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doi: 10.1242/10.1242/dev.00442

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Comparative analysis of neurogenesis in the myriapod Glomeris marginata (Diplopoda) suggests more similarities to chelicerates than to insects

Abteilung für Evolutionsgenetik, Institut für Genetik, Universität zu Köln, Weyertal 121, 50931 Köln, Germany

View larger version (148K): [in a new window]   Fig. 1. (A-D) Comparison of the pattern of invagination sites in the myriapod Glomeris marginata and the spider Cupiennius salei. Confocal micrographs of flat preparations of embryos stained with phalloidin-rhodamine. Anterior is towards the top, the midline towards the left. (A) Pattern of invagination sites in the opisthosoma of the spider. The invagination sites are arranged in seven rows consisting of four to five invagination sites each. The arrows point to two lateral anterior invagination sites that can be easily identified in each hemisegment. (B) A strikingly similar pattern and number of invagination sites is visible in the leg segments of the myriapod. As in the spider, the invagination sites form seven rows consisting of four to five invagination sites each. The arrows point to two lateral anterior invagination sites. (C) Higher magnification of an apical optical section of invagination sites in the ventral neuroectoderm of Glomeris. The arrowheads point to two invagination sites. (D) Basal optical section of the same region shown in C. Basally enlarged cells are visible (asterisks) underneath the dots of high phalloidin-rhodamine staining. o3 to o5, opisthosomal segments 3 to 5; l1 to l3, leg segments 1 to 3. Scale bars: 100 µm in A; 50 µm in B; 10 µm in C,D.

View larger version (122K): [in a new window]   Fig. 3. (A-J) Sequential formation of invagination sites in the myriapod. Confocal micrographs of flat preparations of whole embryos (A-E) and the first two leg segments (F-J). Anterior is towards the top, the midline towards the left in F-J. (A,F) No invagination sites are visible at stage 1. (B,G) When the limb buds form at stage 2, the first invagination sites arise in the medial region of each hemisegment (arrowhead in G). (C,H) New invagination sites arise anterior, posterior and in-between the existing invagination sites (arrowheads in H) during the second wave of neural precursor formation. (D,I) At early stage 4, the next wave generates invagination sites that form a semicircle around the central region where invagination sites have already formed (arrowheads in I). (E,J) At stage 5, the embryo curves inwards and the ventral neuroectoderm stretches along the mediolateral axis. ant, antennal segment; l1 to l7, leg segments 1 to 7; md, mandibular segment; mx, maxillar segment; pmd, premandibular segment; pmx, premaxillar segment. Scale bars: 120 µm in A-E; 50 µm in F-J.

View larger version (81K): [in a new window]   Fig. 2. (A-D) Apical-basal position of invagination sites in Glomeris. Transverse sections of untreated embryos (A,B) and embryos stained for a digoxigenin (DIG)-labelled GmASH probe (C,D). Basal is towards the top. (A) After formation of the first invagination sites, groups of up to 11 cells are visible on the basal side (asterisks) that are attached to the apical surface (arrow). (B) Groups of invaginating cells are located over and above each other (arrowheads) after formation of the third wave of neural precursors (asterisks and crosses). The cells are still attached to the apical surface (arrow). (C,D) GmASH is transiently expressed in the invaginating cell groups, which are located at different apical-basal positions (arrows). Some of the invaginating cell groups form stacks (arrowhead). Scale bar: 10 µm in A-D.

View larger version (74K): [in a new window]   Fig. 7. (A-J) GmASH prefigures the regions where invagination sites arise. Confocal micrographs (A-E) and light micrographs of (F-J) of the first leg hemisegment. Anterior is towards the top, the midline is towards the left. (A,F) GmASH is expressed at heterogeneous levels in the central region of the hemisegment before formation of the invagination sites at stage 1 (arrowheads). (B,G) The first invagination sites (arrowheads in B) arise in the expression domains of GmASH at stage 2. At this time the gene is expressed in distinct regions of the hemisegment (arrowheads in G). In addition, GmASH shows transient expression in the invaginating cells. (C,H) At stage 3, the second wave generates invagination sites in the regions prefigured by GmASH (arrowheads in C, compare to G). At this time, the expression domains of GmASH form a semicircle around the central region where an invagination site have already formed (arrowheads in H). (D,I) Again invagination sites arise in the expression domains of GmASH (arrowheads). At this time the gene is re-expressed in distinct regions (arrowheads in I). (E,J). In these regions the last invagination sites arise during the fourth wave of neural precursor formation at late stage 4. GmASH expression is transiently maintained in the invaginating cell groups (arrowhead in J). Scale bar: 50 µm in A-J.

