The development of the Drosophila genital disc

The imaginal discs of Drosophila melanogaster, which form the adult epidermal structures, are a good experimental model for studying morphogenesis. The genital disc forms the terminalia, which are the most sexually dimorphic structures of the fly. Both sexes of Drosophila have a single genital disc formed by three primordia. The female genital primordium is derived from 8(th) abdominal segment and is located anteriorly, the anal primordium (10 and 11(th) abdominal segments) is located posteriorly, and the male genital primordium from the 9(th) abdominal segment lies between them. In both sexes, only two of these three primordia develop to form the adult terminalia. The anal primordium develops in both sexes but, depending on the genetic sex, will form either male or female analia. However, only one of the genital primordia develops in each sex, forming either the male or the female genitalia. This depends on the genetic sex of the fly. Therefore, the genital disc is a very good experimental model of how the sex-determination and homeotic genes - which determine cell identity - interact to direct the development of a population of cells into male or female terminalia. It has been proposed that the sexually dimorphic development of the genital disc is the result of an integrated genetic input, made up by the sex-determination gene doublesex and the homeotic gene Abdominal-B. This input acts by modulating the response to Hedgehog, Wingless, and Decapentaplegic morphogenetic signals.     

The genital disc of Drosophila melanogaster

 The genital disc of Drosophila, which gives rise to the genitalia and analia of adult flies, is formed by cells from different embryonic segments. To study the organization of this disc, the expressions of segment polarity and homeotic genes were investigated. The organization of the embryonic genital primordium and the requirement of the engrailed and invected genes in the adult terminalia were also analysed. The results show that the three primordia, the female and male genitalia plus the analia, are composed of an anterior and a posterior compartment. In some aspects, each of the three primordia resemble other discs: the expression of genes such as wingless and decapentaplegic in each anterior compartment is similar to that seen in leg discs, and the absence of engrailed and invected cause duplications of anterior regions, as occurs in wing discs. The absence of lineage restrictions in some regions of the terminalia and the expression of segment polarity genes in the embryonic genital disc suggest that this model of compartmental organization evolves, at least in part, as the disc grows. The expression of homeotic genes suggests a parasegmental organization of the genital disc, although these genes may also change their expression patterns during larval development. Received: 4 February 1997 / Accepted: 22 May 1997     

An anterior/posterior communication compartment border in engrailed wing discs: possible implications for Drosophila pattern formation

Our previous studies have suggested that all the known lineage compartment borders in the wing imaginal disc of Drosophila are coincident with boundaries of reduced gap junctional communication (communication compartment borders). Since engrailed discs have a disrupted anterior/posterior (A/P) lineage border (G. Morata and P. A. Lawrence, 1975, Nature (London) 255, 614-617), it was of great interest to determine if their A/P communication restriction boundary is similarly disrupted. Examination of gap-junction-mediated exchange of small fluorescent molecules between cells in the engrailed wing disc revealed a boundary of restricted communication that appeared to be identical to the wild-type A/P communication restriction boundary. This result suggests that lineage compartments are not required for the formation of A/P communication restrictions. Furthermore, we suggest that perhaps communication compartments are the domains within which information is provided for specifying the formation of lineage compartments.     

Effects of engrailed in the genital disc of Drosophila melanogaster

engrailed has been postulated to be the “selector gene” involved in the establishment of the anterior-posterior compartment border in several imaginal discs and in at least the first two abdominal segments of Drosophila melanogaster. Our study of the effects of different mutant engrailed genotypes on genital disc development provided the following major results: All three terminal primordia (female and male genitalia, and analia) were affected. Different heteroallelic combinations showed different expressivities, and the three terminal primordia were differently affected by the same mutant genotype. The engrailed genotypes deleted specific elements of the adult terminalia without causing associated pattern duplications. The reduced morphology of the male engrailed genital disc was analogous to the pattern deletions observed in the adult terminalia. That the engrailed phenotype is stable was demonstrated by culturing in vivo intact and fragmented engrailed genital discs. Cell death was found in a significant number of mature male en²/en³ genital discs. The results are discussed in terms of the segmental organization of the genital disc and in terms of the “selector gene” function postulated for the engrailed locus. The interpretation that each terminal primordium has an anterior and a posterior compartment is presented and it is assumed that in the genital disc engrailed transforms posterior cells into anterior cells that do not develop, thereby causing the deficiency pattern of the engrailed phenotype.     

