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First published online 18 October 2006
doi: 10.1242/dev.02646


Development 133, 4539-4547 (2006)
Published by The Company of Biologists 2006


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brachyenteron is necessary for morphogenesis of the posterior gut but not for anteroposterior axial elongation from the posterior growth zone in the intermediate-germband cricket Gryllus bimaculatus

Yohei Shinmyo*, Taro Mito, Tomohiro Uda, Taro Nakamura, Katsuyuki Miyawaki{dagger}, Hideyo Ohuchi and Sumihare Noji{ddagger}

Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, 2-1 Minami-Josanjima-cho, Tokushima City 770-8506, Japan.


Figure 1
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Fig. 1. Alignment of the conserved T-domain of Bra/Byn homologs in G. bimaculatus and other species. Gb, Gryllus bimaculatus; Tc, Tribolium castaneum; Dm, Drosophila melanogaster; Xl, Xenopus laevis; Mm, Mus musculus. Conserved amino acid residues are highlighted in gray. Arrows indicate the positions of degenerate primers. Arrowheads indicate the 5' and 3' ends of the fragment used for synthesis of dsRNA.

 

Figure 2
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Fig. 2. Gb'byn expression patterns during early embryogenesis of G. bimaculatus. (A-G) Expression patterns of Gb'byn at stages 3.8 (A), 4.3 (B), 4.8 (C), 5.0 (D), 5.8 (E) and 7.5 (F,G). The boxed area in F is enlarged in G. In order to illustrate the segmentation process of the Gryllus embryo, specified segmental regions are labeled based on the appearance of Gb'wg stripes: Mn, mandible; Mx, maxilla; Lb, labium; T1-T3, thoracic segments 1-3; A1-A5, abdominal segments 1-5. Gb'byn expression is first detected as two spots (arrowheads) in the posterior terminal region (A). Gb'byn continues to be expressed in the terminal region until germband elongation is completed (B-E). The proctodeum that expresses Gb'byn starts to invaginate (arrow) after germband elongation and segmentation are completed (G). Anterior is to the left in all embryos. Scale bars: in A, 200 µm for A-F; in G, 100 µm.

 

Figure 3
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Fig. 3. Expression patterns of Gb'byn and Gb'cad during late embryogenesis of G. bimaculatus. (A-D) Expression of Gb'byn at stages 8 (A,B), 9 (C) and 11(D). The boxed area in A is enlarged in B. Gb'byn expression is detected in the ectodermal epithelium of the hindgut (arrows), whereas no Gb'byn expression is detectable in the mesoderm surrounding the hindgut (arrowheads). (E,F) Expression patterns of Gb'cad at stages 9 (E) and 11 (F). Gb'cad is expressed in the region surrounding the orifice of the hindgut, Malpighian tubules (mt), and developing posterior midgut (pmg) of stage 11 embryos. (G,H) Embryo double stained for Gb'cad (brown) and Gb'byn (blue). The boxed area in G is enlarged in H. Very little, if any, overlap can be seen in the expression patterns of the two genes. Anterior is to the left in all embryos. Scale bars: in A, 200 µm; in B,100 µm for B-G; in H, 50 µm.

 

Figure 4
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Fig. 4. Effect of Gb'byn RNAi on G. bimaculatus nymphs. (A,B) Wild-type (A) and Gb'byn RNAi (B) nymphs in the first instar. (C) The alimentary canal of the wild-type nymph in the first instar, consisting of the fore-, mid- and hindgut. Cr, crop; pv, proventriculus; gca, gastric caecum; mt, Malpighian tubules; si, small intestine; li, large intestine; rsc, rectum sac; ce, cercus. (D,E) Representative alimentary canals of Gb'byn RNAi nymphs in the first instar. In most Gb'byn RNAi nymphs, the hindgut and the posterior region of the midgut were severely reduced, and the Malpighian tubules were decreased in size (D). In the more strongly affected nymphs, the Malpighian tubules were severely reduced (E). Scale bars: in A,1 mm for A,B; in C, 500 µm for C-E.

