Developmental Biology

Secretory Wnt trafficking can be studied in the polarized epithelial monolayer of Drosophila wing imaginal discs (WID). In this tissue, Wg (Drosophila Wnt-I) is presented on the apical surface of its source cells before being internalized into the endosomal pathway. Long-range Wg secretion and spread depend on secondary secretion from endosomal compartments, but the exact post-endocytic fate of Wg is poorly understood. Here, we summarize and present three protocols for the immunofluorescence-based visualization and quantitation of different pools of intracellular and extracellular Wg in WID: (1) steady-state extracellular Wg; (2) dynamic Wg trafficking inside endosomal compartments; and (3) dynamic Wg release to the cell surface. Using a genetic driver system for gene manipulation specifically at the posterior part of the WID (EnGal4) provides a robust internal control that allows for direct comparison of signal intensities of control and manipulated compartments of the same WID. Therefore, it also circumvents the high degree of staining variability usually associated with whole-tissue samples. In combination with the genetic manipulation of Wg pathway components that is easily feasible in Drosophila, these methods provide a tool-set for the dissection of secretory Wg trafficking and can help us to understand how Wnt proteins travel along endosomal compartments for short- and long-range signal secretion.

Graphic abstract:

Figure 1. Visualization of extracellular and intracellular Wg trafficking in Drosophila wing imaginal discs. While staining of extracellular Wg without permeabilization exclusively visualizes Wg bound to the extracellular surface (left), Wg uptake and endosomal trafficking can be visualized using an antibody uptake assay (middle). Dynamic Wg release can be visualized by performing a non-permeabilizing staining at a permissive temperature that sustains secretory Wg transport (right).

Organotypic slice culture is a powerful technique for exploring the embryonic development of the mammalian brain. In this protocol we describe a basic slice culture technique we have used for two sets of experiments: axon guidance transplant assays and bead culture assays.

Furin is an evolutionarily conserved proprotein convertase (PC) family enzyme with a broad range of substrates that are essential for developmental, homeostatic, and disease pathways. Classical genetic approaches and in vitro biochemical or cell biological assays identified that precursor forms of most growth factor family proteins are processed by Furin. To quantitatively assess the potential role of Furin in cleaving and modulating intercellular dispersion of a Drosophila signaling protein, we developed a simple assay by combining genetics, ex vivo organ culture, pharmacological treatment, and imaging analyses. The protocol herein describes how to ex vivo culture Drosophila wing imaginal discs expressing a fluorescently tagged Drosophila Fibroblast Growth Factor (FGF, Branchless/Bnl) over a long period of time in the presence of Furin inhibitors and monitor the cleavage and intercellular dispersion of the truncated Bnl parts using microscopy. Although the assay described here is for assessing the effect of Furin inhibition on Bnl cleavage in the Drosophila larval wing imaginal disc, the principle and methodology can easily be adopted for any other signals, tissue systems, or organisms. This strategy and protocol provide an assay for examining Furin activity on a specific substrate by directly visualizing the spatiotemporal distribution of its truncated parts in an ex vivo-cultured organ.

The primary cilium is a non-motile sensory organelle whose assembly and disassembly are closely associated with cell cycle progression. The primary cilium is elongated from the basal body in quiescent cells and is resorbed as the cells re-enter the cell cycle. Dysregulation of ciliary dynamics has been linked with ciliopathies and other human diseases. The in vitro serum-stimulated ciliary assembly/disassembly assay has gained popularity in addressing the functions of the protein-of-interest in ciliary dynamics. Here, we describe a well-tested protocol for transfecting human retinal pigment epithelial cells (RPE-1) and performing ciliary assembly/disassembly assays on the transfected cells.

Notch signaling is an evolutionarily conserved signaling pathway that plays an indispensable role during development, and in the maintenance of homeostatic processes, in a wide variety of tissues (Kopan, 2012; Hori et al., 2013). The multifaceted roles of Notch signaling are stringently regulated at different levels. One of the most important aspects of regulation is the binding of different Notch ligands to each Notch receptor (NOTCH1-NOTCH4). Canonical ligands Delta or Serrate (in Drosophila), and Delta-like (DLL1 and DLL4) or Jagged (JAG1 and JAG2) (in mammals), are transmembrane glycoproteins. Ligands expressed on one cell bind to Notch receptors on an adjacent cell to induce Notch signaling. Glycosylation of Notch receptor extracellular domain by O-fucose and O-GlcNAc glycans is well established as critical regulators for Notch signaling strength (Stanley and Okajima, 2010; Haltom and Jafar-Nejad, 2015; Sawaguchi et al., 2017). In order to characterize Notch ligand binding to Notch receptors in isolated cells, we utilize Notch ligand extracellular domains tagged at the C-terminus by a human Fc domain, and determine binding of fluorescent anti-Fc antibody by flow cytometry.

This protocol describes how to measure interaction between Notch receptors and their ligands by cell-based assay using Dynabeads. We have used the protocol to determine binding capacity between Notch1-transfected HEK293T cells and ligand-coated Dynabeads. Expression of Eogt in Notch1-expressing cells promoted binding toward DLL4-coated beads, but not JAG1-coated beads. The Notch-ligand assay using Dynabeads suggested that Eogt facilitates DLL4-Notch1 interaction (Sawaguchi et al., 2017).

Understanding the dynamic behavior and the continuously changing composition of macromolecular complexes, subcellular structures and organelles is one of areas of active research in both cell and developmental biology, as these changes directly relate to function and subsequently to the development and homeostasis of the organism. Here, we demonstrate the use of the developing Drosophila oocyte to study dynamics of messenger ribonucleoprotein complexes (mRNPs) with high spatiotemporal resolution. The combination of Drosophila genetics with total internal reflection (TIRF) microscopy, image processing and data analysis gives insight into mRNP motility and composition dynamics with unprecedented precision.

This protocol is for testing responses of a candidate cell line/cell lines to Wnt ligands or Wnt pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Wnt pathway responses. Canonical Wnt signaling activity transcriptionally induces Wnt target genes that contain concensus TCF/LEF binding element. Wnt pathway activity responsive cells transiently or stably expressing luciferase proteins under the TCF/LEF promoter element can be used to report stimulus-dependent Wnt-pathway activity. We acquired the TopFlash (TCL/LEF-Firefly luciferase) construct from Addgene.

This protocol is for testing responses of a candidate cell line/cell lines to Hh ligands or Hh pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Hh pathway responses. Canonical Hh signaling activity transcriptionally induces Hh target genes that contain consensus Gli binding element. Hh-responsive cells transiently or stably expressing luciferase protein under the regulation of the Gli promoter element can be used to report stimulus-dependent Hh-pathway activity.