View larger version (106K): [in a new window]   Fig. 4. (A-F) Proliferating cells are associated with invagination sites. Confocal micrographs of flat preparations of embryos stained with phalloidin-rhodamine (red) and anti-phospho-histone 3 antibodies (green). Anterior is towards the top and the midline towards the left in D-F. (A-C) Single mitotic cells are associated with invagination sites that have already formed. The arrow in A points to an invagination site. The arrow in B points to a mitotic cell located at the same position as the invagination site indicated in A. The overlay of A and B is shown in C. (D-F) During formation of the first invagination sites in the central region of the hemisegment (arrowheads and arrow in D), mitotic cells are located at the centre of the invagination sites (arrowheads in E and F) or close to the invagination site (arrows in E and F). l1 to l2, leg segments 1 to 2. Scale bars: 50 µm in A-C; 10 µm in D-F.

View larger version (40K): [in a new window]   Fig. 5. (A,B) Comparison of the deduced amino acid sequences of the conserved domains of GmASH and GmDelta, and their relationships to the same protein regions of other species. (A) The alignment compares the basic domain, the two helices and the loop region of GmASH with the bHLH region of another diplopod, two insects, two vertebrates and two spider sequences. Note that the loop region of Glomeris ASH is reduced to the same extent as in the spider and vertebrate bHLH domains. The GmASH bHLH domain shows the highest similarity to the same region of the millipede Archispirostreptus sp. See text for further details. (B) The alignment compares the highly conserved DSL domain (Delta, Serrate, Lag2) of GmDelta with the DSL domains of the same species used for the alignment in A. The GmDelta DSL domain shows the highest similarity to the same protein region of Anopheles gambiae (67% identical amino acids), Archispirostreptus sp. and Cupiennius salei Delta1 (65% identical amino acids each). Ag, Anopheles gambiae; As, Archispirostreptus sp.; Cs, Cupiennius salei; Gm, Glomeris marginata; Hs, Homo sapiens; Mm, Mus musculus.

View larger version (17K): [in a new window]   Fig. 6. (A-C) Phylogeny of the conserved domains of the achaete-scute, Delta and Notch homologues. The trees were constructed using the neighbour-joining algorithm (see Materials and Methods for further details). The numbers at the nodes are the bootstrap values given in percent (1000 replicates). Nodes without numbers have bootstrap values below 50%. The numbers below the branches are the branch lengths. (A) The tree was constructed from an alignment of the bHLH domains of nine insect, five vertebrate, two spider and two myriapod sequences. Both myriapod homologues group outside the insect genes together with the spider and vertebrate homologues. (B) The tree was created from an alignment of the DSL domains and the adjacent highly conserved EGF-repeats 1 and 2 from two insect species, five vertebrate, two myriapod, an ascidian and the two spider sequences. The insects and the vertebrates form two clear groups, while the myriapods group with the spider sequences. (C) The tree was constructed from an alignment of the obtained GmNotch sequence (5' region up to EGF-repeat 12) with the same region of four vertebrate and three invertebrate Notch homologues. The myriapod sequence groups with the chelicerate Boophilus microplus, while the spider homologue forms a group with the vertebrates. The node joining the chelicerates with the vertebrates and the myriapod has a high bootstrap support (100%). Genes: L-sc, lethal of scute; Sc, scute; Ac, achaete; Ash, achaete-scute homologue; Scal, scalloped wings. Species: Ag, Anopheles gambiae; As, Archispirostreptus sp.; Bm, Boophilus microplus; Cc, Ceratitis capitata; Cs, Cupiennius salei; Csa, Ciona savigny; Cv, Calliphora vicina; Dm, Drosophila melanogaster; Dr, Danio rerio; Gg, Gallus gallus; Gm, Glomeris marginata; Hs, Homo sapiens; Jc, Junonia coenia; Lc, Lucilia cuprina; Mm, Mus musculus; Tc, Tribolium castaneum; Xl, Xenopus laevis.

