Tube Fusion Research Articles

Computational imaging and modeling approaches reveal mechanisms driving myocardial precursor movements

Transformation of planar cardiac progenitor fields into a multilayered tubular heart involves cell and tissue movements in four dimensions. Our objective was to define the forces driving midline myocardial progenitor movements. Imaging of embryos with fluorescence labeled myocardial and endodermal cells demonstrated that myocardial progenitor fields undergo coordinated deformation leading to anterior displacements relative to the endoderm. Microincision mapping implicated endodermal folding during foregut morphogenesis as one force driving midline myocardia movements. Cell shape changes and contraction were also observed at the medial myocardial field border, and pharmacological inhibition of myocardial Rho kinase signaling resulted in decreased endodermal tissue motion. Computational modeling reproduced the myocardial field folding and medial contraction, while simulated incisions reproduced observed post‐surgery cardiac phenotypes. We conclude that antero‐medially directed movement of cardiac progenitors to the site of tubular heart assembly relies on: 1) endoderm folding to drive the convergence at the midline and 2) deformations intrinsic to the myocardial primordia, to mediate movement in the anterior direction and to reposition the myocardia along the dorso‐ventral embryonic axis. Collectively these result in the optimal orientation for myocardial tube fusion at the midline.

Evidence for tryptophan being a signal molecule that inhibits conidial anastomosis tube fusion during colony initiation in Neurospora crassa

Mycelial interconnectedness achieved by hyphal fusion has been hypothesized to facilitate the distribution and sharing of nutrients between different parts of a mycelium, especially when nutrients are heterogeneously distributed in the environment. However, the link between environmental nutrient availability and hyphal fusion is little understood. Here, we report that amino acids and extracellular pH regulate conidial anastomosis tube (CAT) fusion during colony initiation in Neurospora crassa. Quantitative analyses revealed that low extracellular pH and certain amino acids, particularly tryptophan, inhibit CAT fusion. Conidial germination was also inhibited by tryptophan but this inhibition was mitigated by the presence of other amino acids. This provides evidence for tryptophan having a role as a signal molecule that regulates CAT fusion. Tryptophan acts intracellularly because two amino acid permease mutants (Δmtr and Δaap-20) exhibited resistance against tryptophan-mediated inhibition of CAT fusion. Tryptophan and low pH did not significantly affect vegetative hyphal fusion in mature colonies, indicating that the latter is regulated in a different manner to CAT fusion.

Heterokaryon Incompatibility Is Suppressed Following Conidial Anastomosis Tube Fusion in a Fungal Plant Pathogen

It has been hypothesized that horizontal gene/chromosome transfer and parasexual recombination following hyphal fusion between different strains may contribute to the emergence of wide genetic variability in plant pathogenic and other fungi. However, the significance of vegetative (heterokaryon) incompatibility responses, which commonly result in cell death, in preventing these processes is not known. In this study, we have assessed this issue following different types of hyphal fusion during colony initiation and in the mature colony. We used vegetatively compatible and incompatible strains of the common bean pathogen Colletotrichum lindemuthianum in which nuclei were labelled with either a green or red fluorescent protein in order to microscopically monitor the fates of nuclei and heterokaryotic cells following hyphal fusion. As opposed to fusion of hyphae in mature colonies that resulted in cell death within 3 h, fusions by conidial anastomosis tubes (CAT) between two incompatible strains during colony initiation did not induce the vegetative incompatibility response. Instead, fused conidia and germlings survived and formed heterokaryotic colonies that in turn produced uninucleate conidia that germinated to form colonies with phenotypic features different to those of either parental strain. Our results demonstrate that the vegetative incompatibility response is suppressed during colony initiation in C. lindemuthianum. Thus, CAT fusion may allow asexual fungi to increase their genetic diversity, and to acquire new pathogenic traits.

