A dynamic and mosaic basement membrane controls cell intercalation in Drosophila ovaries.

Basement membranes (BM) are extracellular matrices assembled into complex and highly organized networks essential for organ morphogenesis and function. However, little is known about the tissue origin of BM components and their dynamics in vivo Here, we unravel the assembly and role of the BM main component, Collagen type IV (ColIV), in Drosophila ovarian stalk morphogenesis. Stalks are short strings of cells assembled through cell intercalation that link adjacent follicles and maintain ovarian integrity. We show that stalk ColIV has multiple origins and is assembled following a regulated pattern leading to a unique BM organisation. Absence of ColIV leads to follicle fusion, as observed upon ablation of stalk cells. ColIV and integrins are both required to trigger cell intercalation and maintain mechanically strong cell-cell attachment within the stalk. These results show how the dynamic assembly of a mosaic BM controls complex tissue morphogenesis and integrity.

Polycomb controls gliogenesis by regulating the transient expression of the Gcm/Glide fate determinant.

The Gcm/Glide transcription factor is transiently expressed and required in the Drosophila nervous system. Threshold Gcm/Glide levels control the glial versus neuronal fate choice, and its perdurance triggers excessive gliogenesis, showing that its tight and dynamic regulation ensures the proper balance between neurons and glia. Here, we present a genetic screen for potential gcm/glide interactors and identify genes encoding chromatin factors of the Trithorax and of the Polycomb groups. These proteins maintain the heritable epigenetic state, among others, of HOX genes throughout development, but their regulatory role on transiently expressed genes remains elusive. Here we show that Polycomb negatively affects Gcm/Glide autoregulation, a positive feedback loop that allows timely accumulation of Gcm/Glide threshold levels. Such temporal fine-tuning of gene expression tightly controls gliogenesis. This work performed at the levels of individual cells reveals an undescribed mode of Polycomb action in the modulation of transiently expressed fate determinants and hence in the acquisition of specific cell identity in the nervous system.

Asymmetric localisation of cytokine mRNA is essential for JAK/STAT activation during cell invasiveness.

The transition from immotile epithelial cells to migrating cells occurs in all organisms during normal embryonic development, as well as during tumour metastasis. During Drosophila oogenesis, border cells (BCs) are recruited and delaminate from the follicular epithelium. This process is triggered by the polar cells (PCs), which secrete the cytokine Unpaired (Upd) and activate the JAK/STAT pathway in neighbouring cells, turning them into invasive BCs. Interestingly, either a decrease or an increase in BC number alters migration, indicating that mechanisms controlling the level of JAK/STAT signalling are crucial in this process. Here, we show that PCs have a highly stable and polarised network of microtubules along which upd transcripts are asymmetrically transported in a Dynein-dependent manner. We demonstrate that in the absence of upd mRNA localisation the ligand is no longer efficiently secreted, leading to a loss of signalling strength as well as recruitment and migration defects. These findings reveal a novel post-transcriptional regulatory mechanism of JAK/STAT signalling in the control of epithelial cell invasiveness.

A bioinformatics search pipeline, RNA2DSearch, identifies RNA localization elements in Drosophila retrotransposons.

mRNA localization is a widespread mode of delivering proteins to their site of function. The embryonic axes in Drosophila are determined in the oocyte, through Dynein-dependent transport of gurken/TGF-alpha mRNA, containing a small localization signal that assigns its destination. A signal with a similar secondary structure, but lacking significant sequence similarity, is present in the I factor retrotransposon mRNA, also transported by Dynein. It is currently unclear whether other mRNAs exist that are localized to the same site using similar signals. Moreover, searches for other genes containing similar elements have not been possible due to a lack of suitable bioinformatics methods for searches of secondary structure elements and the difficulty of experimentally testing all the possible candidates. We have developed a bioinformatics approach for searching across the genome for small RNA elements that are similar to the secondary structures of particular localization signals. We have uncovered 48 candidates, of which we were able to test 22 for their localization potential using injection assays for Dynein mediated RNA localization. We found that G2 and Jockey transposons each contain a gurken/I factor-like RNA stem-loop required for Dynein-dependent localization to the anterior and dorso-anterior corner of the oocyte. We conclude that I factor, G2, and Jockey are members of a "family" of transposable elements sharing a gurken-like mRNA localization signal and Dynein-dependent mechanism of transport. The bioinformatics pipeline we have developed will have broader utility in fields where small RNA signals play important roles.

mRNA localisation gets more complex.

