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First published online 16 May 2007
doi: 10.1242/dev.004598

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Progressive activation of Delta-Notch signaling from around the blastopore is required to set up a functional caudal lobe in the spider Achaearanea tepidariorum

¹ JT Biohistory Research Hall, 1-1 Murasaki-cho, Takatsuki, Osaka 569-1125, Japan.
² Genome Resource and Analysis Subunit, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
³ Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan.

View larger version (88K): [in this window] [in a new window]   Fig. 1. Internalization of distinct cell types during early stages of Achaearanea embryogenesis. (A) Early cell types defined in terms of gene expression and site of appearance. Note that the two cell types indicated by asterisks are indistinguishable unless site of appearance is considered. (B) Illustration of a late stage 4 embryo. (C-F) A flat-mounted late stage 4 germ disc stained for F-actin (C-E, green), At-Fkh (C-E, purple), At-ß-catenin (F, yellow) and DNA (F, blue). Boxed area in C is magnified in D-F (D, surface; E,F, inside). The pattern obtained by immunostaining for At-Fkh was the same as that obtained by in situ hybridization for its transcript at this stage (compare with Fig. 3D-F). Arrow in D, the closing blastopore; arrow in F, At-ß-catenin concentration. cEND cells are situated just beneath the blastopore. (G) Illustration of a mid-stage 5 embryo. (H-K') A flat-mounted mid-stage 5 germ disc stained for At-fkh transcripts (H-K) and DNA (I'-K'). The upper box in H corresponds to I,I' and the lower one to J,J',K,K'. (I,I') pEND cells are visible near the rim of the germ disc (arrows). (J,J',K,K') The focal plane is adjusted to the surface layer in J and J' and the inner layer in K and K'. cEND and CM (arrows) cells are visible. (L,L') High magnification of the equatorial region of a late stage 5 embryo stained for At-fkh (L, red) and At-twi (L, purple) transcripts and DNA (L'). The boundary between the germ disc and non-germ disc areas is seen. Arrowheads indicate pEND cells located below the surface layer, and arrows indicate probable pMES cell precursors about to internalize. (M) An illustration of an early stage 6 embryo. (N-Q') A flat-mounted early stage 6 germ disc stained for At-fkh (N-Q, red) and At-twi (N-Q, purple) transcripts and DNA (O'-Q'). The boxed area in N is magnified in O-Q,O'-Q'. The focal plane is adjusted to the surface layer in O and O', an intermediate level in P and P' and a one-cell deep level in Q and Q'. Arrows indicate cMES cell precursors, and arrowheads cEND cells. Asterisks mark the same positions in O' and P' to show that At-twi-positive cells are internalizing. (R,S) Illustrations of a late stage 6 embryo viewed from the lateral (R) or caudal (S) side. At-cad expression is shown in red. (T-W) A flat-mounted late stage 6 embryo stained for At-twi (T,U, purple) and At-cad (T,U, red) transcripts and DNA (V,W). The boxed area in T is magnified in U-W. The focal plane is adjusted to the surface layer in V and W, and a one-cell-deep level in W. Arrows indicate likely cMES cell precursors internalizing or about to internalize. Asterisks mark the same positions in U and V. Small open circles indicate pMES cells, and arrowheads cEND or pEND cells. a, anterior; bp, blastopore; cEND, central endoderm cells; cl, forming caudal lobe; CM, cumulus mesenchymal cells; cMES, central mesoderm cells; d, dorsal; ex, extraembryonic area; gd, germ disc; p, posterior; pEND, peripheral endoderm cells; pMES, peripheral mesoderm cells; v, ventral. Scale bars: 20 µm.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Predicted amino acid sequence of At-Delta cDNA in Achaearanea. (A) Domain organization of At-Delta protein. Two non-overlapping regions used to synthesize At-DeltaEB and At-DeltaHH dsRNAs are shown. (B) Alignment of the DSL domains of Delta and other proteins. Only residues not identical to those of At-Delta are displayed. (C) A neighbor-joining tree constructed using 156 amino acid sites in the DSL domain and EGF repeats 1-3. The numbers at the internal branches are bootstrap values (%). Accession numbers of proteins are as follows: At-Delta (AB287420), Cs-Delta1 (CAD45190), Cs-Delta2 (CAD45191), Dm-Delta (NP_477264), Sp-Delta (NP_001027542), Mm-Delta1 (NP_031891), Dr-DeltaD (NP_571030), Dm-Serrate (CAA40148), Mm-Jagged 1 (NP_038850). At, Achaearanea tepidariorum; Cs, Cupiennius salei; Dm, Drosophila melanogaster; Dr, Danio rerio; DSL, Delta/Serrate/LAG-2 domain; EGF, EGF repeat; Mm, Mus musculus; Sp, Strongylocentrotus purpuratus; SP, signal peptide; TM, transmembrane domain.

