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Neurogenesis in the spider Cupiennius salei

Abteilung fuer Evolutionsgenetik, Institut fuer Genetik, Universitaet zu Koeln, Weyertal 121, 50931 Koeln, Germany

View larger version (72K): [in a new window]   Fig. 1. (A-C) Sequential formation of invagination sites in the ventral neuroectoderm of Cupiennius salei embryos stained with phalloidin-rhodamine. (A) Confocal micrograph of a flat preparation of an embryo 130 hours after egg laying. Limb buds have developed in the prosoma (ch, ped, l1 to l4). The first five to eight invagination sites (arrow) are visible in the prosomal hemisegments and the first opisthosomal hemisegment. No invagination can be seen in the remaining opisthosomal hemisegments. (B) Three additional segments have been generated by the posterior growth zone 175 hours after egg laying. Abdominal buds are visible on the second to fifth opisthosomal hemisegments (o2 to o5). New invagination sites are not only added posteriorly but also anteriorly (arrows). (C) Left half of the germ band of a 200 hour embryo. At this stage 9 opisthosomal segments are visible. 30 to 32 invagination sites can be detected in each hemisegment of the prosoma and the opisthosoma. Ch, cheliceral segment; ped, pedipalpal segment; l1 to l4 walking legs 1 to 4, corresponding to prosomal segments 3 to 6; o1 to o6 opisthosomal segments 1 to 6. Scale bars: 200 µm.

View larger version (106K): [in a new window]   Fig. 2. (A-D) Morphology of the invagination sites. (A) Confocal micrograph of an apical optical section of the ventral neuroectoderm between leg 2 and leg 3 of an 175 hour embryo stained with phalloidin-rhodamine. The arrows point to dots of high phalloidin staining. (B) Basal optical section of the same region of the ventral neuroectoderm shown in A. The arrows point to groups of basally enlarged cells that are located underneath the dots of high phalloidin staining. (C) Confocal micrograph of a transverse optical section through two invagination sites (arrows). The cell processes of the basally enlarged cells extend to the apical surface. (D) Light micrograph of a transverse section through an invagination site. This invagination site consists of approximately nine cells (asterisks). The cell processes extend to the apical surface (arrow). Scale bars: 40 µm in A,B; 20 µm in C,D.

View larger version (107K): [in a new window]   Fig. 3. (A,B) Stereotyped pattern of invagination sites. (A,B) Confocal micrographs of the opisthosoma of two different embryos stained with phalloidin-rhodamine. The invagination sites occupy the same positions in each hemisegment. The arrows point to two invagination sites that are located at the lateral anterior edge of each hemisegment. The arrowheads point to high phalloidin staining in the PNS. o1 to o5 opisthosomal segments 1 to 5. Scale bars: 200 µm.

View larger version (118K): [in a new window]   Fig. 4. (A-H) Confocal micrographs of the prosomal hemisegment corresponding to leg 2 (fourth prosomal segment) of embryos stained with phalloidin-rhodamine. Anterior is towards the top, the medial furrow towards the left. The white lines indicate the segmental borders. (A) No invagination sites can be detected up to 120 hours after egg laying. The limb buds are already visible. (B) At 130 hours, the first five to eight invagination sites arise in the anterior most lateral region of the prosomal and the first opisthosomal hemisegments (arrow). (C) At 150 hours, nine to 12 new invagination sites have formed posteriorly and medially to the anterior region, where the first invagination sites occurred (arrows). (D) At 175 hours, the next five to eight invagination sites are visible laterally and medially in the posterior region of the hemisegment (arrows). (E) At 190 hours, seven to ten invagination sites have been added between row three and four and at the posterior end of the hemisegment. (F) At 220 hours, the number of invagination sites decreases. (G) Confocal micrograph of a transverse section through the neuroectoderm of the fourth prosomal segment at 240 hours. Apical is at the bottom. At this time a growing neuropil can be detected basally (arrow). (H) Horizontal optical section through the neuropil of the fourth prosomal segment at 240 hours. The arrowhead points to the neuropil. The outgrowing axons of the invaginated neuroectodermal cells join the developing neuropil (arrow). In C-F, the legs were removed or put to the side. l2, walking leg 2 (corresponding to the fourth prosomal segment). Scale bars : 50 µm in A-F; 20 µm in G; 50 µm in H.

View larger version (170K): [in a new window]   Fig. 5. (A,B) Invaginating cells express HRP antigen. (A) Confocal micrograph of the opisthosoma of a 200 hour embryo stained with phalloidin-rhodamine. All 30 to 32 invagination sites are visible in the opisthosomal hemisegments. The arrows point to the anterior most lateral invagination sites of each hemisegment. (B) Light micrograph of the opisthosoma of a 200 hour embryo labeled with an antibody against HRP. The arrows indicate the anterior most lateral invagination sites of each hemisegment. The arrowhead points to a lateral axon fascicle. o1 to o5, opisthosomal segments 1 to 5. Scale bars: 150 µm.

