Journal articles on the topic 'Fly; Excretory system; Embryonic development'

The c-ret gene encodes a receptor tyrosine kinase that is expressed in the Wolffian duct and ureteric bud of the developing excretory system. Newborn mice homozygous for a mutation in c-ret displayed renal agenesis or severe hypodysplasia, suggesting a critical role for this gene in metanephric kidney development. To investigate the embryological basis of these defects, we characterized the early development of the excretory system in mutant homozygotes, and observed a range of defects in the formation, growth and branching of the ureteric bud, which account for the spectrum of renal defects seen at birth. Co-culture of isolated ureteric buds and metanephric mesenchyme show that the primary defect is intrinsic to the ureteric bud. While the mutant bud failed to respond to induction by wild-type mesenchyme, mutant mesenchyme was competent to induce the growth and branching of the wild-type bud. Furthermore, the mutant metanephric mesenchyme displayed a normal capacity to differentiate into nephric tubules when co-cultured with embryonic spinal cord. These findings suggest a model in which c-ret encodes the receptor for a (yet to be identified) factor produced by the metanephric mesenchyme, which mediates the inductive effects of this tissue upon the ureteric bud. This factor appears to stimulate the initial evagination of the ureteric bud from the Wolffian duct, as well as its subsequent growth and branching.

The sterile insect technique (SIT) is a promising strategy to control the Australian sheep blow fly Lucilia cuprina, a major pest of sheep. We have previously developed a transgenic embryonic sexing system (TESS) for this pest to facilitate the potential SIT application. TESS carry two transgenes, a tetracycline transactivator (tTA) driver and a tTA-activated pro-apoptotic effector. TESS females die at the embryonic stage unless tetracycline is supplied in the diet. However, undesired female sterility was observed in some TESS strains without tetracycline due to expression of tTA in ovaries. Here we investigate if TESS that combine transgenes with relatively low/moderate expression/activity improves the fertility of TESS females. tTA driver lines were evaluated for tTA expression by quantitative real time PCR and/or by crossing with a tTA-activated RFPex effector line. Fertility and lethality tests showed that a TESS strain containing a driver line with moderate tTA expression and an effector line showing moderate pro-apoptotic activity could recover the fertility of parental females and eliminated all female offspring at the embryonic stage. Consequently, such a strain could be further evaluated for an SIT program for L. cuprina, and such a “moderate strategy” could be considered for the TESS development in other pest species.

Members of the orthodenticle gene family are essential for embryonic brain development in animals as diverse as insects and mammals. In Drosophila, mutational inactivation of the orthodenticle gene results in deletions in anterior parts of the embryonic brain and in defects in the ventral nerve cord. In the mouse, targeted elimination of the homologous Otx2 or Otx1 genes causes defects in forebrain and/or midbrain development. To determine the morphogenetic properties and the extent of evolutionary conservation of the orthodenticle gene family in embryonic brain development, genetic rescue experiments were carried out in Drosophila. Ubiquitous overexpression of the orthodenticle gene rescues both the brain defects and the ventral nerve cord defects in orthodenticle mutant embryos; morphology and nervous system-specific gene expression are restored. Two different time windows exist for the rescue of the brain versus the ventral nerve cord. Ubiquitous overexpression of the human OTX1 or OTX2 genes also rescues the brain and ventral nerve cord phenotypes in orthodenticle mutant embryos; in the brain, the efficiency of morphological rescue is lower than that obtained with overexpression of orthodenticle. Overexpression of either orthodenticle or the human OTX gene homologs in the wild-type embryo results in ectopic neural structures. The rescue of highly complex brain structures in Drosophila by either fly or human orthodenticle gene homologs indicates that these genes are interchangeable between vertebrates and invertebrates and provides further evidence for an evolutionarily conserved role of the orthodenticle gene family in brain development.

Information on the pre–hatching development of the platypus, Ornithorhynchus anatinus , is reliant on a small number of specimens, whose precise age is unknown. Material collected for J. P. Hill and now housed in the Hubrecht International Embryological Laboratory, Utrecht, contributes a major source of specimens. This paper presents new observations on developmental stages from the Hill collection, which allow for a more complete description of pre–hatching development. A feature of the pre–embryonic development of the platypus is the incomplete meroblastic cleavage. A column of fine yolk spheres extends from beneath the embryonic blastodisc towards the centre of a yolky vitellus, as seen in birds. The major expansion of extra–embryonic membranes occurs after the formation of the primitive streak. The primitive streak develops within an embryonal area as part of the superficial wall of the yolk–sac, a feature also shared with marsupials, birds and reptiles. The full–term, subspheroidal, intrauterine egg of the platypus has a major axis of about 17 mm and contains a flat, 19 to 20 somite, neurula–stage embryo which has prominent trigeminal ganglion primordia. The embryo at this stage is in a period of rapid modelling of the major early organ primordia of the nervous system, cardiovascular system, excretory system, and somite–derived components of the body wall. Soon after laying, five primary brain vesicles are present, the trigeminal ganglia CN5 as well as CN7, CN8, CN9, CN10, CN11 and CN12 are well developed. The alimentary system has an expanded stomach, pancreatic primordia and a gall bladder. Mesonephric tubules are associated with patent mesonephric ducts, which empty laterally into the cloaca. Extra–embryonic membranes at this stage show an extensive chorioamniotic connection that extends through the greater part of the caudal half of fused amniotic folds. The vascularized yolk–sac consists of a superficial yolk–sac omphalopleura and a deep yolk–sac splanchnopleure. The non–vascularized yolk–sac comprises one–quarter of the aboembryonal pole. Some distinctive monotreme features have developed by the mid–incubation period. The head is bent at an acute angle to the main body axis. The blunt upturned snout marks the site of the future oscaruncle and on the maxilla there is a median primordial papilla representing the egg tooth. The eye is open with a partly pigmented retinal ring. The forelimbs have partly separated digits, and the hindfeet are paddles. Just before hatching the upturned snout contains an oscaruncle and a sharp recurved median egg tooth. Forelimbs are pronated with separate digits possessing claw primordia. Portions of the highly vascularized extra–embryonic membranes are attached to the umbilical region and the flattened vesicular allantois has a distal region fused with the chorion. Prominent features of the hatchling are the presence of a bluntly conical oscaruncle and a translucent, horn–like egg tooth. These structures are thought to enable the hatchling to extricate itself from the egg shell. At hatching, the forelimbs exhibit clawed digits and are capable of digitopalmar prehension. Hindlimbs are still paddles with digital rays. A prominent yolk–sac navel is present. The newly hatched platypus has an external form similar to that of a new–born marsupial. The early development of the platypus has many major differences to the developmental sequence for humans, which has been categorized by the use of Carnegie Stages. The rate of somitogenesis of the platypus is faster in relation to the central nervous system morphogenesis than seen in humans, and the size of the early platypus embryonal area is massive in relation to that of humans. The unique morphology and function of extra–embryonic membranes in the platypus defies comparative staging with human development. Structures adapted for altricial survival of the platypus hatchling require the acquisition of functional competence at an earlier stage of organogenesis than seen in eutherians, although they are reminiscent of those found in new–born marsupials.

