Figure 1.
Repression of Notch signaling by Mef2 at the DV boundary of the wing disc.
When GFP is expressed along the AP boundary of the third instar larva wing disc using the ptc-Gal4 driver (A), Ct, detected by a specific anti-Ct antibody, is normally expressed at the DV boundary (B; overlay in C). In contrast, mis-expression of Mef2 along the AP boundary using the same driver (D; detected using an anti-Mef2 antibody) severely reduces Ct expression at the AP boundary (E, arrowhead; overlay in F).
Figure 2.
Notch repression by Sd and Mef2 in SOPs.
A'–E' are magnifications of A–E. In wild type thoraxes, bristles (A, A' arrows) are aligned along the antero-posterior axis (A', dashed lines). When a dominant negative allele of Notch is expressed using the neur-Gal4 driver, different Notch phenotypes are observed on thoraxes, spanning from wild-type bristles (B, B', arrows), to complete absence of external cells, shaft and socket (B, B', asterisks). Duplicated bristles resulting from Notch inhibition during the pI/pII or pII/pIII asymetric divisions are also present (B', arrowheads). When sd (C, C') or Mef2 (D, D') are expressed alone, no Notch phenotypes are observed: bristles are present and align along the antero-posterior axis (C', D', dotted line). When sd and Mef2 are expressed together, no external cells are observed, corresponding to a strong Notch phenotype (E, E').
Figure 3.
The Notch pathway is repressed during the first asymmetric division of SOPs upon Sd/Mef2 ectopic overexpression.
YFP (A–D) or YFP, sd, and Mef2 (E–H) were overexpressed using the neur-Gal4 driver. In wild type genetic context, cells expressing the YFP (B) express Sens, a marker of the SOP lineage, detected with a specific antibody (C). In two cell clusters (A–D, arrowheads), only one cell corresponding to the pIIb cell expresses Pros (D, arrowhead b). The second cell corresponding to the pIIa cell does not express Pros (D, arrowhead a). When Sd, Mef2 and YFP are co-overexpressed, cells expressing YFP (F), as in wild type, express Sens (G) showing that these cells maintain their SOP identity. The cells of the two cell clusters (E–H, arrowheads) both express Pros (H, arrowhead b), signifying that they correspond to two pIIb cells. A and E correspond to DAPI staining.
Figure 4.
Activation of the Dl2.6 enhancer in vivo monitored by the expression of the Dl2.6-GFP transgene.
E′–H′ are magnifications of the dotted squares in E–H. In 16 h, 24 h, 30 h and 36 h APF pupae, the 22c10 antibody labels muscles and nerves (B, F, F′, J and N respectively), Dl2.6 enhancer is activated in developing fibers of 16 h, 24 h and 30 h APF pupae (C, G, G′, K respectively) but not in myoblasts at 16 h APF (A–D) and 24 h APF (E–H, magnification in E′–H′, arrowheads). Dl2.6 enhancer is not activated in developing fibers of 36 h APF pupa (M-P). DAPI-GFP overlay is shown in D, H, and H′, L and P. Scale bar: 100 µm.
Figure 5.
Reduced activation of the Dl2.6 enhancer in vivo monitored by the expression of the Dl2.6-GFP transgene.
In 21 h APF pupae, muscle fibers are visualized with DAPI staining by the specific arrangement of their nuclei (A, D, asterisks). In a wild-type pupae, Dl2.6 enhancer is activated in developing fibers (B, overlay in C). In pupae overexpressing an RNAi-Mef2 transgene, Dl2.6 enhancer activation is significantly lower than in wild-type (E, overlay in F).
Figure 6.
Activation of the Dl2.6 enhancer in S2 and Dmd8 cells by Sd and Mef2.
A: In S2 cells, the Dl2.6 enhancer was not activated by Sd alone. In contrast, it was significantly activated by Mef2 (p = 0.0003) and Sd/Mef2 (p = 0.0008). Activation with Sd/Mef2 was significantly higher than activation by Mef2 alone (p = 0.003). B: In Dmd8 cells, as in S2 cells, Dl2.6 is not activated by Sd alone. It is significantly activated by Mef2 (p = 7.6×10−6) and Sd/Mef2 (p = 2×10−4). However, activation by Sd/Mef2 was lower than activation by Mef2 alone (p = 4.3×10−6).
Figure 7.
Activation of Dl by Sd/Mef2 in 28 h APF pupa SOPs.
In SOPs, visualized with YFP (green) expressed under the control of the neur-Gal4 driver (arrowheads in A–D), Dl (red) is detectable at the same levels as in non-SOP cells (asterisks in A–D) of the notum. When YFP, sd and Mef2 are overexpressed in SOPs using the same driver (E–H), Dl is up-regulated in SOPs (arrows in G) relative to non-SOP cells (asterisks in G) of the notum. DAPI labeling is shown in gray (A,E). Overlays are shown in D and H.
Figure 8.
Expression of Dl in IFMs of 24 h APF pupae.
Expression of Dl was detected with a anti-Dl antibody in wild-type B, B′–C,C') and 1151-Gal4, UAS-RNAiMef2 (E, E′–F,F') 21 h APF pupae. A'–F' show magnified areas indicated by the dotted squares in A–F respectively. Muscles are visualized with DAPI staining by the specific arrangement of their nuclei (asterisks in A, D, A′, D′; muscles are located between the two dotted lines in A′ and D′). Large nucleus (n in A′) is a larval template nucleus. Myoblasts are located between muscles (m in A′, D′). In wild type pupae, Dl is detected in myoblasts and developing fibers (B, B′; overlay in C, C′). Expression levels in myoblasts and fibers are close (B′). Overexpression of a RNAi-Mef2 construct in myoblasts and fibers using the 1151-Gal4 driver (D–F, D′–F′) lowers Dl expression in developing fibers compared to myoblasts (E′, compare m to asterisks) and in 1151-Gal4, UAS-RNAiMef2 muscles compared to wild type muscles (compare E′, asterisks to B′, asterisk).