Genital Imaginal Disc Research Articles

An insulin-like growth factor-like peptide promotes ovarian development in the silkmoth Bombyx mori

Insulin family peptides are known to be key regulators of growth and metabolism in insects and vertebrates. Insects have two types of insulin family peptides: insulin-like peptides and insulin-like growth factor (IGF)-like peptides (IGFLPs). We recently demonstrated that an IGFLP in the silkmoth, Bombyx mori (BIGFLP) promotes the growth of the genital imaginal disc ex vivo. However, the role of BIGFLP in the regulation of insect growth remains unclear because no in vivo study has been performed. Therefore, we analysed the functions of BIGFLP in vivo by constructing BIGFLP knock-out (KO) B. mori using the clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR-Cas9) system. The KO moths exhibited decreased body weights and size of the appendages compared wild-type (wt) moths. Interestingly, KO females also had drastically lower ovary weights and number of eggs than wt females. However, mutant ovaries that were transplanted into wt host pupae reached a similar weight to wt ovaries that were transplanted into the wt hosts, suggesting that IGFLP in the haemolymph promotes ovarian development. These findings show that BIGFLP regulates the growth and development of adult organs, particularly the ovaries, in B. mori.

Ecdysone-Related Biomarkers of Toxicity in the Model Organism Chironomus riparius: Stage and Sex-Dependent Variations in Gene Expression Profiles

Despite being considered a model organism in toxicity studies, particularly in assessing the environmental impact of endocrine disrupting compounds (EDCs) and other chemicals, the molecular basis of development is largely unknown in Chironomus riparius. We have characterized the expression patterns of important genes involved in the ecdysone pathway from embryos to pupa, but specially during the different phases of C. riparius fourth larval instar, according to the development of genital and thoracic imaginal discs. Real-Time PCR was used to analyze: EcR and usp, two genes encoding the two dimerizing partners of the functional ecdysone receptor; E74, an early response gene induced by ecdysteroids; vg (vitellogenin), an effector gene; hsp70 and hsc70, two heat-shock genes involved in the correct folding of the ecdysone receptor; and rpL13, as a part of the ribosomal machinery. Our results show for the first time stage and sex-dependent variations in ecdysone-responsive genes, specially during the late larval stage of C. riparius. The induction in the expression of EcR and usp during the VII-VIII phase of the fourth instar is concomitant with a coordinated response in the activity of the other genes analyzed, suggesting the moment where larvae prepare for pupation. This work is particularly relevant given that most of the analyzed genes have been proposed previously in this species as sensitive biomarkers for the toxicological evaluation of aquatic ecosystems. Identifying the natural regulation of these molecular endpoints throughout the Chironomus development will contribute to a more in-depth and accurate evaluation of the disrupting effects of EDCs in ecotoxicological studies.

A non-destructive method for identifying the sex of ant larvae

The differences between adult male and female ants are often striking and obvious, yet both sexes appear virtually identical at the larval stage. Current methods for determining larval sex rely on genetic analyses or histology, both of which require killing all larvae examined. Here, we describe a method for identifying larval sex in vivo based on visible differences in genital imaginal discs. Using a light microscope, clear differences in genital disc morphology were observed between male and female larvae of the ponerine ant Harpegnathos saltator. Next, we investigated whether this technique could be broadly applied within ants and found similar differences in genital discs between male and female larvae of Aphaenogaster cockerelli and Camponotus floridanus. Taken together, our results show that genital discs can be used as a reliable indicator of larval sex in species from at least three major ant subfamilies. This technique should facilitate research into topics where information about larval sex is required.

