Yorkie controls tube length and apical barrier integrity during airway development

Epithelial organ size and shape depend on cell shape changes, cell-matrix communication, and apical membrane growth. The Drosophila melanogaster embryonic tracheal network is an excellent model to study these processes. Here, we show that the transcriptional coactivator of the Hippo pathway, Yorkie (YAP/TAZ in vertebrates), plays distinct roles in the developing Drosophila airways. Yorkie exerts a cytoplasmic function by binding Drosophila Twinstar, the orthologue of the vertebrate actin-severing protein Cofilin, to regulate F-actin levels and apical cell membrane size, which are required for proper tracheal tube elongation. Second, Yorkie controls water tightness of tracheal tubes by transcriptional regulation of the δ-aminolevulinate synthase gene (Alas). We conclude that Yorkie has a dual role in tracheal development to ensure proper tracheal growth and functionality

WASH phosphorylation balances endosomal versus cortical actin network integrities during epithelial morphogenesis

Filamentous actin (F-actin) networks facilitate key processes like cell shape control, division, polarization and motility. The dynamic coordination of F-actin networks and its impact on cellular activities are poorly understood. We report an antagonistic relationship between endosomal F-actin assembly and cortical actin bundle integrity during Drosophila airway maturation. Double mutants lacking receptor tyrosine phosphatases (PTP) Ptp10D and Ptp4E, clear luminal proteins and disassemble apical actin bundles prematurely. These defects are counterbalanced by reduction of endosomal trafficking and by mutations affecting the tyrosine kinase Btk29A, and the actin nucleation factor WASH. Btk29A forms protein complexes with Ptp10D and WASH, and Btk29A phosphorylates WASH. This phosphorylation activates endosomal WASH function in flies and mice. In contrast, a phospho-mimetic WASH variant induces endosomal actin accumulation, premature luminal endocytosis and cortical F-actin disassembly. We conclude that PTPs and Btk29A regulate WASH activity to balance the endosomal and cortical F-actin networks during epithelial tube maturation

Genome mapping of quantitative trait loci in Salix with an emphasis on freezing resistance

The current advancement of biotechnology could provide a better understanding of the genetic control and the molecular basis of quantitative traits in plants. The present thesis focuses on the identification of genes affecting freezing resistance and phenological traits. Two genetic maps were constructed for two interspecific Salix families. The first map was based on a backcross (n = 87) ofthe male clone “Björn” (Salix viminalis x Salix schwerinii) with the female clone “78183” (Salix viminalis) and composed of 325 AFLP and 25 RFLP loci, the latter mainly derived from the Populus genome. The second map consisted of 433 AFLPs and it was based on an F? family (n = 92) with grandparents being a frost susceptible female clone (“Jorunn”; S. viminalis) and a frost resistant male clone (“SW901290”; 5. dasyclados like). The average length ofthe maps and the genome coverage were 2483 cM and 72% respectively, while the average distance between loci was about 11 cM.Using the genetic map of the backcross family, nineteen quantitative trait loci (QTL) were identified; eleven for growth-related traits and eight for the timing of bud flush. The estimated magnitude of the QTL effect ranged from 12 to 24% of the total phenotypic variance. One QTL for height growth, one for diameter and one for the height:diameter ratio, were found clustered in the same marker interval. One QTL associated with indoor bud flushing coincided with a QTL controlling timing of bud flush in the field. Little evidence was found for QTL stability in height growth over 3 consecutive years.Ten genomic regions controlling freezing resistance and nine affecting phenological traits were identified during cold acclimation ofthe F? family. The magnitude ofthe phenotypic variation explained by each freezing resistance locus varied over acclimation time (3 - 45%) and there was no time point at which all the QTL could be detected. The single QTL detected for non-acclimated freezing resistance did not reach significance at any time during cold acclimation suggesting an independent relationship between non-acclimated and acclimated freezing resistance in Salix. The determination of QTL position on the map suggested the partial involvement of a common set of genes for autumn freezing resistance and phenology traits. Of the 14 QTL controlling autumn freezing resistance and phenological traits at the indoor experiment, six (43%) were associated with autumn phenology traits i.e. total height increment, dry-to-fresh weight ratio and number of new leaves, as measured in the field. A major locus with multi-trait association in both indoor and outdoor experiments was detected. The presence of genes with large effect for growth and adaptation traits suggests that a marker assisted selection breeding scheme could accelerate the breeding process in Salix

