Dorsal Vessel Research Articles

Phagocytic ability declines with age in adult Drosophila hemocytes.

Most multicellular organisms show a physiological decline in immune function with age. However, little is known about the mechanisms underlying these changes. We examined Drosophila melanogaster, an important model for identifying genes affecting innate immunity and senescence, to explore the role of phagocytosis in age-related immune dysfunction. We characterized the localized response of immune cells at the dorsal vessel to bacterial infection in 1-week- and 5-week-old flies. We developed a quantitative phagocytosis assay for adult Drosophila and utilized this to characterize the effect of age on phagocytosis in transgenic and natural variant lines. We showed that genes necessary for bacterial engulfment in other contexts are also required in adult flies. We found that blood cells from young and old flies initially engulf bacteria equally well, while cells from older flies accumulate phagocytic vesicles and thus are less capable of destroying pathogens. Our results have broad implications for understanding how the breakdown in cellular processes influences immune function with age.

The Stanford Microsurgery and Resident Training (SMaRT) Scale

We previously reported results of our on-line microsurgery training program, showing that residents who had access to our website significantly improved their cognitive and technical skills. In this study, we report an objective means for expert evaluators to reliably rate trainees' technical skills under the microscope, with the use of our novel global rating scale. "Microsurgery Essentials" (http://smartmicrosurgery.com) is our on-line training curriculum. Residents were randomly divided into 2 groups: 1 group reviewed this online resource and the other did not. Pre- and post-tests consisted of videotaped microsurgical sessions in which the trainee performed "microsurgery" on 3 different models: latex glove, penrose drain, and the dorsal vessel of a chicken foot. The SMaRT (Stanford Microsurgery and Resident Training) scale, consisting of 9 categories graded on a 5-point Likert scale, was used to assess the trainees. Results were analyzed with ANOVA and Student t test, with P less than 0.05 indicating statistical significance. Seventeen residents participated in the study. The SMaRT scale adequately differentiated the performance of more experienced senior residents (PGY-4 to PGY-6, total average score=3.43) from less experienced junior residents (PGY-1 to PGY-3, total average score=2.10, P<0.0001). Residents who viewed themselves as being confident received a higher score on the SMaRT scale (average score 3.5), compared to residents who were not as confident (average score 2.1) (P<0.001). There were no significant differences in scoring among all 3 evaluators (P>0.05). Additionally, junior residents who had access to our website showed a significant increase in their graded technical performance by 0.7 points when compared to residents who did not have access to the website who showed an improvement of only 0.2 points (P=0.01). Our SMaRT scale is valid and reliable in assessing the microsurgical skills of residents and other trainees. Current trainees are more likely to use self-directed on-line education because of its easy accessibility and interactive format. Our global rating scale can help ensure residents are achieving appropriate technical milestones.

Myotropic effects of FMRFamide containing peptides on the heart of the mosquito Anopheles gambiae

FMRFamide-like peptides (FLPs) are produced by invertebrate and vertebrate animals, and regulate diverse physiological processes. In insects, several FLPs modulate heart physiology, with some increasing and others decreasing dorsal vessel contraction dynamics. Here, we describe the FMRFamide gene structure in the mosquito, Anopheles gambiae, quantify the developmental and spatial expression of FMRFamide and its putative receptor (FMRFamideR), and show that the peptides FMRFamide and SALDKNFMRFamide have complex myotropic properties. RACE sequencing showed that the FMRFamide gene encodes eight putative FLPs and is alternatively spliced. Of the eight FLPs, only one is shared by A. gambiae, Aedes aegypti and Culex quinquefasciatus: SALDKNFMRFamide. Quantitative PCR showed that peak expression of FMRFamide and FMRFamideR occurs in second instar larvae and around eclosion. In adults, FMRFamide is primarily transcribed in the head and thorax, and FMRFamideR is primarily transcribed in the thorax. Intravital video imaging of mosquitoes injected FMRFamide and SALDKNFMRFamide revealed that at low doses these peptides increase heart contraction rates. At high doses, however, these peptides decrease heart contraction rates and alter the proportional directionality of heart contractions. Taken altogether, these data describe the FMRFamide gene in A. gambiae, and show that FLPs are complex modulators of mosquito circulatory physiology.