A Survey of 6,300 Genomic Fragments for cis-Regulatory Activity in the Imaginal Discs of Drosophila melanogaster

Over 6,000 fragments from the genome of Drosophila melanogaster were analyzed for their ability to drive expression of GAL4 reporter genes in the third-instar larval imaginal discs. About 1,200 reporter genes drove expression in the eye, antenna, leg, wing, haltere, or genital imaginal discs. The patterns ranged from large regions to individual cells. About 75% of the active fragments drove expression in multiple discs; 20% were expressed in ventral, but not dorsal, discs (legs, genital, and antenna), whereas ∼23% were expressed in dorsal but not ventral discs (wing, haltere, and eye). Several patterns, for example, within the leg chordotonal organ, appeared a surprisingly largenumber of times. Unbiased searches for DNA sequence motifs suggest candidate transcription factors that may regulate enhancers with shared activities. Together, these expression patterns provide a valuable resource to the community and offer a broad overview of how transcriptional regulatory information is distributed in the Drosophila genome.

Multinucleated smooth muscles and mononucleated as well as multinucleated striated muscles develop during establishment of the male reproductive organs of Drosophila melanogaster

The adult musculature in D. melanogaster forms during metamorphosis. Much is known about the flight and leg musculature, but not about the muscles surrounding the male reproductive tract. The inner genitalia of males consist of the testes, which emerge from the gonads; the remaining genital organs, i.e., paragonia (or accessory glands), ejaculatory duct, sperm pump, and seminal vesicles, develop out of the genital imaginal disc. We analyzed the myoblasts forming the muscle layers of these organs. In myoblasts derived from the genital imaginal disc, the regulatory region of the transcription factor DMef2 is active. DMef2 is also needed for specification and differentiation of embryonic and adult myoblasts. We could discriminate three different muscle types: (i) multinucleated muscles that resemble vertebrate smooth muscles surround the testes, (ii) multinucleated muscles that resemble striated muscles comprises seminal vesicles and the sperm pump, and (iii) mononucleated striated musculature encloses the paragonia and ejaculatory duct. Members of the immunoglobulin superfamily involved in embryonic myogenesis, Dumbfounded (Duf) and Sticks and Stones (Sns), were also expressed in the genital imaginal disc, in the muscle sheath of the testes during muscle differentiation and in the secretory secondary cells, which are part of the binucleated epithelia enclosing the paragonia.

Multiple Quantitative Trait Loci Influence the Shape of a Male-Specific Genital Structure inDrosophila melanogaster

The observation that male genitalia diverge more rapidly than other morphological traits during evolution is taxonomically widespread and likely due to some form of sexual selection. One way to elucidate the evolutionary forces acting on these traits is to detail the genetic architecture of variation both within and between species, a program of research that is considerably more tractable in a model system. Drosophila melanogaster and its sibling species, D. simulans, D. mauritiana, and D. sechellia, are morphologically distinguishable only by the shape of the posterior lobe, a male-specific elaboration of the genital arch. We extend earlier studies identifying quantitative trait loci (QTL) responsible for lobe divergence across species and report the first genetic dissection of lobe shape variation within a species. Using an advanced intercross mapping design, we identify three autosomal QTL contributing to the difference in lobe shape between a pair of D. melanogaster inbred lines. The QTL each contribute 4.6–10.7% to shape variation, and two show a significant epistatic interaction. Interestingly, these intraspecific QTL map to the same locations as interspecific lobe QTL, implying some shared genetic control of the trait within and between species. As a first step toward a mechanistic understanding of natural lobe shape variation, we find an association between our QTL data and a set of genes that show sex-biased expression in the developing genital imaginal disc (the precursor of the adult genitalia). These genes are good candidates to harbor naturally segregating polymorphisms contributing to posterior lobe shape.

