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doi: 10.1242/10.1242/dev.00109

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Recruitment of cell groups through Delta/Notch signalling during spider neurogenesis

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

View larger version (22K): [in a new window]   Fig. 1. (A,B) Comparison of the spider Delta and Notch structures to the Drosophila proteins and alignment of the DSL domain. (A) The Drosophila Notch protein contains several conserved domains. The sequence obtained so far for Notch encodes two different transcripts. One transcript lacks part of EGF repeat 6 and specifically EGF repeats 7, 8 and 9. (B) CsDelta1 shares the highly conserved DSL domain (Delta, Serrate, Lag2) and eight EGF repeats with the fly sequence. CsDelta2 contains the conserved DSL domain, but has only five EGF repeats. Both spider Delta proteins have a shorter intracellular domain. (C) The DSL domains of both CsDelta1 and CsDelta2 show a higher identity to the same region of the Xenopus X-Delta1 protein (66% and 62% identity, respectively) than to Drosophila (62% and 57% identity, respectively). Ank, ankyrin repeat; DSL, Delta, Serrate, Lag2 domain; EGF-rep, EGF repeats; N/lin12, Notch lin12 repeats; Pest, a region rich in proline, glutamine, serine and threonine residues; RAM, RAM23 domain.

View larger version (89K): [in a new window]   Fig. 2. (A-O) Expression pattern of the neurogenic genes in the prosoma. Whole-mount in situ hybridisation; anterior is towards the top. (A-E) CsDelta1 expression; (F-J) CsDelta2 expression; (K-O) CsNotch expression. (A,D) At 130 hours, CsDelta1 expression is visible in the first five to eight invaginating cell groups that have formed per hemisegment (arrows). (B) Seventeen to 20 additional invagination sites (arrow) were generated at 170 hours, all of them expressing CsDelta1. (C) When the apical cell processes of the invaginating cells lose contact to the apical surface at about 190 hours in the prosoma, CsDelta1 expression decreases, although some of the invaginating cell groups still show a strong expression (arrow). (E) At 180 hours, the final number of 30 to 32 invagination sites is visible. CsDelta1 transcripts can be detected in all invaginating cell groups (arrow). (F,I) CsDelta2 is uniformly expressed in the ventral neuroectoderm before formation of invagination sites (data not shown). At 130 hours, CsDelta2 shows stronger expression in the invaginating cell groups (arrows), although it is still expressed in all neuroectodermal cells. (G,J) The upregulation of CsDelta2 expression is also visible in subsequent invagination groups. (H) Expression of CsDelta2 decreases at the same time (about 190 hours) in the invaginating cells as CsDelta1 expression. (K,N) CsNotch is expressed in the whole ventral neuroectoderm at 130 hours, but shows a slightly stronger expression in the lateral regions of the hemisegments (arrows). (L,O) CsNotch is still expressed in all neuroectodermal cells at 170 and 180 hours, but there is heterogeneity in the expression levels (arrows). (M) The same distribution of CsNotch transcripts is visible at about 190 hours, but in addition a medial group of cells shows strong CsNotch expression (arrow). 11 to 14, walking legs 1 to 4 (corresponding to prosomal segments 3 to 6). Scale bars: in A, 200 µm for A-C,F-H,K-M; in D, 25 µm for D,E,I,J; in N, 50 µm N,O.

View larger version (87K): [in a new window]   Fig. 3. (A-P) Phenotypic analysis of embryos stained with phalloidin-rhodamine after injection of CsDelta1, CsDelta2 and CsNotch dsRNA. (A-J) Confocal micrographs of flat preparations of prosomal segments of 180-hour-old embryos; anterior is towards the top. (K-P) Transverse sections through the fourth prosomal segments; medial is towards the left. Prosomal regions of embryos injected with GFP dsRNA as a control (A,F,K), with CsDelta1 dsRNA (B,G,L), CsDelta2 dsRNA (C,H,M) and CsNotch dsRNA (D-P), respectively. (F-J) Higher magnifications of the fourth prosomal hemisegments. (A,F) After injection of GFP dsRNA, the ventral neuroectoderm shows the normal number of invagination sites (about 30 per hemisegment; dots of high phalloidin-rhodamine staining, arrows). (B,G) After injection of CsDelta1 dsRNA, the number of invagination sites is reduced in individual segments (arrows). (C,H) A more severe reduction of invagination sites can be detected after injection of CsDelta2 dsRNA; invagination sites are absent in the whole ventral neuroectoderm. (D,I,) After injection of CsNotch dsRNA, dots of high phalloidin-rhodamine staining can be detected in the positions that correspond to invagination sites in control injected embryos (arrows), although they are much smaller. (E,J) In a more severely affected embryo there is only diffuse phalloidin-rhodamine staining visible in positions that correspond to invagination sites in control injected embryos (arrow). (K) Confocal micrograph of a transverse optical section through an invagination site (arrowheads) of an embryo injected with GFP dsRNA. The cell processes of the basally enlarged cells extend to the apical surface. (L) Transverse optical section through the fourth prosomal hemisegment of an embryo injected with CsDelta1 dsRNA, showing that in a region where invagination sites are missing the neuroectoermal cells form a bulge (arrowhead). (M) Transverse section through the fourth prosomal hemisegment of an embryo injected with CsDelta2 dsRNA. Two bulges of neuroectermal cells are visible (arrowheads). (O,P) The transverse sections through the fourth prosomal hemisegments of embryos injected with CsNotch dsRNA reveal that, although dots of higher phalloidin-rhodamine staining are visible on the apical surface (arrowheads), there are no bottle-like cells visible underneath these dots. The presence of several cell layers suggests that cells that normally invaginate occupy space in the apical layer so that newly formed cells were pushed basally. l1, l2, walking legs 1 to 2 (corresponding to prosomal segments 3 and 4). Scale bars: in A, 150 µm for A-E; in F, 50 µm for F-J; in K, 20 µm for K-P.