Globozoospermia is a rare form of male infertility where men produce round-headed sperm that are incapable of fertilizing an oocyte naturally. In a previous study where we undertook a whole exome screen to define novel genetic causes of globozoospermia, we identified homozygous mutations in the gene PDCD2L. Two brothers carried a p.(Leu225Val) variant predicted to introduce a novel splice donor site, thus presenting PDCD2L as a potential regulator of male fertility. In this study, we generated a Pdcd2l knockout mouse to test its role in male fertility. Contrary to the phenotype predicted from its testis-enriched expression pattern, Pdcd2l null mice died during embryogenesis. Specifically, we identified that Pdcd2l is essential for post-implantation embryonic development. Pdcd2l−/− embryos were resorbed at embryonic days 12.5-17.5 and no knockout pups were born, while adult heterozygous Pdcd2l males had comparable fertility to wildtype males. To specifically investigate the role of PDCD2L in germ cells, we employed Drosophila melanogaster as a model system. Consistent with the mouse data, global knockdown of trus, the fly ortholog of PDCD2L, resulted in lethality in flies at the third instar larval stage. However, germ cell-specific knockdown with two germ cell drivers did not affect male fertility. Collectively, these data suggest that PDCD2L is not essential for male fertility. By contrast, our results demonstrate an evolutionarily conserved role of PDCD2L in development.

To determine the generality of developmental mechanisms involved in the construction of the insect nervous system, the embryonic development of the peripheral nervous system in the grasshopper Schistocerca gregaria was characterized at the level of identified neurons and nerve branches and then compared to that previously described from the fly Drosophila melanogaster. For this, immunocytochemistry using a neuron-specific antibody was carried out on staged grasshopper embryos. Our results show that initially a simple peripheral nerve scaffolding is established in each segment of the animal. This scaffolding consists of a pair of intersegmental nerves that are formed by identified afferent and efferent pioneer neurons and a pair of segmental nerves that are formed by afferent pioneers situated in limb buds. Subsequently, identified sets of sensory neurons differentiate in a stereotyped spatiotemporal pattern in dorsal, lateral and ventral clusters in each segment and project their axons onto these nerves. Although segment-specific differences exist, serial homologs of the developing nerves and sensory neurons can be identified. A comparison of these results with those obtained from Drosophila shows that virtually the same pattern of peripheral nerves and sensory structures is formed in both species. This indicates that the construction of the peripheral nervous system in extremely divergent modern insects relies on conserved developmental mechanisms that evolved in ancestral insects over 300 million years ago.

ABSTRACT Transforming growth factor β (TGF-β)-activated kinase 1 (TAK1) is a member of the MAPKKK superfamily and has been characterized as a component of the TGF-β/bone morphogenetic protein signaling pathway. TAK1 function has been extensively studied in cultured cells, but its in vivo function is not fully understood. In this study, we isolated aDrosophila homolog of TAK1 (dTAK1) which contains an extensively conserved NH2-terminal kinase domain and a partially conserved COOH-terminal domain. To learn about possible endogenous roles of TAK1 during animal development, we generated transgenic flies which express dTAK1 or the mouseTAK1 (mTAK1) gene in the fly visual system. Ectopic activation of TAK1 signaling leads to a small eye phenotype, and genetic analysis reveals that this phenotype is a result of ectopically induced apoptosis. Genetic and biochemical analyses also indicate that the c-Jun amino-terminal kinase (JNK) signaling pathway is specifically activated by TAK1 signaling. Expression of a dominant negative form of dTAK during embryonic development resulted in various embryonic cuticle defects including dorsal open phenotypes. Our results strongly suggest that in Drosophila melanogaster, TAK1 functions as a MAPKKK in the JNK signaling pathway and participates in such diverse roles as control of cell shape and regulation of apoptosis.

Abstract The Drosophila melanogaster peripheral nervous system (PNS) comprises the sensory organs that allow the fly to detect environmental factors such as temperature and pressure. PNS development is a highly specified process where each sensilla originates from a single sensory organ precursor (SOP) cell. One of the major genetic orchestrators of PNS development is Senseless, which encodes a zinc finger transcription factor (Sens). Sens is both necessary and sufficient for SOP differentiation. Senseless expression and SOP number are regulated by the microRNA miR-9a. However, the reciprocal dynamics of Senseless and miR-9a are still obscure. By coupling single-molecule FISH with immunofluorescence, we are able to visualize transcription of the mir-9a locus and expression of Sens simultaneously. During embryogenesis, we show that the expression of mir-9a in SOP cells is rapidly lost as Senseless expression increases. However, this mutually exclusive expression pattern is not observed in the third instar imaginal wing disc, where some Senseless-expressing cells show active sites of mir-9a transcription. These data challenge and extend previous models of Senseless regulation and show complex co-expression dynamics between mir-9a and Senseless. The differences in this dynamic relationship between embryonic and larval PNS development suggest a possible switch in miR-9a function. Our work brings single-cell resolution to the understanding of dynamic regulation of PNS development by Senseless and miR-9a.

Mutations in the complex gene locus optomotor-blind (omb) can lead to defects in the development of both the optic lobes and external features of the adult fly. We describe here the expression of omb in the developing and adult nervous system using in situ hybridization. During embryogenesis, omb expression is first observed in the optic lobe anlagen. It later expands to a larger part of the developing larval brain and to the gnathal lobes. Cells in the ventral and peripheral nervous systems begin to express omb after completion of germ band extension. Later in embryonic development, expression declines and only persists in the antennomaxillary complex and in part of the brain hemispheres. During the larval and pupal stages, omb expression in the brain is confined to the developing optic lobes and contiguous regions of the central brain. At these stages, only a few cells show expression in the ventral ganglion. In the eye imaginal disc, transcript accumulation is most conspicuous in a group of presumptive glia precursor cells posterior to the morphogenetic furrow and in the optic stalk. In the adult brain, expression is prominent in several regions of the optic lobe cortex and along the border between central brain and optic lobes. In the mutation In(1)ombH31, 40 kb of regulatory DNA, downstream from the transcription unit, are removed from the omb gene. In(1)ombH31 is characterized by the lack of a set of giant interneurons from the lobula plate of the adult optic lobes. We find that, already during embryogenesis, there is a drastic difference between wild type and In(1)ombH31 in the level of the omb transcript in the optic lobe primordia. The adult mutant phenotype may thus be caused by omb misexpression during embryonic development.

ABSTRACT Selenium is implicated in many diseases, including cancer, but its function at the molecular level is poorly understood. BthD is one of three selenoproteins recently identified in Drosophila. To elucidate the function of BthD and the role of selenoproteins in cellular metabolism and health, we analyzed the developmental expression profile of this protein and used inducible RNA interference (RNAi) to ablate function. We find that BthD is dynamically expressed during Drosophila development. bthD mRNA and protein are abundant in the ovaries of female flies and are deposited into the developing oocyte. Maternally contributed protein and RNA persist during early embryonic development but decay by the onset of gastrulation. At later stages of embryogenesis, BthD is expressed highly in the developing salivary gland. We generated transgenic fly lines carrying an inducible gene-silencing construct, in which an inverted bthD genomic-cDNA hybrid is under the control of the Drosophila Gal4 upstream activation sequence system. Duplex RNAi induced from this construct targeted BthD mRNA for destruction and reduced BthD protein levels. We found that loss of BthD compromised salivary gland morphogenesis and reduced animal viability.