Genetic variation in freezing resistance of some Populus and Salix clones

grantor: University of TorontoNineteen (19) clones of Salix and twenty-one (21) clones of Populus were examined for their genetic variability in freezing resistance. A series of laboratory freezing tests were conducted, using visual assessment and electrolyte leakage to detect freezing injury and survival. Clones were tested at predetermined levels of freezing stress and during seven (7) phenological stages: dormant, early spring, spring, flushing of terminal buds, new axillary growth, growing and early-fall stages. Significant clonal variation in freezing resistance was detected at (4) stages: spring, flushing of terminal buds, axillary new growth, and early fall stage. During winter dormancy and early spring, when freezing resistance was greatest, no significant differences in clonal survival were detected. At the growing stage in which clones exhibited the highest susceptibility to freezing stress, only Salix clones showed significant differences in the estimated index of injury which, however, accounted for only a small proportion (2%) of the total variation in index of injury. At the early fall stage, the proportion of within genus variation in index of injury attributable to clonal differences alone, was 22% for Salix and 24% for Populus. The significance of within clones ranking differences from stage to stage and with respect to freezing injury was dependent on the individual clone. Spearman's rank correlation coefficients for clonal rankings between stages were significant, except in the case of the correlation between the spring and early fall stages. Finally, the use of electrolyte leakage was found to provide a simple and rapid screening method for assessing the likely survival and freezing resistance of Populus and Salix clones.M.Sc

Control of airway tube diameter and integrity by secreted chitin-binding proteins in Drosophila.

The transporting function of many branched tubular networks like our lungs and circulatory system depend on the sizes and shapes of their branches. Understanding the mechanisms of tube size control during organ development may offer new insights into a variety of human pathologies associated with stenoses or cystic dilations in tubular organs. Here, we present the first secreted luminal proteins involved in tube diametric expansion in the Drosophila airways. obst-A and gasp are conserved among insect species and encode secreted proteins with chitin binding domains. We show that the widely used tracheal marker 2A12, recognizes the Gasp protein. Analysis of obst-A and gasp single mutants and obst-A; gasp double mutant shows that both genes are primarily required for airway tube dilation. Similarly, Obst-A and Gasp control epidermal cuticle integrity and larval growth. The assembly of the apical chitinous matrix of the airway tubes is defective in gasp and obst-A mutants. The defects become exaggerated in double mutants indicating that the genes have partially redundant functions in chitin structure modification. The phenotypes in luminal chitin assembly in the airway tubes are accompanied by a corresponding reduction in tube diameter in the mutants. Conversely, overexpression of Obst-A and Gasp causes irregular tube expansion and interferes with tube maturation. Our results suggest that the luminal levels of matrix binding proteins determine the extent of diametric growth. We propose that Obst-A and Gasp organize luminal matrix assembly, which in turn controls the apical shapes of adjacent cells during tube diameter expansion

Early development of Drosophila embryos requires Smc5/6 function during oogenesis

Mutations in structural maintenance of chromosomes (Smc) proteins are frequently associated with chromosomal abnormalities commonly observed in developmental disorders. However, the role of Smc proteins in development still remains elusive. To investigate Smc5/6 function during early embryogenesis we examined smc5 and smc6 mutants of the fruit fly Drosophila melanogaster using a combination of reverse genetics and microscopy approaches. Smc5/6 exhibited a maternally contributed function in maintaining chromosome stability during early embryo development, which manifested as female subfertility in its absence. Loss of Smc5/6 caused an arrest and a considerable delay in embryo development accompanied by fragmented nuclei and increased anaphase-bridge formation, respectively. Surprisingly, early embryonic arrest was attributable to the absence of Smc5/6 during oogenesis, which resulted in insufficient repair of pre-meiotic and meiotic DNA double-strand breaks. Thus, our findings contribute to the understanding of Smc proteins in higher eukaryotic development by highlighting a maternal function in chromosome maintenance and a link between oogenesis and early embryogenesis

Overexpression of <i>Obst</i>-family members causes tube dilation.

