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Transcriptomic analysis of developmental features of Bombyx mori wing disc during metamorphosis.

Ou J, Deng HM, Zheng SC, Huang LH, Feng QL, Liu L - BMC Genomics (2014)

Bottom Line: Seventeen transcription factors were significantly up-regulated.Genes responsible for the degradation and de novo synthesis of chitin were significantly up-regulated.These results implied that during the larva-to-pupa metamorphosis, pupal wing development and transition might be mainly controlled by 20E signaling in B. mori.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Developmental Entomology, Guangdong Provincial Key Lab of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou 510631, China. qlfeng@scnu.edu.cn.

ABSTRACT

Background: Wing discs of B. mori are transformed to pupal wings during the larva-to-pupa metamorphosis with dramatic morphological and structural changes. To understand these changes at a transcriptional level, RNA-seq of the wing discs from 6-day-old fifth instar larvae (L5D6), prepupae (PP) and pupae (P0) was performed.

Results: In total, 12,254 transcripts were obtained from the wing disc, out of which 5,287 were identified to be differentially expressed from L5D6 to PP and from PP to P0. The results of comprehensive analysis of RNA-seq data showed that during larvae-to-pupae metamorphosis, many genes of 20E signaling pathway were up-regulated and those of JH signaling pathway were down-regulated. Seventeen transcription factors were significantly up-regulated. Cuticle protein genes (especially wing cuticle protein genes), were most abundant and significantly up-regulated at P0 stage. Genes responsible for the degradation and de novo synthesis of chitin were significantly up-regulated. There were A and B two types of chitin synthases in B. mori, whereas only chitin synthase A was up-regulated. Both trehalose and D-fructose, which are precursors of chitin synthesis, were detected in the hemolymph of L5D6, PP and P0, suggesting de novo synthesis of chitin. However, most of the genes that are related to early wing disc differentiation were down-regulated.

Conclusions: Extensive transcriptome and DGE profiling data of wing disc during metamorphosis of silkworm have been generated, which provided comprehensive gene expression information at the transcriptional level. These results implied that during the larva-to-pupa metamorphosis, pupal wing development and transition might be mainly controlled by 20E signaling in B. mori. The 17 up-regulated transcription factors might be involved in wing development. Chitin required for pupal wing development might be generated from both degradation of componential chitin and de novo synthesis. Chitin synthase A might be responsible for the chitin synthesis in the pupal wing, while both trehalose and D-fructose might contribute to the de novo synthesis of chitin during the formation of pupal wing.

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Expression of the selected 12 transcription factors by qRT-PCR. Expression patterns of the selected 12 transcription factors in the wing disc at indicated six developmental stages, which are 3-day-old fifth instar larvae (L5D3), L5D6, 1-day-old wandering (W1), PP, P0 and 3-day-old pupae (P3). (A) Bm_nscaf2589_261 (probable nuclear hormone receptor HR38). (B) Bm_nscaf1898_501 (D-ETS-4-like isoform X2). (C) Bm_nscaf2876_46 (bHLH protein 15-like). (D) Bm_nscaf2847_349 (protein escargot-like). (E) Bm_nscaf2888_249 (nuclear receptor GRF). (F) Bm_nscaf2902_040 (helix-loop-helix protein delilah-like). (G) Bm_nscaf3090_4 (chorion specific C/EBP). (H) Bm_scaffold316_2 (protein embryonic gonad-like). (I) Bm_nscaf1898_212 (ets DNA-binding protein pokkuri-like). (J) Bm_nscaf2770_58 (transcription factor SOX-4-like). (K) Bm_nscaf2847_110 (Krueppel-like factor 10-like). (L) Bm_nscaf2930_060 (enhancer of split mbeta). The relative expression levels were normalized to the Bmβ-actin levels. The values are mean ± SEM (n = 3). Different letters indicate statistical significance (p < 0.05).
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Fig4: Expression of the selected 12 transcription factors by qRT-PCR. Expression patterns of the selected 12 transcription factors in the wing disc at indicated six developmental stages, which are 3-day-old fifth instar larvae (L5D3), L5D6, 1-day-old wandering (W1), PP, P0 and 3-day-old pupae (P3). (A) Bm_nscaf2589_261 (probable nuclear hormone receptor HR38). (B) Bm_nscaf1898_501 (D-ETS-4-like isoform X2). (C) Bm_nscaf2876_46 (bHLH protein 15-like). (D) Bm_nscaf2847_349 (protein escargot-like). (E) Bm_nscaf2888_249 (nuclear receptor GRF). (F) Bm_nscaf2902_040 (helix-loop-helix protein delilah-like). (G) Bm_nscaf3090_4 (chorion specific C/EBP). (H) Bm_scaffold316_2 (protein embryonic gonad-like). (I) Bm_nscaf1898_212 (ets DNA-binding protein pokkuri-like). (J) Bm_nscaf2770_58 (transcription factor SOX-4-like). (K) Bm_nscaf2847_110 (Krueppel-like factor 10-like). (L) Bm_nscaf2930_060 (enhancer of split mbeta). The relative expression levels were normalized to the Bmβ-actin levels. The values are mean ± SEM (n = 3). Different letters indicate statistical significance (p < 0.05).