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.     

Vein-positioning in the Drosophila wing in response to Hh; new roles of Notch signaling

The Drosophila wing is a classical model for studying the generation of developmental patterns. Previous studies have suggested that vein primordia form at boundaries between discrete sectors of gene expression along the antero-posterior (A/P) axis in the larval wing imaginal disc. Observation that the vein marker rhomboid (rho) is expressed at the centre of wider vein-competent domains led to propose that narrow vein primordia form first, and produce secondary short-range signals activating provein genes in neighbouring cells (see Curr. Opin. Genet. Dev. 10 (2000) 393). Here, we examined how the central L3 and L4 veins are positioned relative to the limits of expression of Collier (Col), a dose-dependent Hedgehog (Hh) target activated in the wing A/P organiser. We found that rho expression is first activated in broad domains adjacent to Col-expressing cells and secondarily restricted to the centre of these domains. This restriction which depends upon Notch (N) signaling sets the L3 and L4 vein primordia off the boundaries of Col expression. N activity is also required to fix the anterior limit of Col expression by locally antagonising Hh activation, thus precisely positioning the L3 vein primordium relative to the A/P compartment boundary. Experiments using Nts mutants further indicated that these two activities of N could be temporally uncoupled. Together, these observations highlight new roles of N in topologically linking the position of veins to prepattern gene expression.     

Permeability of gap junctions at the segmental border in insect epidermis

We have examined cell-cell communication between epidermal cells of fifth-instar larvae of the milkweed bug Oncopeltus fasciatus and those of maggots of the blowfly Calliphora erythrocephala. Ionic coupling and the transfer of injected Lucifer Yellow (molecular weight 450) and lead-EDTA (molecular weight 374) were used to map the pattern of communication. All epidermal cells, regardless of their position with respect to the segmental border, were ionically coupled. In both species Lucifer Yellow was transferred freely between cells lying in the same segment—that is, in the same developmental compartment as defined by cell lineage. Dye injections close to the segmental border showed that Lucifer Yellow was not transferred between cells in adjacent segments—that is, across the compartmental border. In Calliphora failure of Lucifer Yellow transfer at the segmental border was always observed; in Oncopeltus Lucifer Yellow was not transferred in 90% of preparations examined. Injections of PbEDTA^2? in Calliphora showed that this anion was transferred freely from cell to cell and did not respect the segmental boundary. Previous studies of the distribution of gap junctions at and away from the segmental border make it unlikely that the failure of Lucifer Yellow to cross from segment to segment is due to a reduced number of gap-junctional channels at the border. We conclude that gap junctions at the segmental borders may have different permeability properties from those between cells in the same segment.     

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.     

Cell tracing reveals a dorsoventral lineage restriction plane in the mouse limb bud mesenchyme

Regionalization of embryonic fields into independent units of growth and patterning is a widespread strategy during metazoan development. Compartments represent a particular instance of this regionalization, in which unit coherence is maintained by cell lineage restriction between adjacent regions. Lineage compartments have been described during insect and vertebrate development. Two common characteristics of the compartments described so far are their occurrence in epithelial structures and the presence of signaling regions at compartment borders. Whereas Drosophila compartmental organization represents a background subdivision of embryonic fields that is not necessarily related to anatomical structures, vertebrate compartment borders described thus far coincide with, or anticipate, anatomical or cell-type discontinuities. Here, we describe a general method for clonal analysis in the mouse and use it to determine the topology of clone distribution along the three limb axes. We identify a lineage restriction boundary at the limb mesenchyme dorsoventral border that is unrelated to any anatomical discontinuity, and whose lineage restriction border is not obviously associated with any signaling center. This restriction is the first example in vertebrates of a mechanism of primordium subdivision unrelated to anatomical boundaries. Furthermore, this is the first lineage compartment described within a mesenchymal structure in any organism, suggesting that lineage restrictions are fundamental not only for epithelial structures, but also for mesenchymal field patterning. No lineage compartmentalization was found along the proximodistal or anteroposterior axes, indicating that patterning along these axes does not involve restriction of cell dispersion at specific axial positions.     