Surface Tension Driven Instabilities in Single-Component Saturated Fatty Acid Membrane Tubes

Shape instabilities in single-component, saturated fatty acid membrane tubes have been investigated using phase contrast microscopy. These tubes were created in the course of a Langmuir monolayer collapse transition. Two types of shape instabilities were observed: (i) the excitation of a bending mode of a single tube, and (ii) topological changes of an assembly of tubes. The development of tube bending was accompanied by a shape transition from extended amphiphilic globules to confined ones that were transported in the tube. The evolution of bending instability has been analyzed as a balance among the bending energy, the surface tension energy of the tube, and the hydrodynamic dissipation energy by the surrounding fluid. Topological changes of an assembly of tubes were initiated by the formation of a membrane passage connecting two opposing tubes and followed by tube fusion and breaking. These changes were interpreted as a result of surface tension-gradient driven molecules transport on the tube surface.

Arabidopsis thaliana MIRO1 and MIRO2 GTPases Are Unequally Redundant in Pollen Tube Growth and Fusion of Polar Nuclei during Female Gametogenesis

MIRO GTPases have evolved to regulate mitochondrial trafficking and morphology in eukaryotic organisms. A previous study showed that T-DNA insertion in the Arabidopsis MIRO1 gene is lethal during embryogenesis and affects pollen tube growth and mitochondrial morphology in pollen, whereas T-DNA insertion in MIRO2 does not affect plant development visibly. Phylogenetic analysis of MIRO from plants revealed that MIRO 1 and 2 orthologs in dicots cluster in two separate groups due to a gene/genome duplication event, suggesting that functional redundancy may exists between the two MIRO genes. To investigate this possibility, we generated miro1 (+/−)/miro2-2 (−/−) plants. Compared to miro1 (+/−) plants, the miro1 (+/−)/miro2-2 (−/−) plants showed increased segregation distortion. miro1 (+/−)/miro2-2 (−/−) siliques contained less aborted seeds, but more than 3 times the number of undeveloped ovules. In addition, reciprocal crosses showed that co-transmission through the male gametes was nearly absent, whereas co-transmission through the female gametes was severely reduced in miro1 (+/−)/miro2-2 (−/−) plants. Further investigations revealed that loss of MIRO2 (miro2 (−/−)) function in the miro1 (+/−) background enhanced pollen tube growth defects. In developing miro1 (+/−)/miro2 (−/−) embryo sacs, fusion of polar nuclei was further delayed or impaired compared to miro1 plants. This phenotype has not been reported previously for miro1 plants and coincides with studies showing that defects in some mitochondria-targeted genes results in the same phenotype. Our observations show that loss of function in MIRO2 in a miro1 (+/−) background enhances the miro1 (+/−) phenotype significantly, even though miro2 (−/−) plants alone does not display any phenotypes. Based on these findings, we conclude that MIRO1 and MIRO2 are unequally redundant and that a proportion of the miro1 (+/−)/miro2 (−/−) plants haploid gametes displays the complete null phenotype of MIRO GTPase function at key developmental stages.

Grainyhead-like 2 regulates neural tube closure and adhesion molecule expression during neural fold fusion

Defects in closure of embryonic tissues such as the neural tube, body wall, face and eye lead to severe birth defects. Cell adhesion is hypothesized to contribute to closure of the neural tube and body wall; however, potential molecular regulators of this process have not been identified. Here we identify an ENU-induced mutation in mice that reveals a molecular pathway of embryonic closure. Line2F homozygous mutant embryos fail to close the neural tube, body wall, face, and optic fissure, and they also display defects in lung and heart development. Using a new technology of genomic sequence capture and high-throughput sequencing of a 2.5Mb region of the mouse genome, we discovered a mutation in the grainyhead-like 2 gene (Grhl2). Microarray analysis revealed Grhl2 affects the expression of a battery of genes involved in cell adhesion and E-cadherin protein is drastically reduced in tissues that require Grhl2 function. The tissue closure defects in Grhl2 mutants are similar to that of AP-2α null mutants and AP-2α has been shown to bind to the promoter of E-cadherin. Therefore, we tested for a possible interaction between these genes. However, we find that Grhl2 and AP-2α do not regulate each other's expression, E-cadherin expression is normal in AP-2α mutants during neural tube closure, and Grhl2;AP-2α trans-heterozygous embryos are morphologically normal. Taken together, our studies point to a complex regulation of neural tube fusion and highlight the importance of comparisons between these two models to understand more fully the molecular pathways of embryonic tissue closure.

