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First published online 8 November 2006
doi: 10.1242/dev.02689

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Threshold response of C15 to the Dpp gradient in Drosophila is established by the cumulative effect of Smad and Zen activators and negative cues

Department of Biology, New York University, 100 Washington Square East, New York, NY 10003, USA.

View larger version (135K): [in this window] [in a new window]   Fig. 1. The threshold response of C15 to the Mad-P gradient depends on both Smads and Zen. Dorsal views of late stage 5/early stage 6 embryos with anterior to the left. The widest area of expression is delineated with a white line labelled to show the width in number of cells. (A) Wild-type embryo hybridized with C15 probes. Transcripts are present in a five- to ten-cell-wide dorsal strip of cells in the main body region and a faint spot in the presumptive head region. Cells in this region are magnified in the inset, which lies perpendicular to the embryo. (B) Wild-type embryo stained with an antiphospho-Smad1 antibody, which also recognizes Mad-P. Strong staining is observed in a five- to six-cell-wide stripe with less staining in three to four cells to either side of the stripe (see magnified inset). In terminal regions, the Mad-P stripe can be up to 16 cells wide. (C) Wild-type embryo hybridized with Race probes. Transcripts are present in a four- to six-cell-wide stripe. (D) Wild-type embryo hybridized with tup probes. The dorsal stripe is ten to 14 cells wide. (E-H) Mutant embryos hybridized with C15 probes. C15 is absent in dpphr4 homozygotes (E) and zenw36 homozygotes (F). C15 is in a broad domain in sogYS06 hemizygous embryos (G). The C15 pattern broadens to about 26 cells at its widest in a maternal GAL4>UAS-zen embryo (H).

View larger version (24K): [in this window] [in a new window]   Fig. 2. A 650 bp enhancer is sufficient to drive the C15 dorsal-stripe pattern. (A) The C15 gene (yellow arrow) is located in the 93DE homeobox gene cluster (green arrows) (Jagla et al., 2001), which also contains predicted genes (pink boxes). Several overlapping DNA fragments comprising approximately 30 kb of 5', 3' and intronic (I) DNA of the C15 gene were tested in lacZ reporter assays, some of which were capable of activating lacZ expression in blastoderm embryos (blue lines). (B-E) Embryos hybridized with lacZ probes. (B) I-2-lacZ shows a lacZ pattern identical to the endogenous C15 pattern. (C) I-2-4-lacZ shows a broader pattern, suggesting that a negative element is missing. (D) 650-3 shows a lacZ pattern like that of I-2. (E) 350-2 shows weaker lacZ expression than the 650-3.

View larger version (53K): [in this window] [in a new window]   Fig. 3. Zen interacts with the C15 enhancer. (A) Five overlapping fragments comprising the 650 bp enhancer were labeled at one end, incubated with GST-Zen protein, and subjected to DNAse I footprint analysis. Gray boxes at either end represent vector sequences. Only fragments IV (B) and V (C) displayed footprints. The first lane in each gel represents the chemical degradation of the probe at G+As. The second and third lanes contain fragments that were incubated without or with 60 ng of GST-Zen protein extract (denoted by the red rectangle) prior to DNAse I digestion, respectively. The regions protected by Zen are depicted as red ovals. The nucleotide sequence of the protected regions is shown beside the G+A lanes with putative core binding sites highlighted in red. (D) Gel comparing GST-Zen binding to wild-type and mutated oligonucleotides. Arrow, bound probe; two arrows, free probe. (E) DNA sequence of wild-type and mutated oligonucleotides used for mobility shift assays and in vitro mutagenesis. Mutations in Z1m, Z2m and Z3m are denoted in green.

View larger version (34K): [in this window] [in a new window]   Fig. 4. Smads bind to multiple sites in the C15 enhancer. (A) Schematic representation of the DNA fragments comprising the 650 bp enhancer that were labeled for mobility shift assays with GST-Smad proteins. Fragments that formed complexes are highlighted in blue. (B) DNA sequence of wild-type and mutated oligonucleotides containing the putative Smad sites from the C15 enhancer and one from the Race enhancer (R42) that were used for mobility shift assays and in vitro mutagenesis. Putative Smad sites are highlighted in blue, mutated sequences in green. The different types of Smad sites are denoted above the sequences: GC, GC-rich; SBE, Smad-binding element; DSC, Drosophila Smad consensus. (C) Gel of GST-Mad incubated with the labeled wild-type oligonucleotides. The first lane in each section contains free probe. The second and third lanes contain increasing amounts of GST-Mad (10 and 30 ng; indicated by blue steps). (D) Gel of GST-Mad incubated with wild-type (bold) or mutated oligonucleotides. Lanes 1-6, M2 and mutations in one of the four GC-rich sites (denoted 1-4), or all four sites (m). Lanes 7-12, M5 and mutations in the GC (gc), the SBE (sb) or either half of the DSC (ds1, 2), or all of the sites (m). Lanes 13-16, M6, M7 and mutations in all putative sites (m). (E) Summary of Smad-(blue ovals) and Zen-(red ovals) binding sites in the 650 bp enhancer (thick black line). Relative locations of the oligonucleotides and the 350 bp enhancers are delineated by thin horizontal lines.

View larger version (112K): [in this window] [in a new window]   Fig. 5. A combination of the Zen and Smad activators and a repressor are necessary for establishing the C15 domain. Dorsal views of late stage5/early stage 6 embryos carrying the 650 bp C15 enhancer-lacZ transgene with mutations in the Smad- and Zen-binding sites. Embryos were hybridized with lacZ probes. Arrowheads indicate the limits of the expression domain. No effect on the lacZ expression pattern was observed when Smad sites were mutated in the M2 (A) or M5 (B) clusters. However, mutation of the Smad sites in M7 (C) caused a reduction in the intensity of lacZ expression, especially in lateral regions. Arrowheads denote faintly stained cells. Embryos carrying mutations in both the M2 and M5 clusters (D) or the M5 and M7 clusters (E) also showed weaker lacZ expression. Mutation of all Smad sites in M5, M6 and M7 caused severe loss of expression (F); however, the overall size of the domain appears unchanged (arrowheads). Mutation of the putative Zen-binding site in the Z1 sequence caused reduced expression (G). However, mutation of the Zen sites in the Z2 (H) or Z3 (I) sequences caused significant expansion of the lacZ domain, implicating those sites in repression of the C15-lacZ transgene. When the Zen-binding sites were eliminated, expression was drastically reduced (J). Mutation of Z2 and M5 had no effect (K); however, mutation of M2, M5 and Z2 caused lateral expression to be lost (L), presumably because low levels of Mad-P in lateral regions is insufficient to activate transcription even in the absence of the putative repressor.