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The Snail repressor positions Notch signaling in the Drosophila embryo

Department of Molecular and Cell Biology, Division of Genetics and Development, 401 Barker Hall, University of California, Berkeley, CA 94720, USA

View larger version (106K): [in a new window]   Fig. 1. NotchIC induces ectopic activation of sim and m8. Cellularizing embryos express a stripe2-NotchIC transgene and are oriented with dorsal upwards and anterior towards the left. Transgenic embryos were hybridized with digoxigenin-labeled Notch (A), m8 (B) and sim (C) antisense RNA probes, and stained to visualize the indicated gene expression patterns. (A) The Notch probe detects an ectopic stripe of NotchIC expression. General staining of the wild-type Notch RNA is detected throughout the basal cytoplasm. (B) The m8 gene exhibits endogenous lateral lines of expression in the mesectoderm, as well as an ectopic stripe. (C) Ectopic sim expression is restricted to a pyramid pattern in the neurogenic ectoderm.

View larger version (73K): [in a new window]   Fig. 2. NotchIC induces cell fate changes in the neurogenic ectoderm. Transgenic embryos express the stripe2-NotchIC fusion gene and were hybridized with sim or snail antisense RNA probes. (A) The transgene induces the ectopic sim pyramid pattern prior to the onset of the endogenous pattern in the mesectoderm. (B) The ectopic sim pattern is maintained during gastrulation. By this stage sim expression extends into dorsal regions. The endogenous expression pattern is also observed. (C) After gastrulation, the two lines of sim expression converge at the ventral midline to form the mesectoderm. There is also a broad domain of ectopic staining that probably arises from the ectopic pyramid seen in younger embryos. Ectopic sim expression leads to a loss of identifiable neurons, as judged by the snail expression pattern (D). (D) The snail expression pattern is altered in delaminating neuroblasts during germ band elongation. There is a gap in the presumptive ventral nerve cord that coincides with the ectopic sim expression pattern (see C).

View larger version (76K): [in a new window]   Fig. 3. Symmetric stripes of sim and m8 are induced in embryos lacking Dorsal gradients. Embryos were collected from Tollrm9/Tollrm10 (A-C) or gd–/gd– females (D-I). The former mutants contain low, uniform levels of Dorsal in all nuclei. The latter embryos completely lack nuclear Dorsal protein. Different stripe2-NotchIC transgenes were introduced into the mutant backgrounds, and stained with either a digoxigenin-labeled sim (A-F) or m8 probe (G-I). (A-C) Tollrm9/Tollrm10 embryos. (A) sim expression is restricted to the termini of mutant embryos that lack a stripe2-NotchIC transgene. (B) A strong stripe of sim staining is induced by a strongly expressed stripe2-NotchIC transgene. (C) A faint sim stripe is induced by the weakly expressed stripe2-NotchIC transgene. These mutant embryos lack snail expression (Fig. 4E), which might explain why the ectopic sim stripes are symmetrically expressed in dorsal and ventral regions. (D-F) gd–/gd– embryos. (D) sim expression is essentially absent in mutant embryos lacking a stripe2-NotchIC transgene. A weak sim stripe (see arrowhead) is induced by the strongly expressed stripe2-NotchIC transgene (E), whereas the weakly expressed transgene fails to induce sim expression (F). (G-I) gd–/gd– embryos. There is general, weak expression of the m8 gene throughout mutant embryos (G). This staining might be due to the derepression of the staining pattern that is normally restricted to the dorsal ectoderm. Both the strong (H) and weak (I) stripe2-NotchIC transgenes induce stripes of m8 expression.

View larger version (108K): [in a new window]   Fig. 4. Snail represses sog expression. Wild-type (A-D) and Tollrm9/Tollrm10 mutant (E-H) embryos were stained with either a snail (A,B,E,F) or sog (C,D,G,H) hybridization probe. The embryos in A-D and F,H contain a stripe2-snail transgene. (A,B) Lateral and ventral views of wild-type embryos that contain a stripe2-snail transgene. An ectopic stripe of snail expression is detected in addition to the normal pattern in the ventral mesoderm. (C,D) Lateral and ventral views of wild-type embryos expressing the stripe2-snail transgene. There are gaps in the sog expression pattern within the lateral, neurogenic ectoderm near the position of the Snail stripe. (E,F) Tollrm9/Tollrm10 mutant embryos that lack (E) or contain (F) a stripe2-snail transgene. Mutant embryos that lack the transgene exhibit residual snail staining at the poles (E). The stripe2-snail transgene provides the sole source of snail expression in middle body regions (F). This stripe is transiently expressed and rapidly disappears in older embryos (data not shown). (G,H) sog staining patterns in Tollrm9/Tollrm10 mutant embryos that either lack (G) or express (H) a stripe2-snail transgene. In mutant embryos lacking the transgene, sog is uniformly expressed along the anteroposterior axis, with the exception of the extreme termini (G). The stripe2-snail transgene creates a gap in the sog pattern (H).

