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First published online June 8, 2006
doi: 10.1242/10.1242/dev.02428

Development 133, 2565-2574 (2006)
Published by The Company of Biologists 2006
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Neverland is an evolutionally conserved Rieske-domain protein that is essential for ecdysone synthesis and insect growth

Takuji Yoshiyama1, Toshiki Namiki1, Kazuei Mita2, Hiroshi Kataoka1,{dagger} and Ryusuke Niwa1,*,{dagger}

1 Department of Integrated Biosciences, Rm201, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan.
2 Laboratory of Insect Genome, National Institute of Agrobiological Sciences, Owashi 1-2, Tsukuba, Ibaraki 305-8643, Japan.


Figure 1
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  		Fig. 1. Spatiotemporal expression pattern of nvd-Bm. (A) Northern blot analysis showing temporal expression profiles of nvd-Bm. The length of the nvd-Bm cDNA (3747 bp) is similar to the 3.7 kb band detected by northern hybridization. No other bands were detected (data not shown). (B) Tissue expression profiles of nvd-Bm and a control gene rpL3 in the W1 fifth instar larvae by reverse transcription-PCR.

 

Figure 2
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  		Fig. 2. neverland is conserved among several animal species. (A) Predicted polypeptides that are produced from nvd-Bm (Bombyx mori) (GenBank Accession Number AB232986) and its closest relatives from Drosophila melanogaster (AB232987), Anopheles gambiae (XP_309236.1), Caenorhabditis elegans (NP_505629), Ciona intestinalis (EST clones cicl031b24 and cicl077j15 from the Ciona cDNA database; http://ghost.zool.kyoto-u.ac.jp/cgi-bin/getblast1.cgi), Danio rerio (NP_001002612), Gallus gallus (XP_425346), Rhodococcus erythropolis (kshA gene, AAL96829) and Pseudomonas fluorescens (prnD gene, U74493). These polypeptides share a Rieske domain (dark gray) and a highly conserved domain in the C-terminal region (light gray). The bars above each domain indicate the positions of the [2Fe-2S] binding motif (C-X-H-X16-17-C-X2-H) and the non-heme iron-binding motif [Fe(II); E-X3-D-X2-H-X4-H]. Percentages represent amino acid identities between each domain of nvd-Bm and that of others. Also indicated are the total numbers of residues for individual proteins. The Anopheles and Ciona orthologs are incompletely predicted on both ends. A black box indicates the transmembrane domain predicted by TMHMM software (http://www.cbs.dtu.dk/services/TMHMM/). (B) Sequence alignment of the Rieske motif and the non-heme iron-binding motif of Nvd-Bm with its closest relatives. Identical residues in all nine species are white on black, while identical residues in five or more species are boxed. The evolutionally conserved amino acids of the Rieske domain and non-heme iron binding motif are indicated above the alignment.

 

Figure 3
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  		Fig. 3. Expression pattern of nvd-Dm. (A-C) In situ embryonic expression (see also Fig. S4 in the supplementary material). nvd-Dm mRNA was not detected during early stages of embryogenesis, such as preblastoderm stage (A) or stage 8 (B). At stage 17 (C), nvd-Dm expression was detected specifically in the ring gland (arrowhead). (D) Brain-ventral nerve cord-ring gland complex of wandering stage of third instar larva. (D') Higher magnification of D. Expression was detected only in the region of PG cells, but not in either the corpus allatum (CA) or the corpus cardiacum (CC). (E) Quantitative RT-PCR analysis of nvd-Dm transcript in several larval tissues from wandering third instar larvae. nvd-Dm/rp49 indicates the levels of nvd-Dm mRNA normalized to the levels of internal rp49 mRNA. The normalized nvd-Dm mRNA level in the ring gland is set as 1. (F) Cyclic expression of nvd-Dm in the ring glands during the second and third larval stages shown by the quantitative RT-PCR. The normalized nvd-Dm mRNA level from the wandering third instar larvae is set as 1 (±s.e.m.; n=3). (G-I) Ovarian expression. nvd-Dm mRNA was detected in the nurse cells (G,H; arrow), whereas oocytes was devoid of nvd-Dm expression (H; arrowhead). No staining was obtained for the sense RNA probes in ovaries (I), embryos and larvae (data not shown).

