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.

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title: Sex comes in from the cold: the integration of sex and pattern.
abstract: There has recently been a revolution in our understanding of how the Drosophila sex-determination hierarchy generates somatic sexual dimorphism. Most significantly, the sex hierarchy has been shown to modulate the activities of well-known signaling molecules (FGF, Wnt and TGF beta proteins) and transcription factors (BAB and DAC) to direct various sex-specific aspects of growth and differentiation. As some of the genes encoding these proteins are also the targets of Hox gene action, these and other findings are revealing the levels at which the sex determination and Hox patterning pathways are integrated to control growth, morphogenesis and differentiation.

title: Development of the genitalia in Drosophila melanogaster.
abstract: 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.

title: Compartmental organization of the Drosophila genital imaginal discs.
abstract: 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.

title: Organising activities of engrailed, hedgehog, wingless and decapentaplegic in the genital discs of Drosophila melanogaster
abstract: 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.

title: Expression patterns of developmental genes reveal segment and parasegment organization of D. melanogaster genital discs
abstract: We have used the expression patterns of genes known to be important during early Drosophila development to determine the segment-parasegment organization of the genital discs and to localize the three primordia in the male and female genital discs, engrailed (en) and hedgehog (hh) were used to locate posterior compartments in A8-A10, while cubitus interrupts (ci) localized the anterior compartments for each segment, decapentaplegic (dpp) identified the anterior cells that abut en and hh at the anterior-posterior border. abdominal-A (abd-A) identified the anterior compartment for abdominal segment 8 (aA8) in females but was not detected in the repressed female primordium in male discs. Abdominal-B (Abd-B) was expressed throughout the discs except for a small area along the edge of the posterior lobes, leaving open the possibility that A11 may contribute to the genital discs, caudal (cad) was expressed segmentally in the anal primordium of A10, extending through the Abd-B unstained region, wingless (wg) and gooseberry (gsb) may have assumed an added role in the discs perhaps providing proximal-distal cues. Models are presented to show how the segments and parasegments may fuse together during embryogenesis to form the mature male and female genital discs.

title: The genital disc of Drosophila melanogaster : II. Role of the genes hedgehog, decapentaplegic and wingless.
abstract: The genital disc consists of three primordia: the female genital, the male genital and the anal primordia. In individuals of either sex, only one of the two genital primordia grows, whereas the anal primordium develops in both sexes. It is proposed here that the genital disc, which is of ventral origin, is organized in a manner similar to the antennal and leg discs: the expression domains of decapentaplegic (dpp) and wingless (wg) are mostly complementary and abut engrailed (en) expression. An analysis was made of the roles of the genes hedgehog (hh), patched (ptc), dpp and wg in the development of the three primordia that form the genital disc. The morphogenetic alterations produced by ectopic expression of hh mimics lack of ptc function. Both genetic conditions cause derepression of dpp and wg. Ectopic expression of either of these genes causes non-autonomous duplications and/or reductions of genital and anal structures. Some of these alterations are explained by the mutual repression of wg and dpp. In the development of the genital disc, the functional relationships between these genes seem to be analogous to those described for leg and antennal discs: dpp and wg are induced in the anterior compartment by Hh protein blocking the repressive effect of ptc, and the mutual repression of dpp and wg restrict each other to their domains. It may be concluded that dpp and wg act as general organizers for development of the genital disc.

title: Identification and characterization of a parasegment specific regulatory element of the abdominal-B gene of drosophila
abstract: We have characterized mutations of the Abdominal-B gene of the bithorax complex of Drosophila. We conclude that the gene contains two distinct genetic elements: one has a morphogenetic role and acts in parasegments 10, 11, 12, and 13, while the other acts on parasegment 14 and has primarily or exclusively a regulatory function. Evidence indicates that the latter suppresses the activity of the morphogenetic element of Abd-B and of other genes responsible for the development of sclerotic plates. The regulatory element also suppresses those BX-C genes and other homeotics that, in the absence of Polycomb or extra sex combs function, can become active in parasegment 14.

title: Transabdominal, a dominant mutant of the Bithorax Complex, produces a sexually dimorphic segmental transformation in Drosophila.
abstract: Transabdominal (Tab), a dominant mutation in the Bithorax Complex (BX-C) of Drosophila, creates a sexually dimorphic pattern of segmental transformation that has complete penetrance and expressivity. Specific regions within the notum of the second thoracic segment (T2) are transformed into abdominal-like cuticle; thus, the Tab/ + notum has sets of short stripes that are black in males and only bordered with black in females. Also, Tab/ + abdominal tergites, A1-A6, inclusive, have small patches of A7-like tergite cuticle. Tab is inseparable from an 89E/90D inversion, whose DNA breakpoint in 89E is at +188 kb in the infra-abdominal-8 (iab-8) region of the BX-C. When probed with a pupal cDNA from the iab-7 region, labeling above background was not detected in wild-type wing discs but was detected in, and confined to, the notal region of Tab/ + wing discs. The Tab/ + phenotype is assumed to result from cis-overexpression of iab-7 in localized regions of segments T2-A6, inclusive.

title: The molecular genetics of the bithorax complex of Drosophila: cis‐regulation in the Abdominal‐B domain.
abstract: In Drosophila the Abdominal‐B (Abd‐B) domain of the bithorax complex (BX‐C) spans over 100 kb and is responsible for specifying the identities of adult abdominal segments five (A5) to nine (A9), inclusive, and correspondingly, neuromeres 10–14 of the embryonic central nervous system. The domain consists of a region coding for two proteins, ABD‐BI (54 kd) and ABD‐BII (36 kd) and cis‐regulatory regions extending from infra‐abdominal‐5 (iab‐5) to iab‐9, inclusive. We have used a monoclonal anti‐ABD‐B antibody to infer that mutants in iab‐8 eliminate the expression of ABD‐BI in neuromeres 10–13, inclusive, and that mutants in iab‐9 eliminate expression of ABD‐BII in neuromere 14. ABD‐B expression is also analyzed in homozygotes for (i) loss‐of‐function mutants involving the iab‐5, iab‐6 and iab‐7 regions, (ii) gain‐of‐function mutants Miscadastral pigmentation (Mcp) and Superabdominal (Sab), and (iii) a trans‐regulator, Polycomb (Pc). ABD‐B expression along the antero‐posterior axis is colinear with the chromosomal order of the cis‐regulatory regions. The behavior of rearrangement‐associated iab‐6 and iab‐7 mutants suggests that the enhancer‐like region, iab‐5, and possibly also iab‐6, may be shared between the abd‐A and Abd‐B domains. Such sharing is proposed as a factor that tends to keep gene complexes intact during evolution.

title: The abdominal region of the bithorax complex
abstract: The homeotic mutations in the right half of the bithorax complex of Drosophila cause segmental transformations in the second through the eighth segments of the fly. A chromosomal walk in the bithorax complex has now been extended 215 kb through the right half of the complex, and lesions for over 40 mutations have been located on the DNA map. The mutations can be grouped in a series of phenotypic classes, one for each abdominal segment, although each mutation typically affects more than one segment. The mutant lesions of each class are clustered, and they are aligned on the chromosome in the order of the body segments that they affect. Complementation tests suggest interactions between widely spaced DNA regions; indeed, the right half cannot be split anywhere without some loss of function.