View larger version (68K): [in a new window]   Fig. 6. (A-D) Comparison of invagination sites in Cupiennius salei and Pholcus phalangioides. Both embryos were stained with phalloidin-rhodamine. (A) Confocal micrograph of a flat preparation of a Cupiennius salei embryo at 200 hours. The ventral neuroectoderm has developed all 30 to 32 invagination sites. (B) Confocal micrograph of a flat preparation of a Pholcus phalangioides embryo. Despite the fact that the walking legs are longer than those of the Cupiennius embryo shown in A, the Pholcus embryo seems to be somewhat younger because the opisthosoma has not developed all invagination sites yet. (C) Invagination sites of the third opisthosomal segment of Cupiennius at 190 hours. There are six rows of invagination sites at this stage. The arrowheads point to the two anterior most lateral invagination sites. (D) In the third opisthosomal hemisegment of a Pholcus embryo at a stage comparable with the Cupiennius embryo shown in C, six rows of invagination sites can also be counted. The arrowheads point to lateral invagination sites of the hemisegment, which are located at similar positions in Cupiennius and Pholcus. Ch, cheliceral segment; ped, pedipalpal segment; l1 to l4, walking legs 1 to 4 (corresponding to prosomal segments 3 to 6); o1 to o5, opisthosomal segments 1 to 5. Scale bars: 200 µm in A,B; 100 µm in C,D.

View larger version (106K): [in a new window]   Fig. 7. (A-J) Mitotic divisions during neurogenesis. (A-D,F-H) Confocal micrographs of flat preparations of embryos stained with phalloidin-rhodamine and anti-PH3; anterior is towards the top. (A) At 130 hours, when the first five to eight invagination sites (arrow) are visible, only single cells are marked by anti-PH3. (B) The same pattern can be seen until 180 hours after egg laying. (C,D) Higher magnifications of A,B, respectively. The arrows point to single cells marked by anti-PH3. (E) Horizontal optical section of the apical region of the ventral neuroectoderm. Cell nuclei are stained with YOYO. The cells divide perpendicular to the surface. The arrow points to the separated chromosomes of a dividing cell. (F) At 190 hours, when most of the invagination sites have already formed, groups of labelled cells can be seen in the ventral neuroectoderm (arrow). (G) At 200 hours, when all invagination sites have formed, only single labelled cell can be detected (arrow). During decrease of the invagination sites at 220 to 240 hours, groups of labelled cells are visible again (arrow). (I) Transverse optical section of the ventral neuroectoderm of a 190 hour embryo. Cell division is restricted to the apical layer of the ventral neuroectoderm (arrow) with the exception of a few cells (arrowhead). (J) Transverse optical section of the ventral neuroectoderm of a 220 hour embryo. The invaginated cells do not divide (asterisks). Dividing cells can be seen only in the apical layer (arrowhead). The arrows point to invagination sites. Ch, cheliceral segment; ped, pedipalpal segment; l1 to l4, walking legs 1 to 4 (corresponding to prosomal segments 3 to 6); o1 to o6, opisthosomal segments 1 to 6. Scale bars: 200 µm in A,B; 40 µm in C; 50 µm in D; 10 µm in E; 100 µm in F-H; 20 µm in I,J.

View larger version (19K): [in a new window]   Fig. 8. (A,B) Deduced amino acid sequence of the bHLH domain of CsASH1 (A) and CsASH2 (B), and their relationship to other bHLH proteins. The bHLH domain is indicated. The alignment compares the basic domain and the two helices of CsASH1 and CsASH2 with three members of the ASC family of Drosophila and with the genes from other species that show the highest identity to the proneural genes of the spider. CsASH1 shows the highest identity to the chicken proneural protein CASH-1, whereas CsASH2 is highly identical to the proneural protein XASH1 of Xenopus. The loop, which varies in length and in sequence, is not included in the percent amino acid identity calculation. Dashes indicate amino acid identity with CsASH1 and CsASH2, and dots indicate gaps in the protein alignment. GenBank Accession Numbers, AJ309490 and AJ309491.

