in a homogeneous background. Accordingly, the Adh gene from Drosophila grimshawi was introduced into the germ line of Drosophila melanogaster by means of P element-mediated transformation, and transformants carrying this gene were compared to transformants carrying the Adh genes from Drosophila affinidisjuncta and Drosophila hawaiiensis. The results indicate that the D. affinidisjuncta and D. grimshawi genes have relatively higher levels of expression and broader tissue distribution of expression than the D. hawaiiensis gene in larvae. All three genes are expressed at similar overall levels in adults, with differences in tissue distribution of enzyme activity corresponding to the pattern in the donor species. However, certain systematic differences between Adh gene expression in transformants and in the Hawaiian Drosophila are noted along with tissue-specific position effects in some cases. The implications of these findings for the understanding of evolved regulatory variation are discussed.     

Accurate Determination of Zygosity in Transgenic Rice by Real-time PCR Does Not Require Standard Curves or Efficiency Correction

A number of quantitative, real-time PCR methods have been developed for determining transgene copy numbers in plants. Here, we demonstrate that the Roche LightCycler^TM system can be used to determine the zygosity of transgenic lines without the use of standard curves or efficiency correction calculations. We have developed a duplex PCR assay which permits the determination of zygosity, relative to a calibrator sample, in transgenic rice lines containing the gene for a viral glycoprotein. Our data demonstrate that unambiguous 2-fold discrimination of copy number can be attained by calculating relative copy number using the threshold crossing point (Ct) calculated by the LightCycler^TM software combined with delta delta Ct calculations, provided that the appropriate calibrator sample is included in each run. The method presented here is rapid, sensitive, robust and easy to optimise.     

Identification of Regions Interacting with Ovo(d) Mutations: Potential New Genes Involved in Germline Sex Determination or Differentiation in Drosophila Melanogaster

Only a few Drosophila melanogaster germline sex determination genes are known, and there have been no systematic screens to identify new genes involved in this important biological process. The ovarian phenotypes produced by females mutant for dominant alleles of the ovo gene are modified in flies with altered doses of other loci involved in germline sex determination in Drosophila (Sex-lethal(+), sans fille(+) and ovarian tumor(+)). This observation constitutes the basis for a screen to identify additional genes required for proper establishment of germline sexual identity. We tested 300 deletions, which together cover ~58% of the euchromatic portion of the genome, for genetic interactions with ovo(D). Hemizygosity for more than a dozen small regions show interactions that either partially suppress or enhance the ovarian phenotypes of females mutant for one or more of the three dominant ovo mutations. These regions probably contain genes whose products act in developmental hierarchies that include ovo(+) protein.     

Experience-Driven Axon Retraction in the Pharmacologically Inactivated Visual Cortex Does Not Require Synaptic Transmission

Background

Experience during early postnatal development plays an important role in the refinement of specific neural connections in the brain. In the mammalian visual system, altered visual experiences induce plastic adaptation of visual cortical responses and guide rearrangements of afferent axons from the lateral geniculate nucleus. Previous studies using visual deprivation demonstrated that the afferents serving an open eye significantly retract when cortical neurons are pharmacologically inhibited by applying a γ-aminobutyric acid type A receptor agonist, muscimol, whereas those serving a deprived eye are rescued from retraction, suggesting that presynaptic activity can lead to the retraction of geniculocortical axons in the absence of postsynaptic activity. Because muscimol application suppresses the spike activity of cortical neurons leaving transmitter release intact at geniculocortical synapses, local synaptic interaction may underlie the retraction of active axons in the inhibited cortex.

Method and Findings

New studies reported here determined whether experience-driven axon retraction can occur in the visual cortex inactivated by blocking synaptic inputs. We inactivated the primary visual cortex of kittens by suppressing synaptic transmission with cortical injections of botulinum neurotoxin type E, which cleaves a synaptic protein, SNAP-25, and blocks transmitter release, and examined the geniculocortical axon morphology in the animals with normal vision and those deprived of vision binocularly. We found that afferent axons in the animals with normal vision showed a significant retraction in the inactivated cortex, as similarly observed in the muscimol-treated cortex, whereas the axons in the binocularly deprived animals were preserved.