title: Recessive lethal mutations within the bithorax-complex in Drosophila
abstract: SummaryGenetic deficiencies of the bithorax-complex (BX-C) in Drosophila, have been used to recover recessive lethal mutations in this chromosome region following mutagenesis. Complementation analysis separates these lethal mutations into five groups within a smaller deficiency, though to remove the entire BX-C, and into 20 to the left and 4 to the right of the region. Homozygotes for each of only three groups of lethals, Ubx, abdA and AbdB, produce homoeotic segmental transformations in embryos. The functional domains of abdA and AbdB have been defined by changes in the appearance of larval hypodermal structures and of clones in imaginal tissue. The function abdA is required in all the compartments caudal to the anteroposterior border of abdominal segment 1 up to and including the anterior region of abdominal segment 8, whilst AbdB is required in abdominal segments 5 to 9. One allele of AbdB produces a ninth abdominal setal band and structures characteristic of head segments posterior to A8. Rare adult survivors hemizygous for an AbdB allele have eight abdominal segments in both sexes, and lack genitalia in females. Our findings are discussed in the context of the organisation of genetic functions within the BX-C.

title: Expression of the caudal gene in the germ line of Drosophila: Formation of an RNA and protein gradient during early embryogenesis
abstract: The caudal (cad) gene of Drosophila encodes a maternal and a zygotic transcript which have different promoters. Both mRNAs contain the same open reading frame, including a homeo box. In situ hybridization and antibody staining show that the maternal RNA and protein are localized in an anteroposterior gradient during the syncytial blastoderm stage. The protein is found mainly in the nuclei and is also present in the pole cells. Zygotic RNA and protein are localized in the primordia of the terminal abdominal segment, the hindgut, and in the posterior midgut rudiment. In third instar larvae, cad is expressed in the gut, the gonads, and parts of the genital discs. It is the first homeo box-containing gene expressed in the germ line of Drosophila.

title: Gonad formation and development requires the abd-A domain of the bithorax complex in Drosophila melanogaster.
abstract: The abdominal-A (abd-A) gene, a member of the bithorax complex, is required for the correct identity of parasegments (PS) 7 through 13. Mutations in iab-4, one of the cis-regulatory regions of abd-A, transform epidermal structures of PS 9 and also cause loss of gonads in adult flies. Here, we describe a developmental and molecular analysis of the role of iab-4 functions in gonadal development. In flies homozygous for a strong iab-4 allele, gonadogenesis is not initiated in the embryo because the mesodermal cells fail to encapsulate the pole cells. Flies homozygous for weaker iab-4 mutations sometimes form ovaries. The ovary-oviduct junctions are abnormal, however, and egg transfer from the ovary to the uterus is blocked in the adult. To localize the sites that require iab-4 function, we have analyzed animals chimeric for the mutant and wild-type cells. These chimeras were generated by three kinds of transplantation experiments: pole cells, embryonic somatic nuclei or larval ovaries. Our results suggest that iab-4 is required in the somatic cells of the gonadal primordia, but not the germ line. In addition, the formation of functional ovary-oviduct junctions and egg transfer also requires iab-4 functions in the somatic cells of the ovary and in at least one additional somatic tissue.

title: The Drosophila gene escargot encodes a zinc finger motif found in snail-related genes
abstract: Two independent P-element enhancer detection lines were obtained that express lacZ in a pattern of longitudinal stripes early in germband elongation. In this paper, molecular and genetic characterization of a gene located near these transposons is presented. Sequence analysis of a cDNA clone from the region reveals that this gene has a high degree of similarity with the Drosophila snail gene (Boulay et al., 1987). The sequence similarity extends over 400 nucleotides, and includes a region encoding five tandem zinc finger motifs (72% nucleotide identity; 76% amino acid identity). This region is also conserved in the snail homologue from Xenopus laevis (76% nucleotide identity; 83% amino acid identity) (Sargent and Bennett, 1990). We have named the Drosophila snail-related gene escargot (esg), and the region of sequence conservation common to all three genes the 'snailbox'. A number of Drosophila genomic DNA fragments cross-hybridize to a probe from the snailbox region suggesting that snail and escargot are members of a multigene family. The expression pattern of escargot is dynamic and complex. Early in germband elongation, escargot RNA is expressed in a pattern of longitudinal stripes identical to the one observed in the two enhancer detection lines. Later in development, escargot is expressed in cells that will form the larval imaginal tissues, escargot is allelic with l(2)35Ce, an essential gene located near snail in the genome.

title: Localization of thoracic imaginal-disc precursor cells in the early embryo of Drosophila melanogaster

abstract: Our previous cell lineage analysis of the thoracic disc primordia of Drosophila showed that at the blastoderm and early gastrula stage, cells are not yet committed to form either larval or imaginal tissue (Meise and Janning, 1993). We have now refined our studies on the cell lineage and have mapped the imaginal primordia in the thoracic region. Homotopic transplantations of single cells within the thoracic region of blastoderm and early gastrula stages show that the precursor cells of thoracic imaginal discs are locally restricted to a small lateral area of the thoracic region. Clones labelling leg discs frequently included the Keilin's organs. Heterotopic transplantations along the dorsoventral axis indicate that cells within the thoracic region are not yet committed with respect to larval or imaginal tissue, their fate being dependent on the position where the transplanted cell had been deposited. On the other hand, cells taken from the abdominal anlagen and transplanted into the region of thoracic disc primordia could not participate in the formation of imaginal discs. This shows that, in contrast to the dorsoventral axis, determinative events had separated primordia along the anterior-posterior axis.

title: When does determination occur in Drosophila embryos?
abstract: Small groups of blastoderm cells were transplanted from wild-type donor embryos into genetically marked host embryos of the same age. Donor cells were injected either into an homologous or an ectopic region of the recipient, and both donor and recipient embryos were allowed to develop. Donor flies were examined for defects in external structures. Recipients were scored for patches of donor-type marked tissue derived from the injected cells. After ectopic transfer, the donor cells recovered in chimaeric recipients differentiated structures consistent with the donor site of cell removal. No apparent fate change was observed. In the rare cases when both individuals of a donor/host pair survived, a direct correspondence could be made between the deleted region in the donor and the chimaeric patch in the host. The results show that blastoderm cells are stably determined to within a segment.

title: Twisted Perspective New Insights into Extracellular Modulation of BMP Signaling during Development
abstract: TGF-␤-like ligands, the Bone Morphogenetic Proteins (BMPs), is the direct modulation of ligand activity by extracellular factors (reviewed in De Robertis and Sasai, 1996; . In amphibians, opposing activities of BMP4 and its antagonist Chordin (Chd) are responsible for subdivision of the embryonic dorsal–ventral axis. A Brown University similar role for BMP4 and Chd orthologs has been impli-Providence, Rhode Island 02912 cated in dorsal–ventral patterning of the zebrafish embryo (Mullins, 1998). Thus, in the vertebrates, BMP activity specifies ventral fates in the early embryo, and is During the past several decades, considerable research antagonized by localized expression of Chd orthologs efforts have elucidated the central components of many dorsally. In Drosophila, the same relationship exists be-signal transduction pathways in animal systems. More tween the BMP homologs decapentaplegic (dpp) and recently attention has shifted to the importance of mod-screw (scw) and the Chd ortholog short gastrulation ulating intercellular signaling processes during develop-(sog), but the axis is inverted: dpp and scw specify dorsal ment, as it is clear that fine-tuned regulation of signaling fates in the embryo and are antagonized by sog, which systems in vivo results in subtle distinctions at the cellu-is expressed ventrally. In both vertebrates and inverte-lar level. Extracellular modulators have been a particular brates, the activity of Chd orthologs is antagonized by a focus of investigation, including proteins that affect re-family of metalloproteases including Drosophila Tolloid ceptor stability, ligand function, or ligand availability. In (Tld), Xenopus Xolloid (Xol), Human BMP-1, and their these latter types of regulation, proteins such as proteo-orthologs in other vertebrates. Biochemical studies on glycans, heparan sulfate modifying enzymes, and prote-the Xenopus, Drosophila, and mouse proteins have ases have been recognized as key players, as have demonstrated that Tld/Xol/BMP1 cleavage inactivates a diverse class of diffusible modulators that influence Sog/Chd and promotes BMP signaling (Marqué s et al., ligand activity by inhibiting or facilitating its function (see references contained within reviews by Capdevila Xol proteins function as BMP agonists. modulator that influences the availability of BMP ligands, on the Drosophila gene twisted gastrulation (tsg) and a family of the TGF-␤ superfamily of signaling molecules. its vertebrate orthologs reveal another level of complex-It appears that for TGF-␤ signaling in general a major ity in the extracellular modulation of BMP signaling dur-means of signal regulation is the modulation of ligand ing development. tsg was first identified in Drosophila as availability and as yet this system provides …

