Sec24-Dependent Secretion Drives Cell-Autonomous Expansion of Tracheal Tubes in Drosophila

Epithelial tubes in developing organs, such as mammalian lungs and insect tracheae, need to expand their initially narrow lumina to attain their final, functional dimensions [1]. Despite its critical role for organ function, the cellular mechanism of tube expansion remains unclear. Tracheal tube expansion in Drosophila involves apical secretion and deposition of a luminal matrix [2,3,4,5], but the mechanistic role of secretion and the nature of forces involved in the process were not previously clear. Here we address the roles of cell-intrinsic and extrinsic processes in tracheal tube expansion. We identify mutations in the sec24 gene stenosis, encoding a cargo-binding subunit of the COPII complex [6,7,8]. Via genetic-mosaic analyses, we show that stenosis-dependent secretion drives tube expansion in a cell-autonomous fashion. Strikingly, single cells autonomously adjust both tube diameter and length by implementing a sequence of events including apical membrane growth, cell flattening, and taenidial cuticle formation. Known luminal components are not required for this process. Thus, a cell-intrinsic program, rather than nonautonomous extrinsic cues, controls the dimensions of tracheal tubes. These results indicate a critical role of membrane-associated proteins in the process and imply a mechanism that coordinates autonomous behaviors of individual cells within epithelial structures

Lateral Gene Expression in Drosophila Early Embryos Is Supported by Grainyhead-Mediated Activation and Tiers of Dorsally-Localized Repression

The general consensus in the field is that limiting amounts of the transcription factor Dorsal establish dorsal boundaries of genes expressed along the dorsal-ventral (DV) axis of early Drosophila embryos, while repressors establish ventral boundaries. Yet recent studies have provided evidence that repressors act to specify the dorsal boundary of intermediate neuroblasts defective (ind), a gene expressed in a stripe along the DV axis in lateral regions of the embryo. Here we show that a short 12 base pair sequence (“the A-box”) present twice within the ind CRM is both necessary and sufficient to support transcriptional repression in dorsal regions of embryos. To identify binding factors, we conducted affinity chromatography using the A-box element and found a number of DNA-binding proteins and chromatin-associated factors using mass spectroscopy. Only Grainyhead (Grh), a CP2 transcription factor with a unique DNA-binding domain, was found to bind the A-box sequence. Our results suggest that Grh acts as an activator to support expression of ind, which was surprising as we identified this factor using an element that mediates dorsally-localized repression. Grh and Dorsal both contribute to ind transcriptional activation. However, another recent study found that the repressor Capicua (Cic) also binds to the A-box sequence. While Cic was not identified through our A-box affinity chromatography, utilization of the same site, the A-box, by both factors Grh (activator) and Cic (repressor) may also support a “switch-like” response that helps to sharpen the ind dorsal boundary. Furthermore, our results also demonstrate that TGF-β signaling acts to refine ind CRM expression in an A-box independent manner in dorsal-most regions, suggesting that tiers of repression act in dorsal regions of the embryo

Strigolactones inhibit auxin feedback on PIN-dependent auxin transport canalization

Directional transport of the phytohormone auxin is a versatile, plant-specific mechanism regulating many aspects of plant development. The recently identified plant hormones, strigolactones (SLs), are implicated in many plant traits; among others, they modify the phenotypic output of PIN-FORMED (PIN) auxin transporters for fine-tuning of growth and developmental responses. Here, we show in pea and Arabidopsis that SLs target processes dependent on the canalization of auxin flow, which involves auxin feedback on PIN subcellular distribution. D14 receptor- and MAX2 F-box-mediated SL signaling inhibits the formation of auxin-conducting channels after wounding or from artificial auxin sources, during vasculature de novo formation and regeneration. At the cellular level, SLs interfere with auxin effects on PIN polar targeting, constitutive PIN trafficking as well as clathrin-mediated endocytosis. Our results identify a non-transcriptional mechanism of SL action, uncoupling auxin feedback on PIN polarity and trafficking, thereby regulating vascular tissue formation and regeneration

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

Background: There 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 Findings: We 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/Significance: These 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

A Model for the Development of the Rhizobial and Arbuscular Mycorrhizal Symbioses in Legumes and Its Use to Understand the Roles of Ethylene in the Establishment of these two Symbioses

We propose a model depicting the development of nodulation and arbuscular mycorrhizae. Both processes are dissected into many steps, using Pisum sativum L. nodulation mutants as a guideline. For nodulation, we distinguish two main developmental programs, one epidermal and one cortical. Whereas Nod factors alone affect the cortical program, bacteria are required to trigger the epidermal events. We propose that the two programs of the rhizobial symbiosis evolved separately and that, over time, they came to function together. The distinction between these two programs does not exist for arbuscular mycorrhizae development despite events occurring in both root tissues. Mutations that affect both symbioses are restricted to the epidermal program. We propose here sites of action and potential roles for ethylene during the formation of the two symbioses with a specific hypothesis for nodule organogenesis. Assuming the epidermis does not make ethylene, the microsymbionts probably first encounter a regulatory level of ethylene at the epidermis–outermost cortical cell layer interface. Depending on the hormone concentrations there, infection will either progress or be blocked. In the former case, ethylene affects the cortex cytoskeleton, allowing reorganization that facilitates infection; in the latter case, ethylene acts on several enzymes that interfere with infection thread growth, causing it to abort. Throughout this review, the difficulty of generalizing the roles of ethylene is emphasized and numerous examples are given to demonstrate the diversity that exists in plants

