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First published online May 5, 2004
doi: 10.1242/10.1242/dev.01113

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Notch signaling patterns Drosophila mesodermal segments by regulating the bHLH transcription factor twist

Program in Developmental Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, Weill Graduate School of Medical Sciences at Cornell University, 1275 York Avenue, New York, NY 10021, USA

View larger version (145K): [in a new window]   Fig. 1. Notch represses Twist expression. Lateral views of embryos stained with anti-Twist. (A,C,E,G) Whole-mount embryos. In this and all the following figures, the black bracket denotes the mesodermal segments shown at higher magnification in (B,D,F,H). (B,D,F,H) Corresponding close-ups of each embryo in (A,C,E,G). In this and all the following figures, the white bracket demarcates one mesodermal segment. (A,B) Wild-type (wt) stage 9 embryo expresses Twist at high levels uniformly throughout its mesoderm. (C,D) Wt stage 10 embryo exhibits a modulated Twist pattern along its anterior-posterior axis. Each segment consists of a low and high Twist domain. (E,F) Nnull stage 10 embryo maintains high Twist expression throughout its mesoderm. Rather than modulating Twist levels, Nnull mutants display uniform high Twist expression pattern characteristic of wt stage 9 embryos. (G,H) UAS-Nintra stage 10 embryo has fewer high Twist expressing cells than wt.

View larger version (78K): [in a new window]   Fig. 2. Twist expression in Su(H) loss- and gain-of-function embryos. (A,C) Stage 10 whole-mount embryos stained with anti-Twist. (B,D) Corresponding close-ups of embryos in A,C. (A,B) Su(H)null mutant, like wild type, modulates Twist into low and high domains. (C,D) UAS-Su(H)-VP16 embryo ectopically expresses high Twist levels. Black arrowhead points to cells, located in what should be the low Twist domain, that express high amounts of Twist compared to wild type.

View larger version (135K): [in a new window]   Fig. 3. Su(H) is required for Notch to repress Twist. (A,C,E,G,I) Lateral views of stage 10 whole-mount embryos stained with anti-Twist. (B,D,F,H,J) Corresponding close-ups of embryos in A,C,E,G,I. (A,B) Su(H)null; UAS-Nintra embryo has a wild-type-like Twist pattern, similar to Su(H)null. Low and high Twist domains are seen along the anterior-posterior axis; Twist is not strongly repressed as in UAS-Nintra embryos (see Fig. 1). (C,D) Nnull; UAS-FLN embryo exhibits low and high Twist domains. FLN rescues the Twist pattern of Nnull embryos (see Fig. 1). (E,F) Nnull; UAS-Nintra embryo has low and high Twist domains. Nintra rescues the Twist pattern, but represses Twist expression compared to FLN. (G,H) Nnull; UAS-FLNcdc10 maintains uniform high Twist expression, similar to Nnull embryos. FLNcdc10 does not rescue the Twist pattern. (I,J) Nnull; UAS-Su(H)-VP16 embryo no longer maintains uniform high Twist expression. Low and high Twist domains are observed, but high Twist domains appear slightly expanded. Black arrowhead points to cells in the low Twist domain that express higher levels of Twist than wild type. Su(H)-VP16 rescues the Twist pattern but not as strongly as FLN and Nintra.

View larger version (149K): [in a new window]   Fig. 4. Analysis of Notch/Su(H) regulation of a minimal twist promoter. Lateral views of stage 10 embryos stained with anti-GFP. (A,C,E,G) Whole-mount embryos. (B,D,F,H) Corresponding closeups of embryos in A,C,E,G. (A,B) 1428twist embryo exhibits low and high GFP domains. Notice that high domains appear chevron-shaped. (C,D) 1428twist; UAS-Nintra embryo has narrower high GFP domains than 1428twist embryos. GFP expression is repressed so that high GFP domains appear triangle-like in 1428twist; UAS-Nintra embryos. (E,F) 1428twistmutSu(H) embryo displays a modulated pattern of low and high GFP domains. Uniform high GFP expression is not maintained throughout the mesoderm. Additionally, compared with 1428twist, high GFP domains appear to be slightly expanded. (G,H) 1428twistmutSu(H); UAS-Nintra embryo looks different from the three embryos shown above. GFP expression in presumptive high GFP domains, especially laterally, is repressed compared with 1428twist embryos, so 1428twistmutSu(H); UAS-Nintra high GFP domains appear most similar to those seen in 1428twist; UAS-Nintra embryos. However, at the same time, compared with 1428twist embryos, some cells in the presumptive low GFP domains of 1428twistmutSu(H); UAS-Nintra embryos express GFP at high levels; this phenotype is most similar to that of 1428twistmutSu(H) embryos.

