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     

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.     

Sex-specific control of growth and differentiation in the Drosophila genital disc, studied using a temperature-sensitive transformer-2 mutation

Mutations of the transformer-2 (tra-2) locus of Drosophila melanogaster cause chromosomally female (XX) animals to develop as males, but have no effect on the development of chromosomally male (XY) animals. In the female genital disc, such mutations cause repression of growth and inhibition of differentiation in the female genital primordium, while allowing growth and differentiation of the otherwise repressed male genital primordium. We used a temperature-sensitive mutation of this locus (tra-2^ts1) to switch development from one sexual pathway to the other. Following development at the male-determining temperature (29°C), subsequent culture of the XX;tra-2^ts1 genital disc in vivo at the female-determining temperature (16°C) allowed the previously repressed female genital primordium to develop and form female genital structures, whereas the formation of male genital elements was grossly disturbed. Conversely, following development at the female-determining temperature, subsequent culture in vivo at the male-determining temperature allowed the formerly repressed male genital primordium to grow and produce male genital structures, and repressed the formation of female elements from the already fully developed female genital primordium. The experiments indicate that the tra-2 product has to operate during the culture period in order to maintain the female state of sex determination, i.e., to promote the development of female structures, as well as to repress that of male structures. The experimental treatments, as well as the results of temperature shifts on developing larvae, resulted in sexual transformation of the anal plates, and clarified the sexual homologies of these structures. In both genitalia and analia, a switch from the female to the male developmental pathway was accomplished more rapidly and effectively than the reverse change.     

Gynandromorphs reveal two separate primordia for male and female genitalia inDrosophila melanogaster

Summary The derivatives of 110 mosaic genital discs of gynandromorphs have been analysed microscopically. It has been found that theanalia of both sexes are homologous and derive from a single primordium (see Fig. 1a). Whether male or female anal plates are formed depends on the genetic constitution of the cells. This is analogous to the development of male sex combs versus female transversal rows on the forelegs of gynandromorphs. In contrast, the data for thegenitalia (see Fig. 1 b) are best explained if it is assumed that there are two genital primordia in everyDrosophila embryo: a male primordium that will only develop into genitalia if populated by XY (or XO) nuclei, and a female primordium that will only do so if populated by XX nuclei. This model, as depicted in Figure 2, is compatible with all our gynandromorph data and also with observations onMusca andCalliphora where in fact two separate genital primordia are found.     

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     

Development of the Drosophila genital disc requires interactions between its segmental primordia

In both sexes, the Drosophila genital disc comprises three segmental primordia: the female genital primordium derived from segment A8, the male genital primordium derived from segment A9 and the anal primordium derived from segments A10-11. Each segmental primordium has an anterior (A) and a posterior (P) compartment, the P cells of the three segments being contiguous at the lateral edges of the disc. We show that Hedgehog (Hh) expressed in the P compartment differentially signals A cells at the AP compartment border and A cells at the segmental border. As in the wing imaginal disc, cell lineage restriction of the AP compartment border is defined by Hh signalling. There is also a lineage restriction barrier at the segmental borders, even though the P compartment cells of the three segments converge in the lateral areas of the disc. Lineage restriction between segments A9 and A10-11 depends on factors other than the Hh, En and Hox genes. The segmental borders, however, can be permeable to some morphogenetic signals. Furthermore, cell ablation experiments show that the presence of all primordia (either the anal or the genital primordium) during development are required for normal development of genital disc. Collectively, these findings suggest that interaction between segmental primordia is required for the normal development of the genital disc.     

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.     

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.     

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.     

Comparison of Postembryonic Organization of the Genital Segments in Trichoceridae, Tipulidae, and Anisopodidae (Diptera, Nematocera)

The number of abdominal segments in Tipulomorpha and Bibionomorpha larvae and aduts is discussed. For Nematocera, the most primitive number of abdominal segments in both male and female larvae is nine. Reduction of the IX abdominal segment and its subsequent fusion with the VIII segment occurs in different phyletic lines in Nematocera and might have evolved several times. In Trichocera spp. nine abdominal segments are present. In the genital segments the main interrelationships in the position of the anus and some main innervation areas, especially the ventral lobes, and the derivatives of the genital primordia were followed during postembryonic reorganization by studying variously stained serial sections from all developmental stages from the first larval instar to the adult stage. Homologies between male and female derivatives of the IX segment genital primordium were established for Trichoceridae. The trichoceroid male claspers and female ovipositor were found to be of sternal origin and highly specialized structures. They appear to be unique features of the Tipulomorpha. Postembryonic development in Limonia nubeculosa Meigen, 1804 and Sylvicola cinctu (Fab-ricius, 1787) was studied in the same way. In Limonia males the trichoceroid functional system for grasping is present. In Anisopodidae ( Sylvicola ), another functional system for grasping has been evolved by the male, which only includes primordial derivatives. In the adults, fusion of the VIII and IX segments prevents development of outer clasping organs or special structures for egg guidance.     