Transgenerational Stress Memory Is Not a General Response in Arabidopsis

Adverse conditions can trigger DNA damage as well as DNA repair responses in plants. A variety of stress factors are known to stimulate homologous recombination, the most accurate repair pathway, by increasing the concentration of necessary enzymatic components and the frequency of events. This effect has been reported to last into subsequent generations not exposed to the stress. To establish a basis for a genetic analysis of this transgenerational stress memory, a broad range of treatments was tested for quantitative effects on homologous recombination in the progeny. Several Arabidopsis lines, transgenic for well-established recombination traps, were exposed to 10 different physical and chemical stress treatments, and scored for the number of somatic homologous recombination (SHR) events in the treated generation as well as in the two subsequent generations that were not treated. These numbers were related to the expression level of genes involved in homologous recombination and repair. SHR was enhanced after the majority of treatments, confirming previous data and adding new effective stress types, especially interference with chromatin. Compounds that directly modify DNA stimulated SHR to values exceeding previously described induction rates, concomitant with an induction of genes involved in SHR. In spite of the significant stimulation in the stressed generations, the two subsequent non-treated generations only showed a low and stochastic increase in SHR that did not correlate with the degree of stimulation in the parental plants. Transcripts coding for SHR enzymes generally returned to pre-treatment levels in the progeny. Thus, transgenerational effects on SHR frequency are not a general response to abiotic stress in Arabidopsis and may require special conditions

Trafficking through COPII Stabilises Cell Polarity and Drives Secretion during Drosophila Epidermal Differentiation

BACKGROUND: The differentiation of an extracellular matrix (ECM) at the apical side of epithelial cells implies massive polarised secretion and membrane trafficking. An epithelial cell is hence engaged in coordinating secretion and cell polarity for a correct and efficient ECM formation. PRINCIPAL FINDINGS: We are studying the molecular mechanisms that Drosophila tracheal and epidermal cells deploy to form their specific apical ECM during differentiation. In this work we demonstrate that the two genetically identified factors haunted and ghost are essential for polarity maintenance, membrane topology as well as for secretion of the tracheal luminal matrix and the cuticle. We show that they code for the Drosophila COPII vesicle-coating components Sec23 and Sec24, respectively, that organise vesicle transport from the ER to the Golgi apparatus. CONCLUSION: Taken together, epithelial differentiation during Drosophila embryogenesis is a concerted action of ECM formation, plasma membrane remodelling and maintenance of cell polarity that all three rely mainly, if not absolutely, on the canonical secretory pathway from the ER over the Golgi apparatus to the plasma membrane. Our results indicate that COPII vesicles constitute a central hub for these processes

A Functional Genomic Screen Combined with Time-Lapse Microscopy Uncovers a Novel Set of Genes Involved in Dorsal Closure of Drosophila Embryos

Morphogenesis, the establishment of the animal body, requires the coordinated rearrangement of cells and tissues regulated by a very strictly-determined genetic program. Dorsal closure of the epithelium in the Drosophila melanogaster embryo is one of the best models for such a complex morphogenetic event. To explore the genetic regulation of dorsal closure, we carried out a large-scale RNA interference-based screen in combination with in vivo time-lapse microscopy and identified several genes essential for the closure or affecting its dynamics. One of the novel dorsal closure genes, the small GTPase activator pebble (pbl), was selected for detailed analysis. We show that pbl regulates actin accumulation and protrusion dynamics in the leading edge of the migrating epithelial cells. In addition, pbl affects dorsal closure dynamics by regulating head involution, a morphogenetic process mechanically coupled with dorsal closure. Finally, we provide evidence that pbl is involved in closure of the adult thorax, suggesting its general requirement in epithelial closure processes

Identification, Expression and Target Gene Analyses of MicroRNAs in Spodoptera litura

MicroRNAs (miRNAs) are small RNAs widely present in animals and plants and involved in post-transcriptional regulation of gene transcripts. In this study we identified and validated 58 miRNAs from an EST dataset of Spodoptera litura based on the computational and experimental analysis of sequence conservation and secondary structure of miRNA by comparing the miRNA sequences in the miRbase. RT-PCR was conducted to examine the expression of these miRNAs and stem-loop RT-PCR assay was performed to examine expression of 11 mature miRNAs (out of the 58 putative miRNA) that showed significant changes in different tissues and stages of the insect development. One hundred twenty eight possible target genes against the 11 miRNAs were predicted by using computational methods. Binding of one miRNA (sli-miR-928b) with the three possible target mRNAs was confirmed by Southern blotting, implying its possible function in regulation of the target genes

Role of the Arabidopsis PIN6 auxin transporter in auxin homeostasis and auxin-mediated development

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin–regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.Christopher I. Cazzonelli, Marleen Vanstraelen, Sibu Simon, Kuide Yin, Ashley Carron-Arthur, Nazia Nisar, Gauri Tarle, Abby J. Cuttriss¤, Iain R. Searle, Eva Benkova, Ulrike Mathesius, Josette Masle, Jiří Friml, Barry J. Pogso