An exceptionally preserved arthropod cardiovascular system from the early Cambrian

The assumption that amongst internal organs of early arthropods only the digestive system withstands fossilization is challenged by the identification of brain and ganglia in early Cambrian fuxianhuiids and megacheirans from southwest China. Here we document in the 520-million-year-old Chengjiang arthropod Fuxianhuia protensa an exceptionally preserved bilaterally symmetrical organ system corresponding to the vascular system of extant arthropods. Preserved primarily as carbon, this system includes a broad dorsal vessel extending through the thorax to the brain where anastomosing branches overlap brain segments and supply the eyes and antennae. The dorsal vessel provides segmentally paired branches to lateral vessels, an arthropod ground pattern character, and extends into the anterior part of the abdomen. The addition of its vascular system to documented digestive and nervous systems resolves the internal organization of F. protensa as the most completely understood of any Cambrian arthropod, emphasizing complexity that had evolved by the early Cambrian.

Lumbriculus variegatus as a model organism for studying angiogenesis during tissue regeneration (LB696)

Lumbriculus variegatus is an aquatic oligochaete that is often used to study epimorphic regeneration following removal of head or tail segments. In this pattern of regeneration, the cut surface forms a blastema in which undifferentiated cells begin to regenerate the lost tissues. L. variegatus fully regenerates lost tail segments within two to three weeks. While several studies have investigated the regeneration of neural networks during epimorphosis in L. variegatus, little is known about its pattern of angiogenesis during regeneration. We used L. variegatus cultures along with light microscopy and image capturing and analysis for daily in vivo documentation of the structural changes that occur during angiogenesis in regenerating tail segments. The transparent cuticle, closed circulatory system and erythrocruorin pigment in the plasma made the living vasculature visible for microscopic analysis. The branching pattern of new vasculature was easily observed. Pulsations of the dorsal blood vessel allowed tracking of blood flow to determine when new vessels became patent. A brief application of the anesthetic Tricaine reduced locomotion and facilitated the capturing of images. We suggest that this organism is a useful model to study the effects of angiogenic stimulators or inhibitors during wound healing and tissue regeneration.Grant Funding Source: Supported by Baker University Faculty Development Fund

Three-dimensional reconstruction of the musculature of Cossura pygodactylata Jones, 1956 (Annelida: Cossuridae)

The musculature of adult specimens of Cossura pygodactylata was studied by means of F-actin labelling and confocal laser scanning microscopy (CLSM). Their body wall is comprised of five longitudinal muscle bands: two dorsal, two ventral and one ventromedial. Complete circular fibres are found only in the abdominal region, and they are developed only on the border of the segments. Thoracic and posterior body regions contain only transverse fibres ending near the ventral longitudinal bands. Almost-complete rings of transverse muscles, with gaps on the dorsal and ventral sides, surround the terminal part of the pygidium. Four longitudinal bands go to the middle of the prostomium and 5–14 paired dorso-ventral muscle fibres arise in its distal part. Each buccal tentacle contains one thick and two thin longitudinal muscle filaments; thick muscle fibres from all tentacles merge, forming left and right tentacle protractors rooted in the dorsal longitudinal bands of the body wall. The circumbuccal complex includes well-developed upper and lower lips. These lips contain an outer layer of transverse fibres, and the lower lip also contains inner oblique muscles going to the dorsal longitudinal bands. The branchial filament contains two longitudinal muscle fibres that do not connect with the body musculature. The parapodial complex includes strong intersegmental and segmental oblique muscles in the thoracic region only; chaetal retractors, protractors and muscles of the body wall are present in all body regions. Muscle fibres are developed in the dorsal and ventral mesenteries. One semi-circular fibre is developed on the border of each segment and is most likely embedded in the dissepiment. The intestine has thin circular fibres along its full length. The dorsal blood vessel has strong muscle fibres that cover its anterior part, which is called the heart. It consists of short longitudinal elements forming regular rings and inner partitions. The musculature of C. pygodactylata includes some elements that are homologous with similar muscular components in other polychaetes (i.e., the body wall and most parapodial muscles) and several unique features, mostly at the anterior end.