Organising activities of engrailed, hedgehog, wingless and decapentaplegic in the genital discs of Drosophila melanogaster

 The genes engrailed (en), hedgehog (hh), wingless (wg) and decapentaplegic (dpp) have been shown to play vital organising roles in the development and differentiation of thoracic imaginal discs. We have analysed the roles of these genes in organising the development and differentiation of the genital discs, which are bilaterally symmetrical and possess different primordia, namely, the male and female genital primordia and an anal primordium. Our results suggest that the organising activity of en in genital discs programs the normal development and differentiation of the genital disc by regulating the expression of hh. Hh in turn induces wg and dpp, the genes whose products act as secondary signalling molecules. Moreover, the complementary patterns of wg and dpp expression are essential for the bilateral symmetry and are maintained by mutual repression. Received: 20 April 1998 / Accepted 24 June 1998     

Cell lineage restrictions in the genital disc ofDrosophila revealed byMinute gynandromorphs

Summary The genital imaginal disc ofDrosophila differentiates the terminalia, i.e. the genitalia and analia, of both sexes. It represents a composite anlage, containing a female genital primordium, a male genital primordium and an anal primordium. In normal males and females, only one of the two genital primordia differentiates; the other is developmentally repressed. Therefore, cell-lineage relationships between the male and female genital primordia can only be studied in sexual mosaics which differentiate female and male cells. We producedMinute (M)non-Minute(M⁺) gynandromorphs and selected those with sexually mosaic terminalia for a cell-lineage analysis. In these mosaics, either the male (XO) or female (XX) cells wereM ⁺ and thus had a growth advantage. The differential growth rates served as a tool to detect clonal restrictions. In control gynandromorphs (M ⁺M ⁺), the amount of female genitalia differentiated was largely independent of the amount of male genitalia present. In contrast, male and female anal structures, as a rule, added up to one full set. The same was true for the experimentalMM ⁺ gynandromorphs, but the contribution ofXX andXO cells to mosaic terminalia changed drastically due toM ⁺ cells competing successfully against the more slowly growingM cells. Specific subsamples ofMM ⁺ gynandromorphs showed thatM cells in a non-mosaic primordium are shielded from cell competition taking place in the neighbouring mosaic primordium. We conclude that the three primordia of the genital disc represent developmental compartments. In the genital primordia, even developmentally repressedM ⁺ cells compete successfully against developmentally activeM cells.     

Scanning electron microscopy of Drosophila melanogaster embryogenesis: III. Formation of the head and caudal segments

The formation of both the anterior most and posterior most segments in higher dipteran embryos involves complex movements of primordia which can be best visualized with the scanning electron microscope. During head formation, the gnathocephalic segments partially involute through the stomodeum. The labial segment forms the floor of the mouth, and the fused maxillary and mandibular segments form the lateral sides of the mouth. The involuted clypeolabrum forms the roof of the mouth. Invaginations of cells for segmentally derived sense organs can be found prior to involution on all the gnathocephalic and thoracic segments as well as on the labrum. The antennal sense organ derives from the lateral surface of the procephalic lobe. Following involution of the mouth parts, the dorsal ridge, which arises just anterior to the first thoracic segment, is drawn over the dorsal procephalic lobe producing the deep dorsal sac. The optic lobes of the brain invaginate anterior to the dorsal ridge just prior to the covering over of the head. The formation of the anal segment is similarly complex. Two rudimentary segments are found posterior to the eighth abdominal segment. During shortening of the germ band, the posterior most segment is drawn around the posterior tip of the embryo to lie ventrally. Two large anal pads form lateral to the anus from this segment. The next segment, following dorsal closure, produces a pair of anal sense organs and a central tuft of setae. Finally, the eighth abdominal segment gives rise to the posterior spiracles. Following dorsal closure these three segments fuse to produce the terminal (anal) segment of the larva.     

Genesis of the reproductive system of mosquitoes. I. Female of Aedes stimulans (Walker)