View larger version (97K): [in this window] [in a new window]   Fig. 3. Expression patterns of At-Delta transcripts in Achaearanea. (A-C) A flat-mounted mid-stage 4 germ disc stained for At-Delta transcripts (A,B) and DNA (C). The boxed area in A is magnified in B and C. Arrowheads indicate At-Delta-expressing cells near, but not at, the rim of the forming germ disc. (D-F) A flat-mounted late stage 4 embryo stained for At-fkh (red), At-Delta (purple) transcripts and DNA (not shown). Both the germ disc (gd) and non-germ disc (non-gd) areas are presented. Nuclei of non-germ disc cells show a sparse distribution. All nuclei of non-germ disc cells in D were confirmed to associate with the signals of At-Delta (data not shown). The boxed area in D is magnified in E (surface) and F (inside). The At-Delta-expressing cells are distinct from cEND cells. (G,H) A flat-mounted mid-stage 5 germ disc stained for At-fkh (red) and At-Delta (purple) transcripts and DNA. The boxed area in G is magnified in H, which is merged with DNA staining (H, light blue). Paired and unpaired At-Delta-positive cells are evenly spaced. (I-K) Flat-mounted early (I), mid-(J) and late (K) stage 6 embryos stained for At-Delta (purple) and At-cad (red) transcripts. The area displaying interspersed At-Delta expression expands followed by fading from the center and appearance of At-cad expression. At-cad expression is detectable from mid-stage 6. (L,M) A flat-mounted early stage 6 germ disc stained for At-twi (purple) and At-Delta (red) transcripts. The boxed area in L is magnified in M. Arrows point at cells expressing At-twi and At-Delta. (N) Schematic illustrations showing changing expression patterns of At-Delta, At-fkh, At-twi and At-cad transcripts up to caudal lobe formation. (O-Q) Stage 7 (O), stage 8 (P) and stage 9 (Q) embryos stained for At-Delta transcripts. Anterior is to the top. Arrows in O and P indicate a band of At-Delta expression in the anterior region. This anterior expression is visible in K. Note that regularly aligned spotty signals are visible in the central nervous system. cl, caudal lobe; ex, extra-embryonic area; op, opisthosoma; pr, prosoma. Scale bars: 20 µm for A-N; 100 µm for O-Q.

View larger version (61K): [in this window] [in a new window]   Fig. 4. Defects in caudal lobe formation following injection of At-Delta dsRNA. (A,B) Time-lapse recording of live embryos derived from Achaearanea females injected with gfp (A) or At-DeltaEB (B) dsRNA. The time after the start of observation (early stage 5) is shown at the bottom of each image (hour: minute). CM cells and the emerging caudal region are indicated by arrows and brackets, respectively. In B, an unusual invagination is seen at the emerging caudal region. (C-E) Timecourse of phenotype expression in caudal lobe after dsRNA injection. Each graph refers to one female injected with gfp (C), At-DeltaEB (D) or At-DeltaHH (E) dsRNA. #1-8 are identification numbers of individual females. Each vertical bar indicates relative numbers of embryos exhibiting normal (blue), invagination (red), and non-specific (yellow) phenotypes in each egg sac. The number at the top of each bar indicates the total number of embryos examined. Not all egg sacs produced by the females are presented. Unfertilized eggs and embryos that did not form morphologically normal germ disc were excluded from analysis. `Non-specific' phenotypes, which are often observed even in embryos derived from untreated females, include shrinking, thickening and destruction of germ disc. The asterisks in #1/gfp, #3/At-DeltaEB and #7/At-DeltaHH indicate egg sacs used for Fig. 5. Scale bar: 100 µm. ex, extra-embryonic area.