Conclusions

Therefore, the experience-driven axon retraction in the inactivated cortex can proceed in the absence of synaptic transmission. These results suggest that presynaptic mechanisms play an important role in the experience-driven refinement of geniculocortical axons.     

Rapid Male-Specific Regulatory Divergence and Down Regulation of Spermatogenesis Genes in Drosophila Species Hybrids

In most crosses between closely related species of Drosophila, the male hybrids are sterile and show postmeiotic abnormalities. A series of gene expression studies using genomic approaches have found significant down regulation of postmeiotic spermatogenesis genes in sterile male hybrids. These results have led some to suggest a direct relationship between down regulation in gene expression and hybrid sterility. An alternative explanation to a cause-and-effect relationship between misregulation of gene expression and male sterility is rapid divergence of male sex regulatory elements leading to incompatible interactions in an interspecies hybrid genome. To test the effect of regulatory divergence in spermatogenesis gene expression, we isolated 35 fertile D. simulans strains with D. mauritiana introgressions in either the X, second or third chromosome. We analyzed gene expression in these fertile hybrid strains for a subset of spermatogenesis genes previously reported as significantly under expressed in sterile hybrids relative to D. simulans. We found that fertile autosomal introgressions can cause levels of gene down regulation similar to that of sterile hybrids. We also found that X chromosome heterospecific introgressions cause significantly less gene down regulation than autosomal introgressions. Our results provide evidence that rapid male sex gene regulatory divergence can explain misexpression of spermatogenesis genes in hybrids.     

Comparison of Germline Mosaics of Genes in the Polycomb Group of Drosophila Melanogaster

The genes of the Polycomb group (PcG) repress the genes of the bithorax and Antennapedia complexes, among others. To observe a null phenotype for a PcG gene, one must remove its maternal as well as zygotic contribution to the embryo. Five members of the PcG group are compared here: Enhancer of Polycomb [E(Pc)], Additional sex combs (Asx), Posterior sex combs (Psc), Suppressor of zeste 2 [Su(z)2] and Polycomblike (Pcl). The yeast recombinase (FLP) system was used to induce mitotic recombination in the maternal germline. Mutant embryos were analyzed by staining with antibodies against six target genes of the PcG. The loss of the maternal component leads to enhanced homeotic phenotypes and to unique patterns of misexpression. E(Pc) and Su(z)2 mutations had only subtle effects on the target genes, even when the maternal contributions were removed. Asx and Pcl mutants show derepression of the targets only in specific cell types. Psc shows unusual effects on two of the targets, Ultrabithorax and abdominal-A. These results show that the PcG genes do not act only in a common complex or pathway; they must have some independent functions.     

Identification of Homeotic Target Genes in Drosophila Melanogaster Including Nervy, a Proto-Oncogene Homologue

In Drosophila, the specific morphological characteristics of each segment are determined by the homeotic genes that regulate the expression of downstream target genes. We used a subtractive hybridization procedure to isolate activated target genes of the homeotic gene Ultrabithorax (Ubx). In addition, we constructed a set of mutant genotypes that measures the regulatory contribution of individual homeotic genes to a complex target gene expression pattern. Using these mutants, we demonstrate that homeotic genes can regulate target gene expression at the start of gastrulation, suggesting a previously unknown role for the homeotic genes at this early stage. We also show that, in abdominal segments, the levels of expression for two target genes increase in response to high levels of Ubx, demonstrating that the normal down-regulation of Ubx in these segments is functional. Finally, the DNA sequence of cDNAs for one of these genes predicts a protein that is similar to a human proto-oncogene involved in acute myeloid leukemias. These results illustrate potentially general rules about the homeotic control of target gene expression and suggest that subtractive hybridization can be used to isolate interesting homeotic target genes.     