View larger version (87K): [in a new window]   Fig. 8. (A-D) Expression pattern of GmASH. Flat preparations of whole embryos stained for a DIG-labelled GmASH probe. Anterior is towards the top. (A) An anterior to posterior gradient of GmASH expression is visible in the neurogenic regions of the embryo. The mediolateral extension of the GmASH expression domain is smaller in the head segments. An identical GmASH expression pattern is visible in the leg segments 1 and 2 (arrow), while the former expression pattern of anterior segments can be detected in leg segment 3 (arrow; compare to Fig. 7F,G). (B) At stage 3, GmASH expression forms a semicircle around the central region of the hemisegments (arrow). The former expression pattern of the anterior segments is now visible in leg segment 3 (arrow). (C) GmASH expression has extended posteriorly to leg segment 5. The arrow points to the transient expression of GmASH in the invaginating cell groups. (D) After formation of all invagination sites, GmASH is still expressed in about half of them. In addition, the gene is expressed in the precursors of the peripheral nervous system (arrows). ant, antennal segment; l1 to l7, leg segments 1 to 7; md, mandibular segment; mx, maxillar segment; pmd, premandibular segment; pmx, premaxillar segment. Scale bar: 120 µm in A-D.

View larger version (117K): [in a new window]   Fig. 9. (A-F) Expression pattern of GmDelta. Flat preparations of whole embryos (A,B) and leg segments (C-F) stained for a DIG-labelled GmDelta probe. (A,B) GmDelta transcripts can be detected at low levels in all ventral neuroectodermal cells, but they accumulate at higher levels in the invaginating cell groups. In addition, the gene is expressed in groups of cells in the limb buds and dorsal to the limb buds (arrowheads). These regions coincide with the generation sites of the peripheral nervous system. (C) Accumulation of higher levels of GmDelta transcripts is first visible during formation of the first invagination sites at stage 2 (arrow). (D-F) High levels of GmDelta expression correlate with the formation of invagination sites throughout neurogenesis (arrows). The expression is rapidly downregulated during the process of invagination, although the low uniform expression in all neuroectodermal cells is maintained. l1 to l6, leg segments 1 to 6. Scale bars: 120 µm in A,B; 50 µm in C-F.

View larger version (135K): [in a new window]   Fig. 10. (A-F) Expression pattern of GmNotch. Flat preparations of whole embryos (A,B) and leg segments (C-F) stained for a DIG-labelled GmNotch probe. (A,B) At stage 4 GmNotch is expressed in all neuroectodermal cells (arrows) at heterogeneous levels. (C) At stage 1 GmNotch is expressed in segmentally repeated stripes, but shows a stronger expression in the ventral neuroectoderm (arrow). (D) During the first wave of neural precursor formation, GmNotch is expressed uniformly in the head segments and the first three leg segments (arrow). (E) The uniform expression resolves into a heterogeneous expression pattern before formation of the next wave of invagination sites. The arrowhead points to a region of low GmNotch expression, a higher expression is visible in the adjacent region (arrow). (F) GmNotch expression has extended posteriorly and still shows a heterogeneous expression pattern in the neuroectoderm. The expression in the limb buds probably corresponds to the formation of mesodermal tissue (arrowhead). ant, antennal segment; gz, growth zone; l1 to l5, leg segments 11 to 15; md, mandibular segment; mx, maxillar segment; pmd, premandibular segment; pmx, premaxillar segment. Scale bars: 120 µm in A,B; 50 µm in C-F.

View larger version (108K): [in a new window]   Fig. 11. (A-D) The epidermis overgrows the ventral nerve cord after formation of the neuropil. Flat preparations (A,B) and transverse semi-thin sections of the ventral nerve cords at stage 6. (A) Apical optical section of the ventral nerve cord of an embryo stained with phalloidin-rhodamine. The ventral neuromeres sink into the embryo (asterisk) and the epidermis overgrows the nerve cord (arrow). (B) Basal optical section of the same region shown in A. A ladder-like neuropil has been formed by the invaginated cells (arrow). (C) Transverse section of the hemineuromere of leg segment 1 at early stage 6. At this stage a medial thickening forms (arrowhead). The asterisk indicates the midline. (D) At late stage 6 an additional thickening has formed at the lateral border of the hemineuromere and the central part sinks into the embryo. The asterisk indicates the midline. l1 to l3, leg segments 1 to 3. Scale bars: 10 µm.

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