QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

doi: 10.1242/10.1242/dev.00390

This Article Summary Full Text Full Text (PDF) Supplemental Data Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Akiyama-Oda, Y. Articles by Oda, H. Search for Related Content PubMed PubMed Citation Articles by Akiyama-Oda, Y. Articles by Oda, H.

Early patterning of the spider embryo: a cluster of mesenchymal cells at the cumulus produces Dpp signals received by germ disc epithelial cells

Yasuko Akiyama-Oda^*,, and Hiroki Oda^*

JT Biohistory Research Hall, 1-1, Murasaki-cho, Takatsuki, Osaka 569-1125, Japan
^* Tsukita Cell Axis Project, ERATO, JST
PRESTO, JST

View larger version (82K): [in a new window]   Fig. 1. Stages of early embryogenesis of the spider, Achaearanea tepidariorum. Photographs of living embryos at different stages are presented. These embryos were viewed from different angles to show morphologies characteristic of the given stages. Each embryo is schematically illustrated in the lower row. Asterisks indicate the corresponding site of the different stage embryos. White areas in the illustrations indicate yolk. Developmental schedule (at 25°C) is as follows: stage 1, 0-10 hours after egg laying (AEL); stage 2, 10-15 hours AEL; stage 3, 15-25 hours AEL; stage 4, 25-30 hours AEL; stage 5, 30-40 hours AEL; stage 6, 40-45 hours AEL; stage 7, 45-55 hours AEL; stage 8, 55-65 hours AEL. (A) Stage 1 embryo (about 5 hours AEL). Nuclear divisions occur deep in the egg. (B) Early stage 2 embryo (about 11 hours AEL). (C) Late stage 2 embryo (about 15 hours AEL). The energids reach the periphery of the egg, and undergo synchronous cleavages. (D) Stage 3 embryo (about 20 hours AEL). The energids shift toward one pole of the egg along the surface. (E) Stage 4 embryo (about 26 hours AEL). The energids settle and form a germ disc (large shaded circle). The primary thickening (small dark circle) is seen at the center of the germ disc. (F) Early stage 5 embryo (about 32 hours AEL). (G) Late stage 5 embryo (about 36 hours AEL). The cumulus appears from the center of the germ disc and moves straight to the rim (arrow). (H) Stage 6 embryo (about 42 hours AEL). The cumulus disappears, and the germ disc cells are rearranged migrating circumferentially (arrows). (I) Stage 7 embryo (about 50 hours AEL). The previous central region of the germ disc develops into the caudal lobe. Segmentation begins. Metameric morphologies are recognizable. The shape of the embryo is fan-like. (J) Stage 8 embryo (about 60 hours AEL). The germ band is formed, which elongates along the AP axis producing additional segments from the caudal lobe. a, anterior; p, posterior. Scale bar: 200 µm.

View larger version (160K): [in a new window]   Fig. 2. Morphology of the cumulus. The direction of the cumulus movement is shown by the largest arrows (A,B,D-F). (A) SEM image showing the surface view of a cumulus. The epithelium is bulged at the cumulus. (B) SEM image showing the inside view of a cumulus. Asterisks indicate CM cells, which extend lamellipodia-like processes (thick arrows). Note that germ disc epithelial cells extend thin, long cytoplasmic projections like cytonemes (thin arrows) from their basal side onto the surface of the CM cells. A single epithelial cell has a single projection. (C) SEM image showing the inside view of germ disc epithelial cells far from the cumulus in the same embryo as in B. No cytoneme-like projections are seen. (D,E) Laser scanning microscopy (LSM) images of a cumulus stained with phalloidin (green) and TOTO-3 (purple). A series of optical sections separated by intervals of 0.52 µm were obtained to cover the epithelial and mesenchymal layers of the cumulus. D and E were constructed by overlaying 16 and nine successive optical sections, respectively, selected from the data set. D focuses on the CM cells, and E on the interface between the CM cells and the germ disc epithelial cells. Lamellipodia-like processes abundant with F-actin ingress into the spaces between the lateral surfaces of the epithelial cells as indicated by arrows (a and b). (F) LSM image showing a sagittal section of a cumulus stained with phalloidin-fluorescein. Cytoplasmic projections extend from the basal side of the epithelial cells are seen (arrows). Scale bar: 10 µm in A-C; 20 µm in D-F.

