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First published online 8 October 2003
doi: 10.1242/dev.00801

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Stepwise formation of a SMAD activity gradient during dorsal-ventral patterning of the Drosophila embryo

David J. Sutherland^*, Mingfa Li, Xiao-qing Liu, Raymund Stefancsik and Laurel A. Raftery

Cutaneous Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Building 149, 13th Street, Charlestown, MA 02129, USA

View larger version (39K): [in a new window]   Fig. 1. Anti-Medea antiserum is specific. (A) Anti-Medea detected two classes of embryos from a mating of females bearing germline clones of the null allele Med13 with Med13–/+ males. One class, zygotic heterozygotes (M–Z+), showed variable subcellular localization of the antigen, cytoplasmic in ventral and lateral regions, and nuclear in many dorsal cells. The other class lacked detectable staining, the zygotic null homozygotes (M–Z–). (B) Western analysis of larval extracts with affinity-purified anti-Medea antiserum, and actin as a control. Wild-type larvae (WT) and Mad mutant larvae (Mad10/Mad12) give a similar pattern of strongly staining bands of approximately 97 kDa (arrowhead in B-D), 55 kDa and 47 kDa. These bands were missing in protein extracts from Medea mutant larvae (Med1/Df Med). (C,D) Comparison of steady state Medea levels in embryo extracts using Western blot analysis, with tubulin as a control. (C) WT versus embryos from cactusPD74 mothers (cact), to assess the contribution of dorsal tissues to total Medea levels. Total Medea is not decreased when dorsal tissues are absent. (D) WT versus globally increased BMP signaling through either constitutively active (CA) receptor, Sax (nos>Gal4;UAS>saxA) or Tkv (nos>Gal4; UAS>tkvA). Steady state Medea levels are unperturbed by hyperactivation of either BMP receptor.

View larger version (62K): [in a new window]   Fig. 2. Levels of Medea in dorsal nuclei increase sharply at the onset of gastrulation. Optical sections of wild-type (WT) embryos co-stained for Medea (green) and with the DNA dye ToPro3 (red). Dorsal (D) and ventral (V) midline cells from a single embryo are shown as paired images for each stage, with Medea staining alone (top pair) and merged with DNA dye (bottom pair). (A) Most stage 5 embryos have a subcellular distribution of Medea that is indistinguishable between dorsal and ventral cells, but a few (B) show uniform distribution of Medea between the nuclei and cytoplasm within a broad domain of dorsal cells. (C) At the onset of gastrulation, all embryos show strong nuclear accumulation of Medea within a narrow stripe of cells at the dorsal midline.

View larger version (108K): [in a new window]   Fig. 3. Dorsal-midline stripe of SMAD responses expands and intensifies during early gastrulation. (A-F) Confocal projections of wild-type (WT) embryos stained for Medea (A,C,E), or PMAD (B,D,F). (B-G) Dorsal views or slightly rotated. (A,H) Side views, or slightly rotated. (A,B) Strong nuclear SMAD accumulation was co-incident with initial cellular changes in the cephalic furrow. (C-F) Levels of nuclear staining for both antigens intensified during gastrulation and early germband extension, and the midline stripe widened. (E,F) Response levels dropped dramatically over 2-3 cells at the edges of the stripe. (G) Medea nuclear localization was lost in an embryo lacking WT Mad (M-Z-Mad), and induced globally in embryos expressing constitutively activated TKV under the control of nos>Gal4 (H). Note that somatic cells stain more intensely than posterior germ cell primordia, and that this embryo is at stage 5. Anterior is leftwards.

View larger version (121K): [in a new window]   Fig. 4. Low nuclear Medea accumulates in dorsolateral cells during late gastrulation. (A) Confocal projection of Medea staining in a stage 8 embryo, with regions of high magnification, single optical sections (B-D) indicated with yellow boxes. Medea immunofluorescence in white (A) or green (B, B',C,C',D,D') and Topro3 DNA stain in red (B',C' and D'). Nuclei stained with Topro3 are diffuse red with bright spots of condensed chromatin. (A). At the midline, nuclei have higher levels of Medea than the cytoplasm (B,B'). Medea is largely excluded from nuclei in ventral ectoderm cells (C,C'; dorsal here because of germband extension). In dorsolateral cells there is an even distribution of Medea between the cytoplasm and the nucleus. This shoulder of low signal is absent anterior to the cephalic furrow. B-D were collected at the same gain. Anterior is leftwards.

