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Early embryo patterning in the grasshopper, Schistocerca gregaria: wingless, decapentaplegic and caudal expression

Peter K. Dearden^* and Michael Akam

Laboratory for Development and Evolution, University Museum of Zoology, Department of Zoology, Downing Street, Cambridge CB2 3EJ, UK
^* Present address: Zoology Department, University of Western Ontario, B&G Building, London, Ontario N6A 5B7, Canada

View larger version (87K): [in a new window]   Fig. 1. Analysis of the Schistocerca caudal encoded protein. Alignment of Sgcaudal against metazoan caudal proteins. Shading denotes similarity; boxes indicate identity. A.Ga, Anopheles gambiae; B.mori, Bombyx mori; D.mel, Drosophila melanogaster; G.do, Gallus domesticus caudal 1; M.mu, Mus musculus caudal 1; S.greg, Schistocerca gregaria (Sgcad); T.cast1 and T.cast2, Tribolium castaneum caudal proteins 1 and 2; X.Lae2, Xenopus laevis caudal 2.

View larger version (23K): [in a new window]   Fig. 2. RT-PCR analysis of Schistocerca caudal and engrailed expression. (A) Expression of Sgcaudal RNA in ovary and early development. Sgcaudal is expressed in the ovary, and all stages of embryogenesis shown. Timing is in hours. 1 Kb, calibration ladder; -RT, representative control reaction run without reverse transcriptase enzyme; +Cnt, positive control PCR from plasmid clone; Blank, reaction run with no template. Amounts of PCR product are not representative of amounts of Sgcaudal RNA in each stage. The lower band in the experimental lanes is a spurious product produced by using these primers on cDNA. Comparison with the positive control lane identifies the upper band as Sgcaudal transcript. (B) Expression of engrailed RNA at two stages of embryogenesis, 48 hours AEL (late heart-stage, 15% development) and 65 hours AEL. engrailed RNA is detected at 65 hours (arrow) but not 48 hours. –RT, control reaction run with no RT enzyme (on 65 hour RNA); 48 +ve, RT-PCR reaction performed on 48 hour RNA with caudal primers.

View larger version (101K): [in a new window]   Fig. 3. Expression of Sgcaudal during embryogenesis. (A) Ovariole hybridised for caudal RNA. caudal RNA is expressed strongly in the germarium and early vitellarium (left), but staining becomes weaker as oocytes mature. In the most mature eggs, caudal RNA is cortically located. (B) Close up of ovariole seen in A, caudal RNA can be seen in oocytes as they form next to the terminal filament. (C) Egg at 18 hours. caudal RNA is expressed in energids as they move to the posterior pole. Counter staining for DNA shows that all superficial energids express caudal at this stage (data not shown). (D) Expression of caudal RNA in late blastodisc embryo (36-38 hours APF; slightly damaged at posterior (bottom)); caudal expression is graded: low at the anterior, high at the posterior. (This embryo has not been treated with methanol after staining to ensure faint expression is detected, hence the pink colour.) (E) Expression of caudal RNA in early heart-stage embryo (38-40 hours APF). The gradient has a discontinuity forming a curved boundary between higher levels (posterior) and lower levels (anterior; arrowheads). Expression is stronger in the posterior around the terminus of the embryo. (This embryo has also not been treated with methanol.) (F) Expression of caudal at 15% development (48 hours). caudal RNA is restricted to a posterior domain at the end of the germ band. Dots demarcate the anterior boundary of the embryo. Star marks the stomodeum. (G-I) Expression of caudal at 18% (G), 22% (H) and 25% (I) of development. caudal RNA is present in a small posterior domain and absent from the rest of the embryo. (J,K) Expression of caudal at 30% development (J) and 40% (K). As segmentation finishes, caudal is expressed in the posterior of A10 and in A11. (L) Two focal planes of an enlargement of K showing A11 and the invaginating proctodeum. caudal RNA is absent from the invaginating regions of the proctodeum, and present only in the margins of the segment. Faint expression is also seen in a ring of cells at the anterior end of the invaginating hindgut (arrowhead), possibly the Malpighian tubule primordium. (M) Expression of wg RNA in A11 and the proctodeum at 40% development (two focal planes). wg is expressed in a ring around the proctodeal invagination, a pattern almost complementary to that of caudal. Scale bars: 100 µm.