NF-X1 is a novel cytokine-inducible transcription factor that has been implicated in the control of immune responses in humans, presumably by regulating expression of class II major histocompatibility genes. Here we report the cloning and genetic characterization of the first reported NF-X1 homolog, which is encoded by the Drosophila melanogaster shuttle craft (stc) gene. The deduced sequence of the fly and human proteins defines a new family of molecules distinguished by a novel cysteine-rich DNA-binding motif (consisting of seven copies of the consensus sequence Cx3Cx3LxCGx0-5HxCx3CHxGxCx2Cx7-9CxC). We have identified and begun a phenotypic characterization of mutations in the stc gene. stc mutants die at the end of embryogenesis, when they appear to be incapable of coordinating the typical peristaltic contraction waves normally required for embryos to hatch into feeding first instar larvae. Preliminary evidence indicates that the resulting lethality of this behavioral defect is accompanied by subtle morphological abnormalities in the central nervous system, where in wild-type embryos, STC protein is normally localized in the nuclei of repeated cell clusters within each neuromere and brain lobe. Thus, the NF-X1 homolog encoded by the Drosophila stc gene defines a new family of putative transcription factors and plays an essential role in the completion of embryonic development. This study presents the first in vivo genetic analysis of a member of this new protein family.

Adherens and tight junctions play key roles in assembling epithelia and maintaining barriers. In cell culture zonula occludens (ZO)–family proteins are important for assembly/maturation of both tight and adherens junctions (AJs). Genetic studies suggest that ZO proteins are important during normal development, but interpretation of mouse and fly studies is limited by genetic redundancy and/or a lack of null alleles. We generated null alleles of the single Drosophila ZO protein Polychaetoid (Pyd). Most embryos lacking Pyd die with striking defects in morphogenesis of embryonic epithelia including the epidermis, segmental grooves, and tracheal system. Pyd loss does not dramatically affect AJ protein localization or initial localization of actin and myosin during dorsal closure. However, Pyd loss does affect several cell behaviors that drive dorsal closure. The defects, which include segmental grooves that fail to retract, a disrupted leading edge actin cable, and reduced zippering as leading edges meet, closely resemble defects in canoe zygotic null mutants and in embryos lacking the actin regulator Enabled (Ena), suggesting that these proteins act together. Canoe (Cno) and Pyd are required for proper Ena localization during dorsal closure, and strong genetic interactions suggest that Cno, Pyd, and Ena act together in regulating or anchoring the actin cytoskeleton during dorsal closure.

Abstract Genetics is a significant factor contributing to congenital heart disease (CHD), but our understanding of the genetic players and networks involved in CHD pathogenesis is limited. Here, we searched for de novo copy number variations (CNVs) in a cohort of 167 CHD patients to identify DNA segments containing potential pathogenic genes. Our search focused on new candidate disease genes within 19 deleted de novo CNVs, which did not cover known CHD genes. For this study, we developed an integrated high-throughput phenotypical platform to probe for defects in cardiogenesis and cardiac output in human induced pluripotent stem cell (iPSC)-derived multipotent cardiac progenitor (MCPs) cells and, in parallel, in the Drosophila in vivo heart model. Notably, knockdown (KD) in MCPs of RPL13, a ribosomal gene and SON, an RNA splicing cofactor, reduced proliferation and differentiation of cardiomyocytes, while increasing fibroblasts. In the fly, heart-specific RpL13 KD, predominantly at embryonic stages, resulted in a striking ‘no heart’ phenotype. KD of Son and Pdss2, among others, caused structural and functional defects, including reduced or abolished contractility, respectively. In summary, using a combination of human genetics and cardiac model systems, we identified new genes as candidates for causing human CHD, with particular emphasis on ribosomal genes, such as RPL13. This powerful, novel approach of combining cardiac phenotyping in human MCPs and in the in vivo Drosophila heart at high throughput will allow for testing large numbers of CHD candidates, based on patient genomic data, and for building upon existing genetic networks involved in heart development and disease.

Abstract In the housefly, Musca domestica, a single dominant factor, M, determines maleness. Animals hemi-or heterozygous for M are males, whereas those without M develop as females. In certain strains, however, both sexes are homozygous for M, and an epistatic dominant factor, FD, dictates female development. The requirement for these factors was analyzed by producing, with mitotic recombination, mosaic animals consisting of genetically male and female cells. Removal of FD from an M/M;FD/+ cell at any time of larval development, even in the last larval instar, resulted in sex-reversal, i.e., in the development of a male clone in an otherwise female fly. In contrast, when M was removed from M/+ cells, the resulting clones remained male despite their female genotype, even when the removal of M happened at embryonic stages. The occurrence of spontaneous gynandromorphs, however, shows that the loss of M in individual nuclei prior to blastoderm formation causes the affected cells to adopt the female pathway. These results are consistent with the hypothesis that M is the primary sex-determining signal which sets the state of activity of the key gene F at around the blastoderm stage. Parallels and differences to the sex-determining system of Drosophila are discussed.

The choice of sexual identity in somatic tissues of the fruit fly Drosophila melanogaster is determined early in embryogenesis by the X-chromosome-to-autosome (X/A) ratio. The system that signals the X/A ratio selects the sexual development pathway by determining the activity state of the binary switch Sex-lethal (Sxl). In 2X/2A animals, the X/A signalling system turns the Sxl gene on, ultimately activating an RNA-splicing autoregulatory feedback loop which serves to maintain the female state during the remainder of development. In 1X/2A animals, this autoregulatory feedback loop is not activated and the male state is subsequently maintained by the default splicing machinery. In the studies reported here, we have examined how the X/A signalling system controls the initial choice of sexual identity through its action on a special early embryonic Sxl promoter, Sxl-Pe. We show that in the early embryo, the activity of Sxl-Pe is controlled in a highly dose-sensitive fashion by the genes on the X chromosome that function as numerator elements and by genes located on the autosomes that function as denominator elements. Functional dissection of Sxl-Pe indicates that activating the promoter in females requires the cumulative action of multiple numerator genes which appear to exert their effects through reiterated cis-acting target sites in the promoter. Conversely, maintaining the promoter silent in males requires the repressive activities of denominator genes, and at least one of the denominator genes also appears to function through target sequences within the promoter.

ABSTRACT The GCM family of transcription factors consists ofDrosophila melanogaster GCM, an important regulator of gliogenesis in the fly, and its two mammalian homologs, GCMa and GCMb. To clarify the function of these mammalian homologs, we deleted GCMa in mice. Genetic ablation of murine GCMa (mGCMa) is embryonic lethal, with mice dying between 9.5 and 10 days postcoitum. At the time of death, no abnormalities were apparent in the embryo proper. Nervous system development, in particular, was not impaired, as might have been expected in analogy to Drosophila GCM. Instead, placental failure was the cause of death. In agreement with the selective expression of mGCMa in labyrinthine trophoblasts, mutant placentas did not develop a functional labyrinth layer, which is necessary for nutrient and gas exchange between maternal and fetal blood. Only a few fetal blood vessels entered the placenta, and these failed to thrive and branch normally. Labyrinthine trophoblasts did not differentiate. All other layers of the placenta, including spongiotrophoblast and giant cell layer, formed normally. Our results indicate that mGCMa plays a critical role in trophoblast differentiation and the signal transduction processes required for normal vascularization of the placenta.