The Genetic Basis of Rapidly Evolving Male Genital Morphology inDrosophila

The external genitalia are some of the most rapidly evolving morphological structures in insects. The posterior lobe of the male genital arch shows striking differences in both size and shape among closely related species of the Drosophila melanogaster species subgroup. Here, we dissect the genetic basis of posterior lobe morphology between D. mauritiana and D. sechellia, two island endemic species that last shared a common ancestor ∼300,000 years ago. We test a large collection of genome-wide homozygous D. mauritiana genetic introgressions, which collectively cover ∼50% of the genome, for their morphological effects when placed in a D. sechellia genetic background. We find several introgressions that have large effects on posterior lobe morphology and that posterior lobe size and posterior lobe shape can be separated genetically for some of the loci that specify morphology. Using next generation sequencing technology, we perform whole transcriptome gene expression analyses of the larval genital imaginal disc of D. mauritiana, D. sechellia, and two D. mauritiana-D. sechellia hybrid introgression genotypes that each have large effects on either posterior lobe size or posterior lobe shape. Many of the genes we identify as differentially expressed are expressed at levels similar to D. mauritiana in one introgression hybrid, but are expressed at levels similar to D. sechellia in the other introgression hybrid. However, we also find that both introgression hybrids express some of the same genes at levels similar to D. mauritiana, and notably, that both introgression hybrids possess genes in the insulin receptor signaling pathway, which are expressed at D. mauritiana expression levels. These results suggest the possibility that the insulin signaling pathway might integrate size and shape genetic inputs to establish differences in overall posterior lobe morphology between D. mauritiana and D. sechellia.

The female-specific Doublesex isoform regulates pleiotropic transcription factors to pattern genital development inDrosophila

Regulatory networks driving morphogenesis of animal genitalia must integrate sexual identity and positional information. Although the genetic hierarchy that controls somatic sexual identity in the fly Drosophila melanogaster is well understood, there are very few cases in which the mechanism by which it controls tissue-specific gene activity is known. In flies, the sex-determination hierarchy terminates in the doublesex (dsx) gene, which produces sex-specific transcription factors via alternative splicing of its transcripts. To identify sex-specifically expressed genes downstream of dsx that drive the sexually dimorphic development of the genitalia, we performed genome-wide transcriptional profiling of dissected genital imaginal discs of each sex at three time points during early morphogenesis. Using a stringent statistical threshold, we identified 23 genes that have sex-differential transcript levels at all three time points, of which 13 encode transcription factors, a significant enrichment. We focus here on three sex-specifically expressed transcription factors encoded by lozenge (lz), Drop (Dr) and AP-2. We show that, in female genital discs, Dsx activates lz and represses Dr and AP-2. We further show that the regulation of Dr by Dsx mediates the previously identified expression of the fibroblast growth factor Branchless in male genital discs. The phenotypes we observe upon loss of lz or Dr function in genital discs explain the presence or absence of particular structures in dsx mutant flies and thereby clarify previously puzzling observations. Our time course of expression data also lays the foundation for elucidating the regulatory networks downstream of the sex-specifically deployed transcription factors.

Sex- and Developmental Stage-Related Changes in Energy Reserves in Fourth-instar Larvae of the MidgeChironomus ripariusMeigen (Diptera: Chironomidae): Implications for Ecotoxicity Testing

The aim of this study was to examine sex- and age-dependent changes in carbohydrate and lipid reserves throughout the fourth larval instar of Chironomus riparius Meigen. Individuals were sexed using genital discs and aged using phases of development defined through the observation of genital and thoracic imaginal discs. Glycogen concentration was measured in the eviscerated body (fat body, muscles, and cuticle), and the levels of glucose and trehalose were followed in the hemolymph, whereas the amounts of free glycerol and triglycerides were measured in both tissues. Compared with males, females had higher levels of body reserves, whereas the levels of sugars in the hemolymph were similar for both sexes, except for free glucose, which was slightly higher in males. The levels of energy-yielding substrates in both the eviscerated body and hemolymph varied significantly with the phase of development for both sexes, except for the concentration in free glycerol in the hemolymph. Analysis of variability indicated that selection of larvae for both the sex and phase of development significantly reduces uncertainty of biochemical measures. The results are discussed with respect to potential use of the levels of energy-yielding substrates as endpoints in toxicity tests with fourth-instar C. riparius larvae.