Echinoid regulates tracheal morphology and fusion cell fate in Drosophila

Morphogenesis of the Drosophila embryonic trachea involves a stereotyped pattern of epithelial tube branching and fusion. Here, we report unexpected phenotypes resulting from maternal and zygotic (M/Z) loss of the homophilic cell adhesion molecule Echinoid (Ed), as well as the subcellular localization of Ed in the trachea. ed(M/Z) embryos have convoluted trachea reminiscent of septate junction (SJ) and luminal matrix mutants. However, Ed does not localize to SJs, and ed(M/Z) embryos have intact SJs and show normal luminal accumulation of the matrix-modifying protein Vermiform. Surprisingly, tracheal length is not increased in ed(M/Z) mutants, but a previously undescribed combination of reduced intersegmental spacing and deep epidermal grooves produces a convoluted tracheal phenotype. In addition, ed(M/Z) mutants have unique fusion defects involving supernumerary fusion cells, ectopic fusion events and atypical branch breaks. Tracheal-specific expression of Ed rescues these fusion defects, indicating that Ed acts in trachea to control fusion cell fate.

Cephalothoracopagus, janiceps, disymmetros, monoomphalian conjoined twins undiagnosed until early second trimester

Cephalothoracopagus is the less common type of conjoined twins (CTs) with an incidence estimated at one in three million births or one in 58 conjoined twins. Maternal gene Vg1, a member of the TGF-beta family of cell-signalling molecules which are implicated in dorsoanterior development, and specific actions of Hox and Pax genes that are implicated in very early embryogenesis may be identified as aetiologic factors. A prenatal diagnosis of cephalothoracopagus CTs diagnosed at 17 weeks in a woman undergoing amniocentesis for advanced maternal age is reported. Although first-trimester diagnosis of CTs is feasible and has been reported as early as 8 weeks's gestation, CTs may be misdiagnosed with monoamniotic twins, lymphangioma, teratoma, and/or neoplasm and may be undiagnosed until early second trimester. Three-dimensional and color Doppler ultrasound enabled precise prenatal visualization of the fusion site. Ultrafast MR imaging should be considered an adjunct to ultrasound for antenatal characterization of structural anomalies and for planning surgical separation in selected cases. Echocardiography is mandatory in all cases of CTs as congenital heart defects are seen in 20-30% and polyhydramnios in 50-75%. Neural tube and midline fusion defects, diaphragmatic hernia, and imperforate anus are the frequently associated abnormalities. Prognosis among survivors is usually poor (44% die in the neonatal period) and is dependent upon the type of conjunction, degree of involvement of the shared organs, and presence or absence of associated anomalies, with the worst prognosis in case of twins sharing liver and heart.

Physical Blocking Neural Tube Closure Affects Radial Intercalation and Neural Crest Midline-directed Migration in<I>Xenopus</I>Dorsal Explants

Neural tube defects (NTDs) are severe congenital malformation diseases, which occur in 1 out of 1000 births in human. In Xenopus , several tissue movements are involved in the neural tube closure process. Immediately after the neural tube fusion, the neural crest cells get monopolar protrusion toward dorsal midline and migrate to form the roof of the neural tube. At the same time, radial intercalation takes place from the ventral neural tube and forces it to be single-layered. Here, we physically block the neural tube closure to test the cell movements and the following patterning in Xenopus laevis explants. The results show that the single-layered neural tube fails to form and the neural crest cells remain at the lateral regions in the explants with NTDs. However, the patterning of the neural tube is not affected as indicated by the normal expression of the preneural genes. These results indicate a requirement of the neural tube fusion for the radial intercalation and the dorsal midline directed neural crest migration, but not for the dorsal-ventral patterning of the neural tube.