The c-ret proto-oncogene encodes a receptor tyrosine kinase whose normal function has yet to be determined. To begin to investigate the potential role of this gene in vertebrate development, we have isolated cDNA clones representing the murine c-ret gene, and have analyzed the pattern of expression during mouse embryogenesis, using northern blotting, in situ hybridization to histological sections and whole-mount hybridization histochemistry. c-ret transcripts were detected beginning at day 8.5 of embryogenesis, and were observed in a number of cell lineages in the developing peripheral and central nervous systems, as well as in the excretory system. In the cranial region at day 8.5-9.5, c-ret mRNA was restricted to a population of neural crest cells migrating from rhombomere 4 and forming the anlage of the facioacoustic ganglion, as well as to a closely associated domain of surface ectoderm and pharyngeal endoderm. At later stages (10.5-14.5 days), c-ret mRNA was observed in all cranial ganglia. In the peripheral nervous system of the trunk, c-ret was expressed in the autonomic ganglia and in subsets of cells in the dorsal root ganglia. In the enteric nervous system, c-ret was expressed in the presumptive enteric neuroblasts of the vagal crest (day 9.0-11.5), and in the myenteric ganglia of the gut (day 13.5-14.5). c-ret mRNA was observed in several regions of the central nervous system, including the undifferentiated neuroepithelial cells of the ventral neural tube (8.5 days), the motor neurons in the spinal cord and the hindbrain (10.5-14.5 days), the embryonic neuroretina (day 13.5) and the layers of the postnatal retina containing ganglion, amacrine and horizontal cells. Outside the nervous system, c-ret was expressed in the nephric (Wolffian) duct at day 8.5-10.5, the ureteric bud epithelium (but not the surrounding metanephric mesenchyme) at day 11.0-11.5, and the growing tips of the renal collecting ducts (but not the previously formed, subcortical portions of the collecting ducts, or the mesenchyme-derived renal vesicles) at day 13.5-17.5. Our results suggest that the c-ret gene may encode the receptor for a factor involved in the proliferation, migration, differentiation or survival of a variety of neuronal cell lineages, as well as in inductive interactions during organogenesis of the kidney.

Abstract Genetic approaches, including the sterile insect technique (SIT), have previously been considered for control of the Australian sheep blow fly Lucilia cuprina, a major pest of sheep. In an SIT program, females consume 50% of the diet but are ineffective as control agents and compete with females in the field for mating with sterile males, thereby decreasing the efficiency of the program. Consequently, transgenic sexing strains of L. cuprina were developed that produce 100% males when raised on diet that lacks tetracycline. However, as females die mostly at the pupal stage, rearing costs would not be significantly reduced. Here we report the development of transgenic embryonic sexing strains of L. cuprina. In these strains, the Lsbnk cellularization gene promoter drives high levels of expression of the tetracycline transactivator (tTA) in the early embryo. In the absence of tetracycline, tTA activates expression of the Lshid proapoptotic gene, leading to death of the embryo. Sex-specific RNA splicing of Lshid transcripts ensures that only female embryos die. Embryonic sexing strains were also made by combining the Lsbnk-tTA and tetO-Lshid components into a single gene construct, which will facilitate transfer of the technology to other major calliphorid livestock pests.

AbstractHeart failure (HF) and the development of chronic kidney disease (CKD) have a direct association. Both can be cause and consequence of the other. Many factors are known, such as diabetes or hypertension, which can lead to the appearance and/or development of these two conditions. However, it is suspected that other factors, namely genetic ones, may explain the differences in the manifestation and progression of HF and CKD among patients. One candidate factor is Rph, a gene expressed in the nervous and excretory system in mammals and Drosophila, encoding a Rab small GTPase family effector protein implicated in vesicular trafficking. We found that Rph is expressed in the Drosophila heart, and the silencing of Rph gene expression in this organ had a strong impact in the organization of fibers and functional cardiac parameters. Specifically, we observed a significant increase in diastolic and systolic diameters of the heart tube, which is a phenotype that resembles dilated cardiomyopathy in humans. Importantly, we also show that silencing of Rabphilin (Rph) expression exclusively in the pericardial nephrocytes, which are part of the flies' excretory system, brings about a non-cell-autonomous effect on the Drosophila cardiac system. In summary, in this work, we demonstrate the importance of Rph in the fly cardiac system and how silencing Rph expression in nephrocytes affects the Drosophila cardiac system.

AbstractUltrastructural aspects of the advanced embryonic development and cotylocidial morphogenesis of the aspidogastrean Aspidogaster limacoides are described. The posterior or distal regions of the uterus are filled with eggs containing larvae at advanced stages of morphogenesis and fully-formed cotylocidia. Various stages and organs of this larva are described in detail, including the aspects of the developing and fully-differentiated cotylocidium, the body wall (tegument and musculature), glandular regions and the protonephridial excretory system. Blastomere multiplication by means of mitotic divisions takes place simultaneously with the degeneration or apoptosis of some micromeres; this frequently observed characteristic is compared and discussed in relation to corresponding reports for other neodermatans. During the advanced stages of the embryonic development of A. limacoides, the vitelline syncytium disappears and the size of the embryo increases rapidly. Evident polarization of the differentiating larva was observed; towards one pole of the egg, cytodifferentiation of the mouth, surrounded by the oral sucker and cephalic glands, takes place, whereas, towards the opposite pole, differentiation of the posterior sucker (incipient ventral disc) occurs. The oral and posterior suckers are formed from numerous embryonic cells which have differentiated into myocytes. The central part of the oral sucker undergoes invagination and forms the future pharynx and intestine. Fully-developed cotylocidia of A. limacoides have a neodermatan type of tegument, flame cells and two types of glandular structures. These results suggest a sister relationship between the Aspidogastrea and the Digenea, although the systematic position of aspidogastreans in relation to other platyhelminth taxa remains somewhat equivocal.

AbstractEpithelial-mesenchymal transition (EMT) refers to a process in which epithelial cells lose apical-basal polarity and loosen cell–cell junctions to take on mesenchymal cell morphologies and invasive properties that facilitate migration through extracellular matrix. EMT—and the reverse mesenchymal-epithelial transition (MET)—are evolutionarily conserved processes that are used throughout embryonic development to drive tissue morphogenesis. During adult life, EMT is activated to close wounds after injury, but also can be used by cancers to promote metastasis. EMT is controlled by several mechanisms that depend on context. In response to cell–cell signaling and/or interactions with the local environment, cells undergoing EMT make rapid changes in kinase and adaptor proteins, adhesion and extracellular matrix molecules, and gene expression. Many of these changes modulate localization, activity, or expression of cytoskeletal proteins that mediate cell shape changes and cell motility. Since cellular changes during EMT are highly dynamic and context-dependent, it is ideal to analyze this process in situ in living organisms. Embryonic development of model organisms is amenable to live time-lapse microscopy, which provides an opportunity to watch EMT as it happens. Here, with a focus on functions of the actin cytoskeleton, I review recent examples of how live in vivo imaging of embryonic development has led to new insights into mechanisms of EMT. At the same time, I highlight specific developmental processes in model embryos—gastrulation in fly and mouse embryos, and neural crest cell development in zebrafish and frog embryos—that provide in vivo platforms for visualizing cellular dynamics during EMT. In addition, I introduce Kupffer’s vesicle in the zebrafish embryo as a new model system to investigate EMT and MET. I discuss how these systems have provided insights into the dynamics of adherens junction remodeling, planar cell polarity signaling, cadherin functions, and cytoskeletal organization during EMT, which are not only important for understanding development, but also cancer progression. These findings shed light on mechanisms of actin cytoskeletal dynamics during EMT, and feature live in vivo imaging strategies that can be exploited in future work to identify new mechanisms of EMT and MET.