Recent advances in techniques for visualising mRNA movement in living cells have led to rapid progress in understanding the mechanism of mRNA localisation in the cytoplasm. There is an emerging consensus that in many cases the mRNA signals that determine intracellular destination are more complex and difficult to define than was first anticipated. Furthermore, the transacting factors that interpret the mRNA signals are numerous and their combinations change during the life of an mRNA, perhaps allowing the selection of many sub-destinations in the cell. Lastly, an emerging theme over the past few years is that many proteins that determine the destinations of mRNAs are recruited on nascent transcripts in the nucleus. They often function in many different processes in the biogenesis of mRNA and probably act in concert to provide specificity.

gurken and the I factor retrotransposon RNAs share common localization signals and machinery.

Drosophila gurken mRNA is localized by dynein-mediated transport to a crescent near the oocyte nucleus, thus targeting the TGFalpha signal and forming the primary embryonic axes. Here, we show that gurken and the I factor, a non-LTR retrotransposon, share a small consensus RNA stem loop of defined secondary structure, which forms a conserved signal for dynein-mediated RNA transport to the oocyte nucleus. Furthermore, gurken and the I factor compete in vivo for the same localization machinery. I factor transposition leads to its mRNA accumulating near and within the oocyte nucleus, thus causing perturbations in gurken and bicoid mRNA localization and axis specification. These observations further our understanding of the close association of transposable elements with their host and provide an explanation for how I factor transposition causes female sterility. We propose that the transposition of other elements may exploit the host's RNA transport signals and machinery.

glide/gcm: at the crossroads between neurons and glia.

Neurons and glia are generated by multipotent precursors. Recent studies indicate that the choice between the two fates depends on the combined activity of extracellular influences and factors that respond to precise spatial and temporal cues. Drosophila provides a simple genetic model to study the cellular and molecular mechanisms controlling fate choice, mode of precursor division and generation of cell diversity. Moreover, all glial precursors and glial-promoting activities have been identified in Drosophila, which provides us with a unique opportunity to dissect regulatory pathways controlling glial differentiation and specification.

Precocious expression of the Glide/Gcm glial-promoting factor in Drosophila induces neurogenesis.

Neurons and glial cells depend on similar developmental pathways and often originate from common precursors; however, the differentiation of one or the other cell type depends on the activation of cell-specific pathways. In Drosophila, the differentiation of glial cells depends on a transcription factor, Glide/Gcm. This glial-promoting factor is both necessary and sufficient to induce the central and peripheral glial fates at the expense of the neuronal fate. In a screen for mutations affecting the adult peripheral nervous system, we have found a dominant mutation inducing supernumerary sensory organs. Surprisingly, this mutation is allelic to glide/gcm and induces precocious glide/gcm expression, which, in turn, activates the proneural genes. As a consequence, sensory organs are induced. Thus, temporal misregulation of the Glide/Gcm glial-promoting factor reveals a novel potential for this cell fate determinant. At the molecular level, this implies unpredicted features of the glide/gcm pathway. These findings also emphasize the requirement for both spatial and temporal glide/gcm regulation to achieve proper cell specification within the nervous system.

Companion Blood Cells Control Ovarian Stem Cell Niche Microenvironment and Homeostasis.

The extracellular matrix plays an essential role for stem cell differentiation and niche homeostasis. Yet, the origin and mechanism of assembly of the stem cell niche microenvironment remain poorly characterized. Here, we uncover an association between the niche and blood cells, leading to the formation of the Drosophila ovarian germline stem cell niche basement membrane. We identify a distinct pool of plasmatocytes tightly associated with the developing ovaries from larval stages onward. Expressing tagged collagen IV tissue specifically, we show that the germline stem cell niche basement membrane is produced by these "companion plasmatocytes" in the larval gonad and persists throughout adulthood, including the reproductive period. Eliminating companion plasmatocytes or specifically blocking their collagen IV expression during larval stages results in abnormal adult niches with excess stem cells, a phenotype due to aberrant BMP signaling. Thus, local interactions between the niche and blood cells during gonad development are essential for adult germline stem cell niche microenvironment assembly and homeostasis.

In vivo characterization of dynein-driven nanovectors using Drosophila oocytes.

Molecular motors transport various cargoes including vesicles, proteins and mRNAs, to distinct intracellular compartments. A significant challenge in the field of nanotechnology is to improve drug nuclear delivery by engineering nanocarriers transported by cytoskeletal motors. However, suitable in vivo models to assay transport and delivery efficiency remain very limited. Here, we develop a fast and genetically tractable assay to test the efficiency and dynamics of fluospheres (FS) using microinjection into Drosophila oocytes coupled with time-lapse microscopy. We designed dynein motor driven FS using a collection of dynein light chain 8 (LC8) peptide binding motifs as molecular linkers and characterized in real time the efficiency of the FS movement according to its linker's sequence. Results show that the conserved LC8 binding motif allows fast perinuclear nanoparticle's accumulation in a microtubule and dynein dependent mechanism. These data reveal the Drosophila oocyte as a new valuable tool for the design of motor driven nanovectors.

Cell migration: MIM takes the driver's seat.

A recent study reports a novel and conserved function for the I-BAR protein MIM in guiding cell migration: MIM has an anti-endocytic activity that moderates intracellular signalling of guidance cues by sequestration of cortactin.

Novel features of dFMR1, the Drosophila orthologue of the fragile X mental retardation protein.

FMRP belongs to a family of widely expressed proteins that contain RNA-binding domains. Although lack of human FMRP results in mental retardation, correlated with subtle synaptic changes, the precise role of FMRP remains elusive. The Drosophila genome contains a single gene homologous to the FXR family. We show that dFMR1 is subjected to transcriptional and posttranscriptional regulation during development and that it homomerizes, like its human counterpart. dFMR1 profile of expression recapitulates that of the human FXR protein family: it is highly enriched in muscles, in central nervous system and in gonads. In the larval brain, anti-dFMR1 also recognizes mushroom bodies, a centre that mediates learning and memory. These features make the fly an ideal system to analyse the role of the FXR family and to identify genes in the FMRP pathway.

Time-lapse and cell ablation reveal the role of cell interactions in fly glia migration and proliferation.

Migration and proliferation have been mostly explored in culture systems or fixed preparations. We present a simple genetic model, the chains of glia moving along fly wing nerves, to follow such dynamic processes by time-lapse in the whole animal. We show that glia undergo extensive cytoskeleton and mitotic apparatus rearrangements during division and migration. Single cell labelling identifies different glia: pioneers with high filopodial, exploratory, activity and, less active followers. In combination with time-lapse, altering this cellular environment by genetic means or cell ablation has allowed to us define the role of specific cell-cell interactions. First, neurone-glia interactions are not necessary for glia motility but do affect the direction of migration. Second, repulsive interactions between glia control the extent of movement. Finally, autonomous cues control proliferation.

Transcriptional regulation of glial cell specification.

Neuronal differentiation relies on proneural factors that also integrate positional information and contribute to the specification of the neuronal type. The molecular pathway triggering glial specification is not understood yet. In Drosophila, all lateral glial precursors and glial-promoting activity have been identified, which provides us with a unique opportunity to dissect the regulatory pathways controlling glial differentiation and specification. Although glial lineages are very heterogeneous with respect to position, time of differentiation, and lineage tree, they all express and require two homologous genes, glial cell deficient/glial cell missing (glide/gcm) and glide2, that act in concert, with glide/gcm constituting the major glial-promoting factor. Here, we show that glial specification resides in glide/gcm transcriptional regulation. The glide/gcm promoter contains lineage-specific elements as well as quantitative and turmoil elements scattered throughout several kilobases. Interestingly, there is no correlation between a specific regulatory element and the type of glial lineage. Thus, the glial-promoting factor acts as a naive switch-on button that triggers gliogenesis in response to multiple pathways converging onto its promoter. Both negative and positive regulation are required to control glide/gcm expression, indicating that gliogenesis is actively repressed in some neural lineages.