Gradual acquisition of the developmental capacity to differentiate adult structures by the genital disc of Drosophila melanogaster

Summary Diplo-X flies homozygous for the transform-er-2 ^ts (tra-2 ^ts) mutation develop into females at 16° C, while they develop into males at 29° C (Belote and Baker 1982). By means of this conditional mutation, we have carried out a detailed analysis of the development of the genital disc. Temperature shifts between 16 and 29° C, in both directions, and temperature pulses at 29° C, have been applied during the larval growth of tra-2 ^ts homozygous diplo-X flies, and the external derivatives of the genital disc have been analysed. Genital discs shifted from 16 to 29° C rapidly lose their capacity to differentiate female genital structures, while they become able to differentiate male genital structures whose inventory is more complete the earlier in larval development the temperature shift is carried out; moreover, duplicated male genital structures were observed. In the shift from 29 to 16° C, the genital disc loses its capacity to differentiate male genital structures, while it becomes able to differentiate female genital structures. The inventory of male structures is smaller, and the inventory of the female structures is more complete, the earlier in larval development the temperature is shifted. No duplicated female or male genital structures were observed in the downshift experiment. With respect to the analia, the shift from 16 to 29° C resulted in the quick formation of pure male anal plates, while in the opposite shift the formation of pure female anal plates occurred gradually. Moreover, the time course for the dorsal and ventral anal plates to show normal female phenotype was different: when the dorsal anal plates were completely normal, it was still possible to find incomplete ventral anal plates. In the pulse experiment at 29° C, the genital disc is able to differentiate both female and male genital structures, although the inventory of the latter ones was not complete. In addition, the capacity of the genital disc to differentiate male genital structures depended on the duration of the temperature pulse. The anal plates were always female, although they showed a reduction in their size, the ventral female anal plate being more affected than the dorsal one. No male anal plates were observed. The results have revealed that the genital disc follows a sequence in its capacity to differentiate female or male adult structures. We suggest that this sequence reflects the sequence of determination events occurring in the genital disc during its larval growth. In addition, results shown here provide evidence for the existence in the female genital primordium of a set of cells capable of giving rise either to female genital structures (ventral vaginal plates) or to male genital structures (hypandrium and penis apparatus). We also present evidence supporting the previous idea of two primordia for the anal plates.     

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.     

Development of the genitalia in Drosophila melanogaster

The imaginal discs of Drosophila melanogaster are an excellent material with which to analyze how signaling pathways and Hox genes control growth and pattern formation. The study of one of these discs, the genital disc, offers, in addition, the possibility of integrating the sex determination pathway into this analysis. This disc, whose growth and shape are sexually dimorphic, gives rise to the genitalia and analia, the more posterior structures of the fruit fly. Male genitalia, which develop from the ninth abdominal segment, and female genitalia, which develop mostly from the eighth one, display a characteristic array of structures. We will review here some recent findings about the development of these organs. As in other discs, different signaling pathways establish the positional information in the genital primordia. The Hox and sex determination genes modify these signaling routes at different levels to specify the particular growth and differentiation of male and female genitalia.     

The role of the transformer genes in the development of genitalia and analia of Drosophila melanogaster

The autosomal mutations transformer (tra) and transformer-2 (tra-2) of Drosophila convert chromosomal females (X/X) into phenotypical males. Our analysis aims at an understanding of the role which the transformer genes play in the development of the sexually dimorphic genital disc. In each Drosophila embryo, this disc starts development with a male and a female genital primordium, and an anal primordium. Our experiments involved the production of cell clones that were made homozygous for tra or tra-2 at different times of development. Homozygous clones were obtained by inducing mitotic recombination in three types of females heterozygous for tra or tra-2. The cells of the homozygous tra/tra or tra-2/tra-2 clones responded by changing from the female into the male pathway. Male genital structures developed if the clones were induced not later than 81 hr into development. In the analia, male clones appeared up to 120 hr. Our results show that the action of the wild-type alleles of tra⁺ and tra-2⁺ is required until late in larval development to repress the male genital primordium and to support development of the female primordium, as well as to maintain the anal primordium in the female pathway. Our data also suggest that the embryonic genital disc consists of two compartments, one containing the precursors for penis and analia, the other those of the male and female genitalia.     

Developmental genetics of the Drosophila gut: specification of primordia, subdivision and overt-differentiation.

