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Catalogue of epidermal genes: genes expressed in the epidermis during larval molt of the silkworm Bombyx mori.

Okamoto S, Futahashi R, Kojima T, Mita K, Fujiwara H - BMC Genomics (2008)

Bottom Line: We have identified 1,380 genes in epM data set and 13 preferentially expressed genes in epidermis at the molt.The comparison of the epM and other EST libraries clarified the totally different gene expression patterns in epidermis between the molting and feeding stages and many novel tissue- and stage-specifically expressed epidermal genes.These data should further our understanding of cuticle formation and the insect molt.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8562, Japan. kk57516@mail.ecc.u-tokyo.ac.jp

ABSTRACT

Background: The insect cuticle is composed of various proteins and formed during the molt under hormonal regulation, although its precise composition and formation mechanism are largely unknown. The exhaustive catalogue of genes expressed in epidermis at the molt constitutes a massive amount of information from which to draw a complete picture of the molt and cuticle formation in insects. Therefore, we have catalogued a library of full-length cDNAs (designated epM) from epidermal cells during the last larval molt of Bombyx mori.

Results: Of the 10,368 sequences in the library, we isolated 6,653 usable expressed sequence tags (ESTs), which were categorized into 1,451 nonredundant gene clusters. Seventy-one clusters were considered to be isoforms or premature forms of other clusters. Therefore, we have identified 1,380 putative genes. Of the 6,653 expressed sequences, 48% were derived from 92 cuticular protein genes (RR-1, 24; RR-2, 17; glycine-rich, 29; other classes, 22). A comparison of epM with another epidermal EST data set, epV3 (feeding stage: fifth instar, day 3), showed marked differences in cuticular protein gene. Various types of cuticular proteins are expressed in epM but virtually only RR-1 proteins were expressed in epV3. Cuticular protein genes expressed specifically in epidermis, with several types of expression patterns during the molt, suggest different types of responses to the ecdysteroid pulse. Compared with other Bombyx EST libraries, 13 genes were preferentially included in epM data set. We isolated 290 genes for proteins other than cuticular proteins, whose amino acid sequences retain putative signal peptides, suggesting that they play some role in cuticle formation or in other molting events. Several gene groups were also included in this data set: hormone metabolism, P450, modifier of cuticular protein structure, small-ligand-binding protein, transcription factor, and pigmentation genes.

Conclusion: We have identified 1,380 genes in epM data set and 13 preferentially expressed genes in epidermis at the molt. The comparison of the epM and other EST libraries clarified the totally different gene expression patterns in epidermis between the molting and feeding stages and many novel tissue- and stage-specifically expressed epidermal genes. These data should further our understanding of cuticle formation and the insect molt.

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Stage and Tissue specific expression of epidermal genes isolated in epM data set by RT-PCR. (A) Expression profile of several cuticular protein genes by RT-PCR analysis. Relative hemolymph ecdysteroid titer is also shown according to Kiguchi and Agui, 1981 [20], and indicates the stage of epidermis (4A to 5A). The gene for ribosomal protein L3 (rpL3) was used as an internal control (see details in materials & methods). (B) Tissue specific expression of cuticular protein genes and preferentially expressed genes identified in epM data set. Four tissues (epidermis, fat body, hemolymph, and midgut) on fourth E1 stages were used.
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Figure 4: Stage and Tissue specific expression of epidermal genes isolated in epM data set by RT-PCR. (A) Expression profile of several cuticular protein genes by RT-PCR analysis. Relative hemolymph ecdysteroid titer is also shown according to Kiguchi and Agui, 1981 [20], and indicates the stage of epidermis (4A to 5A). The gene for ribosomal protein L3 (rpL3) was used as an internal control (see details in materials & methods). (B) Tissue specific expression of cuticular protein genes and preferentially expressed genes identified in epM data set. Four tissues (epidermis, fat body, hemolymph, and midgut) on fourth E1 stages were used.

Mentions: To determine the detailed expression profiles of several major cuticular protein genes during the last larval molt, we used semiquantitative RT-PCR analysis of epidermal mRNAs at eight developmental stages, A-E2 of the fourth instar, F (just after the fourth ecdysis), and A (day 3) of the fifth instar (Figure 4A, [20]). The expression of most cuticular protein genes is repressed at D1 with the peak of ecdysteroid during the molt, suggesting that a high dose of ecdysteriod suppresses their expression. The expression patterns of cuticular protein genes during the molt can be grouped into four divisions: first group, expressed after the decay of ecdysteroid, as well as in the intermolt phase (BmorCPR3, BmorCPR4, BmorCPR5, BmorCPR38, BmorCPR41, BmorCPR10, BmorCPR39, BmorCPG4, BmorCPG5 and BmorCPG26); second group, expressed after the decay of ecdysteroid with their disappearance at mid fifth instar (BmorCPR83a, BmorCPR91, BmorCPG1, BmorCPG8, and BmorCPG28); third group, not expressed during the molt (C1-E2) (BmorCPFL1a); and fourth group, ubiquitously expressed throughout the fourth and fifth instars (BmorCPR148). We speculate that the first and fourth classes are generally involved in thickening the cuticle layer, but that the second class of dominantly expressed genes during molt are mainly involved in newly synthesized cuticle formation. The second group seems to be induced by the decay of ecdysteroid. We have already reported that the expression of the second class gene BMCPG1 (BmorCPG1 in this study) is induced by an ecdysteroid pulse and may be controlled by the FtzF1 function [26], which supports the idea outlined above. It is noteworthy that the RR-2 and CPG proteins are mainly categorized in the second class, supporting the idea that these proteins form the outer layer of the cuticle, as proposed above.