Fusion Body Formation, Germ Tube Anastomosis, and Nuclear Migration During the Germination of Urediniospores of the Wheat Leaf Rust Fungus, Puccinia triticina

ABSTRACT Vegetative or parasexual recombination is thought to be a key mechanism for the genetic diversity of cereal rust fungi. The process of germ tube fusion leading to hyphal anastomosis and nuclear recombination was analyzed in wheat leaf rust fungus, Puccinia triticina. Germ tube anastomosis was observed in 27 P. triticina isolates, each representing a different virulence phenotype. Germ tube fusion bodies (GFBs), which appeared as viscid globules formed at tips of germ tubes, were essential for germ tube anastomosis. The formation of GFBs was affected by the urediniospore density and the length of illumination during germination. GFBs were formed at the highest frequency when urediniospores were spread to a concentration of 1 x 10(6) urediniospores/ml and incubated in dark for 12 to 24 h during germination. GFB attached to either the side of another germ tube ("tip to side") or to another GFB formed at the tip of a second germ tube ("tip to tip"). In "tip to side" anastomosis, two nuclei in the germ tube bearing the GFB migrated into the second germ tube through the GFB which resulted in four nuclei within this germ tube. In "tip to tip" anastomosis, nuclei in both germ tubes migrated into the fused GFB and all four nuclei came into close proximity. Urediniospores of isolates MBDS-3-115 and TBBJ-5-11 were stained with DAPI (4',6'diamine-2-phenylindole) and Nuclear Yellow (Hoechst S769121), respectively, and then mixed and germinated on water agar. Some fused GFBs contained nuclei stained with DAPI and nuclei stained with Nuclear Yellow in close proximity, demonstrating the fusion between genetically different P. triticina isolates. In some fused GFBs, "bridge-like" structures connecting different nuclei were observed.

CBP/p300 and associated transcriptional co-activators exhibit distinct expression patterns during murine craniofacial and neural tube development.

Mutations in each of the transcriptional co-activator genes - CBP, p300, Cited2, Cart1 and Carm1 - result in neural tube defects in mice. The present study thus furnishes a complete and comparative temporal and spatial expression map of CBP/p300 and associated transcriptional co-activators, Cited2, Cart1 and Carm1 during the period of murine neural tube development (embryonic days 8.5 to 10.5). Each co-activator except Cart1 was expressed in the dorsal neural folds on E8.5. Although CBP and p300 are functionally interchangeable in vitro, their respective expression patterns diverge during embryogenesis before neural fold fusion is complete. CBP gene expression was lost from the neural folds by E8.75 and was thereafter weakly expressed in the maxillary region and limb buds, while p300 exhibited strong expression in the first branchial arch, limb bud and telencephalic regions on E9.5. Cart1 exhibited strong expression in the forebrain mesenchyme from E9.0 through E10.5. Although CBP, p300, Carm1 and Cited2 share temporal expression on E8.5, these co-activators have different spatial expression in mesenchyme and/or the neuroepithelium. Nevertheless, co-localization to the dorsal neural folds on E8.5 suggests a functional role in elevation and/or fusion of the neural folds. Target genes, and pathways that promote cranial neural tube fusion that are activated by CBP/p300/Carm1/Cited2/Cart1-containing transcriptional complexes await elucidation.

EphrinA-EphA receptor interactions in mouse spinal neurulation: implications for neural fold fusion

The molecular basis of neural fold adhesion and fusion is a poorly understood aspect of neurulation. Cell surface glycosyl phosphatidylinositol (GPI)-anchored proteins have been implicated in neural fold adhesion, with ephrinAs particularly attractive candidates in view of the cranial neural tube defects observed in mice lacking ephrinA5 or the EphA7 receptor. Here, we demonstrate that ephrinsA1, A3 and A4, as well as several EphA receptors, are expressed in the closing mouse spinal neural tube. Most ephrinAs and EphA receptors were found to be expressed in multiple tissues in the caudal region, whereas EphA2 receptor was expressed specifically at the apices of the neural folds just prior to onset of neural tube fusion. Using mouse whole embryo culture, we found that cleavage of GPI-anchored molecules from the embryonic cell surface resulted in delay of spinal neural tube closure. Injection of EphA1 and EphA3 fusion proteins intra-amniotically into cultured embryos was used to specifically disrupt ephrinA-EphA receptor interactions, and led to inhibition of spinal neural tube closure, without adverse effects on growth or developmental progression. These treatments did not disturb neural plate bending or neural fold elevation, both of which are critical for spinal neural tube closure. Our findings demonstrate that ephrinA-EphA receptor interactions are required for closure of the mouse spinal neural tube, and support the hypothesis that ephrinA-EphA receptor interactions may participate in the molecular recognition events that culminate in adhesion and fusion of the tips of the neural folds during spinal neurulation.