title: Signaling by the Drosophila epidermal growth factor receptor pathway during development.
abstract: In 1997 we wrote a review entitled "A thousand and one roles for the Drosophila epidermal growth factor (EGF) receptor (DER/EGFR)." We are not there yet in terms of the number of developmental roles assigned to this receptor in Drosophila. Nevertheless, DER has certainly emerged as one of the key players in development, since it is used repeatedly to direct cell fate choices, cell division, cell survival, and migration. A battery of activating ligands and an inhibitory ligand achieves this versatility. For the ligands that are produced as membrane-bound precursors, trafficking and processing are the key regulatory steps, determining the eventual temporal and spatial pattern of receptor activation. In most cases DER is activated at a short range, in the cells adjacent to the ones producing the active ligand. This activation dictates a binary choice. In some instances DER is also activated over a longer range, and multiple cell fate choices may be induced, according to its level of activation. A battery of negative feedback loops assures the limited range of DER induction. The distinct responses to DER activation in the different tissues depend upon combinatorial interactions with other signaling pathways and tissue-specific factors, at the level of target-gene regulation.

title: Divide and conquer: pattern formation in Drosophila embryonic epidermis.
abstract: The pattern of differentiated cell types within tissues and organs is often established by organizers, the localized sources of secreted ligands. Although the mechanisms underlying organizer function have been extensively studied, only in a few cases is it clear how an organizer ultimately controls each individual cell's fate across a field of progenitor cells. One of these cases involves the establishment of a precise pattern of cell differentiation across the embryonic epidermis in Drosophila. Here, we review several recent reports that help to elucidate the regulatory principles used to control this pattern. Because organizers are conserved, the same fundamental principles might operate in other organizers.

title: The Drosophila patched gene encodes a putative membrane protein required for segmental patterning
abstract: The patched (ptc) gene is one of several segment polarity genes required for correct patterning within every segment of Drosophila. The absence of ptc gene function causes a transformation of the fate of cells in the middle part of each segment so that they form pattern elements characteristic of cells positioned around the segment border. Analysis of the mutant phenotype demonstrates that both segment and parasegment borders are included in the duplicated pattern of ptc mutants. We have cloned the ptc gene and deduced that the product is a 1286 amino acid protein with at least seven putative transmembrane alpha helices. ptc RNA is expressed in embryos in broad stripes of segmental periodicity that later split into two stripes per segment primordium. The pattern of expression does not directly predict the transformation seen in ptc mutant embryos, suggesting that ptc participates in cell interactions that establish pattern within the segment.

title: The role of Wg signaling in the patterning of embryonic leg primordium in Drosophila.
abstract: Cellular interaction between the proximal and distal domains of the limb plays key roles in proximal-distal patterning. In Drosophila, these domains are established in the embryonic leg imaginal disc as a proximal domain expressing escargot, surrounding the Distal-less expressing distal domain in a circular pattern. The leg imaginal disc is derived from the limb primordium that also gives rise to the wing imaginal disc. We describe here essential roles of Wingless in patterning the leg imaginal disc. Firstly, Wingless signaling is essential for the recruitment of dorsal-proximal, distal, and ventral-proximal leg cells. Wingless requirement in the proximal leg domain appears to be unique to the embryo, since it was previously shown that Wingless signal transduction is not active in the proximal leg domain in larvae. Secondly, downregulation of Wingless signaling in wing disc is essential for its development, suggesting that Wg activity must be downregulated to separate wing and leg discs. In addition, we provide evidence that Dll restricts expression of a proximal leg-specific gene expression. We propose that those embryo-specific functions of Wingless signaling reflect its multiple roles in restricting competence of ectodermal cells to adopt the fate of thoracic appendages.

title: Hox genes and the evolution of the arthropod body plan 1
abstract: In recent years researchers have analyzed the expression patterns of the Hox genes in a multitude of arthropod species, with the hope of understanding the mechanisms at work in the evolution of the arthropod body plan. Now, with Hox expression data representing all four major groups of arthropods (chelicerates, myriapods, crustaceans, and insects), it seems appropriate to summarize the results and take stock of what has been learned. In this review we summarize the expression and functional data regarding the 10 arthropod Hox genes: labial, proboscipedia, Hox3/zen, Deformed, Sex combs reduced, fushi tarazu, Antennapedia, Ultrabithorax, abdominal‐A, and Abdominal‐B. In addition, we discuss mechanisms of developmental evolutionary change thought to be important for the emergence of novel morphological features within the arthropods.

title: Molecular definition of the morphogenetic and regulatory functions and the cis-regulatory elements of the Drosophila Abd-B homeotic gene.
abstract: The Abdominal-B (Abd-B) gene, a member of the Drosophila bithorax complex, is required during development to specify the identity of parasegments 10-14. Based on genetic studies, Casanova, J., Sánchez-Herrero, E. and Morata, G. (1986) Cell 47, 627-636, proposed that the Abd-B gene consists of two distinct elements that provide a morphogenetic (m) function in PS 10-13 and a regulatory (r) function in PS 14, where it represses m function. Here we present molecular confirmation of this genetic model. Using specific antibodies, we show that the 55 X 10(3) M(r) and 30 X 10(3) M(r) Abd-B proteins, predicted by cDNA analysis, are indeed present in PS 10-13 and PS 14, respectively. We also examine Abd-B mRNA and protein expression patterns in embryos mutant for either the m or r function. These data allow us to unambiguously assign m function to the 55 X 10(3) M(r) protein and r function to the 30 X 10(3) M(r) protein. Furthermore, as postulated by the model, transcription of the mRNA encoding the m protein is derepressed in PS 14 in the absence of r function. We have also studied the effect of mutations mapping in the infra-abdominal (iab) region located downstream of the Abd-B gene. Genetic studies suggest that the iab region contains cis-acting regulatory elements controlling Abd-B expression in PS 10-12. We present molecular evidence for the presence of downstream cis-regulatory elements by analyzing Abd-B mRNA and protein patterns in iab-6 and iab-7 embryos. Our analysis reveals the presence of parasegment and cell-specific regulatory elements of the Abd-B gene within each iab region. The Abd-B gene may provide a model for the understanding of similarly complex homeotic genes in higher organisms.