 

Figure 4
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  		Fig. 4. Developmental arrest of nvd-Dm RNAi animals. (A) RT-PCR analysis of nvd-Dm and control gene rp49 in RNAi and control animals 48 hours AEL. (B) Comparison of the survival rate and developmental progression of nvd-Dm RNAi animals versus control animals. nvd-Dm RNAi animals did not become pupae and died as larvae. (C-G) Comparison of body size between nvd-Dm RNAi and control animals. Anterior is leftwards. RNAi animals (upper) grew at the same rate as control animals (lower) until about 48 hours after egg laying (AEL) (C-E). Forty-eight hours AEL, RNAi animals failed to grow in size (F,G). (H-Q) Dissected mouth hooks of nvd-Dm RNAi animals (H-L) and control animals (M-Q) at different developmental stages. Red arrowheads indicate teeth of the mouth hooks. Control first instar larvae had one-tooth mouth hooks (M,N), control second instar larvae had two to five teeth (O,P), and control third instar larvae had numerous small teeth (Q). By contrast, mouth hooks of all nvd-Dm RNAi animals remained small and showed the characteristic morphology of first instar larvae, even 96 hours AEL (H-L). Scale bar: 1.2 mm for C-G; 10 µm for H-Q.

 

Figure 5
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  		Fig. 5. nvd-Dm RNAi animals show reduced ecdysteroid titer. Larvae were collected 30-42 hours after egg laying (AEL), corresponding to the first instar larval stages. Ecdysteroid titers of control (black box) and the nvd-Dm RNAi animals (shaded box) on food with or without 7-dehydrocholesterol (7dC) were determined by radioimmunoassay. The results are depicted as picograms (pg) of 20-hydroxyecdysone equivalents/mg initial body weight on the vertical axis. Each bar represents the mean±s.e.m. from multiple independent samples. The following number of independent samples was examined: four (control), five (RNAi) and six (control + 7dC and RNAi + 7dC). *P<0.01 by Student's t-test.

 

Figure 6
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  		Fig. 6. Developmental progression of nvd-Dm RNAi animals by feeding 20-hydroxyecdysone, cholesterol and 7-dehydrocholesterol. nvd-Dm RNAi (black arrowheads) and control larvae were fed yeast paste supplemented with either 3.3% ethanol, 1 mg/ml 20-hydroxyecdysone (20E) or 0.5% wet weight of cholesterol (C) or 7-dehydrocholesterol (7dC). All control animals grew to the third instar larvae 96 hours AEL. Red arrowheads indicate teeth of the mouth hooks. (A,B) nvd-Dm RNAi animals fed 20E-containing food during only 30-42 hours after egg laying (AEL) molted to the second instar larvae. However, 96 hours AEL they showed small body size (A) and still possessed the second instar-type mouth hook (B). (C,D) nvd-Dm RNAi animals with 20E 30-42 hours and 54-66 hours AEL molted to the third instar larvae. (E) nvd-Dm RNAi animals fed 20E 30-42 hours, 54-66 hours and 78 hours AEL. After becoming third instar larvae, 45% and 5% of the nvd-Dm animals pupariated and eclosed (E), respectively. (F) nvd-Dm RNAi animals with 7dC. All the animals grew to adults. (G,H) nvd-Dm RNAi animals with excessive C had the second instar-type mouth hook 96 hours AEL (H). Their body size nearly reached the size of control animals (G). Scale bar: 1.0 mm for A,C,E,F,G; 10 µm for B,D,H. (I) Lethality of animals in the feeding experiments. The stage of development at which an animal died is depicted as a percentage of animals that died at that stage. `30-42', `54-66' and `78-' in brackets refer to the periods (AEL) at which animals were fed 20E-treated yeast pastes. `n' refers to the total number of animals.

 

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© The Company of Biologists Ltd 2006