 

Figure 5
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Fig. 5. Expression patterns of marker genes for the posterior gut in Gb'byn RNAi embryos. (A-D) Expression pattern of Gb'byn in wild-type (A,C) and Gb'byn RNAi (B,D) embryos, at stages 5.2 (A,B) and 11 (C,D). Gb'byn expression in the hindgut (hg) is greatly reduced in Gb'byn RNAi embryos, but some Gb'byn-expressing tissues do remain (D). (E,F) Expression patterns of Gb'hh in wild-type (E) and Gb'byn RNAi (F) embryos at stage 11. In wild-type embryos, Gb'hh is strongly expressed in the small intestine (si) and rectum sac (rsc) of the hindgut, and weakly expressed in the large intestine (li) and Malpighian tubules (mt). In Gb'byn RNAi embryos, Gb'hh expression was observed abnormally in the hindgut remnant, probably as a combined pattern from the small intestine and rectum sac. (G,H) Expression of Gb'wg in wild-type (G) and Gb'byn RNAi (H) embryos at stage 11. In wild-type embryos, Gb'wg is expressed in the anterior region of the small intestine (arrow) and in the posterior rectum. Gb'wg expression in these domains was reduced in the Gb'byn RNAi embryos. (I-L) Expression of Gb'cad in wild-type (I,K) and Gb'byn RNAi (J,L) embryos at stages 9 (I,J) and 11 (K,L). In wild-type embryos at stage 9, Gb'cad is expressed in the region surrounding the orifice of the hindgut and in a region adjacent to the hindgut; this expression was much reduced in Gb'byn RNAi embryos (compare I and J, arrow). In wild-type embryos at stage 11, Gb'cad expression was observed in the Malpighian tubules and posterior midgut (pmg; K). In Gb'byn RNAi embryos, Gb'cad expression was observed in a very small remnant of the Malpighian tubules, and Gb'cad expression in the posterior midgut was greatly reduced (L). In all embryos, anterior is to the left. Scale bar in A: 200 µm for A-L.

 

Figure 6
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Fig. 6. Effect of Gb'cad RNAi on posterior terminal patterning. (A-C) Wild-type (A) and Gb'cad RNAi (B,C) embryos at 12 days after egg laying. In the most severe cases (64%, n=178 out of 280), Gb'cad RNAi embryos completely lack gnathum, thorax and abdomen, whereas the anterior head is formed normally (B). In other cases (31%, n=87 out of 280), the normal anterior head and part of the trunk segments are formed (C). (D-H) Expression of Gb'byn in wild-type (D,F) and Gb'cad RNAi (E,G,H) embryos at stages 4 (D,E) and 9 (F-H). At stage 4, Gb'byn expression in the terminal regions was normal in all Gb'cad RNAi embryos (D,E). In wild-type embryos at stage 9, Gb'byn was expressed in the internalized hindgut (F). In the Gb'cad RNAi embryos at this stage, the Gb'byn-expressing cells failed to invaginate, remaining on the outside of the embryo (G,H). (I-K) Expression of Gb'hh in wild-type (I) and Gb'cad RNAi (J,K) embryos at stage 9. In wild-type embryos, Gb'hh expression was observed in the internalized hindgut, whereas, in the Gb'cad RNAi embryos, Gb'hh expression was observed in the external remnant.

 

Figure 7
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Fig. 7. Schematics of the profile of Gb'byn expression and function. (A) A comparison of extrapolated expression patterns of Gb'hh, Gb'wg and Gb'cad in the gut of wild-type (left) and Gb'byn RNAi (right) nymphs. In Gb'byn RNAi nymphs, Gb'hh expression is observed in the severely reduced hindgut as a probable combined pattern from the small intestine and rectum sac, and in the small remnant of the Malpighian tubules. Gb'wg expression seems to be observed in the small intestine and posterior rectum of the severely reduced hindgut of Gb'byn RNAi nymphs. Gb'cad expression in the Gb'byn RNAi nymph seems to be observed in the small remnant of the Malpighian tubules, and is almost completely eliminated in the region surrounding the orifice of the rectum and posterior midgut. The hatched regions indicate overlapping expression patterns. pv, proventriculus; gca, gastric caecum; mg, midgut; mt, Malpighian tubules; si, small intestine; li, large intestine; rsc, rectum sac; rec, rectum. (B) Comparison of the expression of Gb'byn, Gb'wg and Gb'cad in early embryos of Gryllus with those of Drosophila and mouse. In early Gryllus embryos, Gb'wg and Gb'cad expression is detected in the posterior growth zone (Miyawaki et al., 2004Go; Shinmyo et al., 2005Go), whereas Gb'byn expression is restricted in the posterior terminal region (see Fig. 2). In Drosophila, all three genes are expressed in the posterior terminal region at the cellular blastoderm stage (Hoch and Pankratz, 1996Go; Kispert et al., 1994Go; Singer et al., 1996Go; Wu and Lengyel, 1998Go). In mouse, all three genes are expressed in the primitive streak of early embryos (Kispert and Herrmann, 1994Go; Liu et al., 1999Go; Meyer and Gruss, 1993Go).

 

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© The Company of Biologists Ltd 2006