title: Different transcripts of the Drosophila Abd‐B gene correlate with distinct genetic sub‐functions.
abstract: Abdominal‐B (Abd‐B) is a homeotic selector gene required for the correct development of abdominal segments 5‐9. This gene contains at least two different genetic sub‐functions which act as ‘morphogenetic’ or ‘regulatory’ elements within a single lethal complementation group. In an effort to identify the molecular nature of these sub‐functions we have characterized homeobox‐containing transcripts of Abd‐B. The Abd‐B transcripts can be grouped into four major classes whose diversity arises through differential exon splicing. We have isolated cDNAs which represent three of the transcript classes, designated alpha, beta and gamma. Their expression patterns, coupled with previous molecular genetic analyses of Abd‐B, suggest that the alpha transcript class encodes the ‘morphogenetic’ sub‐function while the beta transcript class contributes to the ‘regulatory’ sub‐function.

title: abdA expression in Drosophila embryos.
abstract: The abdominal A (abdA) gene is one of three transcription units in the Bithorax Complex of Drosophila encoding a homeo box protein; it is flanked by Ultrabithorax (Ubx) and Abdominal B (AbdB). The abdA gene is required for segmental identity of the second through eighth abdominal segments. The transcription unit of abdA is approximately 20 kb long and encodes a protein of 330 amino acids. The abdA homeo box is almost identical to the homeo box of Ubx but is quite different from the AbdB homeo box. A polyclonal antibody to abdA protein stains embryonic nuclei in segments A1-A7 (parasegments 7-13). The iab-2, 3, and 4 mutant classes define positive cis-regulatory elements that induce expression of abdA in segments A2-A4 (parasegments 7-9), respectively. Once a pattern of abdA expression is turned on in a given parasegment, it remains on in the more posterior parasegments, so that the complex pattern of expression is built up in the successive parasegments. The abdA product appears to repress expression of Ubx whenever they appear in the same cell, but abdA is repressed by AbdB only in the eighth and ninth abdominal segments.

title: Expression and regulation of the abd-A gene of Drosophila.
abstract: We have developed a specific polyclonal antibody that recognizes the protein products of the abdominal-A (abd-A) gene, a member of the bithorax complex of Drosophila. The normal expression domain extends from parasegments 7 to 13, in good correspondence with previous genetic and molecular results. However, while the anterior border of expression is precisely demarcated by a parasegmental boundary, the posterior border does not coincide with a lineage boundary. Within the normal domain, the expression of abd-A shows intrametameric modulation; the amount of product is higher in posterior compartments and in the most anterior cells of the anterior compartments and then gradually decreases. We have examined the effect on abd-A expression of a number of mutations, some mapping within and others outside the abd-A transcription unit. Those mapping to the transcription unit eliminate or severely reduce the amount of abd-A antigen, while those mapping outside produce an abnormal distribution of abd-A protein. Finally, we show that the abd-A gene is down-regulated in part of the Abdominal-B (Abd-B) domain, precisely in those regions where the Abd-B gene is expressed at high levels.

title: The structure and expression of a hybrid homeotic gene
abstract: We describe the structure and expression of an exceptional bithorax complex mutation, C1. This deletion mutation removes major portions of both the Ultrabithorax (Ubx) and abdominal‐A (abd‐A) protein coding regions, yet retains many of the functions normally executed by these genes. We show that the ends of the C1 deletion map to analogous positions within the Ubx and abd‐A transcription units, and that the deletion chromosome gives rise to a set of novel fusion transcripts that may encode hybrid abd‐A/Ubx proteins. These fusion products are transcribed from the abd‐A promoter, but exhibit a novel spatial pattern of expression that combines aspects of the normal Ubx and abd‐A distributions. This pattern provides evidence for the existence of regulatory elements located in the 3′ region of the Ubx transcription unit that can act on the abd‐A promoter at a distance of at least 20 kb.

title: Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog

abstract: The segment polarity genes engrailed and wingless are expressed in neighboring stripes of cells on opposite sides of the Drosophila parasegment boundary. Each gene is mutually required for maintenance of the other's expression; continued expression of both also requires several other segment polarity genes. We show here that one such gene, hedgehog, encodes a protein targeted to the secretory pathway and is expressed coincidently with engrailed in embryos and in imaginal discs; maintenance of the hedgehog expression pattern is itself dependent upon other segment polarity genes including engrailed and wingless. Expression of hedgehog thus functions in, and is sensitive to, positional signaling. These properties are consistent with the non-cell autonomous requirement for hedgehog in cuticular patterning and in maintenance of wingless expression.

title: Conversion of lacZ enhancer trap lines to GAL4 lines using targeted transposition in Drosophila melanogaster.
abstract: Since the development of the enhancer trap technique, many large libraries of nuclear localized lacZ P-element stocks have been generated. These lines can lend themselves to the molecular and biological characterization of new genes. However they are not as useful for the study of development of cellular morphologies. With the advent of the GAL4 expression system, enhancer traps have a far greater potential for utility in biological studies. Yet generation of GAL4 lines by standard random mobilization has been reported to have a low efficiency. To avoid this problem we have employed targeted transposition to generate glial-specific GAL4 lines for the study of glial cellular development. Targeted transposition is the precise exchange of one P element for another. We report the successful and complete replacement of two glial enhancer trap P[lacZ, ry+] elements with the P[GAL4, w+] element. The frequencies of transposition to the target loci were 1.3% and 0.4%. We have thus found it more efficient to generate GAL4 lines from preexisting P-element lines than to obtain tissue-specific expression of GAL4 by random P-element mobilization. It is likely that similar screens can be performed to convert many other P-element lines to the GAL4 system.

title: Autosomal P[ovoD1] dominant female-sterile insertions in Drosophila and their use in generating germ-line chimeras.
abstract: The 'dominant female-sterile' technique used to generate germ-line mosaics in Drosophila is a powerful tool to determine the tissue specificity (germ line versus somatic) of recessive female-sterile mutations as well as to analyze the maternal effect of recessive zygotic lethal mutations. This technique requires the availability of germ-line-dependent, dominant female-sterile (DFS) mutations that block egg laying but do not affect viability. To date only one X-linked mutation, ovoD1 has been isolated that completely fulfills these criteria. Thus the 'DFS technique' has been largely limited to the X-chromosome. To extend this technique to the autosomes, we have cloned the ovoD1 mutation into a P-element vector and recovered fully expressed P[ovoD1] insertions on each autosomal arm. We describe the generation of these P[ovoD1] strains as well as demonstrate their use in generating germ-line chimeras. Specifically, we show that the Gap1 gene, which encodes a Drosophila homologue of mammalian GTPase-activating protein, is required in somatic follicle cells for embryonic dorsoventral polarity determination.

title: Imaging neuronal subsets and other cell types in whole-mount Drosophila embryos and larvae using antibody probes.
abstract: Publisher Summary This chapter discusses the immunocytochemical procedures used to observe specific tissues and cells within whole-mount preparations of Drosophila embryos and young larvae. A special emphasis is placed on providing examples of antibodies and techniques used to examine the patterns of neurons and axons within the developing nervous system. These methods have two primary applications in the study of Drosophila development. First, once a gene of interest has been cloned and antibodies have been raised to the protein product, these immunocytochemical techniques can be used to determine the pattern of protein distribution during embryogenesis and larval development. Second, antibody probes that recognize defined tissues and cells can be used as markers to analyze phenotypes in mutant embryos. The protocol discussed in the chapter, the secondary antibodies are produced in goats and normal goat serum is used as a blocking agent. This ensures that the secondary antibodies will not recognize any of the immunoglobulins in the normal serum being used as a blocking agent. The procedures outlined in this chapter also work well for immunostaining a wide variety of organisms other than Drosophila.

