Role of svp in Drosophila Pericardial Cell Growth

The Drosophila dorsal vessel is a segmentally repeated linear organ, in which seven-up (svp) is expressed in two pairs of cardioblasts and two pairs of pericardial cells in each segment. Under the control of hedgehog (hh) signaling from the dorsal ectoderm, svp participates in diversifying cardioblast identities within each segment. In this experiment, the homozygous embryos of svp mutants exhibited an increase in cell size of Eve positive pericardial cells (EPCs) and a disarranged expression pattern, while the cardioblasts pattern of svp-lacZ expression was normal. In the meantime, the DA1 muscle founders were absent in some segments in svp mutant embryos, and the dorsal somatic muscle patterning was also severely damaged in the late stage mutant embryos, suggesting that svp is required for the differentiation of Eve-positive pericardial cells and DA1 muscle founders and may have a role in EPC cell growth.     

Role of abd-A and Abd-B in Development of Abdominal Epithelia Breaks Posterior Prevalence Rule

Hox genes that determine anteroposterior body axis formation in all bilaterians are often found to have partially overlapping expression pattern. Since posterior genes dominate over anterior Hox genes in the region of co-expression, the anterior Hox genes are thought to have no function in such regions. In this study we show that two Hox genes have distinct and essential functions in the same cell. In Drosophila, the three Hox genes of the bithorax complex, Ubx, abd-A and Abd-B, show coexpression during embryonic development. Here, we show that in early pupal abdominal epithelia, Ubx does not coexpress with abd-A and Abd-B, while abd-A and Abd-B continue to coexpress in the same nuclei. The abd-A and Abd-B are expressed in both histoblast nest cells and larval epithelial cells of early pupal abdominal epithelia. Further functional studies demonstrate that abd-A is required in histoblast nest cells for their proliferation and suppression of Ubx to prevent first abdominal segment like features in posterior segments while in larval epithelial cells it is required for their elimination. We also observed that these functions of abd-A are required in its exclusive as well as the coexpression domain with that of Abd-B. The expression of Abd-B is required in histoblast nest cells for their identity while it is dispensable in the larval epithelial cells. The higher level of Abd-B in the seventh abdominal segment, that down-regulates abd-A expression, leads this segment to be absent in males or of smaller size in females. We also show that abd-A in histoblast nest cells positively regulates expression of wingless for the formation of the abdominal epithelia. Our study reveals an exception to the rule of posterior prevalence and shows that two different Hox genes have distinct functions in the same cell, which is essential for the development of abdominal epithelia.     

Functional analysis of Ultrabithorax in the silkworm, Bombyx mori, using RNAi

The formation of abdominal appendages in insects is suppressed by the Hox genes Ultrabithorax (Ubx) and abdominal-A (abd-A), but mechanisms of the suppression can differ among species. As the function of Ubx and abd-A has been described in only a few species, more data from various insects are necessary to elucidate the evolutionary transition of regulation on abdominal appendages. We examined the function of Ubx in the silkworm Bombyx mori (Bm-Ubx) by embryonic RNA interference (RNAi). This is the first case in which functional analysis for Ubx is performed in lepidopteran insects. Larvae treated with Bm-Ubx dsRNA displayed an additional pair of thoracic leg-like protuberances in A1, whereas the other abdominal segments had no transformation. Our results suggest that Bm-Ubx is a suppressor of leg development in A1.     

Roles of Hox genes in the patterning of the central nervous system of Drosophila

One of the key aspects of functional nervous systems is the restriction of particular neural subtypes to specific regions, which permits the establishment of differential segment-specific neuromuscular networks. Although Hox genes play a major role in shaping the anterior-posterior body axis during animal development, our understanding of how they act in individual cells to determine particular traits at precise developmental stages is rudimentary. We have used the abdominal leucokinergic neurons (ABLKs) to address this issue. These neurons are generated during both embryonic and postembryonic neurogenesis by the same progenitor neuroblast, and are designated embryonic and postembryonic ABLKs, respectively. We report that the genes of the Bithorax-Complex, Ultrabithorax (Ubx) and abdominal-A (abd-A) are redundantly required to specify the embryonic ABLKs. Moreover, the segment-specific pattern of the postembryonic ABLKs, which are restricted to the most anterior abdominal segments, is controlled by the absence of Abdominal-B (Abd-B), which we found was able to repress the expression of the neuropeptide leucokinin. We discuss this and other examples of how Hox genes generate diversity within the central nervous system of Drosophila.Keyword: development, Hox genes, central nervous system, Drosophila, cell fate specification     

Scanning electron microscopy of the dorsal vessel of Panstrongylus megistus (Burmeister, 1835) (Hemiptera: Reduviidae).

