Heterodimerization-dependent secretion of BMPs in Drosophila

Abstract

Combinatorial signaling is key to instruct context-dependent cell behaviors. During embryonic development, adult homeostasis and disease, Bone Morphogenetic Proteins (BMPs) act as dimers to instruct specific cellular responses. BMP ligands can form both homo- or heterodimers; however, obtaining direct evidence of the endogenous localization and function of each form has proven challenging. Here, we make use of precise genome editing and direct protein manipulation via protein binders to dissect the existence and functional relevance of BMP homo- and heterodimers in the Drosophila wing imaginal disc. This approach revealed in situ the existence of Dpp (BMP2/4)/Gbb (BMP5/6/7/8) heterodimers. We found that Gbb, despite being produced by all the cells of the wing imaginal disc, is only secreted by the cells in which Dpp is expressed. Dpp and Gbb form a gradient of heterodimers whereas neither Dpp nor Gbb homodimers are evident under endogenous physiological conditions. We find that the formation of heterodimers is critical for obtaining optimal signaling and long-range BMP distribution in the developing wing. These results indicate that Dpp/Gbb heterodimers are the active signal required for epithelial patterning and growth.

Diffusible fraction of niche BMP ligand safeguards stem-cell differentiation

“…Next, we conducted trapped HA-tagged Dpp or Gbb using HA-trap (30)(31)(32). Trapping HA-Dpp showed significant reduction of pMad in GSCs similar to trapping mGL-Dpp by mCD8-MT (Figure 5G, H and J).…”

“…First to understand expression pattern of Dpp and Gbb, we conducted anti-HA staining of fully functional HA-tagged Dpp and Gbb (30,31) to detect intracellular fraction of Dpp and Gbb. In a past report, Dpp was shown to be expressed both in hub cells and CySCs by RT-PCR (11).…”

Dally is not essential for Dpp spreading or internalization but for Dpp stability by antagonizing Tkv-mediated Dpp internalization

“…To address this, we first expressed dally or dlp in the dorsal compartment of the wing disc using ap-Gal4 to compare their ability to interact with endogenous Dpp and activate pMad signal in vivo. To visualize endogenous extracellular Dpp distribution, we utilized a functional Ollas-dpp allele 35 . Since Dpp distribution and pMad signal are similar between the dorsal and ventral compartments in discs expressing the Ollas-dpp allele (Fig.…”

“…Flies for experiments were kept at 25°C in standard fly vials containing polenta and yeast. The following fly lines were used: UAS-tkv-RNAi (Bloomington 40937), UAS-dally-RNAi (VDRC 14136), Ollas-dpp 35 , UAS-GFP-HA-dally, UAS-GFP-dally ΔHS (Suzanne Eaton), UAS-dlp (Baeg et al, 2001), HA-dpp 15 , HA-dpp ΔN 40 , Ollas-HA-dpp ΔN (this study), hh-Gal4 (Gift from Dr Manolo Calleja), dally MH32 37 . yw, dpp d8 and dpp d12 are described from Flybase.…”

“…Flies for experiments were kept at 25°C in standard fly vials containing polenta and yeast. The following fly lines were used: UAS-tkv-RNAi (Bloomington 40937), UAS-dally-RNAi (VDRC 14136), Ollas-dpp 35 , UAS-GFP-HA-dally, UAS-GFP-dally ΔHS (Suzanne Eaton), UAS-dlp (Baeg et al, 2001), HA-dpp 15 , HA-dpp ΔN (this study), dlp [ko;attB] (this study), dally[ko;attB] (this study), dally[YFP-dally;attP] (this study), dlp[3xHAdlp;attP] (this study) dally[YFP-dally ΔHS ;attP] (this study), dally[3xHA-dlp;attP] (this study), ap-Gal4 (Bloomington 3041), ap[c1.4b-Gal4] (Michèle Sickmann and Martin Müller, unpublished), JAX 40 , Ollas-HAdpp ΔN (this study), hh-Gal4 (Gift from Dr Manolo Calleja), dally MH32 37 . yw, dpp d8 and dpp d12 are described from Flybase.…”