The feminine dimorph has unique structures that produce eggs, select salubrious sites for the offspring, store sperm, and void the eggs. This paper provides a time table for development of these parts in Aedes stimulans based on preparations examined at 5-hour intervals when reared at 21°C. All growths of imaginal parts proceeds independent of activities in the larval tissues. Ovaries produce the eggs in terminal follicles of the ovarioles. Besides ovarioles each ovary contains sheaths for the ovarioles, pedicels attaching them to a central canal, the calyx, ovarian sheath and muscles. Ovaries are recognizable in newly hatched larvae as caps of cells on larger masses which become part of the delivery system for eggs. Each ovary grows forward from its attachment first as a column of cells that differentiates into the several tissues by the time the insect enters pupal life. Prior accounts have considered the ovary as the whole mass of cells on each side of the hemocoel of segment 6. Only the most anterior cells recognizably distinct at the end of embryogeny are generative. The delivery system for eggs is composed of the lateral oviducts and median or common oviduct. Primordia from which the former are derived are present from the end of embryogeny and throughout larval life as two distinct parts. Two ovoid masses occur in the hemocoel of segment 6. To each of these is attached a filament extending backward to an attachment ventrally and caudally in segment 7. They are rapidly changed into definitive lateral oviducts late in pupal life. The single primordium for generating the median genital tract appears during instar 3 as a caudal ventral plate of cells in segment 8 between a pair of bilateral buds and invaginates during instar 4 to form (1) the common oviduct from a midventral pouch, (2) three spermathecae from two lateral invaginations and (3) the elaborate vaginal area. The bilateral buds form no parts of the female. The post-vaginal area or atrium with its accessory organs is derived in part from the ventral plate of segment 8 and that of segment 9. The imaginal disc in segment 9 is present at the end of embryogeny as primordial buds and ventral plate and development is delayed until early pupal life when it projects inward to form part of the atrium and pouches once to form the common opening for the duct of the accessory gland and the canal to the bursa copulatrix. The buds of this disc produce no feminine parts. During the second larval instar lateral primordia appear as a pair of shields in the anal segment. They develop slowly until pupation when they extend caudally as two flaps called “cerci” in culicid literature and this paper.     

Morphogenesis of an identified leech neuron: segmental specification of axonal outgrowth

We have investigated the development of segmental diversity in an identified leech neuron, the Retzius cell. Retzius cells in the genital segments differ from those in other segments in lacking central axons and contacting different peripheral targets: the genitalia. These differences are not apparent during initial axon outgrowth, when all Retzius cells follow the same morphogenetic pattern. Rather, they first appear about the time the peripheral axons of the genital segment Retzius cells contact the genital primordia. This suggests that the pattern of central and peripheral axonal outgrowth may be modified by an interaction with peripheral targets.     

Genesis of the reproductive system of mosquitoes II. Male of Aedes stimulans (walker)

The imaginal male of mosquitoes bears a combination of organs and appendages that make it morphologically distinctive. Its reproductive organs produce sperm cells, convey and extrude them, provide accessory fluids, and insure copulation and insemination. In Aedes stimulans (Walker) these organs are derived from one of the two sets of primordia provided by the embryo. The second set of primordia is capable of producing the feminine reproductive system under unusual circumstances. Testes are derived from two compact ovoid masses of cells suspended in the hemocoel of abdominal segment 6. Each enlarges slowly throughout larval instars 1–3 and elongates very rapidly late in instar 4. Specialization of the cellular mass into sperm cells proceeds forward from the caudal end early in pupal life. From the beginning, a sheath of nutritive cells or fatbody encases each gonad, and no tracheation of the mass is evident although one small trachea sends branches to the encasing fatbody late in larval life. The efferent canal from each testis is derived from a tenuous filament extending caudally from each gonad to the venter of segment 9 and a small cluster of cells in the wall of the hemocoel on the ental surface of imaginal disc 9. Early in pupal life the filaments become the tubular vasa efferentia. The caudal clusters are primordial terminal parts of the lateral tract that become vasa deferentia, seminal vesicles and associated accessory glands. The ejaculatory canal comes from a short pouch derived from the median genital plate of disc 9. All external parts except the paraprocts are products of disc 9. The bilateral buds begin to proliferate in larval instar 4 and become the basistyles, dististyles and claspettes of the gonapophyses during pupal life. The phallosome is derived from the median genital plate. Primordia of a possible feminine reproductive system and cerci remain undifferentiated and disappear early in pupal life in the normal course of events. Primordia that were recognizable include those of ovaries, parts of lateral oviducts, median genital tract and cerci.     

The evagination of the genital imaginal discs ofDrosophila melanogaster

Summary The morphology of the evaginating female genital disc ofDrosophila melanogaster was examined at different stages of metamorphosis. The observations show that the internal genital organs are derived from the anterior half of the disc and that their morphogenesis is mainly a protrusion of the different primordial areas of the disc epithelium. The external genital and anal derivatives originate from the posterior half of the disc, which undergoes complex rearrangements during metamorphosis. The disc opens along the posterior margin and the dorsal and ventral epithelia evert and thereby completely reverse their anteroposterior orientation. Dramatic elongation has been observed during the formation of the seminal receptacle. The cells of the repressed male genital primordium do not form any recognizable structures and are assumed to be eliminated during metamorphosis.     

