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First published online May 23, 2006
doi: 10.1242/10.1242/dev.02400

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Axis specification in the spider embryo: dpp is required for radial-to-axial symmetry transformation and sog for ventral patterning

¹ JT Biohistory Research Hall, 1-1 Murasaki-cho, Takatsuki, Osaka 569-1125, Japan.
² PRESTO, Japan Science and Technology Agency, Saitama, Japan.

View larger version (28K): [in a new window]   Fig. 1. The deduced amino acid sequences of At-sog, Pp-sog and Af-sog display high similarity to Drosophila Sog and vertebrate chordin. (A) HarrPlot analysis of the entire amino acid sequences of At-Sog and Drosophila Sog (Dm-Sog). The parameter `unit size to compare' was 8 and the parameter `dot plot scores' was 2. The four cysteine-rich domains characteristic of Sog/chordin (CRs 1-4) are indicated by black bars. (B) Amino acid sequence comparisons of At-Sog and the Sog/chordin protein of Pholcus (Pp), Artemia (Af), Anopheles (Ag), Drosophila (Dm) and Xenopus (Xl). Percentage identity and similarity (parentheses) were calculated for each of the CR1-4 domains and the region intervening between the CR1 and CR2 domains (INT) using the BLAST2 sequences tool. Accession numbers of the proteins used are as follows: Ag-Sog, EAA06317.3; Dm-Sog, AAA89117.1; Xl-chordin, AAC42222.

View larger version (91K): [in a new window]   Fig. 2. Changes in the At-sog expression pattern. (A-F) At-sog expression is shown in embryos at mid-stage 5 (A) and stage 6 (B) viewed from the top of the germ disc, in an early stage 7 embryo viewed from the caudal (C) and lateral (D) sides, in a stage 7 embryo viewed from the lateral side (E), and in a stage 8 embryo viewed from the ventrolateral side (F). (A'-F') Schematic illustrations of the embryos shown in A-F. At-sog expression is shown in purple, yolk mass in yellow, the extra-embryonic area in orange, and the At-sog negative embryonic area in gray. Asterisks indicate the posterior of each embryo. Arrows in A and A' indicate the cumulus. Expansion of the extra-embryonic area and convergent extension of the embryonic area (D',E', arrows) transform the germ disc into the germ band. (G) A flat-mounted, stage 9 embryo stained for At-sog. (H,I) A transverse section of a stage 9 embryo stained for At-sog (H) and DNA (I). Arrowheads indicate limb buds, and brackets indicate the ventral midline area. (J) A flat-mounted, stage 5 germ disc stained for At-sog (purple) and pMad (brown). (J') High magnification of the region boxed in J. (K) Lateral view of a stage 7 embryo stained for pMad. The bracket indicates the extra-embryonic area, and the arrow indicates the boundary between the extra-embryonic and embryonic areas. (L) A flat-mounted, stage 8 embryo stained for At-sog (purple) and pMad (brown). (L') High magnification of the region boxed in L. (M,N) Flat-mounted, stage 9 embryos stained for At-sog (M, N, red) and At-fkh (M, purple) or At-sim (N, purple). (O,P) Stage 7 (O) and stage 8 (P) embryos stained for At-sog (red) and At-otd (purple). (Q,R,S,S') Expression of Pp-sog (Q,R) and Af-sog (S,S') transcripts. A ventral view of a Pholcus embryo at the germ band stage (Q). The tail part of a late-stage embryo (R). A ventral view of an Artemia nauplius (S). The region boxed in S is magnified in S'. a, anterior; p, posterior; d, dorsal; v, ventral; Ch, chelicerae; Pp, pedipalps; L1-L4, first to fourth walking legs. If not specified, anterior is to the top. Scale bars: 100 µm.

View larger version (89K): [in a new window]   Fig. 3. Expression of At-sog, At-fkh and At-sim transcripts in embryos at late stage 9 (A,C,E) and stage 10 (B,D,F). (A,B) Embryos stained for At-sog. At-sog expression at the ventral midline area is lost from the medial part (A), splitting into two lines (B). (C,D) Embryos double-stained for At-sog (red) and At-fkh (purple). At-fkh expression is detected at the most medial part, complementary to At-sog expression (C). The At-fkh expression is mostly lost at the ventral midline area, but starts in a pair of cell clusters in every hemisegment (D). Note that the clusters are included in the lines of At-sog. Arrows indicate the expression at the stomodeum. (E,F) Embryos double-stained for At-sog (red) and At-sim (purple). At-sim expression is observed at the most medial part, similar to At-fkh expression (E), then it fades, leaving a pattern different from those of At-sog and At-fkh (F). All embryos were flat-mounted. Anterior is to the top. Ch, chelicerae; Pp, pedipalps; L1 and L2, first and second walking legs. Scale bar: 100 µm.