View larger version (75K): [in a new window]   Fig. 5. E(spl)-C locus regulates Twist modulation into low and high domains. (A-D) Lateral views of stage 10 embryos stained with anti-Twist: (A,C) whole-mount embryo, (B,D) corresponding close-ups of embryos in (A,C). (A,B) Df E(spl)-C mutant maintains high Twist expression uniformly throughout its mesoderm, similar to Nnull embryos. (C,D) Df E(spl)-C, P[gro+] mutant has expanded high Twist domains compared with wild type. Black arrowhead indicates cells, located in a presumptive low Twist domain, expressing higher levels of Twist than wild type.

View larger version (138K): [in a new window]   Fig. 6. Da activity regulates Twist expression. Lateral views of stage 8 (A,B) and stage 10 (C-H) embryos stained with anti-Twist. (A,B,C,E,G) Whole-mount embryos. (D,F,H) Corresponding closeups of embryos in C,E,G. (A) Wild-type (wt) stage 8 embryo expresses Twist uniformly at high levels throughout the mesoderm. (B) da1 embryo, a mutant with reduced maternal and zygotic Da levels, expresses Twist at low levels at stage 8, as well as at later stages (data not shown) (Castanon et al., 2001). (C,D) UAS-da embryo, panmesodermally expressing high levels of Da, ectopically expresses high levels of Twist, similar to Nnull mutants. (E,F) UAS-da embryo, panmesodermally expressing lower levels of Da than the embryo in C-D, ectopically expresses high levels of Twist, but shows a milder phenotype than the embryo in C-D. Black arrowhead indicates cells, located in what should be the low Twist domain, that express higher amounts of Twist than wild type. (G,H) UAS-da-da embryo, panmesodermally expressing the linked Da transgene under the same GAL4 conditions as the embryo in E-F, maintains uniform high Twist expression throughout its mesoderm similar to Nnull mutants (see Fig. 1E,F).

View larger version (99K): [in a new window]   Fig. 7. Notch represses Twist by regulating Emc activity. (A,C,E) Whole-mount embryos. (B,D,F) Corresponding close-ups of embryos in (A,C,E). (A-D) Lateral views of stage 11 embryos stained with anti-Emc. (A,B) Wild-type (wt) embryo shows strong Emc expression around its ectodermal tracheal pits (black arrowheads) and little or no mesodermal Emc expression (white asterisks). (C,D) UAS-Nintra embryo expresses Emc both around its tracheal pits (black arrowheads) and throughout its mesoderm (white asterisks). (E,F) Lateral views of a stage 10 Nnull; UAS-emc embryo stained with anti-Twist. Emc represses Twist in Nnull mutants such that Twist is expressed in low and high domains; compare with Fig. 1E,F and Fig. 3C-J.

View larger version (23K): [in a new window]   Fig. 8. Models of Notch target gene regulation. (A) Notch signaling has multiple inputs into twist; it regulates twist directly and indirectly, through the repressor of twist genes. Both modes of regulation require Notch to act as a transcriptional switch. In the absence of Notch signaling, Su(H) interacts with corepressors [Su(H)rep] to repress transcription of both twist and repressor of twist. Upon activation of the Notch receptor, the intracellular domain of Notch (Nicd) enters the nucleus and associates with Su(H). This interaction displaces corepressors, de-represses Su(H) and allows Su(H) to serve as a transcriptional activator [Su(H)act]. Hence, Notch signaling promotes transcription of both twist and repressor of twist. (B) Notch acts permissively on the twist gene, but instructively on a repressor of twist gene. repressor of twist [E(spl)-C] transcription requires Nicd to alleviate Su(H)-mediated repression and to serve as a coactivator for Su(H). twist transcription is dependent on Nicd, as well as other factors. Nicd is solely required to de-repress Su(H). Su(H) bound to other coactivators and/or other transcriptional activators is necessary for twist activation.