View larger version (68K): [in a new window]   Fig. 4. Expression of dpp during embryogenesis. (A) Expression of dpp RNA in heart-stage (48 hours/15%). dpp RNA is present in a ring of cells that run around the embryo. Arrowheads mark three such cells. (B) Close up view of the specimen in A, stained with Hoechst 33342, to show the dpp staining cells. These are likely to be the necklace cells (Dearden et al., 2000). Arrowheads mark the same cells shown in A. (C) A similar stage to A, but with clearer expression in the U-shaped patch (arrowhead) around the posterior end of the gastrulation furrow (star). Diffuse expression is also seen in the head lobes (arrow). (D) Expression of dpp RNA in a 20% embryo. dpp RNA is located in two pairs of stripes located laterally in each appendage bearing segment, and in a single longitudinal row of cells at the lateral margin of the germ band in the gnathal and thoracic region (arrowheads). Scale bars: 100 µm.

View larger version (105K): [in a new window]   Fig. 5. Expression of wg during embryogenesis and comparison with other markers. (A-C) Expression of wg RNA at 44 hours (A), 48 hours (15%) (B), 50 hours (C). wg first appears in paired domains in the protocephalon, in a single band across the embryo (marking the parasegment 0/1 boundary), faintly in the mesoderm, and in a posterior domain (A). Three thoracic stripes, posterior to the initial one, appear soon afterwards (arrowheads in B). These stripes thicken, and two more stripes (gnathal) appear between them and the initial stripe (C). (D) Expression of wg at 17%. wg RNA is present in the head lobes and proctodeum, and in six stripes across the germ band, representing the gnathal and thoracic parasegment boundaries. Expression is just starting to appear in the antennal segment (arrowheads). (E) Expression of wg at 18%. wg RNA is just appearing in the A1 primordia (arrowheads). (F) Expression of wg (blue) and engrailed protein (brown) at 17% (same stage as D). Engrailed protein is detectable only in the pro- and mesothoracic regions (arrows). (G) wg RNA (blue) and engrailed protein (brown) in a 25% embryo. wg RNA is present in A1, A2 and A3. Engrailed expression has only reached A1 (arrow). (H) Expression of wg RNA (blue) and Hunchback protein (brown) in an early heart-stage embryo (44 hours). The cells expressing wg (marking parasegment 0/1) are the most anterior Hunchback-expressing cells. Hunchback protein is also expressed in the large nuclei of the serosa. (I) Close up of H. Note Hunchback and wg co-expressing cells (star). (J) wg RNA (blue) and Hunchback protein (brown) in a 48-hour embryo. The thoracic stripes of wg lie posterior to the Hunchback domain. (K) Close up of J. Note cells at the parasegment 0/1 boundary express wg and not Hunchback. (L) wg RNA (blue) and Hunchback protein (brown) in a 50 hour embryo. wg RNA is now present in two gnathal stripes inside the Hunchback domain. (M) Close up of L. Note gnathal stripes of wg inside the Hunchback domain (arrowheads). Scale bars: 100 µm.

View larger version (26K): [in a new window]   Fig. 6. Fate map of heart-stage embryos derived from the examination of Sgcaudal, wg and dpp expression patterns. The embryo is shown as a flat projection viewed from the position of the pole of the egg with the dorsal side of the egg (anterior of the embryo) uppermost. Stom., Stomodeum. (A) Early heart-stage embryo (38-40 hours APF). The position of the anterior edge of the high caudal-expressing domain is not precisely defined. It has been drawn as abutting the Hunchback domain, but may not. (B) Late heart-stage embryo (48 hours APF/15% development).