The post-embryonal development of arthropod species, including crustaceans and insects, is characterized by ecdysis or molting. This process defines growth stages and is controlled by a conserved neuroendocrine system. Each molting event is divided in several critical time points, such as pre-molt, molt, and post-molt, and leaves the animals in a temporarily highly vulnerable state while their cuticle is re-hardening. The molting events occur in an immediate ecdysis sequence within a specific time window during the development. Each sub-stage takes only a short amount of time, which is generally in the order of minutes. To find these relatively short behavioral events, one needs to follow the entire post-embryonal development over several days. As the manual detection of the ecdysis sequence is time consuming and error prone, we designed a monitoring system to facilitate the continuous observation of the post-embryonal development of the fruit fly Drosophila melanogaster. Under constant environmental conditions we are able to observe the life cycle from the embryonic state to the adult, which takes about 10 days in this species. Specific processing algorithms developed and implemented in Fiji and R allow us to determine unique behavioral events on an individual level—including egg hatching, ecdysis and pupation. In addition, we measured growth rates and activity patterns for individual larvae. Our newly created RPackage PEDtracker can predict critical developmental events and thus offers the possibility to perform automated screens that identify changes in various aspects of larval development. In conclusion, the PEDtracker system presented in this study represents the basis for automated real-time staging and analysis not only for the arthropod development.

The post-embryonal development of arthropod species, including crustaceans and insects, is characterized by ecdysis or molting. This process defines growth stages and is controlled by a conserved neuroendocrine system. Each molting event is divided in several critical time points, such as pre-molt, molt, and post-molt, and leaves the animals in a temporarily highly vulnerable state while their cuticle is re-hardening. The molting events occur in an immediate ecdysis sequence within a specific time window during the development. Each sub-stage takes only a short amount of time, which is generally in the order of minutes. To find these relatively short behavioral events, one needs to follow the entire post-embryonal development over several days. As the manual detection of the ecdysis sequence is time consuming and error prone, we designed a monitoring system to facilitate the continuous observation of the post-embryonal development of the fruit fly Drosophila melanogaster. Under constant environmental conditions we are able to observe the life cycle from the embryonic state to the adult, which takes about 10 days in this species. Specific processing algorithms developed and implemented in Fiji and R allow us to determine unique behavioral events on an individual level—including egg hatching, ecdysis and pupation. In addition, we measured growth rates and activity patterns for individual larvae. Our newly created RPackage PEDtracker can predict critical developmental events and thus offers the possibility to perform automated screens that identify changes in various aspects of larval development. In conclusion, the PEDtracker system presented in this study represents the basis for automated real-time staging and analysis not only for the arthropod development.

Abstract Background The New World Screwworm fly (NWS), Cochliomyia hominivorax, is an ectoparasite of warm-blooded animals and a major pest of livestock in parts of South America and the Caribbean where it remains endemic. In North and Central America it was eradicated using the Sterile Insect Technique (SIT). A control program is managed cooperatively between the governments of the United States and Panama to prevent the northward spread of NWS from infested countries in South America. This is accomplished by maintaining a permanent barrier through the release of millions of sterile male and female flies in the border between Panama and Colombia. Our research team demonstrated the utility of biotechnology-enhanced approaches for SIT by developing a male-only strain of the NWS. The strain carried a single component tetracycline repressible female lethal system where females died at late larval/pupal stages. The control program can be further improved by removing females during embryonic development as larval diet costs are significant. Results The strains developed carry a two-component system consisting of the Lucilia sericata bottleneck gene promoter driving expression of the tTA gene and a tTA-regulated Lshid proapoptotic effector gene. Insertion of the sex-specifically spliced intron from the C. hominivorax transformer gene within the Lshid gene ensures that only females die when insects are reared in the absence of tetracycline. In several double homozygous two-component strains and in one “All-in-one” strain that had both components in a single construct, female lethality occurred at the embryonic and/or first instar larval stages when raised on diet without tetracycline. Laboratory evaluation for phenotypes that are relevant for mass rearing in a production facility revealed that most strains had fitness characteristics similar to the wild type J06 strain that is currently reared for release in the permanent barrier. Testing of an “All in one” strain under mass rearing conditions showed that the strain maintained the fitness characteristics observed in small-scale rearing. Conclusions The early female lethal strains described here could be selected by the NWS Control Program for testing at large scale in the production facility to enhance the efficiency of the NWS eradication program.

The circulatory system is the first functional organ system that develops during mammalian life. Accumulating evidences suggest that cardiac and endocardial cells can arise from a single common progenitor cell during mammalian cardiogenesis. Notably, these early cardiac progenitors express multiple hematopoietic transcription factors, consistent with previous reports. Indeed, a close relationship among cardiac, endocardial and hematopoietic lineages has been suggested in fly, zebrafish, and embryonic stem cell in vitro differentiation models. However, it is unclear when, where and how this hematopoietic gene program is in operation during in vivo mammalian cardiogenesis. Hematopoietic colony assay suggests that mouse heart explants generate myeloids and erythroids in the absence of circulation, suggesting that the heart tube is a de novo site for the definitive hematopoiesis. Lineage tracing revealed that putative cardiac-derived Nkx2-5+/Isl1+ endocardial cells give rise to CD41+ hematopoietic progenitors that contribute to definitive hematopoiesis in vivo and ex vivo during embryogenesis earlier than in the AGM region. Furthermore, Nkx2-5 and Isl1 are both required for the hemogenic activity of the endocardium. Together, identification of Nkx2-5/Isl1-dependent hemogenic endocardial cells (1) adds hematopoietic component in the cardiogenesis lineage tree, (2) changes the long-held dogma that AGM is the only major source of definitive hematopoiesis in the embryo proper, and (3) represents phylogenetically conserved fundamental mechanism of cardio-vasculo-hematopoietic differentiation pathway during the development of circulatory system.

Abstract Background The invasive fly Drosophila suzukii has become an established fruit pest in Europe, the USA, and South America with no effective and safe pest management. Genetic engineering enables the development of transgene-based novel genetic control strategies against insect pests and disease vectors. This, however, requires the establishment of reliable germline transformation techniques. Previous studies have shown that D. suzukii is amenable to transgenesis using the transposon-based vectors piggyBac and Minos, site-specific recombination (lox/Cre), and CRISPR/Cas9 genome editing. Results We experienced differences in the usability of piggyBac-based germline transformation in different strains of D. suzukii: we obtained no transgenic lines in a US strain, a single rare transgenic line in an Italian strain, but observed a reliable transformation rate of 2.5 to 11% in a strain from the French Alps. This difference in efficiency was confirmed by comparative examination of these three strains. In addition, we used an attP landing site line to successfully established φC31-integrase-mediated plasmid integration at a rate of 10% and generated landing site lines with two attP sequences to effectively perform φC31-Recombinase Mediated Cassette Exchange (φC31-RMCE) with 11% efficiency. Moreover, we isolated and used the endogenous regulatory regions of Ds nanos to express φC31 integrase maternally to generate self-docking lines for φC31-RMCE. Besides, we isolated the promoter/enhancer of Ds serendipity α to drive the heterologous tetracycline-controlled transactivator (tTA) during early embryonic development and generated a testes-specific tTA driver line using the endogenous beta-2-tubulin (β2t) promoter/enhancer. Conclusion Our results provide evidence that the D. suzukii strain AM derived from the French Alps is more suitable for piggyBac germline transformation than other strains. We demonstrated the feasibility of using φC31-RMCE in the cherry vinegar fly and generated a set of lines that can be used for highly efficient integration of larger constructs. The φC31-based integration will facilitate modification and stabilization of previously generated transgenic lines that carry at least one attP site in the transgene construction. An early embryo-specific and a spermatogenesis-specific driver line were generated for future use of the binary expression system tet-off to engineer tissue- and stage-specific effector gene expression for genetic pest control strategies.