<p>(a–c) Overexpression in wild-type embryos of <i>btl</i>>ANF-GFP (a), <i>btl</i>><i>Gasp</i>-GFP (b) and <i>btl</i>><i>Gasp</i>-GFP; <i>Obst-A</i> (c). The simultaneous overexpression of Gasp-GFP and Obst-A in trachea forms tube dilations in dorsal trunk (arrowheads) (c). (d) Quantifications showing the percentage (%) of embryos with dorsal trunk dilations in the following genotypes: <i>btl</i>>ANF-GFP (n = 16), <i>btl</i>><i>Gasp</i>-GFP (n = 12) and <i>btl</i>><i>Gasp</i>-GFP; <i>Obst-A</i> (n = 38). Bars show the means of three independent experiments and error bars show the ± standard error of the means. * and ** denote significant differences (p<0.01 and p<0.0001) between the means of <i>btl</i>><i>Gasp</i>-GFP or <i>btl</i>><i>Gasp</i>-GFP;<i>Obst-A</i> to the <i>btl</i>>ANF-GFP (control) by a two-tailed distribution unpaired Student’s <i>t</i>-test. Scale bar:10 µm.</p

<i>obst-A</i> and <i>gasp</i> mutants show defects in luminal tracheal matrix.

<p>(a–d) Confocal microscopy projections of the trachea labeled with a FITC-conjugated chitin-binding probe (ChtB). The filamentous chitin is detected in the wild-type embryos at stage 16 (a). The chitin staining intensity is weaker in the <i>obst-A</i> (b), <i>gasp</i> (c) and <i>obst-A; gasp</i> (d) mutant embryos. (e–h) Optical sections of tracheal tubes stained for Verm. Verm is secreted to the tracheal lumen in wild-type embryos at stage 16 (e). The amount of secreted Verm is reduced in both <i>obst-A</i> (f) and <i>gasp</i> (g) single mutant embryos and <i>obst-A; gasp</i> (h) double mutant embryos. Scale bar: 10 µm.</p

Lumen diameter expansion requires Obst-A and Gasp.

<p>(a–d) Confocal microscopy projections of the trachea of wild-type (a–a′′′), <i>obst-A</i> (b–b′′′), <i>gasp</i> (c–c′′′) and <i>obst-A; gasp</i> (d–d′′′) embryos labeled with the apical marker Uninflated (Uif) at stage 16.1. Inserts display y-z projections of the DT tubes from 3 different metameres. a′, b′, c′ and d′ inserts are from the middle of metamere 4, a′′, b′′, c′′ and d′′ from the middle of metamere 5 and a′′′, b′′′, c′′′ and d′′′ from the middle of metamere 6. Scale bar: 10 µm (e) Quantification of tracheal diameter of wild-type, <i>obst-A</i>, <i>gasp</i> and <i>obst-A; gasp</i> embryos at stage 16.1. The graph shows diameter measurements of three metameres: 4, 5 and 6. Number of embryos used for measurement of each genotype n = 6. The y-axis represents the diameter in micrometers. Tube diameter values in all mutant embryos were significantly different (p<0.05) from the wild-type by a two-tailed distribution unpaired Student’s <i>t</i>-test.</p

Ultrastructure of the tracheal lumen in <i>obst-A; gasp</i> mutants.

<p>(a–c) Transmission electron micrographs of wild-type and <i>obst-A; gasp</i> dorsal trunks at the end of embryogenesis. The wild-type embryos (a) show uniformly distributed chitin rich procuticle. In <i>obst-A; gasp</i> double mutant (b, c) the taenidial folds are irregular and the procuticle is distorted with granular amorphous accumulations (arrow). epc = epicuticle (arrow heads), pro = procuticle layer (double arrows). Scale bar: 0.5 µm.</p