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Bombyx E75 isoforms display stage- and tissue-specific responses to 20-hydroxyecdysone.

Li K, Guo E, Hossain MS, Li Q, Cao Y, Tian L, Deng X, Li S - Sci Rep (2015)

Bottom Line: At the early 5(th) larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E.At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues.In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level.

View Article: PubMed Central - PubMed

Affiliation: 1] Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding/Guangdong Provincial Sericulture and Mulberry Engineering Research Center, College of Animal Sciences, South China Agricultural University, Guangzhou, 510642, China [2] Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.

ABSTRACT
Resulted from alternative splicing of the 5' exons, the nuclear receptor gene E75 in the silkworm, Bombyx mori, processes three mRNA isoforms, BmE75A, BmE75B and BmE75C. From the early 5(th) larval instar to the prepupal stages, BmE75A mRNA and protein levels in the prothoracic glands display developmental profiles similar to ecdysteroid titer. In the fat body, mRNA levels but not protein levels of all three BmE75 isoforms correlate with ecdysteroid titer; moreover, proteins of all three BmE75 isoforms disappear at the prepupal stages, and a modified BmE75 protein with smaller molecular weight and cytoplasm localization occurs. At the early 5(th) larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E. At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues. In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level.

No MeSH data available.


Developmental profiles of BmE75 in fat body.(A-A”) Developmental profiles of mRNA levels of BmE75A (A), BmE75B (A’) and BmE75C (A”) in the fat body from day 2 of the 5th larval instar (L5D2) to day 2 of the prepupal stage (PP2). L, instar; D, day; EW, early wandering; LW, late wandering; PP, prepupal; P, pupa. (B-B’) Developmental profiles of protein levels of BmE75A, BmE75B and BmE75C in the fat body from L5D2 to P5. The full-length blots are presented in Supplementary Figure 2. (B’) Quantification of BmE75A, BmE75B and BmE75C protein levels in (B). Location of BmE75 proteins in the fat body is detected by immunohistochemistry using the BmE75 antibody from EW to PP2 stage (green). The white box at top-right corner of inset in (L5D2) is merged with DAPI (blue) staining. (D-D”’) dsRNA targeting a common region of all three BmE75 isoforms was injected into Bombyx larvae at EW, and the fat body was isolated 48 hours after treatment. EGFP dsRNA was used as a control. mRNA levels of BmE75A, BmE75B and BmE75C were detected using isoform-specific primers by qPCR (D). Western blotting (D’,D”) and immunohistochemistry (D”’) were performed to detect the small molecular weight BmE75-like protein in the fat body using the BmE75 antibody (green). The white box at top-right corner of inset in (EGFP RNAi) in (D”’) is merged with DAPI (blue) staining.
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f5: Developmental profiles of BmE75 in fat body.(A-A”) Developmental profiles of mRNA levels of BmE75A (A), BmE75B (A’) and BmE75C (A”) in the fat body from day 2 of the 5th larval instar (L5D2) to day 2 of the prepupal stage (PP2). L, instar; D, day; EW, early wandering; LW, late wandering; PP, prepupal; P, pupa. (B-B’) Developmental profiles of protein levels of BmE75A, BmE75B and BmE75C in the fat body from L5D2 to P5. The full-length blots are presented in Supplementary Figure 2. (B’) Quantification of BmE75A, BmE75B and BmE75C protein levels in (B). Location of BmE75 proteins in the fat body is detected by immunohistochemistry using the BmE75 antibody from EW to PP2 stage (green). The white box at top-right corner of inset in (L5D2) is merged with DAPI (blue) staining. (D-D”’) dsRNA targeting a common region of all three BmE75 isoforms was injected into Bombyx larvae at EW, and the fat body was isolated 48 hours after treatment. EGFP dsRNA was used as a control. mRNA levels of BmE75A, BmE75B and BmE75C were detected using isoform-specific primers by qPCR (D). Western blotting (D’,D”) and immunohistochemistry (D”’) were performed to detect the small molecular weight BmE75-like protein in the fat body using the BmE75 antibody (green). The white box at top-right corner of inset in (EGFP RNAi) in (D”’) is merged with DAPI (blue) staining.

