Everything you always wanted to know about sex ... in flies.

'Everything you always wanted to know about sex' is a workshop organized as part of the annual Drosophila Research Conference of the Genetics Society of America. This workshop provides an intellectual venue for interaction among research groups that study sexual dimorphism from the molecular, evolutionary, genomic, and behavioral perspectives. The speakers summarize the key ideas behind their research for people working in other fields, outline unsolved questions, and offer their opinions about future directions. The 2010 workshop highlighted the power of the Drosophila model for understanding sexual dimorphism at levels ranging from cell biology and gene regulation to population genetics and genome evolution, and demonstrated the importance of cross-disciplinary interactions in the study of sex. In this respect, Drosophila sets a good example for research in other organisms, including humans and their mammalian relatives.

Sox100B, a Drosophila group E Sox-domain gene, is required for somatic testis differentiation.

Sex determination mechanisms are thought to evolve rapidly and show little conservation among different animal species. For example, the critical gene on the Y chromosome, SRY, that determines sex in most mammals, is not found in other animals. However, a related Sox domain transcription factor, SOX9, is also required for testis development in mammals and exhibits male-specific gonad expression in other vertebrate species. Previously, we found that the Drosophila orthologue of SOX9, Sox100B, is expressed male-specifically during gonad development. We now investigate the function of Sox100B and find, strikingly, that Sox100B is essential for testis development in Drosophila. In Sox100B mutants, the adult testis is severely reduced and fails to interact with other parts of the reproductive tract, which are themselves unaffected. While a testis initially forms in Sox100B mutants, it fails to undergo proper morphogenesis during pupal stages, likely due to defects in the pigment cells. In contrast, no substantive defects are observed in ovary development in Sox100B mutant females. Thus, as is observed in mammals, a Sox9 homolog is essential for sex-specific gonad development in Drosophila, suggesting that the molecular mechanisms regulating sexually dimorphic gonad development may be more conserved than previously suspected.

Southern cooking and lung cancer.

Dietary associations were examined as part of a case-control study exploring reasons for exceptionally high rates of lung cancer in northeast Florida. Interviews, which included a nationally standardized food frequency questionnaire, were conducted with 507 patients diagnosed with lung cancer during 1993-1996 or their next of kin and 1,007 persons of similar age, race, and gender randomly selected from the general population. A substantial reduction in risk was associated with high consumption of nutrient-dense fruits and vegetables. Risk was nearly doubled among men and women in the highest quartile of fat intake. The effects were most prominent for saturated and monounsaturated fats and not apparent for polyunsaturated fat consumption. Increased risk was linked to consumption of several individual high-fat foods, including some traditional Southern foods or methods of cooking, such as cooking vegetables with lard/fatback/bacon fat. Reported use of vitamin/mineral supplements was associated with decrease risk of lung cancer as well as dietary consumption of vitamins A, C, and E and some carotenoids. The findings are consistent with emerging evidence that risk of lung cancer rises with increasing dietary fat consumption. They indicate the need for further research to determine whether the association between fat intake and lung cancer is causal and, if so, to clarify the relationships with individual fat fractions.

HMG-CoA reductase guides migrating primordial germ cells.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is best known for catalysing a rate-limiting step in cholesterol biosynthesis, but it also participates in the production of a wide variety of other compounds. Some clinical benefits attributed to inhibitors of HMG-CoA reductase are now thought to be independent of any serum cholesterol-lowering effect. Here we describe a new cholesterol-independent role for HMG-CoA reductase, in regulating a developmental process: primordial germ cell migration. We show that in Drosophila this enzyme is highly expressed in the somatic gonad and that it is necessary for primordial germ cells to migrate to this tissue. Misexpression of HMG-CoA reductase is sufficient to attract primordial germ cells to tissues other than the gonadal mesoderm. We conclude that the regulated expression of HMG-CoA reductase has a critical developmental function in providing spatial information to guide migrating primordial germ cells.

Gonadal mesoderm and fat body initially follow a common developmental path in Drosophila.

During gastrulation, the Drosophila mesoderm invaginates and forms a single cell layer in close juxtaposition to the overlying ectoderm. Subsequently, particular cell types within the mesoderm are specified along the anteroposterior and dorsoventral axes. The exact developmental pathways that guide the specification of different cell types within the mesoderm are not well understood. We have analyzed the developmental relationship between two mesodermal tissues in the Drosophila embryo, the gonadal mesoderm and the fat body. Both tissues arise from lateral mesoderm within the eve domain. Whereas in the eve domain of parasegments 10-12 gonadal mesoderm develops from dorsolateral mesoderm and fat body from ventrolateral mesoderm, in parasegments 4-9 only fat body is specified. Our results demonstrate that the cell fate decision between gonadal mesoderm and fat body identity within dorsolateral mesoderm along the anteroposterior axis is determined by the combined actions of genes including abdA, AbdB and srp; while srp promotes fat body development, abdA allows gonadal mesoderm to develop by repressing srp function. Furthermore, we present evidence from genetic analysis suggesting that, before stage 10 of embryogenesis, gonadal mesoderm and the fat body have not yet been specified as different cell types, but exist as a common pool of precursor cells requiring the functions of the tin, zfh-1 and cli genes for their development.

zfh-1 is required for germ cell migration and gonadal mesoderm development in Drosophila.