Mentions: Transcription factors play critical roles in regulation of expression of their target genes. To identify transcription factors that may regulate the development of B. mori wing discs during metamorphosis, transcription factors were identified by KEGG analysis. In total, 147 out of 12,254 assembled transcripts were identified as transcription factors, 60 of which were differentially expressed during metamorphosis. Among the 60 differentially expressed transcription factors, 17 were up-regulated at PP and/or P0 stage (Table 3). Some of them were selected for confirmation by qRT-PCR analysis. The results showed that the transcription factors Bm_nscaf2589_261 (hormone receptor HR38), Bm_nscaf1898_501 (D-ETS-4-like isoform X2), Bm_nscaf2876_46 (bHLH protein 15-like), Bm_nscaf2888_249 (nuclear receptor GRF), Bm_nscaf2902_040 (protein delilah-like), Bm_nscaf3090_4 (chorion specific C/EBP) and Bm_scaffold316_2 (protein embryonic gonad-like) were significantly up-regulated at PP stage, which were well consistent with the RNA-Seq results (Table 3 and Figure 4A-C, E-H). The transcription factors Bm_nscaf1898_212 (protein pokkuri-like), Bm_nscaf2770_58 (SOX-4-like), Bm_nscaf2847_110 (Krueppel-like factor 10-like) and Bm_nscaf2930_060 (enhancer of split mbeta) were significantly up-regulated at P0 stage, which were also consistent with its RNA-Seq results (Table 3 and Figure 4I-L). The transcription factor Bm_nscaf2847_349 (protein escargot-like) was highly expressed at W1 stage, which was not consistent with the RNA-Seq result (Table 3 and Figure 4D). The qRT-PCR results showed that the RNA-Seq results were generally consistent. Bm_nscaf2859_001 (E75 isoform B), Bm_nscaf2964_066 (hormone receptor 3C) and Bm_nscaf2888_433 (transcription factor E74) were significantly up-regulated at PP or P0 stage (Table 3), which was consisted with the reported results[43, 44]. ASH2 was essential for scales formation in B. mori wing during pupal stage[45] and we found this transcription factor (Bm_nscaf1898_167) was significantly up-regulated at P0 stage. The assembled nucleotide sequences of transcripts (RNA-seq ID) in Table 3 were provided in Additional file5, and primers for qRT-PCR were provided in Additional file6.Table 3


Transcriptomic analysis of developmental features of Bombyx mori wing disc during metamorphosis.

Ou J, Deng HM, Zheng SC, Huang LH, Feng QL, Liu L - BMC Genomics (2014)

Expression of the selected 12 transcription factors by qRT-PCR. Expression patterns of the selected 12 transcription factors in the wing disc at indicated six developmental stages, which are 3-day-old fifth instar larvae (L5D3), L5D6, 1-day-old wandering (W1), PP, P0 and 3-day-old pupae (P3). (A) Bm_nscaf2589_261 (probable nuclear hormone receptor HR38). (B) Bm_nscaf1898_501 (D-ETS-4-like isoform X2). (C) Bm_nscaf2876_46 (bHLH protein 15-like). (D) Bm_nscaf2847_349 (protein escargot-like). (E) Bm_nscaf2888_249 (nuclear receptor GRF). (F) Bm_nscaf2902_040 (helix-loop-helix protein delilah-like). (G) Bm_nscaf3090_4 (chorion specific C/EBP). (H) Bm_scaffold316_2 (protein embryonic gonad-like). (I) Bm_nscaf1898_212 (ets DNA-binding protein pokkuri-like). (J) Bm_nscaf2770_58 (transcription factor SOX-4-like). (K) Bm_nscaf2847_110 (Krueppel-like factor 10-like). (L) Bm_nscaf2930_060 (enhancer of split mbeta). The relative expression levels were normalized to the Bmβ-actin levels. The values are mean ± SEM (n = 3). Different letters indicate statistical significance (p < 0.05).
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Related In: Results  -  Collection