Catalogue of epidermal genes: genes expressed in the epidermis during larval molt of the silkworm Bombyx mori.

Okamoto S, Futahashi R, Kojima T, Mita K, Fujiwara H - BMC Genomics (2008)

Stage and Tissue specific expression of epidermal genes isolated in epM data set by RT-PCR. (A) Expression profile of several cuticular protein genes by RT-PCR analysis. Relative hemolymph ecdysteroid titer is also shown according to Kiguchi and Agui, 1981 [20], and indicates the stage of epidermis (4A to 5A). The gene for ribosomal protein L3 (rpL3) was used as an internal control (see details in materials & methods). (B) Tissue specific expression of cuticular protein genes and preferentially expressed genes identified in epM data set. Four tissues (epidermis, fat body, hemolymph, and midgut) on fourth E1 stages were used.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2542385&req=5

Figure 4: Stage and Tissue specific expression of epidermal genes isolated in epM data set by RT-PCR. (A) Expression profile of several cuticular protein genes by RT-PCR analysis. Relative hemolymph ecdysteroid titer is also shown according to Kiguchi and Agui, 1981 [20], and indicates the stage of epidermis (4A to 5A). The gene for ribosomal protein L3 (rpL3) was used as an internal control (see details in materials & methods). (B) Tissue specific expression of cuticular protein genes and preferentially expressed genes identified in epM data set. Four tissues (epidermis, fat body, hemolymph, and midgut) on fourth E1 stages were used.
Mentions: To determine the detailed expression profiles of several major cuticular protein genes during the last larval molt, we used semiquantitative RT-PCR analysis of epidermal mRNAs at eight developmental stages, A-E2 of the fourth instar, F (just after the fourth ecdysis), and A (day 3) of the fifth instar (Figure 4A, [20]). The expression of most cuticular protein genes is repressed at D1 with the peak of ecdysteroid during the molt, suggesting that a high dose of ecdysteriod suppresses their expression. The expression patterns of cuticular protein genes during the molt can be grouped into four divisions: first group, expressed after the decay of ecdysteroid, as well as in the intermolt phase (BmorCPR3, BmorCPR4, BmorCPR5, BmorCPR38, BmorCPR41, BmorCPR10, BmorCPR39, BmorCPG4, BmorCPG5 and BmorCPG26); second group, expressed after the decay of ecdysteroid with their disappearance at mid fifth instar (BmorCPR83a, BmorCPR91, BmorCPG1, BmorCPG8, and BmorCPG28); third group, not expressed during the molt (C1-E2) (BmorCPFL1a); and fourth group, ubiquitously expressed throughout the fourth and fifth instars (BmorCPR148). We speculate that the first and fourth classes are generally involved in thickening the cuticle layer, but that the second class of dominantly expressed genes during molt are mainly involved in newly synthesized cuticle formation. The second group seems to be induced by the decay of ecdysteroid. We have already reported that the expression of the second class gene BMCPG1 (BmorCPG1 in this study) is induced by an ecdysteroid pulse and may be controlled by the FtzF1 function [26], which supports the idea outlined above. It is noteworthy that the RR-2 and CPG proteins are mainly categorized in the second class, supporting the idea that these proteins form the outer layer of the cuticle, as proposed above.

Bottom Line: We have identified 1,380 genes in epM data set and 13 preferentially expressed genes in epidermis at the molt.The comparison of the epM and other EST libraries clarified the totally different gene expression patterns in epidermis between the molting and feeding stages and many novel tissue- and stage-specifically expressed epidermal genes.These data should further our understanding of cuticle formation and the insect molt.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8562, Japan. kk57516@mail.ecc.u-tokyo.ac.jp

ABSTRACT

Background: The insect cuticle is composed of various proteins and formed during the molt under hormonal regulation, although its precise composition and formation mechanism are largely unknown. The exhaustive catalogue of genes expressed in epidermis at the molt constitutes a massive amount of information from which to draw a complete picture of the molt and cuticle formation in insects. Therefore, we have catalogued a library of full-length cDNAs (designated epM) from epidermal cells during the last larval molt of Bombyx mori.

Results: Of the 10,368 sequences in the library, we isolated 6,653 usable expressed sequence tags (ESTs), which were categorized into 1,451 nonredundant gene clusters. Seventy-one clusters were considered to be isoforms or premature forms of other clusters. Therefore, we have identified 1,380 putative genes. Of the 6,653 expressed sequences, 48% were derived from 92 cuticular protein genes (RR-1, 24; RR-2, 17; glycine-rich, 29; other classes, 22). A comparison of epM with another epidermal EST data set, epV3 (feeding stage: fifth instar, day 3), showed marked differences in cuticular protein gene. Various types of cuticular proteins are expressed in epM but virtually only RR-1 proteins were expressed in epV3. Cuticular protein genes expressed specifically in epidermis, with several types of expression patterns during the molt, suggest different types of responses to the ecdysteroid pulse. Compared with other Bombyx EST libraries, 13 genes were preferentially included in epM data set. We isolated 290 genes for proteins other than cuticular proteins, whose amino acid sequences retain putative signal peptides, suggesting that they play some role in cuticle formation or in other molting events. Several gene groups were also included in this data set: hormone metabolism, P450, modifier of cuticular protein structure, small-ligand-binding protein, transcription factor, and pigmentation genes.

Conclusion: We have identified 1,380 genes in epM data set and 13 preferentially expressed genes in epidermis at the molt. The comparison of the epM and other EST libraries clarified the totally different gene expression patterns in epidermis between the molting and feeding stages and many novel tissue- and stage-specifically expressed epidermal genes. These data should further our understanding of cuticle formation and the insect molt.

Show MeSH
Related in: MedlinePlus