View larger version (105K): [in a new window]   Fig. 3. Characterization of amino acid sequences deduced from spider cDNA clones, At.dpp, At.fkh, At.otd and At.cad. Conserved amino acid residues in alignments are highlighted. Gaps introduced to optimize the alignments are indicated by dashes (A,D). (A) Amino acid sequence of the C-terminal domain of At.Dpp aligned with known Dpp/BMP2/4 family proteins and Drosophila 60A and Screw proteins. (Database Accession Numbers: Dm.Dpp, U63857; Tc.Dpp, U63132; Sg.Dpp, AF374725; Bf.BMP2/4, AF068750; Hr.BMPb, D85464; DrBMP-2, AF072456; DrBMP-4, D49972; Dm.60A, M84795; Dm.Screw, U17573.) (B) Amino acid sequence of the winged-helix (forkhead) domain of At.Fkh aligned with known Fkh/HNF-3 family proteins and Drosophila FD1 and FD3-FD5 proteins. (Database Accession Numbers: Dm.Fkh, J03177; Tc.Fkh, AF217810; Bf.HNF3-,1 X96519; Hr.HNF-3, AB007406; DrAxial, Z22762; Dm.FD1 and FD3-FD5, M96440 and M96442-M96444.) (C) Amino acid sequence of the homeodomain and C-terminal hexapeptide of At.Otd aligned with known Otd/Otx family proteins and Drosophila Otp, Prd and Gsb proteins. Dashes indicate the varied numbers of amino acid residues omitted. (Database Accession Numbers: Dm.Otd, X58983; Tc.Otd1 and 2, AJ223627 and AJ223614; Bf.Otx, AF043740; Ci.Otx, AF305499; DrOtx2, D26173; Dm.Otp, NM-079075; Dm.Prd, M14548; Dm.Gsb, NM-079139.) (D) Amino acid sequence of the homeodomain of At.Cad aligned with known Cad/Cdx family proteins and Drosophila Dfd, Scr and Antp proteins. (Database Accession Numbers: Dm.Cad, NM-134301; Bm.Cad, D16683; Tc.CadA and B AJ00542 and AJ005422; DrCad1, X66958, Dm.Dfd, X05136; Dm.Scr, X14475; Dm.Antp, M20704.) Abbreviated species are as follows: Dm, Drosophila melanogaster; Tc, Tribolium castaneum; Sg, Schistocerca gregaria; Bm, Bombyx mori; Bf, Branchiostoma floridae; Hr, Halocynthia roretzi; Ci, Ciona intestinalis; Dr, Danio rerio.

View larger version (66K): [in a new window]   Fig. 4. Expression of At.dpp transcripts in stage 5 embryos revealed by whole-mount in situ hybridization. (A,B) Early stage 5 embryo viewed from the top (A) and the lateral side (B) of the germ disc. The signal is seen as a spot at the center of the germ disc. The rim of the germ disc is marked by dots in B as well as in C and D. (C) Mid stage 5 embryo. A spot of signal is seen at an intermediate position between the center and the rim of the germ disc. (D) Late stage 5 embryo. A spot of signal is seen close to the rim of the germ disc. The spots of dpp expression correspond to the positions of the cumulus in the embryos. (E,F) Close-up of the cumulus in a stage 5 embryo. E focuses on the surface epithelial cell layer and F on the CM cells. The signal is seen in the CM cells (F) but not the surface epithelial cells (E). (G,H) Embryos at about 65 hours (G) and 75 hours (H) AEL. The strong signal of At.dpp is seen in developing limb buds (G) and the tip of the extending limbs (H). The ventral midline is weakly stained (H). Ch, chelicerae; Pp, pedipalp; L1-4, leg segments 1-4.

View larger version (149K): [in a new window]   Fig. 5. Expression of pMad in stage 5 embryos. Embryos were stained with the PS1 antibody. (A) Mid stage 5 embryo viewed from the top of the germ disc. (B) Close-up of the same embryo as in A, flat-mounted. Nuclei of germ disc epithelial cells, but not CM cells, at and around the cumulus are stained. Note that the levels of signal are graded in a concentric manner. (C,D) Mid stage 5 embryo double-labeled for pMad (brown) and At.dpp transcripts (purple). C focuses on the surface epithelial cell layer and D on the CM cells. Note that the highest levels of pMad are observed just above the CM cells, expressing At.dpp transcripts.