View larger version (79K): [in this window] [in a new window]   Fig. 5. Abnormal germ-band configurations following injection of At-Delta dsRNA in Achaearanea. (A-D) Flat preparations of a normal limb-extending embryo derived from #1/gfp (A) and embryos from #7/At-DeltaHH (B-D). All embryos in B-D were fixed at the same time. Prosomal regions of germ bands in A and B display similar shapes. Phenotypes of germ bands resulting from injection of At-Delta dsRNA are categorized as class 1 (B), class 2 (C) and class 3 (D). See text for details. (E) Relative numbers of embryos exhibiting normal, class 1, class 2 and class 3 phenotypes in the egg sacs indicated (see also Fig. 4). Data indicated by #9/At-DeltaHH were collected from an egg sac produced by another female 30 days after the first At-DeltaHH dsRNA injection. Scale bar: 100 µm.

View larger version (93K): [in this window] [in a new window]   Fig. 6. Expression of At-Delta is affected after injection of At-Delta dsRNA in Achaearanea. Expression of At-Delta transcripts in germ discs derived from females injected with gfp (A,B), At-DeltaEB (C,D) or At-DeltaHH (E,F) dsRNA. A smear, not interspersed, pattern of At-Delta expression is seen in C and E. Central areas of germ discs in A,C and E are magnified in B,D and F, respectively. Arrows point at paired dots of nuclear signals implying that the transcription is active. Asterisks indicate the position of cumulus. Scale bars: 20 µm.

View larger version (131K): [in this window] [in a new window]   Fig. 7. Altered gene expression in At-Delta RNAi embryos in Achaearanea. (A-D) Staining for At-fkh transcripts. (A,B) Untreated (A) and At-DeltaHH RNAi (B) germ discs at stage 5. CM cells are indicated by arrows. The number of At-fkh-expressing cells at the periphery of the At-DeltaHH RNAi germ disc is largely reduced. (C,D) gfp (C) and At-DeltaEB (D) RNAi germ discs at stage 6. At-fkh-expressing cells are seen in central and peripheral areas. The dark areas indicated by arrows in D are due to thick folds of the cell sheet, not significant signals. (E-H) gfp (E,G) and At-DeltaEB (F,H) RNAi embryos at late stage 6 stained for At-twi (E,F) or At-cad (G,H) transcripts. The emerging caudal region is indicated by brackets in C-H. In At-DeltaEB RNAi embryos, At-twi-expressing cells were overproduced at the expense of At-cad-expressing cells. (I-N) gfp (I,K,M) and At-DeltaEB (J,L,N) RNAi embryos at early germ band stage (stage 8) stained for At-twi (I,J) or At-hh (K,L) transcripts, or for cell death (M,N). The caudal region of At-DeltaEB RNAi germ bands was grossly affected. In K and L, dots indicate segments corresponding to the four pairs of walking legs. In M and N, arrowheads indicate the anterior and posterior ends of the germ band. Anterior is to the top in C-N. Scale bars: 100 µm.

View larger version (73K): [in this window] [in a new window]   Fig. 8. Phenotypes of At-Notch and At-Su(H) RNAi embryos are similar to those of At-Delta RNAi embryos. (A,C,E,G) At-Notch RNAi embryos. (B,D,F,H) At-Su(H) RNAi embryos. (A,B) Live embryos at late stage 6. At the central area of each germ disc, an invagination such as seen in At-Delta RNAi embryos is formed (brackets). (C,D) Stage 5 germ discs stained for At-Delta transcripts; compare with Fig. 6A,C,E. The evenly spaced pattern of central At-Delta expression was partially disrupted in C, and almost completely in D. Asterisks indicate the position of cumulus. (E-H) Embryos at late stage 6 stained for At-twi (E,F) or At-cad (G,H) transcripts; compare with Fig. 7E-H. The emerging caudal region of each embryo is indicated by brackets. In E, but not in F, At-twi-expressing cells are seen at the peripheral areas. In G and H, only small patches of At-cad expression are visible. ex, extra-embryonic area. Scale bars: 100 µm.