In Drosophila as well as many vertebrate systems, germ cells form extraembryonically and migrate into the embryo before navigating toward gonadal mesodermal cells. How the gonadal mesoderm attracts migratory germ cells is not understood in any system. We have taken a genetic approach to identify genes required for germ cell migration in Drosophila. Here we describe the role of zfh-1 in germ cell migration to the gonadal mesoderm. In zfh-1 mutant embryos, the initial association of germ cells and gonadal mesoderm is blocked. Loss of zfh-1 activity disrupts the development of two distinct mesodermal populations: the caudal visceral mesoderm and the gonadal mesoderm. We demonstrate that the caudal visceral mesoderm facilitates the migration of germ cells from the endoderm to the mesoderm. Zfh-1 is also expressed in the gonadal mesoderm throughout the development of this tissue. Ectopic expression of Zfh-1 is sufficient to induce additional gonadal mesodermal cells and to alter the temporal course of gene expression within these cells. Finally, through analysis of a tinman zfh-1 double mutant, we show that zfh-1 acts in conjunction with tinman, another homeodomain protein, in the specification of lateral mesodermal derivatives, including the gonadal mesoderm.

Identification of genes controlling germ cell migration and embryonic gonad formation in Drosophila.

Gonadogenesis in the Drosophila embryo is a complex process involving numerous cellular migratory steps and cell-cell interactions. The mechanisms guiding germ cells to move through, recognize and adhere to specific cell types are poorly understood. In order to identify genes that are required for these processes, we have conducted an extensive mutagenesis of the third chromosome and screened for mutations disrupting germ cell migration at any point in embryonic development. Phenotypic analysis of these mutants demonstrates that germ cell migration can be broken down into discrete developmental steps, with each step requiring a specific set of genes. Many of these genes are involved in the development of gonadal mesoderm, the tissue that associates with germ cells to form the embryonic gonad. Moreover, mutations that we isolated affecting embryonic patterning as well as germ cell migration suggest that the origin of gonadal mesoderm lies within the eve domain of the developing mesoderm.

Regulation of zygotic gene expression in Drosophila primordial germ cells.

Activation of the zygotic genome is a prerequisite for the transition from maternal to zygotic control of development. The onset of zygotic transcription has been well studied in somatic cells, but evidence suggests that it is controlled differently in the germline. In Drosophila, zygotic transcription in the soma has been detected as early as one hour after egg laying (AEL) [1]. In the germline, general RNA synthesis is not detected until 3.5 hours AEL (stage 8) [2] and poly(A)-containing transcripts are not observed in early germ cell nuclei [3]. However, rRNA gene expression has been demonstrated at this time [4]. Therefore, either there is a general, low level activation of the genome in early germ cells, or specific classes of genes, such as those transcribed by RNA polymerase (RNAP) II, are repressed. We addressed this issue by localizing the potent transcriptional activator Gal4-VP16 to the germline, and we find that Gal4-VP16-dependent gene expression is repressed in early germ cells. In addition, localization of germ plasm to the anterior reveals that it is sufficient to repress Bicoid-dependent gene expression. Thus, even in the presence of known transcriptional activators, RNAP II dependent gene expression is actively repressed in early germ cells. Furthermore, once the germ cell genome is activated, we find that vasa is expressed specifically in germ cells. This expression does not require proper patterning of the soma, indicating that it is likely to be controlled by the germ plasm.

Germ plasm assembly and germ cell migration in Drosophila.

Many aspects of germ cell behavior, migration, and gonad formation are shared between vertebrate and invertebrate species. For example, a specialized germ plasm has been observed in many species including Caenorhabditis elegans and Xenopus. Furthermore, the fact that Vasa marks germ cells in many species suggests that even certain molecular aspects of germ cells may be common between different organisms. In most organisms, germ cells initially form at a location away from their target mesodermal tissues and have to migrate to reach the mesoderm. Further genetic studies will reveal the extent to which molecular aspects of germ cell migration and gonad formation are conserved.

