Engrailed acts with Nejire to control decapentaplegic expression in the Drosophila ovarian stem cell niche.

Homeostasis of adult tissues is maintained by a small number of stem cells, which are sustained by their niches. In the Drosophila female germline stem cell (GSC) niche, Decapentaplegic (Dpp) is the primary factor that promotes GSC self-renewal. However, the mechanism regulating dpp expression in the niche is largely unknown. Here, we identify a 2.0 kb fragment located in a 5' cis-regulatory region of the dpp locus containing enhancer activity that drives its expression in the niche. This region is distinct from a previously characterized 3' cis-regulatory enhancer responsible for dpp expression in imaginal discs. Our data demonstrate that Engrailed, a homeodomain-containing transcription factor that serves as a cap cell marker, binds to this region and regulates dpp expression in cap cells. Further data suggest that En forms a complex with Nejire (Nej), the Drosophila ortholog of histone acetyltransferase CBP/p300, and directs Nej to this cis-regulatory region where Nej functions as the co-activator for dpp expression. Therefore, our study defines the molecular pathway controlling dpp expression in the Drosophila ovarian stem cell niche.

Dpp/Gbb signaling is required for normal intestinal regeneration during infection.

Maintaining tissue homeostasis is a critical process during infection and inflammation. Tissues with a high intrinsic turnover, such as the intestinal epithelium, must launch a rapid response to infections while simultaneously coordinating cell proliferation and differentiation decisions. In this study, we searched for genes required for regeneration of the Drosophila intestine, and thereby affecting overall organism survival after infection with pathogenic bacteria. We found that Dpp/Gbb (BMP) signaling is essential for normal midgut regeneration, and that infection induces the BMP signaling ligands Dpp and Gbb. We demonstrate that Dpp is induced in visceral muscle and required for signaling activation. Subsequently, Gbb is induced in enterocytes after oral infection. Loss-of Dpp signaling in ISCs and transient committed progenitors called enteroblasts (EBs), or in EBs alone, led to a blockage in EC differentiation or maturation. Furthermore, our data show that down-regulation of Dpp signaling in the precursor cells including EBs also resulted in an increased number of abnormally small Pdm1-positive cells, suggesting a role of Dpp/Gbb signaling in EC growth. In addition, we show that Dpp/Gbb signaling acted downstream or in parallel to the Notch pathway to promote EC differentiation and growth. Our results suggest that Dpp/BMP signaling plays an important role in EBs to maintain tissue integrity and homeostasis during pathogenic infections.

Canonical Wnt Signaling Promotes Formation of Somatic Permeability Barrier for Proper Germ Cell Differentiation.

Morphogen-mediated signaling is critical for proper organ development and stem cell function, and well-characterized mechanisms spatiotemporally limit the expression of ligands, receptors, and ligand-binding cell-surface glypicans. Here, we show that in the developing Drosophila ovary, canonical Wnt signaling promotes the formation of somatic escort cells (ECs) and their protrusions, which establish a physical permeability barrier to define morphogen territories for proper germ cell differentiation. The protrusions shield germ cells from Dpp and Wingless morphogens produced by the germline stem cell (GSC) niche and normally only received by GSCs. Genetic disruption of EC protrusions allows GSC progeny to also receive Dpp and Wingless, which subsequently disrupt germ cell differentiation. Our results reveal a role for canonical Wnt signaling in specifying the ovarian somatic cells necessary for germ cell differentiation. Additionally, we demonstrate the morphogen-limiting function of this physical permeability barrier, which may be a common mechanism in other organs across species.

Wnt ligands regulate Tkv expression to constrain Dpp activity in the Drosophila ovarian stem cell niche.

Stem cell self-renewal versus differentiation is regulated by the niche, which provides localized molecules that favor self-renewal. In the Drosophila melanogaster female germline stem cell (GSC) niche, Decapentaplegic (Dpp), a fly transforming growth factor ß molecule and well-established long-range morphogen, acts over one cell diameter to maintain the GSCs. Here, we show that Thickveins (Tkv; a type I receptor of Dpp) is highly expressed in stromal cells next to Dpp-producing cells and functions to remove excess Dpp outside the niche, thereby spatially restricting its activity. Interestingly, Tkv expression in these stromal cells is regulated by multiple Wnt ligands that are produced by the niche. Our data demonstrate a self-restraining mechanism by which the Drosophila ovarian GSC niche acts to define its own boundary.

Coordinated niche-associated signals promote germline homeostasis in the Drosophila ovary.

Stem cell niches provide localized signaling molecules to promote stem cell fate and to suppress differentiation. The Drosophila melanogaster ovarian niche is established by several types of stromal cells, including terminal filament cells, cap cells, and escort cells (ECs). Here, we show that, in addition to its well-known function as a niche factor expressed in cap cells, the Drosophila transforming growth factor ß molecule Decapentaplegic (Dpp) is expressed at a low level in ECs to maintain a pool of partially differentiated germline cells that may dedifferentiate to replenish germline stem cells upon their depletion under normal and stress conditions. Our study further reveals that the Dpp level in ECs is modulated by Hedgehog (Hh) ligands, which originate from both cap cells and ECs. We also demonstrate that Hh signaling exerts its function by suppressing Janus kinase/signal transducer activity, which promotes Dpp expression in ECs. Collectively, our data suggest a complex interplay of niche-associated signals that controls the development of a stem cell lineage.

Immunostaining of germline stem cells and the niche in Drosophila ovaries.

Stem cells have the ability to switch between proliferative (self-renewal) and differentiation modes. The Drosophila germarium is a well-established in vivo model for the study of communication between stem cells and their niche. One commonly used technique for such study is immunostaining that allows examination of protein localization at a fixed time point. This chapter provides a detailed protocol for immunofluorescence staining of Drosophila ovaries. This protocol has been optimized to enable explicit visualization of the niche structure, as well as to maximize the degree of multiplexing for protein labeling and detection.