γCOP Is Required for Apical Protein Secretion and Epithelial Morphogenesis in Drosophila melanogaster

BackgroundThere is increasing evidence that tissue-specific modifications of basic cellular functions play an important role in development and disease. To identify the functions of COPI coatomer-mediated membrane trafficking in Drosophila development, we were aiming to create loss-of-function mutations in the γCOP gene, which encodes a subunit of the COPI coatomer complex.Principal FindingsWe found that γCOP is essential for the viability of the Drosophila embryo. In the absence of zygotic γCOP activity, embryos die late in embryogenesis and display pronounced defects in morphogenesis of the embryonic epidermis and of tracheal tubes. The coordinated cell rearrangements and cell shape changes during tracheal tube morphogenesis critically depend on apical secretion of certain proteins. Investigation of tracheal morphogenesis in γCOP loss-of-function mutants revealed that several key proteins required for tracheal morphogenesis are not properly secreted into the apical lumen. As a consequence, γCOP mutants show defects in cell rearrangements during branch elongation, in tube dilation, as well as in tube fusion. We present genetic evidence that a specific subset of the tracheal defects in γCOP mutants is due to the reduced secretion of the Zona Pellucida protein Piopio. Thus, we identified a critical target protein of COPI-dependent secretion in epithelial tube morphogenesis.Conclusions/SignificanceThese studies highlight the role of COPI coatomer-mediated vesicle trafficking in both general and tissue-specific secretion in a multicellular organism. Although COPI coatomer is generally required for protein secretion, we show that the phenotypic effect of γCOP mutations is surprisingly specific. Importantly, we attribute a distinct aspect of the γCOP phenotype to the effect on a specific key target protein.

Examinations of nuclear behaviour during sexual cycle in the diplontic oomycete Phytophthora palmivora (Butl.) Butl.

Nuclear behaviour during gametangial- and oospore development in Phytophthora palmivora was studied by light-, fluorescence-, and electron microscopy. After contact between oogonial and anther-idial initials, the antheridial initial is penetrated by the oogonial initial. The latter expands and nuclei and cytoplasm stream into it. After the delimitation of gametangia, a number of nuclei degenerate, the rest proceed through two gametic meiotic divisions both in the antheridium and the oogonium. Three of the meiotic products from each nucleus degenerate. The oogonium is penetrated by the antheridial fertilization tube and nuclear fusion occurs. The oosphere forms, then the periplasm degenerates, and the oospore wall builds up. Upon release of zoospores diploid mycelia are produced.

Annual Meeting, Saskatoon, Saskatchewan, 2007 / Réunion annuelle, Saskatoon, Saskatchewan, 2007

Annual Meeting, Saskatoon, Saskatchewan, 2007 / Réunion annuelle, Saskatoon, Saskatchewan, 2007

Heparan sulfate Ndst1 gene function variably regulates multiple signaling pathways during mouse development

Disruption of heparan sulfate (HS) synthesis in vertebrate development causes malformations that are composites of those caused by mutations of multiple HS binding growth factors and morphogens. We previously reported severe developmental defects of the forebrain and the skull in mutant mice bearing a targeted disruption of the heparan sulfate-generating enzyme GlcNAc N-deacetylase/GlcN N-sulfotransferase 1 (Ndst1). Here, we further characterize the molecular mechanisms leading to frontonasal dysplasia in Ndst1 mutant embryos and describe additional malformations, including impaired spinal and cranial neural tube fusion and skeletal abnormalities. Of the numerous proteins that bind HS, we show that impaired fibroblast growth factor, Hedgehog, and Wnt function may contribute to some of these phenotypes. Our findings, therefore, suggest that defects in HS synthesis may contribute to multifactor types of congenital developmental defects in humans, including neural tube defects.