Allocation and specification of the genital disc precursor cells in Drosophila

The adult structures of Drosophila melanogaster are derived from larval imaginal discs, which originate as clusters of cells within the embryonic ectoderm. The genital imaginal disc is composed of three primordia (female genital, male genital, and anal primordia) that originate from the embryonic tail segments A8, A9, and A10, respectively, and produce the sexually dimorphic genitalia and analia. We show that the genital disc precursor cells (GDPCs) are first detectable during mid-embryogenesis as a 22-cell cluster in the ventral epidermis. Analysis of mutant and double mutant phenotypes of embryonic patterning genes in the GDPCs, together with their expression patterns in these cells, revealed the following with respect to the origins and specification of the GDPCs. The allocation of the GDPCs from the ventral epidermis requires the function of ventral patterning genes, including the EGF receptor and the spitz group of genes. The ventral localization of the GDPCs is further restricted by the action of dorsal patterning genes. Along the anterior–posterior axis, several segment polarity genes ( wingless, engrailed, hedgehog, and patched) are required for the proper allocation of the GDPCs. These segment polarity genes are expressed in some, but not all of the GDPCs, indicating that anterior and posterior compartments are not fully established in the GDPCs. In addition, we found that the three primordia of the larval genital disc have already been specified in the GDPCs by the coordinated actions of the homeotic (Hox) genes, abdominal-A, Abdominal-B, and caudal. By identifying how these different patterning networks regulate the allocation and primordial organization of the 22 embryonic precursors of the compound genital disc, we demonstrate that at least some of the organization of the larval disc originates as positional information in the embryo, thus providing a context for further studies on the development of the genital disc.

Novel Behavioral and Developmental Defects Associated with Drosophila single-minded

In Drosophila, the development of the midline cells of the embryonic ventral nerve cord depends on the function of the bHLH-PAS transcription factor Single-minded (Sim). The expression domain of sim, however, is also found anterior and posterior to the developing ventral cord throughout the germ band. Indeed, mutations in sim were identified based on their characteristic cuticle phenotype. Eight abdominal segments (A1–A8) can be easily seen in the larval cuticle, while three more can be identified during embryogenesis. Cells located in A8–A10 give rise to the formation of the genital imaginal discs, and a highly modified A11 segment gives rise to the anal pads that flank the anus. sim is expressed in all these segments and is required for the formation of both the anal pads and the genital imaginal discs. A new temperature-sensitive sim allele allowed an assessment of possible postembryonic function(s) of sim. Reduction of sim function below a 50% threshold leads to sterile flies with marked behavioral deficits. Most mutant sim flies were only able to walk in circles. Further analyses indicated that this phenotype is likely due to defects in the brain central complex. This brain region, which has previously been implicated in the control of walking behavior, expresses high levels of nuclear Sim protein in three clusters of neurons in each central brain hemisphere. Additional Sim localization in the medullary and laminar neurons of the optic lobes may correlate with the presence of ectopic axon bundles observed in the optic lobes of sim mutant flies.

Sex-Specific Deployment of FGF Signaling in Drosophila Recruits Mesodermal Cells into the Male Genital Imaginal Disc

A central issue in developmental biology is how the deployment of generic signaling proteins produces diverse specific outcomes. We show that Drosophila FGF is used, only in males, to recruit mesodermal cells expressing its receptor to become part of the genital imaginal disc. Male-specific deployment of FGF signaling is controlled by the sex determination regulatory gene doublesex. The recruited mesodermal cells become epithelial and differentiate into parts of the internal genitalia. Our results provide exceptions to two basic tenets of imaginal disc biology—that imaginal disc cells are derived from the embryonic ectoderm and belong to either an anterior or posterior compartment. The recruited mesodermal cells migrate into the disc late in development and are neither anterior nor posterior.

Ecdysteroid synthesis and imaginal disc development in the midge Chironomus riparius as biomarkers for endocrine effects of tributyltin