Molecular characterization and possible biological roles of allatotropin in Rhodnius prolixus

Allatotropins (ATs) are a family of neuropeptides that have been shown to stimulate the biosynthesis of juvenile hormone in certain insect species, and to have stimulatory activity on some visceral muscles. Here, we have examined the AT in Rhodnius prolixus. Molecular analysis revealed a cDNA fragment of 973bp encoding one mature amidated AT (Rhopr-AT) with transcript levels observed in the central nervous system (CNS) and pool of fat body, trachea and associated peripheral nerves. AT-like immunoreactive neurons were found throughout the CNS and AT-like immunoreactive processes were present on some peripheral tissues. Bioassays based upon changes in hindgut and dorsal vessel contractions failed to demonstrate any myotropic effects of Rhopr-AT on these tissues; however Rhopr-AT stimulated contractions of muscles surrounding the salivary glands and secretion of saliva, as judged by the reduction in content of the cherry red saliva from the salivary glands. Serotonin stimulated an increase in peristaltic contractions of the gland though no secretion was observed. Co-application of Rhopr-AT and serotonin resulted in a more rapid secretion than either chemical alone.

Characterization of spermatogonial markers in the mature testis of the dogfish (Scyliorhinus canicula L.)

In dogfish, spermatogenesis progresses from a restricted germinative zone, which lines the dorsal testicular vessel. Single spermatogonia (A(s)), including the spermatogonial stem cells (SSCs), produce successively paired (A(p)), undifferentiated (A(u4) to A(u512)), and differentiated (A(d1) to A(d8)) spermatogonia and preleptotene (PL) spermatocytes through 13 mitoses. Dogfish spermatogonial subpopulations present classical morphological characteristics but cannot be distinguished on the basis of molecular markers. This characterization has been initiated in mammals despite the difficulty to separate each spermatogonial subpopulation. For instance, both glial cell-derived neurotrophic factor family receptor alpha 1 (GFRα1) and promyelocytic leukemia zinc finger protein (PLZF) are markers of undifferentiated spermatogonia, whereas receptor tyrosine kinase C-kit is a marker of differentiated spermatogonia. The aim of this study is to characterize spermatogonial markers and to differentiate several spermatogonial subpopulations. Dogfish cDNA sequences have been identified and validated by phylogenetic analyses for gfrα1, plzf, pou2, as well as for high-mobility group box proteins 2 and 3 (hmgb2 and 3) and for mini-chromosome maintenance protein 6 (mcm6). We have used the anatomical advantage of the polarized dogfish testis to analyze the expression of those markers by RT-PCR and in situ hybridization. gfrα1, pou2, and plzf have been detected in the testicular germinative zone, suggesting that spermatogonial markers are relatively well conserved among vertebrates but with a less restricted expression for plzf. Moreover, hmgb3 and mcm6 have been identified as new markers of differentiated spermatogonia. Finally, this first molecular characterization of spermatogonial subpopulations in a chondrichthyan model will be useful for further studies on the SSC niche evolution.

Interactions of PPAR-alpha and adenosine receptors in hypoxia-induced angiogenesis

Hypoxia and adenosine are known to upregulate angiogenesis; however, the role of peroxisome proliferator-activated receptor alpha (PPARα) in angiogenesis is controversial. Using transgenic Tg(fli-1:EGFP) zebrafish embryos, interactions of PPARα and adenosine receptors in angiogenesis were evaluated under hypoxic conditions. Epifluorescent microscopy was used to assess angiogenesis by counting the number of intersegmental (ISV) and dorsal longitudinal anastomotic vessel (DLAV) at 28h post-fertilization (hpf). Hypoxia (6h) stimulated angiogenesis as the number of ISV and DLAV increased by 18-fold (p<0.01) and 100±8% (p<0.001), respectively, at 28hpf. Under normoxic and hypoxic conditions, WY-14643 (10μM), a PPARα activator, stimulated angiogenesis at 28hpf, while MK-886 (0.5μM), an antagonist of PPARα, attenuated these effects. Compared to normoxic condition, adenosine receptor activation with NECA (10μM) promoted angiogenesis more effectively under hypoxic conditions. Involvement of A2B receptor was implied in hypoxia-induced angiogenesis as MRS-1706 (10nM), a selective A2B antagonist attenuated NECA (10μM)-induced angiogenesis. NECA- or WY-14643-induced angiogenesis was also inhibited by miconazole (0.1μM), an inhibitor of epoxygenase dependent production of eicosatrienoic acid (EET) epoxide. Thus, we conclude that: activation of PPARα promoted angiogenesis just as activation of A2B receptors through an epoxide dependent mechanism.