View larger version (93K): [in a new window]   Fig. 5. Division 14 mitotic domains are positioned relative to threshold levels of SMAD responses. Cephalic regions of embryos stained for Medea (A,C) or PMAD (B,D,E,F) (both green), and ToPro3 (red), dorsal views except C. ToPro3 DNA dye intensely stains highly condensed mitotic chromosomes. One of each paired domain is outlined in white. (A,B) The dorsal edges of mitotic domains 141 (1) and 145 (5) abut the lateral edges of the midline stripe of high-level SMAD response, either nuclear Medea (A) or PMAD (B). Mitotic domain 143 (3) has the same width as the stripe. (C-E) These domains retain their position relative to the altered stripe in embryos with altered dpp gene dosage. (C) Domains 141 and 145 still abut the narrow, weak Medea stripe in dppH46/+ embryos (1Xdpp). (D) Each pair of domains 141 and 145 is wider apart, but each domain still abuts the broad PMAD stripe in Dp(2;2)DTD48/Dp(2;2)DTD48 embryos (4Xdpp). Domain 143 fully spans the broader stripe. (E) Domains 141 and 145 fuse at the midline in dppH46/dppH46 embryos (dpp null), PMAD staining and 143 are lost. (F) PMAD staining is more intense in embryos expressing high levels of Medea from a transgene (ubi>Medea), but the mitotic domains still abut the stripe. Anterior is leftwards.

View larger version (144K): [in a new window]   Fig. 6. Both BMP ligands are required for nuclear SMAD responses. Confocal projections of stage 6/7 embryos stained for Medea (A,A',C,C') or PMAD (B,B',D,D'), dorsolateral views except C',D' are dorsal. Mutants at left (A-D), with gain-matched wild-type (WT) controls at right (A'-D'). For each mutant-WT pair (e.g. A and A'), contrast was manipulated coordinately; contrast and confocal gain differ between different pairs. (A,B) In dppH46 embryos, neither Medea nor PMAD is detectable in nuclei in any region. (C) In scws12 embryos, Medea does not accumulate in nuclei, although apical staining is slightly more intense in approximately the dorsal 40% of the embryo. (D) PMAD levels are at background in somatic cells of scws12 mutants. Anterior is leftwards.

View larger version (71K): [in a new window]   Fig. 7. The final level of SMAD response is sensitive to dpp gene dosage. Confocal projections of embryos stained for Medea (E,F) or PMAD (A-D). 2Xdpp embryos are wild-type (WT) controls, 1Xdpp and 3Xdpp are dppH46/dpp+ and dpp+ P{dppH+}/dpp+, respectively. (A) Stage 7, 3Xdpp embryos show an expanded dorsal region of PMAD staining, which broadens in the trunk region as amnioserosa cells flatten. (B) Stage 7 1Xdpp embryos have weaker PMAD staining. Stage 8 1Xdpp embryos (D,F) have substantially weaker SMAD responses than WT. (D) Stage 8, 1Xdpp embryo with undetectable PMAD staining and partially ventralized phenotype. (E) In stage 9 WT has strong nuclear Medea accumulation in the amnioserosa and dorsal-midline of cephalic region. (F) Stage 9 1Xdpp embryos often have a weak stripe of nuclear Medea accumulation in the cephalic region and variable but low levels in amnioserosa nuclei.

View larger version (56K): [in a new window]   Fig. 8. SOG reduces the width and increases the level of SMAD response. Embryos from sog heterozygous parents stained for Medea (A,C,E) or PMAD (B,D,F), here shown for sogU2 (C-F) compared with wild type (A,B). (C,D) One aberrant class, inferred to be sogU2/+, has a variably broader dorsal stripe of nuclear SMAD responses. (E,F) The other aberrant class, inferred to be sogU2 hemizygous males, has a low-level response over the dorsal half of the embryo. A region of cells with predominantly nuclear Medea is indicated (E, arrow). Anterior is leftwards.

View larger version (45K): [in a new window]   Fig. 9. Patterns of SMAD responses reveal stepwise changes in BMP activity. (A) When nuclear Medea was detected during stage 5, it was at uniformly low levels across about 24 dorsal nuclei, and then declined across four nuclei at each edge. (B) At the beginning of stage 6, a narrow stripe of more intense nuclear Medea staining was detected at the dorsal midline. Nuclear Medea was no longer detected in dorsolateral regions, even though staining levels rose at the dorsal midline. (C) Levels of nuclear Medea peaked in dorsal midline cells during stage 7, and adjacent domains of low nuclear Medea became detectable on either side. At this stage, the entire nuclear Medea response domain was not as wide as the initial response domain during stage 5. (D) Stage 6 sog hemizygous embryos (no Sog) had a broad domain with low levels of nuclear Medea and PMAD. Levels were higher than wild-type stage 5 embryos, but did not reach the peak levels seen in wild type. (E) Heterozygous dpp embryos (1X dpp) formed a narrow dorsal midline stripe, but the levels of nuclear Medea and PMAD did not reach the peak levels seen in wild type. During stages 7 and 8, a narrower stripe was sometimes evident in the cephalic region of these embryos.

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