View larger version (73K): [in a new window]   Fig. 4. Depletion of At-sog by dsRNA injection results in unseparated limb buds. (A-C) Flat preparations of DNA-stained stage 9 embryos derived from females injected with At-sog dsRNA (A,B) or gfp dsRNA (C). The limb bud defects were classified as severe (A) and mild (B) (see Materials and methods). (D,E) Comparison of At-sog expression by whole-mount in situ hybridization in stage 9 embryos derived from females injected with At-sog (D) or gfp (E) dsRNA. (F-P) Time course of phenotype expression in limb buds following dsRNA injection. Each graph refers to one female injected with At-sog (F-M) or gfp (N-P) dsRNA. Each vertical bar indicates the relative numbers of embryos exhibiting severe (red), mild (yellow), and normal (blue) phenotype in each egg sac. Unfertilized eggs or embryos exhibiting non-specific defects before germ band formation were not considered. The day when the first dsRNA injection was performed was defined as day 0. The numbers in parentheses indicate how many times dsRNA injection was performed. Asterisks indicate unhealthy egg sacs (see Materials and methods). Eggs from the egg sacs labeled D,E,Q in graphs F and N were used for the experiments shown in D,E,Q, respectively. (Q) RT-PCR comparison of the levels of At-sog, At-sim, ef1 and histone H3 transcripts in At-sog RNAi (sog Ri) and untreated (u.t.) embryos at stage 9. `+' and `-' indicate reactions with and without reverse transcriptase, respectively. Note that faint bands of At-sog and At-sim transcripts are visible in the `+' lanes of the At-sog RNAi sample. Ch, chelicerae; Pp, pedipalps; L1-L4, first to fourth walking legs; sog Ri, At-sog RNAi, gfp Ri, gfp RNAi. Scale bars: 100 µm.

View larger version (102K): [in a new window]   Fig. 5. The germ bands are dorsalized in sog RNAi embryos. (A) An At-sog RNAi embryo at stage 8 stained for DNA and At-otd transcripts. (B,C) Ventral views of untreated (B) and At-sog RNAi-treated (C) stage 8 embryos stained for pMad. (D-F) Flat preparations of untreated (D) and At-sog RNAi (E,F) stage 9 embryos stained for At-sog (red) and At-sim (purple). The arrow in E indicates the L1 segment, in which a patch of At-sog and At-sim expression intervenes between separated limb buds. (G-I) Flat preparations of untreated (G) and At-sog RNAi (H,I) late stage 9 embryos stained for At-sog (red) and At-pros (purple). Arrows in G indicate dorsal-most neural cell clusters. (J-Q) Prosomal (J,L,N,P) and opisthosomal (K,M,O,Q) regions of untreated (J,K,N,O) and At-sog RNAi (L,M,P,Q) stage 9 embryos stained for At-omb (J-M) or At-twi (N-Q) transcripts. a, anterior; p, posterior; Ch, chelicerae; Pp, pedipalps; L1-L4, first to fourth walking legs; sog Ri, At-sog RNAi. If not specified, anterior is to the top. Scale bars: 100 µm.

View larger version (90K): [in a new window]   Fig. 6. The At-dpp RNAi embryos exhibit defects in formation of the extra-embryonic area and germ band. (A-E,A'-E') Time course of a developing At-dpp RNAi embryo. (F-H,F'-H') Time course of a developing normal embryo. Images were taken at the time points indicated (day: hour: minute). For both embryos, time point 0:00:00 is adjusted to mid-stage 5, when the cumulus (arrows) is midway through shifting. The images in A-E, F-H show posterior views (or the top of the germ disc), and those in A',B',F'-H' show lateral views with the posterior to the bottom. The DV orientation of the embryo shown in C'-E' could not be determined, but the posterior side is to the bottom. Arrows in A,A',B,B',F,F' indicate the cumulus, and arrowheads in G,G',H indicate the expanding extra-embryonic area (ex) in the normal embryo. Since the cells at the extra-embryonic area are very thin, the yolk mass is seen through them. By contrast, the embryonic area or the germ band (gb) is seen as opaque (or white) material. Note that the extra-embryonic area is not formed in the At-dpp RNAi embryo (C-E,C'-E'). (I) RT-PCR comparison of the levels of At-dpp, At-sog, ef1 and histone H3 transcripts in At-dpp RNAi (dpp Ri) and untreated (u.t.) embryos at stage 5. `+' and `-' indicate reactions with and without reverse transcriptase, respectively. Note that a faint band of At-dpp transcripts is visible in the `+' lane of the At-dpp RNAi sample. Scale bar: 100 µm.

View larger version (51K): [in a new window]   Fig. 7. At-dpp RNAi embryos fail to break the radial symmetry. (A-C) DNA staining of untreated (A) and At-dpp RNAi (B,C) embryos at stage 8. All these embryos were also stained for At-otd transcripts. (D-R) Expression of At-en (D-F), At-otd (G-I), At-caudal (J-L), At-sog (M-O), and At-twi (P-R) transcripts in untreated (D,G,J,M,P) and At-dpp RNAi (E,F,H,I,K,L,N,O,Q,R) embryos at stage 9 (D-F,M-R) or stage 8 (G-L). D,G,J and P are lateral views and M is a ventral view; F,I,L,O,R are viewed from the directions indicated by arrows in E,H,K,N,Q. (S,S1,S2) Expression of At-pros transcripts in untreated (left in S) and At-dpp RNAi (right in S) late stage 9 embryos. The two boxed regions in S are magnified in S1 and S2. a, anterior; p, posterior; dpp Ri, At-dpp RNAi. Scale bars: 100 µm.

View larger version (27K): [in a new window]   Fig. 8. Schematic representation summarizing developmental events in the normal Achaearanea embryo and primary defects in the At-dpp and At-sog RNAi embryos. The state of the embryo symmetry is shown at the top. Time (hours; h) after egg laying at 25°C and the stages of development are also shown. Each developmental event takes place during the period indicated by the solid bar (faded areas indicate uncertain start or end points). In illustrations for the respective stages, the yolk area, the embryonic area, the extra-embryonic area and the limb buds are outlined. Stages 6 and 8 are shown in yellow and blue, respectively, when primary defects were observed in At-dpp RNAi and At-sog RNAi embryos (see the insides of the boxes). a, anterior; p, posterior; d, dorsal; v, ventral.