Rab11 Is Required for Epithelial Cell Viability, Terminal Differentiation, and Suppression of Tumor-Like Growth in the Drosophila Egg Chamber

The Drosophila egg chamber provides an excellent system in which to study the specification and differentiation of epithelial cell fates because all of the steps, starting with the division of the corresponding stem cells, called follicle stem cells, have been well described and occur many times over in a single ovary.Here we investigate the role of the small Rab11 GTPase in follicle stem cells (FSCs) and in their differentiating daughters, which include main body epithelial cells, stalk cells and polar cells. We show that rab11-null FSCs maintain their ability to self renew, even though previous studies have shown that FSC self renewal is dependent on maintenance of E-cadherin-based intercellular junctions, which in many cell types, including Drosophila germline stem cells, requires Rab11. We also show that rab11-null FSCs give rise to normal numbers of cells that enter polar, stalk, and epithelial cell differentiation pathways, but that none of the cells complete their differentiation programs and that the epithelial cells undergo premature programmed cell death. Finally we show, through the induction of rab11-null clones at later points in the differentiation program, that Rab11 suppresses tumor-like growth of epithelial cells. Thus, rab11-null epithelial cells arrest differentiation early, assume an aberrant cell morphology, delaminate from the epithelium, and invade the neighboring germline cyst. These phenotypes are associated with defects in E-cadherin localization and a general loss of cell polarity.While previous studies have revealed tumor suppressor or tumor suppressor-like activity for regulators of endocytosis, our study is the first to identify such activity for regulators of endocytic recycling. Our studies also support the recently emerging view that distinct mechanisms regulate junction stability and plasticity in different tissues

(Re)Blurring Coast: Architecture Within the Coastal Threshold of the Waitematā Harbour

The coast is the threshold between two distinct zones: land and sea. In an island nation, architecture so often finds itself in a position where, through time, it can occupy both. Hence, the architecture of the foreshore is subjected to the duality of these zones. Time is the contingency which regularly transforms the surrounding site of the architecture and thus regularly transforms its context. This research asks to what extent architecture can expand on the temporal condition of the coastal threshold, and make it spatially appreciable to people? Architecture is a key tool in creating static boundaries, but what if buildings could call attention to blurred boundaries? How might it respond to the dynamics of the foreshore? How might it be blurred by the coast? This research builds on the work of Jacky Bowring, Nancy Vance and Mick Abbott in ‘Living with the Sea: Knowledge Awareness and Action’ (Brown & Peters, 2018). The authors discuss how coast- situated architecture can challenge the binary oppositions of land and sea, by treating the shared space as a liminal zone. This thesis also draws on the ideas of David Leatherbarrow and Mohsen Mostafavi, whose work discusses how material weathering indicates time and change. To expand upon this theoretical base, an iterative design process of drawing and physical model-making is employed. A sequence of designed experiments are tested to illustrate how architecture may contribute to a greater collective understanding of the temporal coastal space. This design-led research discusses how key architectural characteristics could modify temporal conditions of the coast, such as rhythm, reflection translucency and weathering. These characteristics are examined as interactive phenomena through three design stages. These stages are iterative, progressively introducing scale and architectural complexity. An installation explores qualities of the coastal threshold through a design test at the scale of the body. A pavilion series highlights the possibilities of mid-scale architecture, while a larger scale building tests ideas through the design of a residence, amphitheatre and jetty. This research begins with a macro-level analysis of the New Zealand coast, then develops a particular focus on the shallow bays of the Waitematā Harbour in Tāmaki Makaurau (Auckland). The design propositions in the second and third stage are sited amid the foreshore of Fitzpatrick Bay in the Waitematā. This research proposes that architecture can resonate with the temporal phenomena of the coastal zone, which invests it with a contingency of time and a liminal presence. This resonation is distilled as an architecture, with design explorations engaging form, journey, materiality and temporality. Consequently, a dialogue about the architecture, the coast and person’s passage through its temporal and spatial dimensions is communicated.</p

A two-step Notch-dependant mechanism controls the selection of the polar cell pair in Drosophila oogenesis.

International audienceOrganisers control the patterning and growth of many tissues and organs. Correctly regulating the size of these organisers is crucial for proper differentiation to occur. Organiser activity in the epithelium of the Drosophila ovarian follicle resides in a pair of cells called polar cells. It is known that these two cells are selected from a cluster of equivalent cells. However, the mechanisms responsible for this selection are still unclear. Here, we present evidence that the selection of the two cells is not random but, by contrast, depends on an atypical two-step Notch-dependent mechanism. We show that this sequential process begins when one cell becomes refractory to Notch activation and is selected as the initial polar cell. This cell then produces a Delta signal that induces a high level of Notch activation in one other cell within the cluster. This Notch activity prevents elimination by apoptosis, allowing its selection as the second polar cell. Therefore, the mechanism used to select precisely two cells from among an equivalence group involves an inductive Delta signal that originates from one cell, itself unable to respond to Notch activation, and results in one other cell being selected to adopt the same fate. Given its properties, this two-step Notch-dependent mechanism represents a novel aspect of Notch action

Tight Coordination of Growth and Differentiation between Germline and Soma Provides Robustness for Drosophila Egg Development