View larger version (139K): [in a new window]   Fig. 4. (A-D) Distribution of phospho-histone 3 antigen in embryos injected with CsDelta1, CsDelta2 and CsNotch dsRNA. Confocal micrographs of flat preparations of opisthosomal segments of 160-hour embryos; anterior is towards the top. (A) The anti-phospho-histone 3 antibody stains single mitotic cells (arrow; green) that are uniformly distributed over the whole ventral neuroectoderm throughout neurogenesis in embryos injected with GFP dsRNA as a control. (B-D) The overall mitotic pattern is unchanged after injection of CsDelta1, CsDelta2 and CsNotch, respectively (arrows). Scale bar: 50 µm.

View larger version (151K): [in a new window]   Fig. 5. (A-C) Expression pattern of the proneural gene CsASH1 and the neural HRP antigen after injection of CsDelta2 and CsNotch dsRNA. Light micrographs of flat preparations of the second to fourth opisthosomal segments; anterior is towards the top. (A) At about 180 hours, CsASH1 is expressed in a medial stripe and a patch of cells in the lateral region of the opisthosomal hemisegments (arrows). This pattern is unchanged after injection of GFP dsRNA. (B,C) In embryos injected with CsDelta2 dsRNA (B) and CsNotch dsRNA (C), a strong upregulation of CsASH1 expression can be observed (arrows), while after injection of CsDelta1 only a minor change in the expression pattern is visible (data not shown). (D) The anti-HRP antibody stains the cell processes of the invaginating cells (arrow) in an embryo injected with GFP dsRNA as a control. (E) After injection of CsNotch dsRNA, the HRP antigen is upregulated in the apical cells. A stronger staining (arrow) is visible in the regions where invagination sites normally form (compare with Fig. 3J). (F) Higher magnification of a neuroectodermal region, where no invagination sites have been formed after injection of CsDelta2 dsRNA. Apical located cells are stained with the anti-HRP antibody (arrow). o2-o4, opisthosomal segments 2 to 4. Scale bars: in A, 100 µm for A-C; inD, 50 µm for D,E; in F, 20 µm for F.

View larger version (70K): [in a new window]   Fig. 6. (A-E) Expression patterns of the neural anti-HRP and anti-acetylated tubulin antibodies (green) and morphology of the neuromeres; double-staining with phalloidin-rhodamine (red). Confocal micrographs of flat preparations of prosomal neuromeres (A,B,D,E), an opisthosomal neuromere (C) and light (F,H,I) and electron micrographs (G) of transverse sections through the fourth prosomal neuromere at 240 hours (A-C), 280 hours (D,E), 250 hours (F,G,I) and 300 hours (H); anterior is towards the top (A-E); medial is towards the left (F-I). (A,E) The HRP antigen is expressed on the axons of the invaginated cells and in addition in the apical cells (arrows). (B,C) Acetylated tubulin is expressed in the cells that remain in the outer layer, but not in the axons of the invaginated cells (arrows). (D,F) The epidermis overgrows the neuromeres at the end of neurogenesis (arrows in D; arrowheads in F). (G) Higher magnification of a similar region framed in F. Ultrastructural analysis reveals that the neurones and axons (asterisks) are not covered with epidermal cells. (H) At 300 hours the epidermal cells have completely overgrown the ventral neuromers. (I) At 250 hours the epidermoblasts (asterisks) can be distinguished morphologically from the surrounding cells. Neurones at the border of the ventral neuromer are marked with black dots. 12, walking leg 2 corresponding to the fourth prosomal segment; o2, second opisthosomal segment; 1c, longitudinal connective; c, commissure; vn, ventral neuropil. Scale bars: in A, 100 µm for A,B; in C, 50 µm for C-E; in F, 20 µm for F; in G, 4 µm for G; in H, 20 µm for H; in I, 15 µm for I.

View larger version (116K): [in a new window]   Fig. 7. (A-D) Expression pattern of the proneural gene CsASH1 and the neurogenic genes during the second phase of neurogenesis. Whole-mount in situ hybridisation; anterior is towards the top. (A) The proneural gene CsASH1 is re-expressed in the apical neuroectderm after the process of invagination is completed. (B) CsDelta1 is still expressed in a subset of the invaginated cells. Transcripts are visible in about 7 axon fascicles per hemisegement (arrow). (C,D) CsDelta2 (C) and CsNotch (D) are uniformly expressed in the neuroectoderm (arrows). 11 to 14, walking legs 1 to 4. Scale bar: 200 µm.

View larger version (49K): [in a new window]   Fig. 8. Comparison of recruitment of neural precursors in fly and spider. (Top) In Drosophila, Delta/Notch signalling specifies differences in a single cell within a field of initially equivalent cells leading to the recruitment of one neural precursor (neuroblast) from a proneural cluster. (Bottom) In the spider, groups of cells that are located within a field of initially equivalent cells adopt the neural fate simultaneously. Dark blue, cells that have the competence to adopt the neural fate; light blue, cells that have not adopted the neural fate.

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