Discussing the interaction between representation and narrative structures, Anthony Mandal argues that the Gothic has always been “an intrinsically intertextual genre” (Mandal 350). From its inception, the intertextuality of the Gothic has taken many and varied incarnations, from simple references and allusions between texts—dates, locations, characters, and “creatures”—to intricate and evocative uses of style and plot organisation. And even though it would be unwise to reduce the Gothic “text” to a simple master narrative, one cannot deny that, in the midst of re-elaborations and re-interpretations, interconnections and interpolations also appear, a collective gathering of ideas and writing practices that construct what is known as “the Gothic intertext” (Mishra 235). As far as storytelling, characterisation, and symbolism are concerned, the Gothic finds strength in its ability to develop as well as negate expectation, re-moulding the culturally known and the aesthetically acceptable in order to present its audience with a multi-faceted and multi-layered narrative. Although the Gothic has traditionally found fertile ground in literary works—a connection that is now a legacy as much as an origin—other contemporary media, such as animation, have offered the Gothic a privileged chance for growth and adaptation. An evocative example of the mergence between the Gothic mode and the animated medium is Alex Hirsch’s Gravity Falls. This visual text provides an example of the reach of the Gothic within popular culture, where intersecting hideous creatures and interconnected narrative structures, although simple and “for children” on the surface, reveal the presence of a dense and intertextual Gothic network. Those interlacings are, of course, never disconnected from the wider cultural framework, and clearly occupy an important part in unravelling the insidious aspects of human nature, from the difficulties of finding “oneself” to the loneliness of the everyday. Gravity Falls is an animated television series created by Alex Hirsch. It premiered on the Disney Channel in the United States on 15 June 2012. Now scheduled for its second season of running, Gravity Falls follows the adventures of 12-year-old twin siblings Dipper and Mabel Pines while on their summer vacation in the small town of Gravity Falls, Oregon. The choice of “twins” as main characters reveals, even at such an embryonic level, a connection to Gothicised structures, as the mode itself, as Vijay Mishra suggests, finds an affinity with doublings and “specular identifications” that “confuse the norm” (63). The presence of twins makes the double nature of character, traditionally a metaphorical and implicit idea in the Gothic, a very obvious and explicit one. Dipper and Mabel are staying with their eccentric and money-grabbing Great Uncle Stan—often referred to as “Grunkle Stan”—who runs the local curiosity shop known as the Mystery Shack. It becomes very obvious from the very beginning that an air of mystery truly surrounds the Shack, which quickly lives up to its name, and the eponymous town. In an aptly Gothic manner, things are definitely not what they seem and the twins are caught in odd plots, eerily occurrences, and haunted/haunting experiences on a daily basis. The instigator for the twins’ interest in the odd manifestations is the finding of a mysterious journal, a manual the relays detailed descriptions of the creatures that inhabit the forest in the town of Gravity Falls. The author of the journal remains unknown, and is commonly known only as “3”, an unexplained number that marks the cover of the book itself. Although the connection between the Gothic and animation may be obscure, it is in fact possible to identify many common and intersecting elements—aesthetically, narratively, and conceptually—that highlight the two as being intrinsically connected. The successful relation that the Gothic holds with animation is based in the mode’s fundamental predilection for not only subversion, excess and the exploration of the realm of the “imagination”, but also humour and self-reflexivity. These aspects are shared with animation which, as a medium, is ideally placed for exploring and presenting the imaginative and the bizarre, while pushing the boundaries of the known and the proper. Julia Round suggests that the Gothic “has long been identified as containing a dual sense of play and fear” (7). The playfulness and destabilisation that are proper to the mode find a fertile territory in animation in view of not only its many genres, but also its style and usually sensational subject matter. This discourse becomes particularly relevant if one takes into consideration matters of audienceship, or, at least, receivership. Although not historically intended for younger viewers, the animation has evolved into a profoundly children-orientated medium. From cinema to television, animated features and series are the domain of children of various ages. Big production houses such as Disney and Warner Bros have capitalised on the potential of the medium, and established its place in broadcasting slots for young viewers. Not unlike comics—which is, in a way, its ancestral medium—animation is such a malleable and contextual form that it requires a far-reaching and inclusive approach, one that is often interdisciplinary in scope; within this, where the multi-faceted nature of the Gothic opens up the way for seeing animated narratives as the highly socio-historical mediums they are. And not unlike comics, animation shares a common ground with the Gothic in requiring a vast scope of analysis, one that is intrinsically based on the conceptual connections between “texts”. Round has also aptly argued that, like comics, animated series lend themselves to malleable and mouldable re-elaboration: “from the cultural to the aesthetic, the structural to the thematic”, graphic media always reflect the impact of “intertextual and historical references” (8). Animation’s ability to convey, connect, and revolutionise ideas is, therefore, well-matched to the aesthetic and conceptual idiosyncrasies of Gothic tropes. Dipper and Mabel’s vacation in the town of Gravity Falls is characterised by the appearance of numerous super- and preter-natural creatures. The list of “monsters” encountered by the twins is long and growing, from gnomes, goblins, mermaids and zombies, to ghosts, clones, and a wide and colourful variety of demons. And although, at first glance, this list would appear to be a simple and simplistic grouping of bizarre and creatively assembled creatures, it is made quickly apparent that these “monsters” are all inspired, often very directly, by “existing”—or, at least, well-known—Gothic creatures, and their respective contexts of development. Indeed, the links to the Gothic in contemporary popular culture are unavoidable. The creatures in Gravity Falls are presented with subtle references to Gothic literature and cinema, from John Carpenter’s Halloween (1978) and Joe Dante’s Gremlins (1984), to Stephen King’s The Shining (1977) and Needful Things (1991). Borrowing from these texts, the creatures in the series all have strange names that rely on play-on-words and re-inventions, and the rubric twists that they undertake are part of a system of both homage and conceptual interdependency. One can find, for instance, “Manotaurs”—creatures that are half-bull and half-man, and that value “manliness” in their society above all else—and the “Gremlobin” – a gigantic monster somewhere in between, we are told, a “gremlin” and a “goblin”, whose eyes can show “your worst nightmares”. But the range extends to other bizarre “creatures” that are clearly very spooky, such as the “Summewrween Trickster”—a large, shadowy, purple/orange monster with a “jack-o’-melon” mask – the living “mailbox”—a sentient and omniscient object—and the truly haunting Bill Cipher—a mind demon that can be summoned through an incantation and enter a person’s subconscious. The connection to the Gothic in popular culture is instrumental for the construction of the Gothic intertext in Gravity Falls. In episode One, “Tourist Trapped” (1.01), Mabel is kidnapped by a tribe of gnomes, who are set on making her their queen. The gnomes are incongruous creatures: on the one hand, they are vengeful and spiteful, recalling the horror monsters found in