Rex-Induced Recombination Implies Bipolar Organization of the Ribosomal RNA Genes of Drosophila Melanogaster

Rex-induced mitotic recombination was used to determine whether nucleolus organizers can pair in both inverted and noninverted orientations. Two target chromosomes, each duplicated for the rDNA region, were exposed to maternal Rex activity. Recombination in one orientation should yield deletion of the material between the two nucleolus organizers, recombination in the other orientation should yield inversion of the same material. Both products were recovered from both target chromosomes. The generality of using Rex-mediated recombination for analysis of the rDNA is considered.     

Obervations on the Induction of Position Effect Variegation of Euchromatic Genes in Drosophila Melanogaster

In the T(1;2)dor(var7) translocation, the 1A-2B7-8 segment of the X chromosome is brought to the vicinity of 2R-chromosome heterochromatin resulting in position effect variegation of dor, BR-C and more distal genes, as well as compaction of chromatin in this segment. By irradiation of T(1;2)dor(var7), nine reversions (rev) to a normal phenotype were recovered. In two cases (rev27, rev226), the 1A-2B7-8 section is relocated to the 19A region of the X chromosome, forming free duplications (1A-2B7-8/19A-20F-X-het). Modifiers of position effect do not change the normal expression of the BR-C and dor genes in these duplications. In five reversions (rev3, rev40, rev60, rev167, rev175), free duplications have formed from the 1A-2B7-8 fragment and X chromosome heterochromatin. In these rearrangements, modifiers of position effect (low temperature, removal of Y and 2R-chromosome heterochromatin and a genetic enhancer (E-var(3)201) induce position-effect again. Two reversions (rev45 and rev110) are associated with additional inversions in the original dor(var7) chromosomes. The inversions relocate part of the heterochromatin adjacent to the 1A-2B7-8 section into new positions. In T(1;2)dor(rev45), position-effect is seen in the 2B7-8-7A element as compaction spreading from 2B7-8 proximally in some cases as far as the 5D region. Thus, in rev45 the pattern of euchromatin compaction is reciprocal to that of the initial dor(var7) strain. Apparently, it is due to the same variegation-evoking center near the 2R centromere in both cases. In all nine revertants, weakening or complete disappearance of the position-effect is observed despite retention of the 20- kb heterochromatic segment adjacent to the 1A-2B7-8 region. Thus, a 20-kb heterochromatic sequence does not inactivate euchromatin joined to it.     

Seven Genes of the Enhancer of Split Complex of Drosophila Melanogaster Encode Helix-Loop-Helix Proteins

Enhancer of split [E(spl)] is one of the neurogenic loci of Drosophila and, as such, is required for normal segregation of neural and epidermal cell progenitors. Genetic observations indicate that the E(spl) locus is in fact a gene complex comprising a cluster of related genes and that other genes of the region are also required for normal early neurogenesis. Three of the genes of the complex were known to encode helix-loop-helix (HLH) proteins and to be transcribed in nearly identical patterns. Here, we show that four other genes in the vicinity also encode HLH proteins and, during neuroblast segregation, three of them are expressed in the same pattern. We show by germ-line transformation that these three genes are also necessary to allow epidermal development of the neuroectodermal cells.     

Identification of Trans-Acting Genes Necessary for Centromere Function in Drosophila Melanogaster Using Centromere-Defective Minichromosomes

Deletions in the Drosophila minichromosome Dp1187 were used to investigate the genetic interactions of trans-acting genes with the centromere. Mutations in several genes known to have a role in chromosome inheritance were shown to have dominant effects on the stability of minichromosomes with partially defective centromeres. Heterozygous mutations in the ncd and klp3A kinesin-like protein genes strongly reduced the transmission of minichromosomes missing portions of the genetically defined centromere, but had little effect on the transmission of minichromosomes with intact centromeres. Using this approach, ncd and klp3A were shown to require only the centromeric region of the chromosome for their roles in chromosome segregation. Increased gene dosage also affected minichromosome transmission and was used to demonstrate that the nod kinesin-like protein gene interacts genetically with the centromere, in addition to interacting with extracentromeric regions as demonstrated previously. The results presented in this study strongly suggest that dominant genetic interactions between mutations and centromere-defective minichromosomes could be used effectively to identify novel genes necessary for centromere function.