In this study we analyzed the microanatomy of the dorsal vessel of the triatomine Panstrongylus megistus. The organ is a tubule anatomically divided into an anterior aorta and a posterior heart, connected to the body wall through 8 pairs of alary muscles. The heart is divided in 3 chambers by means of 2 pairs of cardiac valves. A pair of ostia can be observed in the lateral wall of each chamber. A bundle of nerve fibers was found outside the organ, running dorsally along its major axis. A group of longitudinal muscular fibers was found in the ventral portion of the vessel. The vessel was found to be lined both internally and externally by pericardial cells covered by a thin laminar membrane. Inside the vessel the pericardial cells were disposed in layers and on the outside they formed clusters or rows.     

Morphology of the dorsal vessel in the abdomen of the blood-feeding insect Rhodnius prolixus

The dorsal vessel (DV) in the abdomen of the blood-feeding insect Rhodnius prolixus was divided functionally into two regions, the heart, into which haemolymph entered the DV through four pairs of ostia located in abdominal segment VII, and the aorta, along which the haemolymph was propelled from abdominal segment VI to the thorax. Osmium-fixed whole mounts revealed the DV to consist of spirally arranged striated muscle fibers and to possess two rows of ventrally attached longitudinal fibers extending the length of the abdomen. Seven pairs of alary muscles were found attached to the DV in the posterior abdominal segments. Contractions of the alary muscles attached to the ventral surface of abdominal segments VII and VIII served to expand the heart. Electron microscopy revealed the DV to consist of a thin layer of contractile elements surrounded by an inner (intima) and outer (adventitia) connective tissue layer. Embedded in the intima along each lateral side of the DV were two large groups of endocardial cells extending the length of the DV. A small group of pericardial cells was embedded in the adventitia along the mid-ventral side of the DV, and clusters of pericardial cells were found attached to the alary muscles. Nerve terminals were found only on the heart: they contained agranular synaptic vesicles approximately 30 nm in diameter and densely stained granules approximately 100-120 nm in diameter. These structural components are discussed in relation to the role of the DV in circulation.     

Balancing sex chromosome expression and satisfying the sexes

One of the key aspects of functional nervous systems is the restriction of particular neural subtypes to specific regions, which permits the establishment of differential segment-specific neuromuscular networks. Although Hox genes play a major role in shaping the anterior-posterior body axis during animal development, our understanding of how they act in individual cells to determine particular traits at precise developmental stages is rudimentary. We have used the abdominal leucokinergic neurons (ABLKs) to address this issue. These neurons are generated during both embryonic and postembryonic neurogenesis by the same progenitor neuroblast, and are designated embryonic and postembryonic ABLKs, respectively. We report that the genes of the Bithorax-Complex, Ultrabithorax (Ubx) and abdominal-A (abd-A) are redundantly required to specify the embryonic ABLKs. Moreover, the segment-specific pattern of the postembryonic ABLKs, which are restricted to the most anterior abdominal segments, is controlled by the absence of Abdominal-B (Abd-B), which we found was able to repress the expression of the neuropeptide leucokinin. We discuss this and other examples of how Hox genes generate diversity within the central nervous system of Drosophila.     

Embryonic origin and differentiation of the Drosophila heart

We have followed the normal development of the different cell types associated with the Drosophila dorsal vessel, i.e. cardioblasts, pericardial cells, alary muscles, lymph gland and ring gland, by using several tissue-specific markers and transmission electron microscopy. Precursors of pericardial cells and cardioblasts split as two longitudinal rows of cells from the lateral mesoderm of segments T2-A7 (cardiogenic region) during stage 12. The lymph gland and dorsal part of the ring gland (corpus allatum) originate from clusters of lateral mesodermal cells located in T3 and T1/dorsal ridge, respectively. Cardioblast precursors are strictly segmentally organized; each of T2-A6 gives rise to six cardioblasts. While moving dorsally during the stages leading up to dorsal closure, cardioblast precursors become flattened, polarized cells aligned in a regular longitudinal row. At dorsal closure, the leading edges of the cardioblast precursors meet their contralateral counterparts. The lumen of the dorsal vessel is formed when the trailing edges of the cardioblast precursors of either side bend around and contact each other. The amnioserosa invaginates during dorsal closure and is transiently attached to the cardioblasts; however, it does not contribute to the cells associated with the dorsal vessel and degenerates during late embryogenesis. We describe ultrastructural characteristics of cardioblast differentiation and discuss similarities between cardioblast development and capillary differentiation in vertebrates. Correspondence to: V. Hartenstein     