movies such as the questionable Blood Gnome (2004). On the other, however, they wear red pointy hats and white beards, and their friendly smiles recall the harmless appearance of actual garden gnomes. When the gnomes grow upset, they throw up rainbows; this strange fact destroys their potential as a Gothic horror icon, and makes them accessible and amusing. This subversion of iconography takes place with a number of other “creatures” in Gravity Falls, with the Summerween Trickster—subverting the “terror” of Hallowe’en—being another fitting example. When the gnomes are attempting to woe Mabel, they do not appear to her in their real form: they camouflage themselves into a teenage boy— one who is moody, brooding, and mysterious—and become Mabel’s boyfriend; the “boy’s” interest in her, however, is so intense, that Dipper suspects him to be a member of “The Undead”, a category of monster that is closely described in 3’s journal: due to their “pale skin” and “bad attitudes”, they are often mistaken for “teenagers”. Clues to Dipper’s doubts include the teenage boy’s hand “falling off” while he is hugging Mabel, a clear sign—it would seem—that the boy is obviously a decaying, zombie corpse. The intertextual connection to several horror visual narratives where limbs “fall off” the undead and the monstrous is clear here, with apt film examples being Dawn of the Dead (1978), The Fly (1986), horror comedy Army of Darkness (1992), and, more recently, television’s The Walking Dead (2010-). The references to well-known horror films are scattered throughout the series, and comprise the majority of the lampooned cultural context in which the creatures appear. In spite of Dipper’s suspicions, the situation is revealed to have a rather different outcome. When the boyfriend tells Mabel he has a big secret to reveal, her mind wanders into another direction, choosing a different type of undead, as she expectantly thinks: “Please be a vampire…please be a vampire”. It is not difficult to spot the conceptual connection here to narratives such as Stephenie Meyer’s Twilight saga (2005-2008), both in its literary and cinematic variations, where brooding and mysterious teenage boys find ideal incarnations as the undead creature. The romanticised nature of teenage fictional narratives such as the Twilight saga is also mirrored in Mabel’s distinctive love-centred interest in the potential vampire, revealing an intertextual and highly contextual association to seeing the creature as part of an amorous relationship, as opposed to a blood-thirsty murderer. Mabel’s dreams of vampric love are unfortunately shattered when the boyfriend is revealed to be several gnomes carefully assembled to operate a human-like body, rather than one immortal lover. Irrespective of its desire to parody the Gothic, however, Gravity Falls still maintains unavoidable links to the notion of terror. Clear evidence of this is to be found in the fact that all “creatures” in the series present a level of anthropomorphism about them, and this is interpreted by the characters—and the viewers—as one of their scariest aspects. Leigh Blackmore suggests that a special brand of terror can be found in “anthropomorphic beings” that are in fact not human (Blackmore 95). Most of the creatures in the series are humanoid in shape, and can speak like humans. From gnomes to mermaids, mailboxes and demons, the creatures act as humans, but they are in fact something “other”, something that only recalls the human itself. This idea of being “almost human”, but “not quite”, is disturbing in itself, and connects the presentation of the creatures to the Gothic via the notion of the uncanny: “a crisis of the natural, touching upon everything that one might have thought was ‘part of nature’ […] human nature, the nature of reality and the world” (Royle 1). The uncanny nature of the creatures in Gravity Falls is maintained through their profound inhumanity, and their simultaneous links to human ways of acting, speaking, and even thinking. Indeed, most of the creatures are presented as petty, bitter, and childish, and often seen as greedy and sulking. In a way, the creatures lampoon some of the most intrinsic qualities of the human species, what separates us from animals. The supernatural creatures operate here as a critique of the humans themselves, exposing, as the Gothic often does, the most disturbing parts of humanity. The creatures are presented initially as scary, recalling—albeit very briefly—notions of terror and horror, but that façade is quickly destroyed as their “real nature” is exposed. They are de-terrorised by not only making them common, but also ridiculing their habits and de-constructing their thinly-veiled Gothic personas. The creatures in Gravity Falls are a subversion of the subversion, a re-thinking of the Gothic through parody that allows their conceptual, and culturally relevant, function to be rapidly exposed. The impact of the Gothic intertext in Gravity Falls is not only visible in its representational forms—its monsters and “creatures”—but also extends to its structural organisation. Jerrold Hogle has argued that, although they maintain a heterogeneous construction of texts and contexts, there are certain qualities applicable to “Gothic texts”: an antiquated space (often decaying); a concealed secret from the historical past; a physical or psychological haunting; and an oscillation between “reality” and the “supernatural” (3). Although Hogle’s pinpointing of what he calls the “Gothic matrix” (3) is mainly focused on the literary world, a broader and more wide-reaching understanding of the Gothic text allows these qualities to be clearly identifiable in other narrative mediums, such as an animated series. Indeed, Gravity Falls presents the main elements of the “Gothic matrix”: the Mystery Shack is an old and isolated place, physically crumbling and in constant state of disrepair; it is made clear that the Shack harbours many secrets—filled as it is with hidden passageways and underground vaults—connected to the shady past of Grunkle Stan and its unresolved connections to mysticism and magic; there are plenty of hauntings to be found in the series: from physical ones—in the form of demons and ghosts—to psychological ones, condensed in Dipper and Mabel’s difficulties with their approaching puberties and “growing up”; finally, the line between reality and supernatural is constantly challenged by the appearance of multiple creatures that are clearly not of this world, and even though several characters doubt their existence within the story, their very presence challenges the stability of the boundaries between real and unreal. On the surface, the series is presented as a standard linear narrative, where the linear journey of each 20-minute episode culminates with the resolution of the main “haunting”, and the usual destruction or appeasing of the “creature”. And while the series’ use of cliff-hangers is, in true television style, a common presence, they also expose and recall the unresolved nature of the narrative. Indeed, the story’s structure in Gravity Falls is reliant on narrative undertellings and off-shoots that often lie underneath the logical “line” of the plot. Sub-plots reign supreme, and multiple motives for the characters’ actions are introduced but not expanded upon, leaving the series impregnated with an aura of uncertainty and chaos. The focus of the storytelling is also denied; one moment, it appears to be Dipper’s desire to discover the “secrets” of the forest; the other, it is Grunkle Stan’s long-time battle with his arch-nemesis Gideon over the ownership of the Shack. This plot confusion in Gravity Falls continues to expose its narrative debt to the Gothic intertext, since “structural multiplicity”, as Round suggests, is “a defining feature of the Gothic” (19). The series’ narrative structure is based on numerous multiplicities, an open denial of linear journeys that is dependant, paradoxically, on the illusion of resolution. The most evocative example of Gravity Falls’ denial of clear-cut structures is arguably to be found in the narrative underlayers added by 3’s monster manual. It is obvious from the beginning that 3’s stay in the town of Gravity Falls was riddled with strange experiences, and that his sojourn intersected, at one point or the other, with the lives and