The Drosophila gut is composed of three major parts, the foregut, midgut and hindgut, which arise from anterior and posterior invaginations of the early blastoderm. We review the process of the specification of the gut primordia, subsequent subdivision and region-specific cell differentiation in terms of developmental genetics. Graded activities of maternal signals at anterior and posterior terminal domains of the blastoderm, being mediated by activities of two zygotic gap genes, tailless and huckebein, lead to the activation of key genes that determine the gut primordia: serpent (GATA factor gene) for the endodermal midgut; brachyenteron (Brachyury homolog) for the ectodermal hindgut. fork head (HNF-3 homolog) and caudal (Cdx homolog) are also essential for the development of all gut primordia or hindgut primordium, respectively. Subdivision of the midgut epithelium is regulated by inductive signals emanating from the visceral mesoderm, which is under the control of HOM-C genes. In contrast, pattern formation of the ectodermal foregut and hindgut is regulated by secreted signaling molecules, such as Wingless (Wnt homolog), Hedgehog and Decapentaplegic (Bmp-4 homolog), as in the case of segmented structures and imaginal discs. Finally, the gut is subdivided into at least 36 compartments that are recognized asminimum tissue units of regional differentiation. A few genes that are responsible for determining and maintaining the state of overt-differentiation of the compartments have also been reported. A marked feature of the genetic mechanism of the gut development is the unexpectedly wide spectrum of the similarities of relevant genes and regulatory pathways of gene expression between Drosophila and vertebrates, which may imply a prototypic style of body plan common to protostomes and deuterostomes.     

Identification of a novel target of D/V signaling in Drosophila wing disc: Wg-independent function of the organizer

Growth and patterning during Drosophila wing development are mediated by signaling from its dorso-ventral (D/V) organizer. Wingless is expressed in the D/V boundary and functions as a morphogen to activate target genes at a distance. Wingless pathway and thereby D/V signaling is negatively regulated by the homeotic gene Ultrabithorax (Ubx) to mediate haltere development. In an enhancer-trap screen to identify genes that show differential expression between wing and haltere discs, we identified CG32062, which codes for a RNA-binding protein. In wing discs, CG32062 is expressed only in non-D/V cells. CG32062 expression in non-D/V cells is dependent on Notch-mediated signaling from the D/V boundary. However, CG32062 expression is independent of Wingless function, thus providing evidence for a second long-range signaling mechanism of the D/V organizer. In haltere discs, CG32062 is negatively regulated by Ubx. The non-cell autonomous nature of Ubx-mediated repression of CG32062 expression suggests that the novel component of D/V signaling is also negatively regulated during haltere specification.     

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.     

Comparative approach to developmental analysis: the case of the dalyellid flatworm, Gieysztoria superba

Dalyellida represents a taxon of small rhabdocoel flatworms that occur in freshwater habitats all over the world. Combining histology and electron microscopy we have analyzed the embryonic development of a new dalyellid species, Gieysztoria superba, in order to obtain more comparative data pertaining to morphogenesis and organogenesis in flatworms. We have used a morphological staging system that we recently introduced for another rhabdocoel, Mesostoma lingua (Younossi-Hartenstein et al., 2000). Our data show that in many fundamental respects, such as the irregular cleavage, mesenchymal embryonic primordium, and lack of gastrulation movements, Gieysztoria is highly similar to Mesostoma. During cleavage (stages 1 and 2) the embryo is located in the center of the egg where it is surrounded by a layer of yolk cells. Cleavage leads up to a solid, disc shaped cell cluster. During stage 3, the embryo migrates to the ventral side of the egg and acquires bilateral symmetry. Stages 4/5 sees the emergence of the first organ primordia, the brain, epidermis and pharynx. A peculiar invagination of the epidermal layer pushes the embryo back into the center of the yolk ("embryonic invagination"). Organogenesis takes place during stages 5 and 6 while the embryo is invaginated. A junctional complex, consisting initially of small septate junctions, followed later by a more apically located zonula adherens, is formed in all epithelial tissues, including epidermis, protonephridia, and pharynx. During late stages (6-8), Gieysztoria embryos evert back to the surface where the epidermal primordium expands and grows around the yolk to close dorsally. During this phase of development cytodifferentiation of the different organ systems takes place. Stage 7 is characterized by the appearance of eye pigmentation, brain condensation and spindle shaped myocytes. Stage 8 describes the fully dorsally closed and differentiated embryo. In comparison to other rhabdocoels, including Mesostoma, Gieysztoria exhibits a precocious differentiation of an intestinal epithelium and male genital apparatus. In Mesostoma, these structures are formed post hatching.