Delta activity independent of its activity as a ligand of Notch

Abstract

Background: Delta, Notch, and Scabrous often function together to make different cell types and refine tissue patterns during Drosophila development. Delta is known as the ligand that triggers Notch receptor activity. Scabrous is known to bind Notch and promote Notch activity in response to Delta. It is not known if Scabrous binds Delta or Delta has activity other than its activity as a ligand of Notch. It is very difficult to clearly determine this binding or activity in vivo as all Notch, Delta, and Scabrous activities are required simultaneously or successively in an inter-dependent manner.

Results: Using Drosophila cultured cells we show that the full length Delta promotes accumulation of Daughterless protein, fringe RNA, and pangolin RNA in the absence of Scabrous or Notch. Scabrous binds Delta and suppresses this activity even though it increases the level of the Delta intracellular domain. We also show that Scabrous can promote Notch receptor activity, in the absence of Delta.

Conclusion: Delta has activity that is independent of its activity as a ligand of Notch. Scabrous suppresses this Delta activity. Scabrous also promotes Notch activity that is dependent on Delta's ligand activity. Thus, Notch, Delta, and Scabrous might function in complex combinatorial or mutually exclusive interactions during development. The data reported here will be of significant help in understanding these interactions in vivo.

Figure 1

Sca associates with Dl. A-C. Fluorescent photomicrographs of different cell lines treated with Sca-GFP medium for 30 minutes. Cells simultaneously fixed and rinsed in 4% paraformaldehyde/1X PBS are shown in the insets. Experiments were repeated three times. D. Western blots showing recovery of Dl in Sca immuno-precipitates from total protein extracts prepared from S2-Dl cells treated with S2-Sca cells. S2-Sca cells were used instead of Sca conditioned medium to maximize the ratio of bound to unbound Sca. Cross-linker = membrane insoluble and cleavable 3,3'- Dithiobis (sulfosuccinimidylpropionate) (DTSSP), which cross-links proteins interacting at the cell surface. IP Ab = antibody used for immunoprecipitation; W Ab = antibody used on the western blot; ppt = immunoprecipitate; super = supernatant. Experiments were repeated two times.

Figure 2

Dl down-regulates Daughterless protein expression, and N up-regulates E(spl)C m3 gene expression, in response to Scabrous. A. Northern blots of total RNA from the indicated cell mixtures extracted at 0 or 45 minutes after treatment with medium containing Sca (+) or not (-). Gene probes used are shown on the right. m3 = E(spl)C m3 and rp 49 = a ribosomal protein gene used to show the levels of total RNA in the lanes in all northern blots. Sca = conditioned medium prepared from the S2-Sca stable cell line in all experiments here onwards. The control medium used along side Sca medium (-) was prepared from heat shocked S2 cells. Experiments were repeated two times. For unknown reasons, the medium collected from heat shocked S2 cells (used in lanes 1, 3, and 5) produced higher background levels of E(spl)C m3 RNA in S2-N cells (lane 1). B. Western blots showing the levels of Da and Dl in different Dl cell lines. S2-Dl, S2-Dl(1) and S2-Dl(2) are independently established hsDl cell lines. Ui = un-induced (i.e., not heat shocked). Hsp70 = heat shock 70 protein used to show the levels of proteins in the lanes of all western blots. Dl and DlΔI were detected with αDlEC. Da signals here (and the indicated signals elsewhere) were quantified relative to Hsp70 (western blots), rp49 (northern blots), or other indicated molecules, using the NIH Image 1.63 program. These experiments were repeated more than ten times. C. Western blots showing Da levels in the indicated cell mixtures, with (+) or without (-) Sca. These experiments were repeated five times.

Figure 3

The levels of cleaved Dl intracellular domain is not associated with high levels of Da. A. Western blots (from a 8% SDS-PAGE) showing the level of Dl and DlIC in the indicated cell mixtures, with (+) or without (-) Sca. B. Western blots (from a 12% gel) showing the levels of Dl and DlIC in S2-Dl cells treated medium containing different levels of Sca. C. Western blots showing the levels of Da in the indicated cell mixtures. D. Western blots showing the levels of Da, Dl, and DlIC at different times following heat shock induction of Dl in S2-Dl cells. All experiments were repeated at least three times.

Figure 4

Dl promotes expression of fng and pan. A. Northern blots showing fng and pan expression in the indicated cell mixtures at 0 and 45 minutes after cell mixing. B. Northern blots showing fng and pan expression in the indicated cell lines two hours after induction of expression. C. Northern blots showing fng and pan expression in two other independently established S2-Dl cell lines. Cells used for lanes 1–2 were uninduced (ui); cells used for lanes 3–4 were heat shock induced. D. Northern blots showing fng and pan expression in S2-Dl cells that were either untreated or treated with Sca medium. All experiments were repeated at least three times. The fng band marked with an asterisk corresponds to the published mRNA [35]. Only this band was used for fng quantification. The pan band shown is consistent with the information described in van de Wetering et al. [40] and Brunner et al. [39].