title: Sex-specific apoptosis regulates sexual dimorphism in the Drosophila embryonic gonad.
abstract: Sexually dimorphic development of the gonad is essential for germ cell development and sexual reproduction. We have found that the Drosophila embryonic gonad is already sexually dimorphic at the time of initial gonad formation. Male-specific somatic gonadal precursors (msSGPs) contribute only to the testis and express a Drosophila homolog of Sox9 (Sox100B), a gene essential for testis formation in humans. The msSGPs are specified in both males and females, but are only recruited into the developing testis. In females, these cells are eliminated via programmed cell death dependent on the sex determination regulatory gene doublesex. Our work furthers the hypotheses that a conserved pathway controls gonad sexual dimorphism in diverse species and that sex-specific cell recruitment and programmed cell death are common mechanisms for creating sexual dimorphism.

title: Establishment of ventral cell fates in the Drosophila embryonic ectoderm requires DER, the EGF receptor homolog.
abstract: The embryonic ectoderm in Drosophila displays a highly organized arrangement of specific structures along the dorsal-ventral axis. To establish this characteristic design, cells must receive instructive cues regarding their position. We present evidence that during stages 8-9 of embryonic development, the Drosophila EGF receptor homolog (DER) is essential for determining the identity of cells within the ventral ectoderm. In the absence of DER activity at this phase, alterations in cell fate are observed: Ventral cells acquire more dorsal fates, as visualized by the expression profile of specific markers. The ventralizing effect of DER appears to function later than that of the dorsalizing dpp pathway, and the spatial overlap between them is minimal. A model for the determination of cell fates along the dorsal-ventral axis involving the two pathways is presented. Some aspects of the mutant ectodermal and CNS phenotypes of the DER locus (faint little ball, flb) resemble the phenotype of mutations from the spitz group. Synergistic interactions between flb and spitz or Star mutations suggest that these genes participate in a common signaling pathway.

title: The argos gene encodes a diffusible factor that regulates cell fate decisions in the drosophila eye
abstract: The argos gene encodes a protein that is required for viability and that regulates the determination of cells in the Drosophila eye. A developmental analysis of argos mutant eyes indicates that the mystery cells, which are usually nonneuronal, are transformed into extra photoreceptors, and that supernumerary cone cells and pigment cells are also recruited. Clonal analysis indicates that argos acts nonautonomously and can diffuse over the range of several cell diameters. Conceptual translation of the argos gene suggests that it encodes a secreted protein.

title: The Drosophila embryonic midline is the site of Spitz processing, and induces activation of the EGF receptor in the ventral ectoderm.
abstract: The Drosophila EGF receptor (DER) is activated by secreted Spitz to induce different cell fates in the ventral ectoderm. Processing of the precursor transmembrane Spitz to generate the secreted form was shown to be the limiting event, but the cells in which processing takes place and the mechanism that may generate a gradient of secreted Spitz in the ectoderm were not known. The ectodermal defects in single minded (sim) mutant embryos, in which the midline fails to develop, suggested that the midline cells contribute to patterning of the ventral ectoderm. This work shows that the midline provides the site for Spitz expression and processing. The Rhomboid and Star proteins are also expressed and required in the midline. The ectodermal defects of spitz, rho or Star mutant embryos could be rescued by inducing the expression of the respective normal genes only in the midline cells. Rho and Star thus function non-autonomously, and may be required for the production or processing of the Spitz precursor. Secreted Spitz is the only sim-dependent contribution of the midline to patterning the ectoderm, since the ventral defects observed in sim mutant embryos can be overcome by expression of secreted Spitz in the ectoderm. While ectopic expression of secreted Spitz in the ectoderm or mesoderm gave rise to ventralization of the embryo, increased expression of secreted Spitz in the midline did not lead to alterations in ectoderm patterning. A mechanism for adjustment to variable levels of secreted Spitz emanating from the midline may be provided by Argos, which forms an inhibitory feedback loop for DER activation. The production of secreted Spitz in the midline, may provide a stable source for graded DER activation in the ventral ectoderm.

title: Inhibition of Drosophila EGF receptor activation by the secreted protein Argos
abstract: THE Drosophila homologue of the mammalian epidermal growth factor (EGF) receptor (DER)1,2 is a receptor tyrosine kinase involved in many stages of fly development, including photoreceptor determination, and wing-vein formation39. Its primary activating ligand is the Spitz protein10,11, which is similar to mammalian TGF-α (ref. 12). Argos is a secreted protein that, like Spitz, contains a single EGF motif1315. It is a represser of cell determination in the eye, and acts in other tissues, including the wing1618. Because Argos has the opposite effects to DER in the eye (the former blocks photoreceptor determination, the latter promotes it) we have tested whether it acts by blocking the DER pathway. We show that Argos does indeed repress this pathway in vivo and find that, in vitro, Argos protein can inhibit the activation of DER by Spitz. Thus the determination of cells by the DER pathway is regulated by a balance between extracellular activating and inhibiting signals. This is the first in vivo example of an extracellular inhibitor of a receptor tyrosine kinase.

title: Specification of the embryonic limb primordium by graded activity of Decapentaplegic.
abstract: Two thoracic limbs of Drosophila, the leg and the wing, originate from a common cluster of cells that include the source of two secreted signaling molecules, Decapentaplegic and Wingless. We show that Wingless, but not Decapentaplegic, is responsible for initial specification of the limb primordia with a distal identity. Limb formation is restricted to the lateral position of the embryo by negative control of the early function of Decapentaplegic and the EGF receptor homolog that determine the global dorsoventral pattern. Late function of Decapentaplegic locally determines two additional cell identities in a dosage dependent manner. Loss of Decapentaplegic activity results in a deletion of the proximal structures of the limb, which is in contrast to the consequence of decapentaplegic mutations in the imaginal disc, which cause a deletion of distal structures. The results indicate that the limb pattern elements are added in a distal to proximal direction in the embryo, which is opposite to what is happening in the growing imaginal disc. We propose that Wingless and Decapentaplegic act sequentially to initiate the proximodistal axis.

title: 
 decapentaplegic acts as a morphogen to organize dorsal-ventral pattern in the Drosophila embryo
abstract: Zygotic expression of the Drosophila TGF beta family member decapentaplegic (dpp) is required for the development of the dorsal embryonic structures. By injecting dpp transcripts into young embryos, we find that 2- to 4-fold increases in the concentration of injected RNA elicit progressively more dorsal cell fates: only low levels of dpp permit development of ventral ectoderm, intermediate dpp levels drive dorsal epidermal development, and high dpp levels drive cells to differentiate as the most dorsal pattern element, the amnioserosa. Localized dpp RNA injections into embryos that lack all known maternal and zygotic dorsal-ventral polarity indicate that dpp can both define embryonic polarity and organize detailed patterning within the ectoderm. We infer that dpp acts as an extracellular morphogen and that the graded activity of dpp specifies the pattern of ectodermal cell fates in the Drosophila embryo.

title: Direct and Long-Range Action of a DPP Morphogen Gradient
abstract: During development of the Drosophila wing, the decapentaplegic (dpp) gene is expressed in a stripe of cells along the anteroposterior compartment boundary and gives rise to a secreted protein that exerts a long-range organizing influence on both compartments. Using clones of cells that express DPP, or in which DPP receptor activity has been constitutively activated or abolished, we show that DPP acts directly and at long range on responding cells, rather than by proxy through the short-range induction of other signaling molecules. Further, we show that two genes, optomotor-blind and spalt are transcriptionally activated at different distances from DPP-secreting cells and provide evidence that these genes respond to different threshold concentrations of DPP protein. We propose that DPP acts as a gradient morphogen during wing development.

title: Allocation of the thoracic imaginal primordia in the Drosophila embryo.
abstract: The primordia of the thoracic imaginal discs of the Drosophila embryo originate as groups of cells spanning the parasegment boundary. We present evidence that the thoracic imaginal primordia are allocated in response to signals from the wingless (wg) and decapentaplegic (dpp) gene products. Rows of cells that express wg intersect rows of cells that express dpp to form a ladder-like pattern in the ectoderm of the germ band extended embryo. The imaginal primordia originate as groups of cells which lie near these intersection points. We have used a molecular probe derived from the Distal-less (Dll) gene to show that this population contains progenitor cells for both the dorsal (i.e. wing) and ventral (i.e. leg) discs. Although we show that Dll function is not required for allocation of imaginal cells, activation of an early Dll enhancer may serve as a molecular marker for allocation. A group of cells, which includes the imaginal progenitors, activate this enhancer in response to intercellular signals from wg and perhaps from dpp. We have used a conditional allele of wg to show that wg function is transiently required for both allocation of the imaginal primordia and for initiation of Dll expression in these cells during the brief interval when wg and dpp form the ladder-like pattern. Allocation of the imaginal primordium and activation of Dll expression appear to be parallel responses to a single set of positional cues.

title: Imaginal discs can be recovered from cultured embryos mutant for the segment-polarity genes engrailed, naked and patched but not from wingless.
abstract: Drosophila embryos homozygous for strong mutations in each of the segment-polarity genes wingless (wg), engrailed (en), naked (nkd) and patched (ptc) form a larval cuticle in which there is a deletion in every segment. The mutant embryos normally fail to hatch but by in vivo culture we were able to show which could produce adult structures. Cultured wg⁻ embryos did not produce any adult structures. Cultured en⁻ embryos produced eye-antennal derivatives and rarely produced partial thoracic structures. nkd⁻ and ptc⁻ embryos produced eye-antennal and thoracic derivatives. The nkd⁻ and ptc⁻ thoracic imaginal discs developed with an abnormal morphology and abnormal pattern of en-expression. Our findings are consistent with the idea that the thoracic imaginal discs derive from two adjacent groups of cells that express wg and en respectively in the embryo.

title: The Drosophila homology of the mouse mammary oncogene int-1 is identical to the segment polarity gene wingless

abstract: We have isolated the Drosophila melanogaster homolog (Dint-1) of int-1, a conserved cellular oncogene implicated in viral mammary tumorigenesis in mice. The deduced Dint-1 protein sequence contains 468 amino acids and starts with a hydrophobic leader; it is 54% identical to the int-1 sequence, and all 23 cysteine residues are conserved. The putative Drosophila protein has an extra sequence of 85 amino acids, encoded on an additional exon. Dint-1 is expressed throughout development, but transcripts are barely detectable in adult flies. Hybridization in situ to embryos reveals a segmented pattern of expression. We show that Dint-1 and the segment polarity gene wingless are identical and map to the same location. The sequence of the gene suggests that the Dint-1/wingless protein functions in morphogenesis as a signal in cell-cell communication.

title: Regulation of wingless transcription in the Drosophila embryo.
abstract: The segment polarity gene wingless (wg) is expressed in a complex pattern during embryogenesis suggesting that it plays multiple roles in the development of the embryo. The best characterized of these is its role in cell pattening in each parasegment, a process that requires the activity of other segment polarity genes including patched (ptc) and hedgehog (hh). Here we present further evidence that ptc and hh encode components of a signal transduction pathway that regulate the expression of wg transcription following its activation by pair-rule genes. We also show that most other aspects of wg expression are independent of this regulatory network.

title: Ectopic expression of homeotic genes caused by the elimination of the Polycomb gene in Drosophila imaginal epidermis.
abstract: The morphological patterns in the adult cuticle of Drosophila are determined principally by the homeotic genes of the bithorax and Antennapedia complexes. We find that many of these genes become indiscriminately active in the adult epidermis when the Pc gene is eliminated. By using the Pc3 mutation and various BX-C mutant combinations, we have generated clones of imaginal cells possessing different combinations of active homeotic genes. We find that, in the absence of BX-C genes, Pc- clones develop prothoracic patterns; this is probably due to the activity of Sex combs reduced which overrules Antennapedia. Adding contributions of Ultrabithorax, abdominal-A and Abdominal-B results in thoracic or abdominal patterns. We have established a hierarchical order among these genes: Antp less than Scr less than Ubx less than abd-A less than Abd-B. In addition, we show that the engrailed gene is ectopically active in Pc- imaginal cells.

title: The Abdominal‐B gene of Drosophila melanogaster: overlapping transcripts exhibit two different spatial distributions
abstract: The Abdominal‐B (Abd‐B) gene of Drosophila controls the specification of segment identities in the posterior abdomen. We describe here the spatial and temporal distribution of Abd‐B transcripts. At least five RNA species are detected on Northern blots with probes within the region of the Abd‐B homeobox. We have identified probes specific for subsets of these transcripts and have used these probes to study the distribution of each subset by in situ hybridization to embryonic tissue sections. The transcripts can be divided into two groups: one group is expressed maximally in parasegment (PS)13 and extends anteriorly to PS10; the other is expressed only in PS14 and 15. These two different patterns of expression correspond to the anatomical domains defined by two classes of mutations in Abd‐B. These results help explain the complex genetic interactions and phenotypes of mutations within this gene.

title: Development of the D. melanogaster caudal segments involves suppression of the ventral regions of A8, A9 and A10.
abstract: Whereas the segmental organization of the thorax and anterior abdomen is morphologically delineated in both the Drosophila larva and adult, segments in the head and caudal regions lack such well-defined boundaries. Consequently, the organization of these regions has been difficult to decipher. In this study, transformations caused by the bithorax-complex homeotic mutants 48, M3, Ultraabdominal-1 (Uab1) and tumorous-head-3 (tuh-3), as well as the patterns of engrailed gene expression have been analyzed to investigate the segmental organization of the caudal segments. A special emphasis was placed on sense organs appearing in abdominal segments 8, 9 and 10 (A8-A10): We find that: (1) transformations in the caudal segments obey parasegmental borders; (2) the sense organs on A8, A9, and A10 are probably homologous to the pits and hairs in anterior A1-A7; (3) except for the larval anal tuft and the anterior side of A8, all structures in larval segments A8, A9 and A10 are dorsal/lateral in origin; and (4) dorsalization of embryonic A8 and A9 cells leaves space ventrally for A10, as it follows the contracting ventral nervous system during the embryological process of germ band contraction.

title: The genital disc of Drosophila melanogaster. : I. Segmental and compartmental organization.
abstract: 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.