Organs often need to coordinate the growth of distinct tissues during their development. Here, we analyzed the coordination between germline cysts and the surrounding follicular epithelium during Drosophila oogenesis. Genetic manipulations of the growth rate of both germline and somatic cells influence the growth of the other tissue accordingly. Growth coordination is therefore ensured by a precise, two-way, intrinsic communication. This coordination tends to maintain constant epithelial cell shape, ensuring tissue homeostasis. Moreover, this intrinsic growth coordination mechanism also provides cell differentiation synchronization. Among growth regulators, PI3-kinase and TORC1 also influence differentiation timing cell-autonomously. However, these two pathways are not regulated by the growth of the adjacent tissue, indicating that their function reflects an extrinsic and systemic influence. Altogether, our results reveal an integrated and particularly robust mechanism ensuring the spatial and temporal coordination of tissue size, cell size, and cell differentiation for the proper development of two adjacent tissues

Drosophila LKB1 is required for the assembly of the polarized actin structure that allows spermatid individualization

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Multiple functions of the scaffold protein Discs large 5 in the control of growth, cell polarity and cell adhesion in Drosophila melanogaster

International audienceBackground: Scaffold proteins support a variety of key processes during animal development. Mutant mouse for the MAGUK protein Discs large 5 (Dlg5) presents a general growth impairment and moderate morphogenetic defects. Results: Here, we generated null mutants for Drosophila Dlg5 and show that it owns similar functions in growth and epithelial architecture. Dlg5 is required for growth at a cell autonomous level in several tissues and at the organism level, affecting cell size and proliferation. Our results are consistent with Dlg5 modulating hippo pathway in the wing disc, including the impact on cell size, a defect that is reproduced by the loss of yorkie. However, other observations indicate that Dlg5 regulates growth by at least another way that may involve Myc protein but nor PI3K neither TOR pathways. Moreover, epithelia cells mutant for Dlg5 also show a reduction of apical domain determinants, though not sufficient to induce a complete loss of cell polarity. Dlg5 is also essential, in the same cells, for the presence at Adherens junctions of N-Cadherin, but not E-Cadherin. Genetic analyses indicate that junction and polarity defects are independent. Conclusions: Together our data show that Dlg5 own several conserved functions that are independent of each other in regulating growth, cell polarity and cell adhesion. Moreover, they reveal a differential regulation of E-cadherin and Ncadherin apical localization

Generation of <i>dLkb1</i> mutant germline clones in testis germ cells.

<p>A-A’) Expression level of Mst77F-GFP fusion protein increases as spermatid nuclei become more elongated. B-B’) WT clones do not express GFP showing that Mst77F-GFP is a suitable marker of mitotic recombination. C-C’ and D-D’) <i>Lkb1</i>^x5 mutant clones were generated and detected by the absence of GFP. Arrows indicate WT nuclei and arrowheads mutant clones. Scale bars = 10μm.</p

Analysis of endogenous and transgene LKB1 expression in the testis.

<p><b>(A)</b> Schematic representation of the two different mRNAs of the <i>Drosophila Lkb1</i> gene. Primers used for the RT-PCR assay shown in B are indicated below with arrows: a, primers in the 5’UTR and b, primers in the 3’ UTR. The primers in the first exons amplify only a male-specific transcript (amplicon of 592bp), while the primers in the 3’UTR amplify all transcripts (amplicon of 131bp). <b>(B)</b> RT-PCR assay with primers that amplify the 5’UTR (a) or the 3’ UTR (b). T: testis RNA; O: ovary RNAs. <b>(C)</b> Schematic representation of the different transgenes that express dLkb1 or hLKB1. Numbers on the left correspond to the lanes on the western blot shown in D. On the right column is indicated whether the transgene can rescue male sterility. <b>(D)</b> Testis protein extracts from adult flies that express dLkb1 or hLKB1 were analyzed by western blotting with an anti-GFP antibody. The lane numbers correspond to the transgenic lines shown in C: 1) dLkb1>dLkb1-GFP, 2) Ubi>dLkb1-GFP, 3) Ubi>hLKB1L-GFP, and 4) Ubi>hLKB1S-GFP. The anti-tubulin antibody was used as loading control.</p

Myosin V loss of function mimics LKB1 loss of function.

<p>A) Spermatid nuclei from bam-GAL4–driven <i>didum</i> (myosin V) RNAi flies were stained with phalloidin (F-actin, red) and Hoechst (DNA, blue). B-B’) <i>didum</i>^<i>154</i> mutant clones are detected by the absence of protamine B-GFP expression. Arrows, WT bundle of nuclei; arrowheads, mutant clones. Upon myosin V loss of function, no actin cone is formed. C) Spermatid cells from <i>didum</i> RNAi clones incubated with an anti-GFP antibody (Lkb1-GFP, green) and stained with Hoechst (DNA, white). D) <i>Lkb1</i> RNAi does not prevent myosin V (red) accumulation in spermatid nuclei (Hoechst, white). E, F) Differently from wild type (E), Lkb1^x5/x5 germline clones, which are identified by the lack of MST77F-GFP expression (green), accumulate myosin V (red) in spermatid nuclei (white). Arrows, WT bundles of nuclei; arrowheads, mutant clones. Scale bars = 10μm.</p

The AMPK-related kinases AMPK, KP78b and NUAK are involved in actin cone shaping and migration.

<p>Representative images of actin cone formation and migration in spermatid bundles from wild type (WT) (A and A’), or from the following bam-GAL4–driven UAS RNAi lines: (B) <i>Lkb1</i>, (C) <i>Kp78b</i>, (D) <i>Ampk</i> and (E and E’) <i>Nuak</i>. Arrows indicate migrating actin cones; brackets indicate formation of actin cones on nuclear bundles. Scale bars = 10μm.</p