Homeotic gene function in the muscles of Drosophila larvae

The segmental musculature of Drosophila melanogaster larvae consists of 24-30 muscles per segment. Unique patterns of muscles are found in the three thoracic segments and the first and last abdominal segments; the remaining abdominal segments share the same pattern. Mutations in Ultrabithorax (Ubx) cause partial transformation of the muscle pattern of larval abdominal segments towards metathorax. The muscles of the thorax are not affected. In the first two abdominal segments the changes include the loss of at least 11 `abdominal' muscles and the gain of 11 `thoracic' muscles. Less extensive transformations are seen in more posterior abdominal segments. Anterobithorax, bithorax, postbithorax and bithoraxoid mutations also induce transformations of the larval musculature. Each allelic group affects a domain that is a subset of the entire Ubx domain but these domains are not restricted to compartments or segments and may extend through as many as five segments. In the muscles the segmental distribution of Ubx antigen correlates with the segments affected by Ubx mutations. The different domains of Ubx in mesoderm and ectoderm argue that the segmental diversity of the muscle pattern is not simply induced by the overlying epidermis and that Ubx function in the mesoderm is required for the correct development of abdominal segments.     

Negative regulation ofUltrabithorax expression byengrailed is required for proper specification of wing development inDrosophila melanogaster

In both vertebrates and invertebrates, homeotic selector genes confer morphological differences along the antero-posterior axis. However, insect wing development is independent of all homeotic gene functions, reflecting the ground plan of an ancestral pterygote, which bore wings on all segments. Dipteran insects such asDrosophila are characterized by a pair of wings in the mesothoracic segment. In all other segments, wing development is essentially repressed by different homeotic genes, although in the metathorax they are modified into a pair of halteres. This necessitates that during development all homeotic genes are to be maintained in a repressed state in wing imaginal discs. In this report we show that (i) the function of the segment polarity geneengrailed (en) is critical to keep the homeotic selector geneUltrabithorax (Ubx) repressed in wing imaginal discs, (ii) normal levels of En in the posterior compartment of haltere discs, however, are not enough to completely repressUbx, and (iii) the repression ofUbx byen is independent of Hedgehog signalling through which the long-range signalling ofen is mediated during wing development. Finally we provide evidence for a possible mechanism by whichen repressesUbx. On the basis of these results we propose thaten has acquired two independent functions during the evolution of dorsal appendages. In addition to its well-known function of conferring posterior fate and inducing long-range signalling to pattern the developing appendages, it maintains wing fate by keepingUbx repressed.     

Hox genes are essential for the development of eyespots in Bicyclus anynana butterflies

The eyespot patterns found on the wings of nymphalid butterflies are novel traits that originated first in hindwings and subsequently in forewings, suggesting that eyespot development might be dependent on Hox genes. Hindwings differ from forewings in the expression of Ultrabithorax (Ubx), but the function of this Hox gene in eyespot development as well as that of another Hox gene Antennapedia (Antp), expressed specifically in eyespots centers on both wings, are still unclear. We used CRISPR-Cas9 to target both genes in Bicyclus anynana butterflies. We show that Antp is essential for eyespot development on the forewings and for the differentiation of white centers and larger eyespots on hindwings, whereas Ubx is essential not only for the development of at least some hindwing eyespots but also for repressing the size of other eyespots. Additionally, Antp is essential for the development of silver scales in male wings. In summary, Antp and Ubx, in addition to their conserved roles in modifying serially homologous segments along the anterior–posterior axis of insects, have acquired a novel role in promoting the development of a new set of serial homologs, the eyespot patterns, in both forewings (Antp) and hindwings (Antp and Ubx) of B. anynana butterflies. We propose that the peculiar pattern of eyespot origins on hindwings first, followed by forewings, could be due to an initial co-option of Ubx into eyespot development followed by a later, partially redundant, co-option of Antp into the same network.     

Peptidergic control of the heart of the stick insect, Baculum extradentatum

The dorsal vessel of the Vietnamese stick insect, Baculum extradentatum, consists of a tubular heart and an aorta that extends anteriorly into the head. Alary muscles, associated with the heart, are anchored to the body wall with attachments to the dorsal diaphragm. Alary muscle contraction draws haemolymph into the heart through incurrent ostia. Excurrent ostia lie on the dorsal vessel in the last thoracic and in each of the first two abdominal segments. Muscle fibers are associated with these excurrent ostia. Crustacean cardioactive peptide (CCAP)- and proctolin-like immunoreactivity is present in axons of the segmental nerves that project to the dorsal vessel, and in processes extending over the heart and alary muscles. Proctolin-like immunoreactive processes are also localized to the valves of the incurrent ostia and to the excurrent ostia. Neither the link nerve neurons, nor the lateral cardiac neurons, stain positively for these peptides. Physiological assays reveal dose-dependent increases in heart beat frequency in response to CCAP and proctolin. Isolating the dorsal vessel from the ventral nerve cord led to a change in the pattern of heart contractions, from a tonic, stable heart beat, to one which was phasic. The tonic nature was restored by the application of CCAP.     