View larger version (88K): [in a new window]   Fig. 5. The stripe2-snail transgene induces complex changes in the m8 and sim expression patterns. Wild-type (A-F) and Tollrm9/Tollrm10 (G-L) were stained with m8 (A-C;G-I) or sim (D-F;J-L) hybridization probes. (A-C) Lateral views of wild-type embryos that either lack (A) or carry (B,C) the stripe2-snail transgene. In early embryos (just after cellularization), the transgene creates a gap in the normal lateral lines within the presumptive mesectoderm (arrowhead, B; compare with A). In older embryos (gastrulation) there is both a gap in the pattern and ectopic staining in lateral regions (arrowhead, C). (D-F). Lateral views of wild-type embryos that either lack (D) or carry (E,F) the stripe2-snail transgene. There is a gap in the pattern in early embryos (arrowhead in E; compare with D), but the top of the gap is filled to produce a continuous bump of staining in older embryos (arrowhead, F). The asterisk indicates a gap in the sim pattern that is due to normal discontinuities in the initial sim pattern, not to the stripe2-snail transgene. (G-I) Mutant embryos obtained from Tollrm9/Tollrm10 females. There is no m8 expression in middle body regions of mutant embryos (G), although there is expression at the posterior pole. Mutants that express the stripe2-snail transgene exhibit broad stripes of m8 staining (H). Mutants that express the stripe2-snail/hairy transgene also exhibit ectopic m8 staining (I). (J-L) Mutant embryos derived from Tollrm9/Tollrm10 embryos. sim expression is restricted to the termini of mutant embryos lacking the stripe2-snail transgene (J). By contrast, patchy stripes of sim expression are observed both in mutants that contain the stripe2-snail transgene (K) and the stripe2-snail/hairy transgene (L).

View larger version (71K): [in a new window]   Fig. 6. Snail represses potential inhibitors of Notch signaling. Wild-type (A,B) and Tollrm9/Tollrm10 (C,D) mutant embryos were stained with either a Delta (A,B) or T3 hybridization probe (C,D). Embryos have completed cellularization and are oriented with anterior to the left. (A,B) Delta is expressed in lateral and dorsal regions of wild-type embryos (A). Staining is excluded from the ventral mesoderm, possibly by the Snail repressor as the Delta pattern is derepressed in sna–/sna– mutant embryos (not shown). The stripe2-snail transgene (B) causes a slight weakening of the normal Delta pattern (arrowhead). (C,D) T3 is expressed in a series of stripes along the entire dorsoventral axis of Tollrm9/Tollrm10 mutant embryos (C). The stripe2-snail transgene (D) creates a gap in the T3 expression pattern (arrowhead).

View larger version (56K): [in a new window]   Fig. 7. A Snail repressor gradient helps localize Notch signaling. Embryos were collected from females that contain an hsp83-Toll10b-bcd transgene and thereby express a broad anterior-posterior Dorsal nuclear gradient. These embryos were derived from gd–/gd– females, and therefore lack the normal dorsoventral Dorsal gradient. Mutants were stained with either a snail (A,B) or sim (C) hybridization probe. (A,B) snail staining pattern in precellular (A) and cellularized (B) embryos. snail is activated by high levels of the ectopic anteroposterior Dorsal nuclear gradient in anterior regions of mutant embryos. The snail pattern is initially broad and fuzzy (A), but refines during cellularization (B) and exhibits the very sharp border seen for the normal snail pattern at the boundary between the mesoderm and mesectoderm. (C) sim expression is not detected until the onset of gastrulation. Staining is detected in cells that reside just posterior of the sharp snail expression pattern. These sim-positive cells exhibited weak, transient snail expression at earlier stages (A). (D) A model for the positioning of Notch signaling by the Snail repressor. The top and bottom circles represent cross-sections through precellular (top) and cellularized (bottom) embryos. snail is initially expressed in a broad pattern in ventral and ventrolateral regions that encompass the presumptive mesoderm and mesectoderm. At this early stage Snail might repress a number of inhibitors of Notch signaling, such as Delta and T3. At later stages, the snail expression pattern is refined and restricted to the mesoderm. Notch signaling is activated in the cells that transiently expressed the Snail repressor.

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