Negative regulation of proneural gene activity: hairy is a direct transcriptional repressor of achaete.

hairy (h) acts as a negative regulator in both embryonic segmentation and adult peripheral nervous system (PNS) development in Drosophila. Here, we demonstrate that h, a basic-helix-loop-helix (bHLH) protein, is a sequence-specific DNA-binding protein and transcriptional repressor. We identify the proneural gene achaete (ac) as a direct downstream target of h regulation in vivo. Mutation of a single, evolutionarily conserved, high-affinity h binding site in the upstream region of ac results in the appearance of ectopic sensory organs in adult flies, in a pattern that strongly resembles the phenotype of h mutants. This indicates that direct repression of ac by h plays an essential role in pattern formation in the PNS. Our results demonstrate that HLH proteins negatively regulate ac transcription by at least two distinct mechanisms.

Spatial regulation of proneural gene activity: auto- and cross-activation of achaete is antagonized by extramacrochaetae.

The spatially restricted activities of achaete (ac) and scute (sc) are thought to define proneural clusters of potential sensory organ precursor cells in the imaginal discs of Drosophila. These genes encode transcriptional regulators of the basic helix-loop-helix (bHLH) class. We have found that direct, positive transcriptional autoregulation by the ac protein and cross-regulation by sc are essential for high-level expression of the ac promoter in the proneural cluster pattern and that autoactivation is important for the bristle-promoting function of the ac gene. These auto- and cross-regulatory activities are antagonized in a dose-dependent manner by the inhibitory HLH protein encoded by the extramacrochaetae (emc) gene. We have found that emc is expressed in the wing imaginal disc in a pattern strongly complementary to that of the proneural clusters. Our results indicate that emc plays an essential early role in defining territories of bristle-forming potential.

The Drosophila extramacrochaetae protein antagonizes sequence-specific DNA binding by daughterless/achaete-scute protein complexes.

In Drosophila, a group of regulatory proteins of the helix-loop-helix (HLH) class play an essential role in conferring upon cells in the developing adult epidermis the competence to give rise to sensory organs. Proteins encoded by the daughterless (da) gene and three genes of the achaete-scute complex (AS-C) act positively in the determination of the sensory organ precursor cell fate, while the extramacrochaetae (emc) and hairy (h) gene products act as negative regulators. In the region upstream of the achaete gene of the AS-C, we have identified three 'E box' consensus sequences that are bound specifically in vitro by hetero-oligomeric complexes consisting of the da protein and an AS-C protein. We have used this DNA-binding activity to investigate the biochemical basis of the negative regulatory function of emc. Under the conditions of our experiments, the emc protein, but not the h protein, is able to antagonize specifically the in vitro DNA-binding activity of da/AS-C and putative da/da protein complexes. We interpret these results as follows: the heterodimerization capacity of the emc protein (conferred by its HLH domain) allows it to act in vivo as a competitive inhibitor of the formation of functional DNA-binding protein complexes by the da and AS-C proteins, thereby reducing the effective level of their transcriptional regulatory activity within the cell.

Regulation of 6-phosphofructo-1-kinase activity in ras-transformed rat-1 fibroblasts.

Fructose 2,6-bisphosphate (F-2,6-P2) stimulated glycolysis in cell-free extracts of both normal and ras-transfected rat-1 fibroblasts. The extract of the transformed cell glycolyzed more rapidly in both the absence and the presence of F-2,6-P2 than the extract of the parent fibroblast. Addition of mitochondrial ATPase (F1) or inorganic phosphate (Pi) further stimulated lactate production in both cell lines. F-2,6-P2 stimulated the 6-phosphofructo-1-kinase (PFK-1) activity in extracts of normal and transfected cells. The activity in extracts of transformed cells tested with a fructose 6-phosphate regenerating system was considerably higher than in the extract of normal cells. Stimulation of PFK-1 activity by cAMP of both cell lines was not as pronounced as that by F-2,6-P2. In the absence of F-2,6-P2 the PFK-1 activity was strongly inhibited in the transformed cell by ATP concentrations higher than 1 mM, whereas in the normal cell only a marginal inhibition was noted even at 2 or 3 mM ATP. F-2,6-P2 reversed the inhibition of PFK-1 by ATP. Nicotinamide adenine dinucleotide (NAD) at 100 microM (in the presence of 2 mM ATP and 1 microM F-2,6-P2) stimulated PFK-1 activity only in the transformed cell, whereas nicotinamide adenine dinucleotide phosphate (NADP) inhibited PFK-1 activity (in the presence or absence of 1 microM F-2,6-P2) in extracts of both cell lines. No previous observations of stimulation or inhibition by NAD or NADP on PFK-1 activity appear to have been reported. A threefold increase in the intracellular concentration of F-2,6-P2 was observed after transfection of rat-1 fibroblast by the ras oncogene. We conclude from these data that the PFK-1 activity of ras-transfected rat-1 fibroblasts shows a greater response to certain stimulating and inhibitory regulating factors than that of the parent cell.