Tracheal tube fusion in Drosophila involves release of extracellular vesicles from multivesicular bodies

Extracellular vesicles (EVs) comprise diverse types of cell-released membranous structures that are thought to play important roles in intercellular communication. While the formation and functions of EVs have been investigated extensively in cultured cells, studies of EVs in vivo have remained scarce. We report here that EVs are present in the developing lumen of tracheal tubes in Drosophila embryos. We defined two distinct EV subpopulations, one of which contains the Munc13-4 homologue Staccato (Stac) and is spatially and temporally associated with tracheal tube fusion (anastomosis) events. The formation of Stac-positive luminal EVs depends on the tracheal tip-cell-specific GTPase Arl3, which is also required for the formation of Stac-positive multivesicular bodies, suggesting that Stac-EVs derive from fusion of Stac-MVBs with the luminal membrane in tip cells during anastomosis formation. The GTPases Rab27 and Rab35 cooperate downstream of Arl3 to promote Stac-MVB formation and tube fusion. We propose that Stac-MVBs act as membrane reservoirs that facilitate tracheal lumen fusion in a process regulated by Arl3, Rab27, Rab35, and Stac/Munc13-4.

eNOS controls angiogenic sprouting and retinal neovascularization through the regulation of endothelial cell polarity

The roles of nitric oxide (NO) and endothelial NO synthase (eNOS) in the regulation of angiogenesis are well documented. However, the involvement of eNOS in the sprouting of endothelial tip-cells at the vascular front during sprouting angiogenesis remains poorly defined. In this study, we show that downregulation of eNOS markedly inhibits VEGF-stimulated migration of endothelial cells but increases their polarization, as evidenced by the reorientation of the Golgi in migrating monolayers and by the fewer filopodia on tip cells at ends of sprouts in endothelial cell spheroids. The effect of eNOS inhibition on EC polarization was prevented in Par3-depleted cells. Importantly, downregulation of eNOS increased the expression of polarity genes, such as PARD3B, PARD6A, PARD6B, PKCΖ, TJP3, and CRB1 in endothelial cells. In retinas of eNOS knockout mice, vascular development is retarded with decreased vessel density and vascular branching. Furthermore, tip cells at the extremities of the vascular front have a marked reduction in the number of filopodia per cell and are more oriented. In a model of oxygen-induced retinopathy (OIR), eNOS deficient mice are protected during the initial vaso-obliterative phase, have reduced pathological neovascularization, and retinal endothelial tip cells have fewer filopodia. Single-cell RNA sequencing of endothelial cells from OIR retinas revealed enrichment of genes related to cell polarity in the endothelial tip-cell subtype of eNOS deficient mice. These results indicate that inhibition of eNOS alters the polarity program of endothelial cells, which increases cell polarization, regulates sprouting angiogenesis and normalizes pathological neovascularization during retinopathy.

Two new calcicolous caloplacoid lichens from South Korea, with a taxonomic key to the species of Huriella and Squamulea

Pyrenodesmia rugosa Lee & Hur and Huriella aeruginosa Lee & Hur are described as new lichen-forming fungi from a calcareous mountain of South Korea. Pyrenodesmia rugosa is distinguishable from Pyrenodesmia micromontana (Frolov, Wilk & Vondrák) Hafellner & Türk, the most similar species, by thicker thallus, rugose areoles, larger apothecia, shorter hymenium, shorter hypothecium and narrower tip cells of paraphyses. Huriella aeruginosa, the second new species, differs from ‘Squamulea’ chelonia Bungartz & Søchting by dark greenish-grey to grey thallus without pruina, gold to yellow-brown epihymenium, larger ascospores and thallus K– and KC– reaction. Molecular analyses employing internal transcribed spacer (ITS), mitochondrial small subunit (mtSSU) and nuclear large subunit ribosomal RNA (LSU) sequences strongly support the two caloplacoid species to be distinct in their genera. A surrogate key is provided to assist in the identification of all 20 taxa in Huriella and Squamulea.

The histone acetyltransferase HBO1 (KAT7) promotes efficient tip cell sprouting during angiogenesis

Blood vessel growth and remodelling are essential during embryonic development and disease pathogenesis. The diversity of endothelial cells (ECs) is transcriptionally evident and ECs undergo dynamic changes in gene expression during vessel growth and remodelling. Here, we investigated the role of the histone acetyltransferase HBO1 (KAT7), which is important for activating genes during development and histone H3 lysine 14 acetylation (H3K14ac). Loss of HBO1 and H3K14ac impaired developmental sprouting angiogenesis and reduced pathological EC overgrowth in the retinal endothelium. Single-cell RNA-sequencing of retinal ECs revealed an increased abundance of tip cells in Hbo1 deleted retinas, which lead to EC overcrowding in the retinal sprouting front and prevented efficient tip cell migration. We found that H3K14ac was highly abundant in the endothelial genome in both intra- and intergenic regions suggesting that the role of HBO1 is as a genome organiser that promotes efficient tip cell behaviour necessary for sprouting angiogenesis.

Tracheal tube fusion in Drosophila involves release of luminal exosomes from multivesicular bodies

Fusion of endothelial or epithelial tubes is essential for the development of organs like the vertebrate vasculature or the insect tracheal system, but the mechanisms underlying the formation of tubular connections (anastomoses) are not well understood. Tracheal tube fusion in Drosophila is mediated by tip cells that transform into lumenized toroids to connect adjacent tubes. This process depends on the Munc13-4 orthologue Staccato (Stac), which localizes to tip-cell-specific lysosome-related organelles (LROs). We show that tracheal LROs display features of multivesicular bodies (MVBs) and that the tracheal lumen contains membranous extracellular vesicles (EVs), a subset of which carries Stac/Munc13-4 and is associated with tracheal anastomosis sites. The presence of LROs and luminal Stac-EVs depends on the tip-cell-specific GTPase Arl3, suggesting that Stac-EVs derive from fusion of MVBs with the luminal membrane in tip cells during anastomosis formation. The GTPases Rab27 and Rab35 cooperate downstream of Arl3 to promote Stac-MVB formation and tube fusion. We propose that Stac-MVBs act as membrane reservoirs that facilitate lumen fusion in tip cells, in a process regulated by Arl3, Rab27, Rab35, and Stac/Munc13-4.

Impact of Bisphenol A on seed germination, radicle length and cytogenetic alterations in Pisum sativum L

Bisphenol A (BPA) is a global transpiring pollutant and an endocrine disruptor present in the environment which has a substantial harmful effect on plants. In the present study, its effects on seed germination, radicle length and cytogenetic alterations were investigated in Pisum sativum L root tip cells. Pisum sativum L seeds were germinated after treating with various concentrations of BPA (2 mg/L, 5 mg/L, 10 mg/L, 15 mg/L, 20 mg/L and 25 mg/L) at 24±1°C for 72 hours and the cytogenetic variations were assessed. The investigation showed that BPA reduced the percentage of seed germination, mitotic index, radicle length (at higher concentrations) and instigated a rise in chromosomal anomalies in a dose-related manner. In total, there is an enhanced occurrence of c-mitosis, stickiness, bridges, fragments and laggards in the BPA treated root tip cells of Pisum sativum L seeds.