title: Development of embryonic pattern in D. melanogaster as revealed by accumulation of the nuclear engrailed protein
abstract: Engrailed is required to establish and maintain developmental compartments within each segment of the fly. To understand the role of the engrailed protein in this process, we have raised antibodies against engrailed and have visualized an engrailed protein in embryos by indirect immunofluorescence. The protein accumulates in the nucleus, supporting the notion that engrailed is a regulatory factor. The first pattern of expression is in alternating segments followed by expression in every segment, suggesting that engrailed may be responding to pair-rule segmentation gene products. Overall, engrailed protein levels peak in areas undergoing morphogenesis. Finally, the complex final form of the head and terminalia derive from earlier simple subdivision of these areas into developmental fields by engrailed.

title: Molecular organization and embryonic expression of the hedgehog gene involved in cell-cell communication in segmental patterning of Drosophila.
abstract: hedgehog is a segment polarity gene necessary to maintain the proper organization of each segment of the Drosophila embryo. We have identified the physical location of a number of rearrangement breakpoints associated with hedgehog mutations. The corresponding hh RNA is expressed in a series of segmental stripes starting at cellular blastoderm in the posterior portion of each segment. This RNA is localized predominantly within nuclei until stage 10, when the localization becomes primarily cytoplasmic. Expression of hh RNA in the posterior compartment is independent of most other segment polarity genes, including en, until the late extended germ-band stage (stage 11). Sequence analysis of the hedgehog locus suggests the protein product is a transmembrane protein, which may, therefore, be directly involved in cell-cell communication.

title: The embryonic origin of imaginal discs in Drosophila.
abstract: The thoracic imaginal discs of Drosophila melanogaster can be observed during embryogenesis as clusters of cells with particular shapes, sizes and behaviours. These structures can be detected soon after germ band shortening and their development appears to be tightly linked to that of the larval epidermis.

title: Morphogens, Compartments, and Pattern: Lessons from Drosophila?
abstract: We thank Javier Sampedro for planning Figure 4Figure 4 and Dan Barbash, Paul Vincent for help with the manuscript. The advice and support of Hugh Pelham and Jim Smith is much appreciated.

title: The role of buttonhead and Sp1 in the development of the ventral imaginal discs of Drosophila
abstract: The related genes buttonhead (btd) and Drosophila Sp1 (the Drosophila homologue of the human SP1 gene) encode zinc-finger transcription factors known to play a developmental role in the formation of the Drosophila head segments and the mechanosensory larval organs. We report a novel function of btd and Sp1: they induce the formation and are required for the growth of the ventral imaginal discs. They act as activators of the headcase (hdc) and Distal-less (Dll) genes, which allocate the cells of the disc primordia. The requirement for btd and Sp1 persists during the development of ventral discs: inactivation by RNA interference results in a strong reduction of the size of legs and antennae. Ectopic expression of btd in the dorsal imaginal discs (eyes, wings and halteres) results in the formation of the corresponding ventral structures (antennae and legs). However, these structures are not patterned by the morphogenetic signals present in the dorsal discs; the cells expressing btd generate their own signalling system, including the establishment of a sharp boundary of engrailed expression, and the local activation of the wingless and decapentaplegic genes. Thus, the Btd product has the capacity to induce the activity of the entire genetic network necessary for ventral imaginal discs development. We propose that this property is a reflection of the initial function of the btd/Sp1 genes that consists of establishing the fate of the ventral disc primordia and determining their pattern and growth.

title: The engrailed locus of drosophila: In situ localization of transcripts reveals compartment-specific expression
abstract: The engrailed locus plays a unique and critical role in organizing the segmented body plan of Drosophila. Embryos lacking engrailed function die with fused, abnormal segments. Adult mosaics with patches of engrailed cells similarly suffer defects in all of their segments, but only with mutant cells that are in the posterior developmental compartment of each segment. The non-uniform requirement for engrailed function reflects the position-dependent expression of the engrailed locus and we demonstrate it here unambiguously by directly visualizing engrailed transcripts in frozen sections of embryos and larvae and in whole imaginal discs. These results demonstrate that developmental compartments subdivide the embryonic insect segments. In these and in the compartments of the later developmental stages the engrailed locus is expressed in the posterior but not the anterior compartments. With its role in controlling the developmental pathway of the posterior compartment cells, the engrailed locus may be an example of a binary developmental switch.

title: Compartments in the wing of Drosophila: a study of the engrailed gene.
abstract: It has recently been suggested that the wildtype alleles of homeotic genes are responsible for controlling the development of compartments. Because the mutation engrailed gives the posterior wing compartment anterior characteristics, it can be regarded as such a homeotic gene. Our experiments confirm the role of the engrailed gene in development of the posterior wing compartment, results which strongly support and extend the compartment hypothesis. Clonal analysis reveals that the state of the engrailed gene is immaterial to the entire anterior compartment, and crucial to the normal development of the posterior compartment, where it controls the pattern of veins and bristles. The presence of a straight and precisely positioned compartment border is dependent on the activity of the engrailed gene until late in development. We suggest that this is due to the gene's effects on cell affinities of the posterior compartment. The engrailed mutation increases the size and changes the shape of the posterior compartment. engrailed clones cause local wing enlargement only if they are dorsal and include the posterior margin of the wing. Wildtype cells outside the clone contribute to this change of shape. This result suggests that the postero-dorsal margin is primarily responsible for the control of shape, and that the ventral compartment is, to some extent, modeled on the dorsal.

title: Analysis of the genes involved in organizing the tail segments of the Drosophila melanogaster embryo
abstract: The metameric organization of the Drosophila melanogaster tail is obscured by developmental events that partially suppress or fuse some of its regions. To better define the developmental origins and segmental identities in the tail of the Drosophila embryo, we documented expression patterns and mutant phenotypes of several genes that play important roles in its morphogenesis. We documented the domains of engrailed (en), Abdominal-B (Abd-B) and caudal (cad) expression in the tail region. The staining pattern of cut (ct) was used to correlate the embryonic sense organs with their respective positions on the larval cuticle. The en patterns in different Bithorax-Complex (BX-C) Abd-B morphogenetic (m) and regulatory (r) mutants demonstrated that Abd-B functions to, among other things, suppress embryonic ventral epidermal structures on the posterior side of A8 to A9. Ventral epidermal structures were not added back into the en pattern in r- or BX-C- mutants, indicating that although the BX-C functions extend through A10, other non-BX-C genes must be required for development of this segment.

title: Specification, migration and assembly of the somatic cells of the Drosophila gonad.
abstract: The adult ovaries and testes contain several specialized somatic cell types that support the differentiation of germ cells into mature gametes. Each of these cell types arise from mesodermal cells that constitute the embryonic gonad. To explore the mechanisms governing the development and differentiation of these cells, we focus on the formation of the gonad during Drosophila development. Using markers for the precursors of the somatic cells of the gonad, we identify discrete steps in the development of the gonad. Our results suggest the existence of different populations of gonadal precursors at early stages in gonadogenesis that represent precursors of cell types found within the adult gonad. The functions of the homeotic genes abdominal A and Abdominal B are required for the development of gonadal precursors, however, here we provide evidence that each plays a distinct role. abd A activity alone specifies anterior gonadal precursor fates, whereas abd A and Abd B act together to specify a posterior subpopulation of gonadal precursors. Once specified, gonadal precursors born within posterior parasegments must move to the site of gonad formation. Here, we show that the proper regional identities, as established by homeotic gene function, are required for the arrest of migration at the correct position. Finally, our analysis of late stages of gonadogenesis suggests that abd A is required in a population of cells within parasegments 10 and 11 that partially ensheath the coalescing gonad. Mutations in iab-4 abolish expression of abd A within these cells, and as a result block the coalescence of the gonad.

title: Maintenance of the engrailed expression pattern by Polycomb group genes in Drosophila.
abstract: The stable maintenance of expression patterns of homeotic genes depends on the function of a number of negative trans-regulators, termed the Polycomb (Pc) group of genes. We have examined the pattern of expression of the Drosophila segment polarity gene, engrailed (en), in embryos mutant for several different members of the Pc group. Here we report that embryos mutant for two or more Pc group genes show strong ectopic en expression, while only weak derepression of en occurs in embryos mutant for a single Pc group gene. This derepression is independent of two known activators of en expression: en itself and wingless. Additionally, in contrast to the strong ectopic expression of homeotic genes observed in extra sex combs- (esc-) mutant embryos, the en expression pattern is nearly normal in esc- embryos. This suggests that the esc gene product functions in a pathway independent of the other genes in the group. The data indicate that the same group of genes is required for stable restriction of en expression to a striped pattern and for the restriction of expression of homeotic genes along the anterior-posterior axis, and support a global role for the Pc group genes in stable repression of activity of developmental selector genes.

title: Ten different Polycomb group genes are required for spatial control of the abdA and AbdB homeotic products.
abstract: Mutations in genes of the Polycomb (Pc) group cause abnormal segmental development due to ectopic expression of the homeotic products of the Antennapedia and bithorax complexes. Here the requirements for Pc group genes in controlling the abdA and AbdB products of the bithorax complex are described. Embryos containing mutations in the genes Polycomb (Pc), extra sex combs (esc), Enhancer of zeste [E(z)], polyhomeotic (ph), Sex comb on midleg (Scm), Polycomb-like (Pcl), Sex comb extra (Sce), Additional sex combs (Asx), Posterior sex combs (Psc) and pleiohomeotic (pho) were examined. In every case, both abdA and AbdB are expressed outside of their normal domains along the anterior-posterior (A-P) axis, consistent with these Pc group products acting in a single pathway or molecular complex. The earliest detectable ectopic expression is highest in the parasegments immediately adjacent to the normal expression boundary. Surprisingly, in the most severe Pc group mutants, the earliest ectopic AbdB is distributed in a pair-rule pattern. At all stages, ectopic abdA in the epidermis is highest along the anterior edges of the parasegments, in a pattern that mimics the normal abdA cell-specific pattern. These examples of highly patterned mis-expression show that Pc group mutations do not cause indiscriminate activation of homeotic products. We suggest that the ectopic expression patterns result from factors that normally activate abdA and AbdB only in certain parasegments, but that in Pc group mutants these factors gain access to regulatory DNA in all parasegments.

title: Visualization of Gene Expression in Living Adult Drosophila
abstract: To identify genes involved in the patterning of adult structures, Gal4-UAS (upstream activating site) technology was used to visualize patterns of gene expression directly in living flies. A large number of Gal4 insertion lines were generated and their expression patterns were studied. In addition to identifying several characterized developmental genes, the approach revealed previously unsuspected genetic subdivisions of the thorax, which may control the disposition of pattern elements. The boundary between two of these domains coincides with localized expression of the signaling molecule wingless.

title: Hox genes, homeosis and the evolution of segment identity: no need for hopeless monsters.
abstract: Significant changes have occurred in the developmental role of Hox genes, even within groups of arthropods that already have complex body plans and many different segment types. This is hard to reconcile with the 'selector gene' model for Hox gene function. Selector genes act as stable binary switches that direct lineages of cells to adopt alternative developmental fates. This model suggests that the regulation of selector genes can only evolve through mutations that alter the identity of whole developmental compartments -in the case of Hox genes, whole segments. Once segments have evolved distinct morphology and function, such mutations will result in dramatic homeotic transformations that are unlikely to be tolerated by natural selection. Thus we would expect the developmental role of these "master control genes" to become frozen as body plans become more complex. I argue for a revised model for the role and regulation of the Hox genes. This provides alternative mechanisms for evolutionary change, that may lead to incremental changes in segment morphology. The summation of such changes over long periods of time would result in differences in Hox gene function between taxa comparable to the effects of gross homeotic mutations, without the need to invoke the selective advantage of hopeful monsters.

title: Drosophila terminalia as an appendage-like structure
abstract: 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.

title: Genetic control and evolution of sexually dimorphic characters in Drosophila
abstract: Sexually dimorphic abdominal pigmentation and segment morphology evolved recently in the melanogaster species group of the fruitfly Drosophila . Here we show that these traits are controlled by the bric-a-brac (bab) gene, which integrates regulatory inputs from the homeotic and sex-determination pathways. bab expression is modulated segment- and sex-specifically in sexually dimorphic species, but is uniform in sexually monomorphic species. We suggest that bab has an ancestral homeotic function, and that regulatory changes at the bab locus played a key role in the evolution of sexual dimorphism. Pigmentation patterns specified by bab affect mating preferences, suggesting that sexual selection has contributed to the evolution of bab regulation.

title: Gene interactions in gonadal development.
abstract: The acquisition of a sexually dimorphic phenotype is a critical event in mammalian development. Although the maturation of sexual function and reproduction occurs after birth, essentially all of the critical developmental steps take place during embryogenesis. Temporally, these steps can be divided into two different phases: sex determination, the initial event that determines whether the gonads will develop as testes or ovaries; and sexual differentiation, the subsequent events that ultimately produce either the male or the female sexual phenotype. A basic tenet of sexual development in mammals is that genetic sex--determined by the presence or absence of the Y chromosome--directs the embryonic gonads to differentiate into either testes or ovaries. Thereafter, hormones produced by the testes direct the developmental program leading to male sexual differentiation. In the absence of testicular hormones, the pathway of sexual differentiation is female. This chapter reviews the anatomic and cellular changes that constitute sexual differentiation and discusses SRY and other genes, including SF-1, WT1, DAX-1, and SOX9, that play key developmental roles in this process. Dose-dependent interactions among these genes are critical for sex determination and differentiation.

title: Hoxa 11 structure, extensive antisense transcription, and function in male and female fertility.
abstract: Hoxa 11 is a murine Abdominal-B-type homeobox gene. The structure of this gene is presented, including genomic and cDNA sequence. The cDNA includes the complete open reading frame and based on primer extension results is near full length. Surprisingly, the antisense strand of Hoxa 11 was found to be transcribed. Moreover, these antisense transcripts were processed and polyadenylated. The developmental expression patterns for both sense and antisense transcripts were examined using serial section and whole-mount in situ hybridizations. Hoxa 11 transcription patterns were defined in the limbs, kidney and stromal cells surrounding the Mullerian and Wolffian ducts. Of particular interest, in the developing limbs, the sense and antisense transcripts showed complementary expression patterns, with antisense RNAs increasing in abundance in regions where sense RNAs were diminishing in abundance. Furthermore, targeted mutation of Hoxa 11 is shown to result in both male and female sterility. The female mutants produce normal ova, which develop properly post-fertilization when transferred to wild-type surrogate mothers. The Hoxa 11 homozygous mutants are shown to provide a defective uterine environment. The mutant males exhibited a malformation of the vas deferens that resembles a partial homeotic transformation to an epididymis. In addition, the mutant testes fail to descend properly into the scrotum and, likely as a result, spermatogenesis is perturbed.