Activation of knot (kn) specifies the 3-4 intervein region in the Drosophila wing

Hedgehog (Hh) plays an important role in Drosophila wing patterning by inducing expression of Dpp, which serves to organize the wing globally across the A-P axis. We show here how Hh signalling also plays a direct role in patterning the medial wing through the activation of the Hh-target gene, knot (kn). kn is expressed in Hh-responsive cells near the A-P compartment boundary, where its expression is dependent on fu, a component of Hh signalling. kn is required for the proper positioning of veins 3 and 4 and to prevent ectopic venation between them. Furthermore, the expansion anteriorly of the normal kn expression domain causes an associated anterior shift in the position of vein 3 in the resultant wing. Ectopic expression of kn elsewhere in the wing imaginal disc results in the failure to properly activate the vein initiation genes, rho and Dl. Expression of the gene encoding the EGF-receptor (EGFR), which is required for vein initiation and subsequent differentiation, is normally depressed in the 3-4 intervein region. This downregulation of EGFR in the medial portion of the imaginal disc is dependent on kn activity and ectopic expression of kn inactivates EGFR elsewhere in the wing primordium. We propose kn expression in Hh-responsive cells of the wing blade anlagen during the late third instar creates a zone of cells in the medial wing in which vein primordia cannot be induced. The primordia for veins 3 and 4 are laid down adjacent to the kn-imposed vein-free zone, presumably by a signalling factor (such as Vn) also synthesized in the medial region of the wing.     

Sex determination genes control the development of the Drosophila genital disc, modulating the response to Hedgehog, Wingless and Decapentaplegic signals

In both sexes, the Drosophila genital disc contains the female and male genital primordia. The sex determination gene doublesex controls which of these primordia will develop and which will be repressed. In females, the presence of Doublesex(F) product results in the development of the female genital primordium and repression of the male primordium. In males, the presence of Doublesex(M) product results in the development and repression of the male and female genital primordia, respectively. This report shows that Doublesex(F) prevents the induction of decapentaplegic by Hedgehog in the repressed male primordium of female genital discs, whereas Doublesex(M) blocks the Wingless pathway in the repressed female primordium of male genital discs. It is also shown that Doublesex(F) is continuously required during female larval development to prevent activation of decapentaplegic in the repressed male primordium, and during pupation for female genital cytodifferentiation. In males, however, it seems that Doublesex(M) is not continuously required during larval development for blocking the Wingless signaling pathway in the female genital primordium. Furthermore, Doublesex(M) does not appear to be needed during pupation for male genital cytodifferentiation. Using dachshund as a gene target for Decapentaplegic and Wingless signals, it was also found that Doublesex(M) and Doublesex(F) both positively and negatively control the response to these signals in male and female genitalia, respectively. A model is presented for the dimorphic sexual development of the genital primordium in which both Doublesex(M) and Doublesex(F) products play positive and negative roles.     

Drosophila terminalia as an appendage-like structure.

In Drosophila, the homeotic gene Distal-less (Dll) has a fundamental role in the establishment of the identity of ventral appendages such as the leg and antenna. This study reports the expression pattern of Dll in the genital disc, the requirement of Dll activity for the development of the terminalia and the activation of Dll by the combined action of the morphogenetic signals Wingless (Wg) and Decapentaplegic (Dpp). During the development of the two components of the anal primordium - the hindgut and the analia - only the latter is dependent on Dll and hedgehog (hh) functions. The hindgut is defined by the expression of the homeobox gene even-skipped. The lack of Dll function in the anal primordia transforms the anal tissue into hindgut by the extension of the eve domain. Meanwhile targeted ectopic Dll represses eve expression and hindgut formation. The Dll requirement for the development of both anal plates in males and only for the dorsal anal plate in females, provides further evidence for the previously held idea that the analia arise from two primordia. In addition, evaluation was made of the requirement for the optomotor-blind (omb) gene which, as in the leg and antenna, is located downstream to Dpp. These results suggest that the terminalia show similar behaviour to the leg disc or the antennal part of the eye-antennal disc consistent with both the proposed ventral origin of the genital disc and the evolutive consideration of the terminalia as an ancestral appendage.