Diversification of muscle types: Recent insights from Drosophila

Myogenesis is a highly conserved process ending up by the formation of contracting muscles. In Drosophila embryos, myogenesis gives rise to a segmentally repeated array of thirty distinct fibres, each of which represents an individual muscle. Since Drosophila offers a large range of genetic tools for easily testing gene functions, it has become one of the most studied and consequently best-described model organisms for muscle development. Over the last two decades, the Drosophila model system has enabled major advances in our understanding of how the initially equivalent mesodermal cells become competent for entering myogenic differentiation and how each distinct type of muscle is specified. Here we present an overview of Drosophila muscle development with a special focus on the diversification of muscle types and the genes that control acquisition of distinct muscle properties.     

Hox gene duplications correlate with posterior heteronomy in scorpions

The evolutionary success of the largest animal phylum, Arthropoda, has been attributed to tagmatization, the coordinated evolution of adjacent metameres to form morphologically and functionally distinct segmental regions called tagmata. Specification of regional identity is regulated by the Hox genes, of which 10 are inferred to be present in the ancestor of arthropods. With six different posterior segmental identities divided into two tagmata, the bauplan of scorpions is the most heteronomous within Chelicerata. Expression domains of the anterior eight Hox genes are conserved in previously surveyed chelicerates, but it is unknown how Hox genes regionalize the three tagmata of scorpions. Here, we show that the scorpion Centruroides sculpturatus has two paralogues of all Hox genes except Hox3, suggesting cluster and/or whole genome duplication in this arachnid order. Embryonic anterior expression domain boundaries of each of the last four pairs of Hox genes (two paralogues each of Antp, Ubx, abd-A and Abd-B) are unique and distinguish segmental groups, such as pectines, book lungs and the characteristic tail, while maintaining spatial collinearity. These distinct expression domains suggest neofunctionalization of Hox gene paralogues subsequent to duplication. Our data reconcile previous understanding of Hox gene function across arthropods with the extreme heteronomy of scorpions.     

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of α-actinin in larval hearts. Additionally, we perform labeling of F-actin and α-actinin in myosin-GFP expressing adult flies and of α-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.Download video file.^{(130M, mp4)}     

Over-expression of Ultrabithorax alters embryonic body plan and wing patterns in the butterfly Bicyclus anynana

In insects, forewings and hindwings usually have different shapes, sizes, and color patterns. A variety of RNAi experiments across insect species have shown that the hox gene Ultrabithorax (Ubx) is necessary to promote hindwing identity. However, it remains unclear whether Ubx is sufficient to confer hindwing fate to forewings across insects. Here, we address this question by over-expressing Ubx in the butterfly Bicyclus anynana using a heat-shock promoter. Ubx whole-body over-expression during embryonic and larvae development led to body plan changes in larvae but to mere quantitative changes to adult morphology, respectively. Embryonic heat-shocks led to fused segments, loss of thoracic and abdominal limbs, and transformation of head limbs to larger appendages. Larval heat-shocks led to reduced eyespot size in the expected homeotic direction, but neither additional eyespots nor wing shape changes were observed in forewings as expected of a homeotic transformation. Interestingly, Ubx was found to be expressed in a novel, non-characteristic domain – in the hindwing eyespot centers. Furthermore, ectopic expression of Ubx on the pupal wing activated the eyespot-associated genes spalt and Distal-less, known to be directly repressed by Ubx in the fly?s haltere and leg primordia, respectively, and led to the differentiation of black wing scales. These results suggest that Ubx has been co-opted into a novel eyespot gene regulatory network, and that it is capable of activating black pigmentation in butterflies.     

The Border Between the Ultrabithorax and abdominal-A Regulatory Domains in the Drosophila Bithorax Complex

The bithorax complex in Drosophila melanogaster includes three homeobox-containing genes—Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B)—which are required for the proper differentiation of the posterior 10 segments of the body. Each of these genes has multiple distinct regulatory regions; there is one for each segmental unit of the body plan where the genes are expressed. One additional protein- coding gene in the bithorax complex, Glut3, a sugar-transporter homolog, can be deleted without phenotype. We focus here on the upstream regulatory region for Ubx, the bithoraxoid (bxd) domain, and its border with the adjacent infraabdominal-2 (iab-2) domain, which controls abdA. These two domains can be defined by the phenotypes of rearrangement breakpoints, and by the expression patterns of enhancer traps. In D. virilis, the homeotic cluster is split between Ubx and abd-A, and so the border can also be located by a sequence comparison between species. When the border region is deleted in melanogaster, the flies show a dominant phenotype called Front-ultraabdominal (Fub); the first abdominal segment is transformed into a copy of the second abdominal segment. Thus, the border blocks the spread of activation from the bxd domain into the iab-2 domain.