View larger version (107K): [in a new window]   Fig. 9. (A-T) Expression pattern of CsASH1. (A-O) Whole mounts; anterior is towards the top. (A-E) Cephalic lobe; (F-J) prosoma; (K-O) opisthosoma; (P-T) flat preparations of the fourth prosomal segments; anterior is towards the top, the black lines indicate the segmental borders. (A,F,K) At 120 hours, CsASH1 staining is visible in the cephalic lobe (arrow in A), in the anterior-most lateral regions of all hemisegments of the prosoma (F,P) and in the first opisthosomal segment (K). (B,G,L) At 130 hours, CsASH1 staining has extended posteriorly and medially in the prosoma (G, arrows in Q). The staining in the anterior-most lateral region that was visible in the prosoma and the first opisthosomal hemisegments before can now be seen in opisthosomal segments o2 to o4 (L). Additional expression domains can be also detected in the cephalic lobe. (C,H,M) At 160 hours, CsASH1 expression has decreased in the cephalic lobe (C) and in the second to sixth opisthosomal hemisegments (M). The RNA is now expressed in a lateral and a medial stripe of cells in the prosomal and the first opisthosomal hemisegments (H,M,arrows in R). (D,I,N) At 180 hours, new expression domains can be seen in the cephalic lobe (D, arrows). The former CsASH1 expression has disappeared in the prosoma and the first opisthosomal segment and a new medial expression domain is visible (I, arrow in S). The expression pattern in the opisthosoma reflects that seen before in the prosoma (N). CsASH1 is expressed weakly in the invaginating neuroectodermal cells (S, arrowheads). (E,J,O) At 220 hours, the medial expression domain has disappeared with the exception of the last three segments of the opisthosoma (O). CsASH1 is only weakly expressed in the invaginating cells (T, arrowhead). ped, pedipalpal segment; l1 to l4, walking legs 1 to 4 (corresponding to prosomal segments 3 to 6); o1 to o5, opisthosomal segments 1 to 5. Scale bar: 200 µm in A-O; 150 µm in P-T.

View larger version (102K): [in a new window]   Fig. 10. (A-P) Expression pattern of CsASH2. (A-L) Whole mounts; anterior is towards the top. (A-D) Cephalic lobe; (E-H) prosoma; (I-L) opisthosoma; (M,N) flat preparations of the fourth prosomal hemisegment; ; (O,P) flat preparations of the second opisthosomal segment; anterior is towards the top. (A,E,I) At 130 hours, CsASH2 expression can be seen only in two lateral and one median cell cluster in the cephalic lobe (A, arrows). (B,F,J) At 175 hours, CsASH2 transcripts are visible in the invaginating neuroectodermal cells in the cephalic lobe (B, arrow) and in the ventral neuroectoderm (F,J, arrows). Expression has decreased in the two lateral cell clusters of the cephalic lobe, which were labelled before, while the median cell cluster still expresses the gene (B). (C,G,K) At 190 hours, CsASH2 expression has decreased in the cephalic lobe, but now two lateral cell clusters show CsASH2 expression (C, arrows). There is also a decrease of expression in the prosomal (G, arrow) and the first to second opisthosomal segments (K), while expression can still be seen in the remaining opisthosomal segments (K, arrow). (D,H,L) At 220 hours, additional regions of CsASH2 expression can be detected in the cephalic lobe (D, arrow). In the ventral neuroectoderm, expression has disappeared in all segments with the exception of the last (H, arrows in L). (M) Higher magnification of the fourth prosomal hemisegment of an 175 hour embryo hybridised with the CsASH2 probe. CsASH2 is expressed in the invaginating cell groups (arrows). N shows the same region as M of a 175 hour embryo stained with phalloidin-rhodamine. The arrows point to the two anterior-most lateral invagination sites of the hemisegment (compare with arrows in M). (O) Higher magnification of the second opisthosomal hemisegment of an 175-hour embryo. Only five invaginating cell groups in the anterior-most lateral region show strong expression of CsASH2, whereas the remaining invaginating cells express the gene only weakly at this time. P shows the same region as in O of an embryo stained with phalloidin-rhodamine. The arrows point to two invagination sites, which show strong expression in O (arrows). l1 to l4, walking legs 1 to 4 (corresponding to prosomal segments 3 to 6); o1 to o4, opisthosomal segments 1 to 5. Scale bar: 200 µm in A-L; 100 µm in M-P.

View larger version (81K): [in a new window]   Fig. 11. (A-D) Expression pattern of CsASH1 and CsASH2 in the PNS. Whole mounts; anterior is towards the top. (A) At 200 hours, CsASH1 is expressed in four clusters of cells each in the pedipalps and the legs (arrows). (B) At the same time, CsASH2 is also expressed in the PNS in non overlapping cell clusters (arrows). (C) At 220 hours, CsASH1 expression in the PNS disappears. There is only a weak staining left at the base of the extremities. (D) At the same time, CsASH2 is expressed in 11 new cell clusters (arrows). Scale bar: 200 µm.