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Fig4: Expression of the selected 12 transcription factors by qRT-PCR. Expression patterns of the selected 12 transcription factors in the wing disc at indicated six developmental stages, which are 3-day-old fifth instar larvae (L5D3), L5D6, 1-day-old wandering (W1), PP, P0 and 3-day-old pupae (P3). (A) Bm_nscaf2589_261 (probable nuclear hormone receptor HR38). (B) Bm_nscaf1898_501 (D-ETS-4-like isoform X2). (C) Bm_nscaf2876_46 (bHLH protein 15-like). (D) Bm_nscaf2847_349 (protein escargot-like). (E) Bm_nscaf2888_249 (nuclear receptor GRF). (F) Bm_nscaf2902_040 (helix-loop-helix protein delilah-like). (G) Bm_nscaf3090_4 (chorion specific C/EBP). (H) Bm_scaffold316_2 (protein embryonic gonad-like). (I) Bm_nscaf1898_212 (ets DNA-binding protein pokkuri-like). (J) Bm_nscaf2770_58 (transcription factor SOX-4-like). (K) Bm_nscaf2847_110 (Krueppel-like factor 10-like). (L) Bm_nscaf2930_060 (enhancer of split mbeta). The relative expression levels were normalized to the Bmβ-actin levels. The values are mean ± SEM (n = 3). Different letters indicate statistical significance (p < 0.05).
Mentions: Transcription factors play critical roles in regulation of expression of their target genes. To identify transcription factors that may regulate the development of B. mori wing discs during metamorphosis, transcription factors were identified by KEGG analysis. In total, 147 out of 12,254 assembled transcripts were identified as transcription factors, 60 of which were differentially expressed during metamorphosis. Among the 60 differentially expressed transcription factors, 17 were up-regulated at PP and/or P0 stage (Table 3). Some of them were selected for confirmation by qRT-PCR analysis. The results showed that the transcription factors Bm_nscaf2589_261 (hormone receptor HR38), Bm_nscaf1898_501 (D-ETS-4-like isoform X2), Bm_nscaf2876_46 (bHLH protein 15-like), Bm_nscaf2888_249 (nuclear receptor GRF), Bm_nscaf2902_040 (protein delilah-like), Bm_nscaf3090_4 (chorion specific C/EBP) and Bm_scaffold316_2 (protein embryonic gonad-like) were significantly up-regulated at PP stage, which were well consistent with the RNA-Seq results (Table 3 and Figure 4A-C, E-H). The transcription factors Bm_nscaf1898_212 (protein pokkuri-like), Bm_nscaf2770_58 (SOX-4-like), Bm_nscaf2847_110 (Krueppel-like factor 10-like) and Bm_nscaf2930_060 (enhancer of split mbeta) were significantly up-regulated at P0 stage, which were also consistent with its RNA-Seq results (Table 3 and Figure 4I-L). The transcription factor Bm_nscaf2847_349 (protein escargot-like) was highly expressed at W1 stage, which was not consistent with the RNA-Seq result (Table 3 and Figure 4D). The qRT-PCR results showed that the RNA-Seq results were generally consistent. Bm_nscaf2859_001 (E75 isoform B), Bm_nscaf2964_066 (hormone receptor 3C) and Bm_nscaf2888_433 (transcription factor E74) were significantly up-regulated at PP or P0 stage (Table 3), which was consisted with the reported results[43, 44]. ASH2 was essential for scales formation in B. mori wing during pupal stage[45] and we found this transcription factor (Bm_nscaf1898_167) was significantly up-regulated at P0 stage. The assembled nucleotide sequences of transcripts (RNA-seq ID) in Table 3 were provided in Additional file5, and primers for qRT-PCR were provided in Additional file6.Table 3

Bottom Line: Seventeen transcription factors were significantly up-regulated.Genes responsible for the degradation and de novo synthesis of chitin were significantly up-regulated.These results implied that during the larva-to-pupa metamorphosis, pupal wing development and transition might be mainly controlled by 20E signaling in B. mori.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular and Developmental Entomology, Guangdong Provincial Key Lab of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou 510631, China. qlfeng@scnu.edu.cn.

ABSTRACT

Background: Wing discs of B. mori are transformed to pupal wings during the larva-to-pupa metamorphosis with dramatic morphological and structural changes. To understand these changes at a transcriptional level, RNA-seq of the wing discs from 6-day-old fifth instar larvae (L5D6), prepupae (PP) and pupae (P0) was performed.

Results: In total, 12,254 transcripts were obtained from the wing disc, out of which 5,287 were identified to be differentially expressed from L5D6 to PP and from PP to P0. The results of comprehensive analysis of RNA-seq data showed that during larvae-to-pupae metamorphosis, many genes of 20E signaling pathway were up-regulated and those of JH signaling pathway were down-regulated. Seventeen transcription factors were significantly up-regulated. Cuticle protein genes (especially wing cuticle protein genes), were most abundant and significantly up-regulated at P0 stage. Genes responsible for the degradation and de novo synthesis of chitin were significantly up-regulated. There were A and B two types of chitin synthases in B. mori, whereas only chitin synthase A was up-regulated. Both trehalose and D-fructose, which are precursors of chitin synthesis, were detected in the hemolymph of L5D6, PP and P0, suggesting de novo synthesis of chitin. However, most of the genes that are related to early wing disc differentiation were down-regulated.

Conclusions: Extensive transcriptome and DGE profiling data of wing disc during metamorphosis of silkworm have been generated, which provided comprehensive gene expression information at the transcriptional level. These results implied that during the larva-to-pupa metamorphosis, pupal wing development and transition might be mainly controlled by 20E signaling in B. mori. The 17 up-regulated transcription factors might be involved in wing development. Chitin required for pupal wing development might be generated from both degradation of componential chitin and de novo synthesis. Chitin synthase A might be responsible for the chitin synthesis in the pupal wing, while both trehalose and D-fructose might contribute to the de novo synthesis of chitin during the formation of pupal wing.

Show MeSH
Related in: MedlinePlus