secrets of Grunkle Stan and his enemies. It is also made clear that 3’s journal is not a solitary presence in the narrative, but is in fact only one in a triad of mystical books—these books, it is suggested, have great power once put together, but the resolution to this mystery is yet to be revealed. As Grunkle Stan and Gideon fight (secretly) over the possession of the three books, it is openly suggested that several uncovered stories haunt the main narrative in the series and, unknown Dipper and Mabel, are responsible for many of the strange occurrences during their stay at the Shack. Jean-Jacques Lecercle has long argued that one of the defining characteristics of the Gothic, and its intertextual structure, is the presence of “embedded narratives” (72). In Gravity Falls, the use of 3’s manual as not only an initiator of the plot, but also as a continuous performative link to the “haunted” past, uncovers the series’ re-elaboration of the traditional structure of Gothic narratives. As a paratextual presence in the story—one that is, however, often responsible for the development of the main narrative—3’s manual draws attention to the importance of constructing layered stories in order to create the structures of terror, and subsequent horror, that are essential to the Gothic itself. Although it often provides Dipper with information for solving the mysteries of the Shack, and subduing the supernatural creatures that overtake it, 3’s manual is, in reality, a very disruptive presence in the story. It creates confusion as it begins storytelling without concluding it, and opens the way to narrative pathways that are never fully explored. This is of course in keeping with the traditional narrative structures of the Gothic mode, where ancient books and stories— belonging to “antiquity”—are used as a catalyst for the present narrative to take place, but are also strangely displaced from it. This notion recalls Victor Sage’s suggestion that, in Gothic narratives, ancient books and stories paradoxically “disrupt” the main narrative, starting a separate dialogue with a storytelling structure that is inevitably unexplored and left unanswered (86). Canonical examples such as Ann Radcliffe’s The Mysteries of Udolpho (1794) and Emily Brontë’s Wuthering Heights (1847) inevitably come to mind here, but also more recent cinematic examples such as the Evil Dead franchise (1978-), where ancient books and old storytellers uncover hoary secrets that instigate, as well as obscure, the main narrative. In Gravity Falls, the interaction with 3’s manual is inherently performative, and continuously intertextual, but it is also deeply confusing, adding to the feeling of strangeness and mystery that is the conceptual basis for the series itself. The intertextual connections that drive the narrative in Gravity Falls construct lampooned versions of both the traditional concepts of Gothic horror and Gothic terror. Hogle has suggested that Gothic terror is apparent in the construction of suspense, achieved through an exploration of psychological hauntings, human nature and its un/limitations, and that which is kept out sight, the expected “hidden secrets” (3). Gothic horror, on the other hand, is characterised by the consequences of these occurrences; the physical manifestation of the “haunting”, so to speak, is achieved through the presentation of something repulsive and horrific, the monstrous in its various incarnations (Hogle 3). In Gravity Falls, the connection to the traditional Gothic intertext is made clear through both elicitations of “terror”, and subsequent manifestations of “horror”. Indeed, the “hidden secrets” of the Shack, and to some extent, the fears and insecurities of the characters, are mediated through the appearance of horrific machinery and creatures. The Shack always conceals something hidden, a magical element of sort that is kept secret by intricate passageways. The shadowy nature of the building – evoking the psychological hauntings of Gothic terror – inevitably causes the appearance of something physically disturbing, finding its apogee in a Gothic horror experience. A clear example of this can be found in the episode “Double Dipper” (1.07). Desperate to impress his co-worker and secret love-interest Wendy, and “haunted” by his lack of self-worth, Dipper roams the rooms of the Shack and discovers a very old and enchanted photocopier machine; the machine copies “people”, making clones of the original. The “clones” themselves are a manifestation of horror, a presence that breaks the boundaries of propriety, and worries its viewers in view of its very existence. The cloning copy machine is strongly intertextual as it not only provides conceptual links to numerous cinematic and literary examples where a “haunted machine” threats to destroy humanity— in examples such as Stephen King’s Christine (1983) —but also evokes the threat of “doubles”, another powerfully Gothic conduit (Royle). As it is often the case in Gravity Falls, Dipper loses control of the situation, and the dozens of clones he unwittingly created take over his life and threaten to annihilate him. Dipper must destroy the “horror” —the clones—and confront the “terror”—his haunting insecurities and personal secrets—in order to restore the original balance. This intertextual dynamic validates Hogle’s contention that, in Gothic narratives, both the physical and the psychological “hauntings” rise from view “within the antiquated space” and “manifest unresolved conflicts that can no longer be successfully buried from view” (Hogle 2). The “hidden secrets” of Gravity Falls, and their manifestations through both Gothic horror and terror, are clearly connected to explorations of human nature and deeply existentialist crises that are put forward through humour and parody. These range from Grunkle Stan’ inability to commit to a relationship—and his feeling that life is slipping away in his old age—to the twins’ constant insecurities about pre-teen amorous encounters. Not to mention the knowledge that, in reality, Dipper and Mabel were “abandoned” by their mother in the care of Stan, as she had other plans for the summer. As Round has argued, the Gothic’s most significant development seems to have been the “transvaluation of moral issues”, as notions of “monsters have become less clear cut” (18). The series’ successful engagement with the wider “monstrous” intertext, and its connection to moral issues and “hidden” preoccupations, uncovers the ability of the Gothic, as Catherine Spooner puts it, to act as “commodity”, no longer a marginalised cultural presence, but a fully purchasable item in consumer-capitalist systems (Spooner 2007). The evocations of both horror and terror in Gravity Falls are, naturally, unavoidably diluted, a homage as much as a direct encounter. The use of the monstrous and the haunted in the series is domesticated, made accessible so that it can be presented to a younger and more commercial audience. The profound interlacings with the Gothic intertext remain, however, unchanged, as the series reconciles its subversive, uncanny elements with the inevitably conventional, Disney-fied context in which it is placed. References Blackmore, Leigh. “Marvels and Horrors: Terry Dowling’s Clowns at Midnight”. 21st Century Gothic: Great Gothic Novels Since 2000, ed. Danel Olson. Lanham: Rowman & Littlefield, 2011. 87-97. Gravity Falls. Disney Television. Disney Channel, Los Angeles. 2012-2014. Hogle, Jerrold. “Introduction: The Gothic in Western Culture”. The Cambridge Companion of Gothic Fiction, ed. Jerrold Hogle. Cambridge: Cambridge University Press, 2002. 1-20. Lecercle, Jean-Jacques. “The Kitten’s Nose: Dracula and Witchcraft”. The Gothic, ed. Fred Botting. D.S Brewer: Cambridge, 2001. 71-86. Mandal, Anthony. “Intertext”. The Encyclopaedia of the Gothic, ed. David Punter, Bill Hughes and Andrew Smith. Basingstoke: Wiley, 2013. 350-355. Mishra, Vijay. The Gothic Sublime. Albany: State University of New York Press, 1994. Round, Julia. Gothic in Comics and Graphic Novels. Jefferson: McFarland, 2014. Royle, Nicholas. The Uncanny. Manchester: Manchester University Press, 2003. Sage, Victor. “Irish Gothic: C.R. Maturin and J.S. LeFanu. A Companion to the Gothic, ed. David Punter. Oxford; Blackwell, 2001. 81-93. Spooner, Catherine. Contemporary Gothic. London: Reaktion, 2007.