View larger version (84K): [in a new window]   Fig. 12. (A-E) Phenotypic analysis of embryos stained with phalloidin-rhodamine after injection of CsASH1 dsRNA. (A) Opisthosoma of an embryo injected with GFP dsRNA as a control. The embryo shows the normal number of invagination sites in the ventral neuroectoderm. The arrows point to the anterior-most lateral invagination sites. (B) Opisthosoma of an embryo injected with CsASH1 dsRNA. No invagination sites can be detected in the ventral neuroectoderm, with the exception of two lateral invagination sites (arrows). (C) Transverse optical section of an embryo injected with GFP dsRNA as a control; apical is towards the top. The invagination sites have decreased (arrow) and a second layer (bracket) of invaginated cells has formed. A developing neuropil is visible basally (arrowhead). (D,E) Transverse optical section of embryos injected with CsASH1 dsRNA; apical is towards the top. (D) The morphology of the neuroectodermal cells is disturbed, the cells have a rounded appearance (arrow). (E) In this segment, where only a reduced number of invagination sites has developed (arrows), the process of invagination seems to be blocked: no second cell layer is visible. o2 to o5, opisthosomal segments 1 to 5. Scale bars: 100 µm in A,B; 20 µm in C-E.

View larger version (82K): [in a new window]   Fig. 13. (A-D) Phenotypic analysis of embryos stained with an antibody against HRP after injection of CsASH1 dsRNA; anterior is towards the top. (A,D) The prosoma and opisthosoma, respectively, of an embryo injected with GFP dsRNA as a control. The embryo shows the normal HRP staining. The arrows point to staining in the PNS. (B) Prosoma of an embryo injected with CsASH1 dsRNA. Several segments of the prosoma show no HRP staining (arrows). (C) Prosoma of an embryo injected with CsASH1 dsRNA. This embryo shows an asymmetric effect: only one half of the germ band shows a reduced staining. (E) Opisthosoma of an embryo injected with CsASH1 dsRNA. All invagination sites are missing in the opisthosoma. (F) Opisthosoma of an embryo injected with CsASH1 dsRNA. Invagination sites are reduced in the opisthosoma. Only the anterior-most lateral invagination sites are visible (arrow). Ch, cheliceral segment; ped, pedipalpal segment; l1 to l4, walking legs 1 to 4 (corresponding to prosomal segments 3 to 6); o1 to o4, opisthosomal segments 1 to 4. Scale bar: 200 µm.

View larger version (114K): [in a new window]   Fig. 14. (A-F) Phenotypic analysis of embryos stained with phalloidin-rhodamine after injection of CsASH2 dsRNA. (A) Flat preparation of an embryo injected with GFP dsRNA as a control. The embryo shows the normal number of invagination sites in the ventral neuroectoderm and the cephalic lobe; anterior is towards the left. (B) Flat preparation of an embryo injected with CsASH2 dsRNA. The embryo shows the normal number of invagination sites in the ventral neuroectoderm and the cephalic lobe; anterior is towards the left. (C) Flat preparation of the second and third prosomal hemisegements of an embryo injected with GFP dsRNA as a control. The arrowheads point to the developing neuropils of the hemisegments, which are connected by an axon fascicle (arrows). (D) Flat preparation of the second and third prosomal hemisegments of an embryo injected with CsASH2 dsRNA. The arrowheads point to the reduced neuropils. The axon fascicle connecting the neuropils in the control embryos are missing in CsASH2-injected embryos. (E) Higher magnification of the third prosomal segment of an embryo injected with GFP. The arrow points to the neuropil. (F) Higher magnification of the third prosomal segment of an embryo injected with CsASH2 dsRNA. The neuropil is strongly reduced (arrow). Ch, cheliceral segment; ped, pedipalpal segment; l1 to l4, walking legs 1 to 4 (corresponding to prosomal segments 3 to 6). Scale bars: 200 µm in A,B; 150 µm in C,D; 40 µm in E,F.

View larger version (84K): [in a new window]   Fig. 15. (A-D) Phenotypic analysis of embryos stained against HRP after injection of CsASH2 dsRNA; anterior is towards the top. (A,C) The prosoma and opisthosoma, respectively, of an embryo injected with GFP dsRNA as a control. The embryo shows the normal HRP staining. The arrow indicates staining in the PNS. (B) Prosoma of an embryo injected with CsASH2 dsRNA. The embryo shows a reduced HRP staining in several prosomal segments (arrow). (D) Opisthosoma of an embryo injected with CsASH2 dsRNA. HRP staining is completely lost in the opisthosoma. l1 to l4, walking legs 1 to 4 (corresponding to prosomal segments 3 to 6); o2 to o5, opisthosomal segments 2 to 5. Scale bar: 200 µm.