Comparative anatomy of the heart–glomerulus complex of Cephalodiscus gracilis (Pterobranchia): structure, function, and phylogenetic implications

Cephalodiscus gracilis Harmer, 1905 is a semi-sessile deuterostome that shares with fish-like chordates pharyngeal gill slits and a dorsally situated brain. In order to reveal structures potentially homologous among deuterostomes and to infer their functional roles, we investigated the axial complex, associated blood vessels and structures of C. gracilis using transmission electron microscopy, light microscopy, and digital 3D reconstructions. We describe the smooth, bipartite cephalic shield retractor muscles that originate as solid compact muscles and fan out to traverse the protocoel as individual muscle cells. The axial complex consists of a cap-shaped coelomic sac, the pericardium that surrounds the central heart. The pericardium is constituted of myoepithelial cells, with the cells facing the heart being thicker and richer in myofilaments. A prominent dorsal median blood vessel opens into the heart, which gives rise to a short median ventral vessel that opens into the paired glomeruli connected to the ventral side of the stomochord. The tip of the curved stomochord rests precisely above the connection of the dorsal median vessel with the heart, a position that would allow the stomochord to function as a valve facilitating unidirectional blood flow. Glomeruli are lined by podocytes of the spacious protocoel and are considered to be the site of ultrafiltration. Two pairs of blood vessels enter the median dorsal blood vessel from the tentacles. The median dorsal blood vessel is separated from the brain by a thin basement membrane. This arrangement is consistent with the hypothesis that blood vessels in the tentacles increase oxygen supply for the brain. Based on detailed similarities, the heart–glomerulus complex of C. gracilis is considered homologous with the heart–glomerulus complex in Rhabdopleura spp., and Enteropneusta, and the axial complex in Echinodermata. In addition, we hypothesize homology to the excretory complex including Hatschek’s nephridium in Cephalochordata. Thus, the heart–glomerulus complex does not support a sister-group relationship between Echinodermata and Hemichordata, whereas the organization of the cephalic shield retractor muscles is consistent with the evolution of pterobranchs within enteropneusts.

Different regulation of physiological and tumor angiogenesis in zebrafish by protein kinase D1 (PKD1)

Protein kinase D isoenzymes (PKDs, Prkds) are serine threonine kinases that belong to the CAMK superfamily. PKD1 is expressed in endothelial cells and is a major mediator of biological responses downstream of the VEGFRs that are relevant for angiogenesis such as endothelial cell migration, proliferation and tubulogenesis in vitro. PKDs also play a critical role in tumor development and progression, including tumor angiogenesis. However, given the plethora of signaling modules that drive angiogenesis, the precise role of PKD1 in both physiological and tumor angiogenesis in vivo has not been worked out so far. This study aimed at dissecting the contribution of PKD1 to physiological blood vessel formation, PKD1 was found to be widely expressed during zebrafish development. As far as physiological angiogenesis was concerned, morpholino-based silencing of PKD1 expression moderately reduced the formation of the intersomitic vessels and the dorsal longitudinal anastomotic vessel in tg(fli1:EGFP) zebrafish. In addition, silencing of PKD1 resulted in reduced formation of the parachordal lymphangioblasts that serves as a precursor for the developing thoracic duct. Interestingly, tumor angiogenesis was completely abolished in PKD1 morphants using the zebrafish/tumor xenograft angiogenesis assay. Our data in zebrafish demonstrate that PKD1 contributes to the regulation of physiological angiogenesis and lymphangiogenesis during zebrafish development and is essential for tumor angiogenesis.

