A Late Role for bmp2b in the Morphogenesis of Semicircular Canal Ducts in the Zebrafish Inner Ear

BACKGROUND:The Bone Morphogenetic Protein (BMP) genes bmp2 and bmp4 are expressed in highly conserved patterns in the developing vertebrate inner ear. It has, however, proved difficult to elucidate the function of BMPs during ear development as mutations in these genes cause early embryonic lethality. Previous studies using conditional approaches in mouse and chicken have shown that Bmp4 has a role in semicircular canal and crista development, but there is currently no direct evidence for the role of Bmp2 in the developing inner ear. METHODOLOGY/PRINCIPAL FINDINGS:We have used an RNA rescue strategy to test the role of bmp2b in the zebrafish inner ear directly. Injection of bmp2b or smad5 mRNA into homozygous mutant swirl (bmp2b(-/-)) embryos rescues the early patterning defects in these mutants and the fish survive to adulthood. As injected RNA will only last, at most, for the first few days of embryogenesis, all later development occurs in the absence of bmp2b function. Although rescued swirl adult fish are viable, they have balance defects suggestive of vestibular dysfunction. Analysis of the inner ears of these fish reveals a total absence of semicircular canal ducts, structures involved in the detection of angular motion. All other regions of the ear, including the ampullae and cristae, are present and appear normal. Early stages of otic development in rescued swirl embryos are also normal. CONCLUSIONS/SIGNIFICANCE:Our findings demonstrate a critical late role for bmp2b in the morphogenesis of semicircular canals in the zebrafish inner ear. This is the first demonstration of a developmental role for any gene during post-embryonic stages of otic morphogenesis in the zebrafish. Despite differences in the early stages of semicircular canal formation between zebrafish and amniotes, the role of Bmp2 in semicircular canal duct outgrowth is likely to be conserved between different vertebrate species

Wingless Signalling Alters the Levels, Subcellular Distribution and Dynamics of Armadillo and E-Cadherin in Third Instar Larval Wing Imaginal Discs

Background: Armadillo, the Drosophila orthologue of vertebrate beta-catenin, plays a dual role as the key effector of Wingless/Wnt1 signalling, and as a bridge between E-Cadherin and the actin cytoskeleton. In the absence of ligand, Armadillo is phosphorylated and targeted to the proteasome. Upon binding of Wg to its receptors, the "degradation complex'' is inhibited; Armadillo is stabilised and enters the nucleus to transcribe targets. Methodology/Principal Findings: Although the relationship between signalling and adhesion has been extensively studied, few in vivo data exist concerning how the "transcriptional'' and "adhesive'' pools of Armadillo are regulated to orchestrate development. We have therefore addressed how the subcellular distribution of Armadillo and its association with E-Cadherin change in larval wing imaginal discs, under wild type conditions and upon signalling. Using confocal microscopy, we show that Armadillo and E-Cadherin are spatio-temporally regulated during development, and that a punctate species becomes concentrated in a subapical compartment in response to Wingless. In order to further dissect this phenomenon, we overexpressed Armadillo mutants exhibiting different levels of activity and stability, but retaining E-Cadherin binding. Arm(S10) displaces endogenous Armadillo from the AJ and the basolateral membrane, while leaving E-Cadherin relatively undisturbed. Surprisingly, Delta NArm(1-155) caused displacement of both Armadillo and E-Cadherin, results supported by our novel method of quantification. However, only membrane-targeted Myr-Delta NArm(1-155) produced comparable nuclear accumulation of Armadillo and signalling to Arm(S10). These experiments also highlighted a row of cells at the A/P boundary depleted of E-Cadherin at the AJ, but containing actin. Conclusions/Significance: Taken together, our results provide in vivo evidence for a complex non-linear relationship between Armadillo levels, subcellular distribution and Wingless signalling. Moreover, this study highlights the importance of Armadillo in regulating the subcellular distribution of E-CadherinPublisher PDFPeer reviewe

Directed-Backbone Dissociation Following Bond-Specific Carbon-Sulfur UVPD at 213 nm

Ultraviolet photodissociation or UVPD is an increasingly popular option for tandem-mass spectrometry experiments. UVPD can be carried out at many wavelengths, and it is important to understand how the results will be impacted by this choice. Here, we explore the utility of 213 nm photons for initiating bond-selective fragmentation. It is found that bonds previously determined to be labile at 266 nm, including carbon-iodine and sulfur-sulfur bonds, can also be cleaved with high selectivity at 213 nm. In addition, many carbon-sulfur bonds that are not subject to direct dissociation at 266 nm can be selectively fragmented at 213 nm. This capability can be used to site-specifically create alaninyl radicals that direct backbone dissociation at the radical site, creating diagnostic d-ions. Furthermore, the additional carbon-sulfur bond fragmentation capability leads to signature triplets for fragmentation of disulfide bonds. Absorption of amide bonds can enhance dissociation of nearby labile carbon-sulfur bonds and can be used for stochastic backbone fragmentation typical of UVPD experiments at shorter wavelengths. Several potential applications of the bond-selective fragmentation chemistry observed at 213 nm are discussed. Graphical Abstract ᅟ

Experimental infection of Leishmania (L.) chagasi in a cell line derived from Lutzomyia longipalpis (Diptera:Psychodidae)

The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37ºC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6%) and on day 4 in the J774 cells (51%). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite