RASSF8-mediated transport of Echinoid via the exocyst promotes Drosophila wing elongation and epithelial ordering

Cell-cell junctions are dynamic structures that maintain cell cohesion and shape in epithelial tissues. During development, junctions undergo extensive rearrangements to drive the epithelial remodelling required for morphogenesis. This is particularly evident during axis elongation, where neighbour exchanges, cell-cell rearrangements and oriented cell divisions lead to large-scale alterations in tissue shape. Polarised vesicle trafficking of junctional components by the exocyst complex has been proposed to promote junctional rearrangements during epithelial remodelling, but the receptors that allow exocyst docking to the target membranes remain poorly understood. Here, we show that the adherens junction component Ras Association domain family 8 (RASSF8) is required for the epithelial re-ordering that occurs during Drosophila pupal wing proximo-distal elongation. We identify the exocyst component Sec15 as a RASSF8 interactor. RASSF8 loss elicits cytoplasmic accumulation of Sec15 and Rab11-containing vesicles. These vesicles also contain the nectin-like homophilic adhesion molecule Echinoid, whose depletion phenocopies the wing elongation and epithelial packing defects observed in RASSF8 mutants. Thus, our results suggest that RASSF8 promotes exocyst-dependent docking of Echinoid-containing vesicles during morphogenesis.

Cell Dissociation from Butterfly Pupal Wing Tissues for Single-Cell RNA Sequencing

Butterflies are well known for their beautiful wings and have been great systems to understand the ecology, evolution, genetics, and development of patterning and coloration. These color patterns are mosaics on the wing created by the tiling of individual units called scales, which develop from single cells. Traditionally, bulk RNA sequencing (RNA-seq) has been used extensively to identify the loci involved in wing color development and pattern formation. RNA-seq provides an averaged gene expression landscape of the entire wing tissue or of small dissected wing regions under consideration. However, to understand the gene expression patterns of the units of color, which are the scales, and to identify different scale cell types within a wing that produce different colors and scale structures, it is necessary to study single cells. This has recently been facilitated by the advent of single-cell sequencing. Here, we provide a detailed protocol for the dissociation of cells from Bicyclus anynana pupal wings to obtain a viable single-cell suspension for downstream single-cell sequencing. We outline our experimental design and the use of fluorescence-activated cell sorting (FACS) to obtain putative scale-building and socket cells based on size. Finally, we discuss some of the current challenges of this technique in studying single-cell scale development and suggest future avenues to address these challenges.

Overexpressed Gliotactin activates BMP signaling through interfering with the Tkv–Dad association

Epithelial junctions ensure cell–cell adhesion and establish permeability barriers between cells. At the corners of epithelia, the tricellular junction (TCJ) is formed by three adjacent epithelial cells and generates a functional barrier. In Drosophila, a key TCJ protein is Gliotactin (Gli) where loss of Gli disrupts barrier formation and function. Conversely, overexpressed Gli spreads away from the TCJ and triggers apoptosis, delamination, and cell migration. Thus, Gli protein levels are tightly regulated and by two mechanisms, at the protein levels by tyrosine phosphorylation and endocytosis and at the mRNA level through microRNA-184. Regulation of Gli mRNA is mediated through a Gli–BMP–miR184 feedback loop. Excessive Gli triggers BMP signaling pathway through the activation of Tkv type-I BMP receptor and Mad. Elevated level of pMad induces micrRNA-184 expression which in turn targets the Gli 3′UTR and mRNA degradation. Gli activation of Tkv is not through its ligand Dpp but rather through the inhibition of Dad, an inhibitory-Smad. Here, we show that ectopic expression of Gli interferes with Tkv–Dad association by sequestering Dad away from Tkv. The reduced inhibitory effect of Dad on Tkv results in the increased Tkv–pMad signaling activity, and this effect is continuous through larval and pupal wing formation.

Mechano-chemical feedback leads to competition for BMP signalling during pattern formation

Developmental patterning is thought to be regulated by conserved signalling pathways. Initial patterns are often broad before refining to only those cells that commit to a particular fate. However, the mechanisms by which pattern refinement takes place remain to be addressed. Using the posterior crossvein (PCV) of the Drosophila pupal wing as a model, into which bone morphogenetic protein (BMP) ligand is extracellularly transported to instruct vein patterning, we investigate how pattern refinement is regulated. We found that BMP signalling induces apical enrichment of Myosin II in developing crossvein cells to regulate apical constriction. Live imaging of cellular behaviour indicates that changes in cell shape are dynamic and transient, only being maintained in those cells that retain vein fate after refinement. Disrupting cell shape changes throughout the PCV inhibits pattern refinement. In contrast, disrupting cell shape in only a subset of vein cells can result in a loss of BMP signalling. In addition, we observed that expressing the constitutively active form of the BMP type I receptor in clones caused apical constriction autonomously and often induced BMP signalling loss in the PCV region in a non-autonomous manner. We propose that the cell shape changes of future PCV cells allow them to compete more efficiently for the basally localised BMP signal by forming a mechano-chemical feedback loop. This study highlights a new form of competition among the cells: competing for a signal that induces cell fate.

DAnkrd49 and Bdbt act via Casein Kinase Iε to regulate planar polarity in Drosophila

The core planar polarity proteins are essential mediators of tissue morphogenesis, controlling both the polarised production of cellular structures and polarised tissue movements. During development the core proteins promote planar polarisation by becoming asymmetrically localised to opposite cell edges within epithelial tissues, forming intercellular protein complexes that coordinate polarity between adjacent cells. Here we describe a novel protein complex that regulates the asymmetric localisation of the core proteins in the Drosophila pupal wing. DAnkrd49 (an ankyrin repeat protein) and Bride of Doubletime (Bdbt, a non-canonical FK506 binding protein family member) physically interact, and regulate each other’s levels in vivo. Loss of either protein results in a reduction in core protein asymmetry and disruption of the placement of trichomes at the distal edge of pupal wing cells. Post-translational modifications are thought to be important for the regulation of core protein behaviour and their sorting to opposite cell edges. Consistent with this, we find that loss of DAnkrd49 or Bdbt leads to reduced phosphorylation of the core protein Dishevelled and to decreased Dishevelled levels both at cell junctions and in the cytoplasm. Bdbt has previously been shown to regulate activity of the kinase Discs Overgrown (Dco, also known as Doubletime or Casein Kinase Iε), and Dco itself has been implicated in regulating planar polarity by phosphorylating Dsh as well as the core protein Strabismus. We demonstrate that DAnkrd49 and Bdbt act as dominant suppressors of Dco activity. These findings support a model whereby Bdbt and DAnkrd49 act together to modulate the activity of Dco during planar polarity establishment.Author summaryIn many animal tissues, sheets of cells are polarised in the plane of the tissue, which is evident by the production of polarised structures, such as hairs on the fly wing that point in the same direction or cilia that beat in the same direction. One group of proteins controlling this coordinated polarity are the core planar polarity proteins, which localise asymmetrically within cells such that some core proteins localise to one cell end and others to the opposite cell end. It is thought that modifications such as phosphorylation may locally regulate core protein stability, and this promotes sorting of proteins to different cell ends. We identify two proteins, DAnkrd49 and Bdbt, that form a complex and regulate core protein asymmetry. Loss of either protein causes a reduction in overall levels of the core protein Dishevelled (Dsh), and a reduction in its phosphorylation. We provide evidence that the effect on core protein asymmetry is mediated via regulation of the kinase activity of Discs overgrown (Dco, also known as Doubletime/Casein Kinase Iε) by DAnkrd49 and Bdbt. We propose that modulation of Dco activity by DAnkrd49 and Bdbt is a key step in the sorting of core proteins to opposite cell ends.

Reciprocal action of Casein Kinase Iε on core planar polarity proteins regulates clustering and asymmetric localisation

The conserved core planar polarity pathway is essential for coordinating polarised cell behaviours and the formation of polarised structures such as cilia and hairs. Core planar polarity proteins localise asymmetrically to opposite cell ends and form intercellular complexes that link the polarity of neighbouring cells. This asymmetric segregation is regulated by phosphorylation through poorly understood mechanisms. We show that loss of phosphorylation of the core protein Strabismus in the Drosophila pupal wing increases its stability and promotes its clustering at intercellular junctions, and that Prickle negatively regulates Strabismus phosphorylation. Additionally, loss of phosphorylation of Dishevelled – which normally localises to opposite cell edges to Strabismus – reduces its stability at junctions. Moreover, both phosphorylation events are independently mediated by Casein Kinase Iε. We conclude that Casein Kinase Iε phosphorylation acts as a switch, promoting Strabismus mobility and Dishevelled immobility, thus enhancing sorting of these proteins to opposite cell edges.

Real-Time In Vivo Imaging of the Developing Pupal Wing Tissues in the Pale Grass Blue Butterfly Zizeeria maha: Establishing the Lycaenid System for Multiscale Bioimaging

To systematically analyze biological changes with spatiotemporal dynamics, it is important to establish a system that is amenable for real-time in vivo imaging at various size levels. Herein, we focused on the developing pupal wing tissues in the pale grass blue butterfly, Zizeeria maha, as a system of choice for a systematic multiscale approach in vivo in real time. We showed that the entire pupal wing could be monitored throughout development using a high-resolution bright-field time-lapse imaging system under the forewing-lift configuration; we recorded detailed dynamics of the dorsal and ventral epithelia that behaved independently for peripheral adjustment. We also monitored changes in the dorsal hindwing at the compartmental level and directly observed evaginating scale buds. We also employed a confocal laser microscopy system with multiple fluorescent dyes for three-dimensional observations at the tissue and cellular levels. We discovered extensive cellular clusters that may be functionally important as a unit of cellular communication and differentiation. We also identified epithelial discal and marginal dents that may function during development. Together, this lycaenid forewing system established a foundation to study the differentiation process of epithelial cells and can be used to study biophysically challenging mechanisms such as the determination of color patterns and scale nanoarchitecture at the multiscale levels.