Optical tweezer micromanipulation of filamentous fungi

Optical tweezers have been little used in experimental studies on filamentous fungi. We have built a simple, compact, easy-to-use, safe and robust optical tweezer system that can be used with brightfield, phase contrast, differential interference contrast and fluorescence optics on a standard research grade light microscope. We have used this optical tweezer system in a range of cell biology applications to trap and micromanipulate whole fungal cells, organelles within cells, and beads. We have demonstrated how optical tweezers can be used to: unambiguously determine whether hyphae are actively homing towards each other; move the Spitzenkörper and change the pattern of hyphal morphogenesis; make piconewton force measurements; mechanically stimulate hyphal tips; and deliver chemicals to localized regions of hyphae. Significant novel experimental findings from our study were that germ tubes generated significantly smaller growth forces than leading hyphae, and that both hyphal types exhibited growth responses to mechanical stimulation with optically trapped polystyrene beads. Germinated spores that had been optically trapped for 25 min exhibited no deleterious effects with regard to conidial anastomosis tube growth, homing or fusion.

Reference guide to the stages of chick heart embryology

Cardiac progenitors of the splanchnic mesoderm (primary and secondary heart field), cardiac neural crest, and the proepicardium are the major embryonic contributors to chick heart development. Their contribution to cardiac development occurs with precise timing and regulation during such processes as primary heart tube fusion, cardiac looping and accretion, cardiac septation, and the development of the coronary vasculature. Heart development is even more complex if one follows the development of the cardiac innervation, cardiac pacemaking and conduction system, endocardial cushions, valves, and even the importance of apoptosis for proper cardiac formation. This review is meant to provide a reference guide (Table 1) on the developmental timing according to the staging of Hamburger and Hamilton (1951) (HH) of these important topics in heart development for those individuals new to a chick heart research laboratory. Even individuals outside of the heart field, who are working on a gene that is also expressed in the heart, will gain information on what to look for during chick heart development. This reference guide provides complete and easy reference to the stages involved in heart development, as well as a global perspective of how these cardiac developmental events overlap temporally and spatially, making it a good bench top companion to the many recently written in-depth cardiac reviews of the molecular aspects of cardiac development.

The cap 'n' collar family member NF-E2-related factor 3 (Nrf3) is expressed in mesodermal derivatives of the avian embryo

NF-E2-related factor 3 (Nrf3) is a recently identified member of a family of transcription factors homologous to the Drosophila "cap 'n' collar" or CNC protein. The cnc gene is located immediately 3' to the Drosophila homeobox gene cluster and has been shown to regulate at least one of those genes, deformed. Likewise, human and mouse CNC homologues are located immediately 3' to each of the four Hox complexes, although no genetic interactions have yet been demonstrated in vertebrates. Work presented here demonstrates that Nrf3, adjacent to the Hox A cluster, is expressed during early development of the chicken embryo. Expression begins in the presumptive heart myocardium from the time of cardiac tube fusion through the looping process. Nrf3 transcripts then disappear from the heart and are next observed in the myotomal compartment of maturing somites, restricted to the medial portion along the rostrocaudal axis and fading after muscle precursors migrate. Central nervous system expression appears gradually and persists at low levels in ventricular neuroepithelial cells until at least embryonic day 6. Strong expression is observed in the early epiphysis, in the collecting ducts of the developing kidney and in individual cells of the yolk sac, underlying blood islands. This is the first description using in situ hybridization of the expression of a CNC family member and its dynamics through the course of early development.

Roles of JUMONJI in mouse embryonic development

Cardiac development is a complex biological process requiring the integration of cell specification, differentiation, migration, proliferation, and morphogenesis. Although significant progress has been made recently in understanding the molecular basis of cardiac development, mechanisms of transcriptional control of cardiac development remain largely unknown. In search for the developmentally important genes, the jumonji gene (jmj) was identified by gene trap technology and characterized as a critical nuclear factor for mouse embryonic development. Jmj has been shown to play important roles in cardiovascular development, neural tube fusion process, hematopoiesis, and liver development in mouse embryos. The amino acid sequence of the JUMONJI protein (JMJ) reveals that JMJ belongs to the AT-rich interaction domain transcription factor family and more recently has been described as a member of the JMJ transcription factor family. Here, we review the roles of jmj in multiple organ development with a focus on cardiovascular development in mice.