Seizure-Sensitivity in Drosophila Is Ameliorated by Dorsal Vessel Injection of the Antiepileptic Drug Valproate

Drosophila is a powerful model organism that can be used for the development of new drugs directed against human disease. A limitation is the ability to deliver drugs for testing. We report on a novel delivery system for treating Drosophila neurological mutants, direct injection into the circulatory system. Using this method, we show that injection of the antiepileptic drug valproate can ameliorate seizure-sensitive phenotypes in several mutant genotypes in the bang-sensitive (BS) paralytic mutant class, sda, eas, and parabss1. This drug-injection method is superior to drug-feeding methods that we have employed previously, presumably because it bypasses potent detoxification systems present in the fly. In addition, we find that utilizing blood-brain barrier mutations in the background may further improve the injection results under certain circumstances. We propose that this method of drug delivery is especially effective when using Drosophila to model human pathologies, especially neurological syndromes.

Abstract 172: Notch Signaling Induces Endocardial Hematopoiesis And Endocardial Cushion Formation Downstream Of Nkx2-5

A subset of cardiac progenitors display multipotency to give rise to major cardiovascular lineages during cardiogenesis. In addition, we have recently identified Nkx2-5+ endocardial cells in the outflow and atria that contribute to definitive hematopoietic cells in vivo, which represents phylogenetically conserved pathway of cardio-vasculo-hematopoietic differentiation. Here, we have examined the molecular mechanism underlying the endocardial hematopoiesis. Immunostaining and colony assay suggest that Nkx2-5 mutant mice showed significant decrease in CD31+/41+ hemogenic endocardial cells, while lineage tracing on Nkx2-5-null background revealed that Nkx2-5-derived endocardial cells are present. These data indicate that Nkx2-5 is required not for the formation of the Nkx2-5-derived endocardium but for the hemogenic activity of the endocardium. Notch signaling is also active in cushion endocardium and its mutant embryos show a similar defect with Nkx2-5 mutants as previously reported. Inspired by these similarities between the function of Nkx2-5 and Notch signaling during cardiogenesis, we next investigated the impact of Notch activation on the endocardial hematopoiesis. In vivo forced activation of Notch in the developing heart using Nkx2-5-Cre mice led to massive increase in the endocardial hematopoiesis. As Nkx2-5 binding sites are found in the regulatory region of Notch1, RBPjk, and Jag1 genes, we tested whether Notch regulates endocardial hematopoiesis downstream of Nkx2-5. Surprisingly, Notch activation restored the defective endocardial hematopoiesis as well as endocardial cushion formation in Nkx2-5 knockout mice, suggesting that Notch signaling mediates the Nkx2-5-dependent endocardial hematopoiesis and cushion development. Drosophila dorsal vessel and lymph gland are closely related and regulated by tinman and notch. Therefore, Nkx2-5-Notch regulatory pathway represents phylogenetically conserved fundamental mechanism of cardio-vasculo-hematopoietic differentiation pathway.

TOPOGRAPHY OF CIRCULATORY SYSTEM OF GLOSSOSCOLEX PAULISTUS MICHAELSEN 1926 (OLIGOCHAETA GLOSSOSCOLECIDAE)

SUMMARY In segment 24 and successive ones the vascular topography of Glossoscolex paulistus Michaelsen, 1926 is repeated metamerically. The afferent system is more similar to those of Allolobophora and Eisenia than that of Lumbricus. The efferent system is similar to that of Pontodrilus. In segments 17 to 23 the position of the vessels is slightly modified due to a pair of copulatory chambers, whose circulation is like that of the prostates of Pheretima. In segments 7 to 9 the ventro-tegumentary vessels arise from the lateral hearts. Those of 9 which supply the genital organs and anastomose with the ventro-tegumentary vessels of 14, are especially voluminous forming a safety system. From the gizzard forwards the dorsal vessel is replaced by a vessel formed by the coalescence of branches of the ventro-tegumentary vessels of 6. Longitudinal neural vessels are absent throughout the whole body.