Acute effects of the endocrine disruptor bis(tri-n-butyltin) oxide (TBTO) on molting-hormone biosynthesis and imaginal-disc development were investigated in larvae of the midge Chironomus riparius (Meigen). Ecdysteroid synthesis was measured by 24-h incubation of molting-hormone-synthesizing tissues (prothoracic glands) in vitro with or without the addition of TBTO. The amount of ecdysteroids produced was analyzed by radioimmunoassay. Developmental effects in vivo were investigated by determining the developmental phase of the genital imaginal discs before and after a 48-h exposure to TBTO in water. Sex-specific effects were found with both endpoints. Ecdysteroid synthesis was significantly reduced (analysis of variance [ANOVA], p < or = 0.005) in female larvae at all concentrations (TBTO-Sn at 50, 500, and 5,000 ng/L), whereas a significant elevation of the biosynthesis rate occurred in male larvae in the 500-ng/L treatment (ANOVA, p < or = 0.05). In vivo experiments with development of the genital imaginal disc within a 48-h exposure period revealed a significantly slower development in female larvae and a significantly faster development in male larvae (contingency tables, p < or = 0.001) at all concentrations tested (TBTO-Sn at 10, 50, 200, and 1,000 ng/L). These results partly coincided with the in vitro effects on molting-hormone synthesis. The 48-h median lethal concentration (LC50) was 25 microg/L (20-30 microg/L 95% confidence intervals). The combination of in vitro and in vivo methods has proven to be a useful approach for the detection of endocrine effects of TBTO in C. riparius at levels 2,000-fold below the LC50 value. High sensitivity and short test duration suggest that chironomids may have potential as freshwater sentinel organisms for endocrine-disrupting chemicals.

The Sex Determination Gene doublesex Regulates the A/P Organizer to Direct Sex-Specific Patterns of Growth in the Drosophila Genital Imaginal Disc

Each Drosophila genital imaginal disc contains primordia for both male and female genitalia and analia. The sexually dimorphic development of this disc is governed by the sex-specific expression of doublesex ( dsx). We present data that substantially revises our understanding of how dsx controls growth and differentiation in the genital disc. The classical view of genital disc development is that in each sex, dsx autonomously “represses” the development of the inappropriate genital primordium while allowing the development of the appropriate primordium. Instead, we show that dsx regulates the A/P organizer to control growth of each genital primordium, and then directs each genital primordium to differentiate defined adult structures in both sexes.

The Drosophila sex determination hierarchy modulates wingless and decapentaplegic signaling to deploy dachshund sex-specifically in the genital imaginal disc.

The integration of multiple developmental cues is crucial to the combinatorial strategies for cell specification that underlie metazoan development. In the Drosophila genital imaginal disc, which gives rise to the sexually dimorphic genitalia and analia, sexual identity must be integrated with positional cues, in order to direct the appropriate sexually dimorphic developmental program. Sex determination in Drosophila is controlled by a hierarchy of regulatory genes. The last known gene in the somatic branch of this hierarchy is the transcription factor doublesex (dsx); however, targets of the hierarchy that play a role in sexually dimorphic development have remained elusive. We show that the gene dachshund (dac) is differentially expressed in the male and female genital discs, and plays sex-specific roles in the development of the genitalia. Furthermore, the sex determination hierarchy mediates this sex-specific deployment of dac by modulating the regulation of dac by the pattern formation genes wingless (wg) and decapentaplegic (dpp). We find that the sex determination pathway acts cell-autonomously to determine whether dac is activated by wg signaling, as in females, or by dpp signaling, as in males.

Compartmental organization of the Drosophila genital imaginal discs.

We have investigated the anterior and posterior compartmental organization of the genital imaginal disc. Unlike the thoracic discs, the genital disc is a compound disc consisting of three primordia--the female genital, male genital, and anal primordia. Here we provide evidence that each primordium is divided into anterior and posterior compartments. Genes that are known to be expressed in compartment-specific manners in other discs (engrailed, hedgehog, patched, decapentaplegic, wingless and cubitus interruptus) are expressed in analogous patterns in each primordium of the genital disc. Specifically, engrailed and cubitus interruptus are expressed in complementary domains, while patched, decapentaplegic and wingless are expressed along the border between the two domains. Mitotic clones induced at the beginning of the second larval instar do not cross the boundary between the engrailed-expressing and cubitus interruptus-expressing domains, indicating that these domains are true genetic compartments. Furthermore, we examined the phenotypes of mutant clones of the cAMP-dependent protein kinase A and engrailed-invected, genes that are known to play compartment-specific functions in other discs. These experiments demonstrate that the anterior/posterior patterning functions of these genes are conserved in the genital disc. The adult clonal phenotypes of protein kinase A and engrailed-invected mutants also provide a more detailed map of the adult genitalia and analia with respect to the anterior/posterior compartmental subdivision. Our results lead us to propose a new model to describe the anterior and posterior compartmental organization of the genital disc.