View larger version (70K): [in a new window]   Fig. 6. Expression of At.fkh transcripts revealed by whole-mount in situ hybridization. (A) Stage 4 embryo viewed from the top of the germ disc. fkh transcripts are found around the blastopore (white arrow). (B) Early stage 5 embryo. Expression of fkh transcripts are seen in cells dispersing from the center of the germ disc and in cells encircling the germ disc. (C,D) Mid-stage 5 embryo flat mounted. (D) High magnification of the boxed area in C, focused on the mesenchymal cell layer. The At.fkh-positive cells are mesenchymal cells located below the surface epithelial cell layer. (E,F) Late stage 5 embryo. The cumulus is magnified in F. In addition to the dispersing mesenchymal cells and the peripheral cells, expression of fkh transcripts is observed in epithelial cells at the cumulus (arrow in E). (G,H) Stage 6 (G) and 7 (H) embryos. Expression of fkh transcripts is observed in the emerging dorsal region of the embryos (arrows), where the At.fkh-positive cells appear to spread over the yolk. The peripheral At.fkh-positive cells appear to migrate circumferentially to the emerging anterior region of the embryo (H, a). a, anterior; p, posterior; v, ventral.

View larger version (52K): [in a new window]   Fig. 7. Expression of At.otd and At.cad transcripts. Embryos stained by whole-mount in situ hybridization with the At.otd probe (A-D), At.otd and At.dpp mixed probes (E,F) or At.cad probe (G-J). (A,B) Stage 5 embryo viewed from the top (A) and the lateral side (B) of the germ disc. Expression of At.otd transcripts is observed in peripheral cells of the germ disc epithelium, three or four cells wide. (C) Stage 6 embryo viewed from the top of the germ disc. The pattern of At.otd expression is changed to an `open' circle. (D) Stage 7 embryo viewed laterally. Expression of At.otd is seen at the emerging anterior region of the embryo. (E,F) Late stage 5 (E) and early stage 6 (F) embryos. The pattern of the At.otd-positive cells becomes the `open' circle at almost the same time when the CM cells expressing At.dpp (arrowheads) reach the rim of the germ disc. (G,H) Stage 7 embryos viewed posteriorly (G) and laterally (H). Expression of At.cad transcripts is found at the most posterior region of the embryo corresponding to the caudal lobe. (I,J) High magnifications of the posterior end of a stage 7 embryo flat-mounted. I focuses on the surface of the embryo, and J on the inside. A pit is found at the center of the At.cad-expressing region (arrow in I). Below the pit, there is a population of cells expressing At.cad transcripts (arrow in J). Mesenchymal cells not expressing At.cad transcripts are also found. The margin of the forming germ band is indicated by dots in D and H. a, anterior; p, posterior; d, dorsal; v, ventral.

View larger version (22K): [in a new window]   Fig. 8. Illustrations showing different modes of axial patterning of the Drosophila and spider embryo. The Drosophila cellular blastoderm stage (A) and the spider germ disc stage (B) are compared. a, anterior; p, posterior; v, ventral; d, dorsal. In each illustration, the otd- or At.otd-expressing anterior region of the embryo is indicated by yellow, and the posterior end of the embryo by purple. The area shown in gray indicate yolk (B). (A) In the Drosophila embryo, dpp is expressed in blastoderm cells at the 40% most dorsal region (green). This expression initiates simultaneously along the AP axis. In the same cell population that dpp is expressed, phosphorylation of Mad is induced. (B) In the spider embryo, future anterior cells (yellow) are located at the rim, whereas future posterior cells (purple) that form the caudal lobe exist around the center of the germ disc. The CM cells, which express At.dpp (green), migrate from the center to the rim of the germ disc (black arrow), progressively inducing phosphorylation of Mad in epithelial cells (light green). The area that once expressed pMad is shaded in green (right panel). It develops into the dorsal tissues. The site diametrically opposite to the cumulus is defined as the anterior pole (a, right panel). Broken line indicates the ventral midline; blue arrows indicate the directions of cell movement during stage 6.