Different Regulation of Physiological and Tumor Angiogenesis in Zebrafish by Protein Kinase D1 (PKD1)

Protein kinase D isoenzymes (PKDs, Prkds) are serine threonine kinases that belong to the CAMK superfamily. PKD1 is expressed in endothelial cells and is a major mediator of biological responses downstream of the VEGFRs that are relevant for angiogenesis such as endothelial cell migration, proliferation and tubulogenesis in vitro. PKDs also play a critical role in tumor development and progression, including tumor angiogenesis. However, given the plethora of signaling modules that drive angiogenesis, the precise role of PKD1 in both physiological and tumor angiogenesis in vivo has not been worked out so far. This study aimed at dissecting the contribution of PKD1 to physiological blood vessel formation, PKD1 was found to be widely expressed during zebrafish development. As far as physiological angiogenesis was concerned, morpholino-based silencing of PKD1 expression moderately reduced the formation of the intersomitic vessels and the dorsal longitudinal anastomotic vessel in tg(fli1:EGFP) zebrafish. In addition, silencing of PKD1 resulted in reduced formation of the parachordal lymphangioblasts that serves as a precursor for the developing thoracic duct. Interestingly, tumor angiogenesis was completely abolished in PKD1 morphants using the zebrafish/tumor xenograft angiogenesis assay. Our data in zebrafish demonstrate that PKD1 contributes to the regulation of physiological angiogenesis and lymphangiogenesis during zebrafish development and is essential for tumor angiogenesis.

Identification and Expression of the CCAP Receptor in the Chagas’ Disease Vector, Rhodnius prolixus, and Its Involvement in Cardiac Control

Rhodnius prolixus is the vector of Chagas’ disease, by virtue of transmitting the parasite Trypanosoma cruzi. There is no cure for Chagas’ disease and therefore controlling R. prolixus is currently the only method of prevention. Understanding the physiology of the disease vector is an important step in developing control measures. Crustacean cardioactive peptide (CCAP) is an important neuropeptide in insects because it has multiple physiological roles such as controlling heart rate and modulating ecdysis behaviour. In this study, we have cloned the cDNA sequence of the CCAP receptor (RhoprCCAPR) from 5th instar R. prolixus and found it to be a G-protein coupled receptor (GPCR). The spatial expression pattern in 5th instars reveals that the RhoprCCAPR transcript levels are high in the central nervous system, hindgut and female reproductive systems, and lower in the salivary glands, male reproductive tissues and a pool of tissues including the dorsal vessel, trachea, and fat body. Interestingly, the RhoprCCAPR expression is increased prior to ecdysis and decreased post-ecdysis. A functional receptor expression assay confirms that the RhoprCCAPR is activated by CCAP (EC50 = 12 nM) but not by adipokinetic hormone, corazonin or an extended FMRFamide. The involvement of CCAP in controlling heartbeat frequency was studied in vivo and in vitro by utilizing RNA interference. In vivo, the basal heartbeat frequency is decreased by 31% in bugs treated with dsCCAPR. Knocking down the receptor in dsCCAPR-treated bugs also resulted in loss of function of applied CCAP in vitro. This is the first report of a GPCR knock-down in R. prolixus and the first report showing that a reduction in CCAPR transcript levels leads to a reduction in cardiac output in any insect.

DIFERENTIAL DNA SYNTHESIS IN Anopheles albimanus TISSUES INDUCED BY IMMUNE CHALLENGE WITH DIFFERENT MICROORGANISMS

The induction of DNA synthesis in various tissues of Anopheles albimanus, in response to challenge with Saccharomyces cerevisiae, Micrococcus luteus, and Serratia marcescens, was analyzed by 5-bromo-2-deoxy-uridine (BrdU) incorporation. Microorganism-inoculated mosquitoes were fed with a sucrose solution containing BrdU and maintained alive for 5 days. Alternatively, abdominal carcasses of microorganisms-inoculated mosquitoes were cultivated in Roswell Park Memorial Institute (RPMI) medium supplemented with BrdU for 5 days. Control groups were inoculated with RPMI alone. In both experiments, DNA synthesis, evidenced by epifluorescence with an anti-BrdU fluorescein-labeled antibody, occurred in fat body, epithelial cells of pleural membranes, dorsal vessel, and the oviducts. Relative quantification of DNA synthesis, evaluated by ELISA using an anti-BrdU peroxidase-labeled antibody, was higher in abdomen tissues of microorganisms-inoculated mosquitoes than controls in in vitro and in vivo experiments. The intensity of DNA synthesis varied among the different microorganism challenges, but was higher in in vivo experiments, compared to cultured samples. These differences in DNA synthesis suggest a compartmentalization of the immune response, probably mediated by different signaling pathways.