Sexually dimorphic sterility phenotypes in Hoxa10-deficient mice.

The Abdominal B (AbdB) genes constitute a distinct subfamily of homeobox genes that exhibit posterior domains of expression, including the genital imaginal disc in Drosophila and the developing urogenital system in vertebrates. We have mutated the AbdB gene Hoxa10 in mice. We report here that homozygotes are fully viable and show an anterior homeotic transformation of lumbar vertebrae. All male homozygotes manifest bilateral cryptorchidism resulting in severe defects in spermatogenesis and increasing sterility with age. Female homozygotes ovulate normally, but about 80% are sterile because of death of embryos between days 2.5 and 3.5 post coitum. This coincides spatially and temporally with expression of maternal Hoxa10 in distal oviductal and uterine epithelium. These results indicate a role for AbdB Hox genes in male and female fertility and suggest that maternal Hoxa10 is required to regulate the expression of a factor that affects the viability of preimplantation embryos.

Genital tract growth factors from a moth, the tobacco budworm, Heliothis virescens.

Development and maturation of the genital tract of the moth, Heliothis virescens, takes place within a few days in the pupal stage. The insect steroid hormone, 20-hydroxyecdysone (20E), essential during this period, stimulated testis sheath and fat body tissue to secrete factors that, in turn, stimulated growth and development of pupal spermducts and genital imaginal discs in vitro. Factors could be extracted in aqueous solution from tissues incubated for 24 h in media containing 1000pg/microliters 20E, and partially purified by chromatography on polyacrylamide gels. Ten active molecular weight fractions were separated from testis sheath extracts, and 9 from fat body extracts. Most fractions were labile to protease, although the activity of six of the fractions was also destroyed by lipase. Testis sheath, fat body tissue, and active fractions, caused partial development of the genital tract in vitro, as well as increased incorporation of [3H]methionine into precipitable protein and [3H]thymidine into nuclear material.

Control of imaginal cell development by the escargot gene of Drosophila

Mutations in the escargot (esg) locus, which codes for a zinc-finger-containing protein with similarity to the product of the snail gene, cause a variety of defects in adult structures such as loss of abdominal cuticle and malformation of the wings and legs. esg RNA is expressed in wing, haltere, leg and genital imaginal discs and in abdominal histoblast nests in the embryo. Expression in imaginal tissues is also found in third instar larvae. In esg mutant larvae, normally diploid abdominal histoblasts replicate their DNA without cell division and become similar in appearance to the polytene larval epidermal cells. A similar phenotype was also found in imaginal discs of larvae mutant for both esg and the Drosophila raf gene. These results suggest that one of the normal functions of esg may be the maintenance of diploidy in imaginal cells.

Development of genital imaginal discs ofHeliothis virescensculturein vitrowith 20-hydroxyecdysone and fat body or testis sheaths

Summary Genital imaginal discs were dissected from Heliothis virescens pupae just after diapause break and cultured in vitro. The discs continued to grow and differentiate when maintained in media containing sufficiently high titers of 20-hydroxyecdysone (20HE) and co-cultured fat body or testis sheath tissue, or extract of fat body previously exposed to 20HE in vitro. Continuous changes were observed for approximately 30 days. Other tissues provided only temporary (5–8 day) developmental support. Genetal discs did not develop in the absence of 20 HE. Discs co-cultured with fat body in 20 HE medium synthesized more protein and DNA than discs co-cultured with fat body but without 20 HE. Fat body and testis sheath tissue, cultured with sufficient titers of 20 HE, produced factors essential to the development of genital imaginal discs of H. virescens.