Frazzled/DCC facilitates cardiac cell outgrowth and attachment during Drosophila dorsal vessel formation

Drosophila embryonic dorsal vessel (DV) morphogenesis is a highly stereotyped process that involves the migration and morphogenesis of 52 pairs of cardioblasts (CBs) in order to form a linear tube. This process requires spatiotemporally-regulated localization of signaling and adhesive proteins in order to coordinate the formation of a central lumen while maintaining simultaneous adhesion between CBs. Previous studies have shown that the Slit/Roundabout and Netrin/Unc5 repulsive signaling pathways facilitate site-specific loss of adhesion between contralateral CBs in order to form a luminal space. However, the concomitant mechanism by which attraction initiates CB outgrowth and discrete localization of adhesive proteins remains poorly understood. Here we provide genetic evidence that Netrin signals through DCC (Deleted in Colorectal Carcinoma)/UNC-40/Frazzled (Fra) to mediate CB outgrowth and attachment and that this function occurs prior to and independently of Netrin/UNC-5 signaling. fra mRNA is expressed in the CBs prior to and during DV morphogenesis. Loss-of-fra-function results in significant defects in cell shape and alignment between contralateral CB rows. In addition, CB outgrowth and attachment is impaired in both fra loss- and gain-of-function mutants. Deletion of both Netrin genes (NetA and NetB) results in CB attachment phenotypes similar to fra mutants. Similar defects are also seen when both fra and unc5 are deleted. Finally we show that Fra accumulates at dorsal and ventral leading edges of paired CBs, and this localization is dependent upon Netrin. We propose that while repulsive guidance mechanisms contribute to lumen formation by preventing luminal domains from coming together, site-specific Netrin/Frazzled signaling mediates CB attachment.

Transitions in the heartbeat pattern during pupal diapause and adult development in the flesh fly, Sarcophaga crassipalpis

The heartbeat of diapausing pupae of the flesh fly Sarcophaga crassipalpis was investigated using electrocardiographic methods including gravimetry, thermography, and optocardiography. During deep diapause, characterized by minimum metabolic activity, the heart exhibited discontinuous bouts of exclusively unidirectional, anterograde pulsations (40–60contractions/min) that lasted only a few seconds. These bouts of cardiac pulsations were separated by periods of rest lasting 5–30min. During infradian peaks of metabolic activity (4-day cycles) that occur throughout diapause, periods of rest were shortened and frequency of the anterograde heartbeat increased more than two-fold. Throughout diapause, the heart consistently exhibited a simple, bi-phasic pattern of pulsations generated by bouts of anterograde heartbeats interspersed with periods of cardiac rest. When the fly broke diapause and initiated pharate adult development, a new tri-phasic pattern was observed: the new pattern incorporated heartbeat reversal, as noted by the appearance of retrograde pulsations that directed hemolymph in a posterior direction. These retrograde heartbeats occurred exclusively in the abdominal portion of the dorsal vessel and were not observed in the head or thorax. The transition to pharate adult development was also accompanied by the appearance of extremely strong extracardiac pulsations that served a respiratory function. Although these pulsations made it more challenging to record heartbeat patterns in pharate adults, we observed progressive shortening of the resting periods and a continual increase in the rate of both anterograde and retrograde pulsations, a trend that was further magnified in the adult fly (anterograde contractions up to 300/min and retrograde contractions approximately 125/min). These results imply that the circulatory function of the heart is homeostatically regulated and is responsive to developmental changes and the diverse metabolic rate demands of larvae, diapausing pupae, pharate adults and adult flies.

The Chicken Foot Dorsal Vessel as a High-Fidelity Microsurgery Practice Model

The Chicken Foot Dorsal Vessel as a High-Fidelity Microsurgery Practice Model