Mentions: The fat body is an organ analogue to vertebrate adipose tissue and liver and functions as a major organ for nutrient storage and energy metabolism in insects27. BmE75A mRNA level in the fat body is under detectable during the feeding larval stages, and reaches a small peak on L5D7 and a very high peak on PP1 and PP2 (Fig. 5A). BmE75B and BmE75C mRNA levels show similar developmental profiles to BmE75A mRNA level, while they gradually increase from L5D2 to L5D6 (Fig. 5A’). The results show that, in the fat body, mRNA levels of all three BmE75 isoforms correlate with ecdysteroid titer.


Bombyx E75 isoforms display stage- and tissue-specific responses to 20-hydroxyecdysone.

Li K, Guo E, Hossain MS, Li Q, Cao Y, Tian L, Deng X, Li S - Sci Rep (2015)

Developmental profiles of BmE75 in fat body.(A-A”) Developmental profiles of mRNA levels of BmE75A (A), BmE75B (A’) and BmE75C (A”) in the fat body from day 2 of the 5th larval instar (L5D2) to day 2 of the prepupal stage (PP2). L, instar; D, day; EW, early wandering; LW, late wandering; PP, prepupal; P, pupa. (B-B’) Developmental profiles of protein levels of BmE75A, BmE75B and BmE75C in the fat body from L5D2 to P5. The full-length blots are presented in Supplementary Figure 2. (B’) Quantification of BmE75A, BmE75B and BmE75C protein levels in (B). Location of BmE75 proteins in the fat body is detected by immunohistochemistry using the BmE75 antibody from EW to PP2 stage (green). The white box at top-right corner of inset in (L5D2) is merged with DAPI (blue) staining. (D-D”’) dsRNA targeting a common region of all three BmE75 isoforms was injected into Bombyx larvae at EW, and the fat body was isolated 48 hours after treatment. EGFP dsRNA was used as a control. mRNA levels of BmE75A, BmE75B and BmE75C were detected using isoform-specific primers by qPCR (D). Western blotting (D’,D”) and immunohistochemistry (D”’) were performed to detect the small molecular weight BmE75-like protein in the fat body using the BmE75 antibody (green). The white box at top-right corner of inset in (EGFP RNAi) in (D”’) is merged with DAPI (blue) staining.
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Related In: Results  -  Collection

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Show All Figures
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f5: Developmental profiles of BmE75 in fat body.(A-A”) Developmental profiles of mRNA levels of BmE75A (A), BmE75B (A’) and BmE75C (A”) in the fat body from day 2 of the 5th larval instar (L5D2) to day 2 of the prepupal stage (PP2). L, instar; D, day; EW, early wandering; LW, late wandering; PP, prepupal; P, pupa. (B-B’) Developmental profiles of protein levels of BmE75A, BmE75B and BmE75C in the fat body from L5D2 to P5. The full-length blots are presented in Supplementary Figure 2. (B’) Quantification of BmE75A, BmE75B and BmE75C protein levels in (B). Location of BmE75 proteins in the fat body is detected by immunohistochemistry using the BmE75 antibody from EW to PP2 stage (green). The white box at top-right corner of inset in (L5D2) is merged with DAPI (blue) staining. (D-D”’) dsRNA targeting a common region of all three BmE75 isoforms was injected into Bombyx larvae at EW, and the fat body was isolated 48 hours after treatment. EGFP dsRNA was used as a control. mRNA levels of BmE75A, BmE75B and BmE75C were detected using isoform-specific primers by qPCR (D). Western blotting (D’,D”) and immunohistochemistry (D”’) were performed to detect the small molecular weight BmE75-like protein in the fat body using the BmE75 antibody (green). The white box at top-right corner of inset in (EGFP RNAi) in (D”’) is merged with DAPI (blue) staining.
Mentions: The fat body is an organ analogue to vertebrate adipose tissue and liver and functions as a major organ for nutrient storage and energy metabolism in insects27. BmE75A mRNA level in the fat body is under detectable during the feeding larval stages, and reaches a small peak on L5D7 and a very high peak on PP1 and PP2 (Fig. 5A). BmE75B and BmE75C mRNA levels show similar developmental profiles to BmE75A mRNA level, while they gradually increase from L5D2 to L5D6 (Fig. 5A’). The results show that, in the fat body, mRNA levels of all three BmE75 isoforms correlate with ecdysteroid titer.

Bottom Line: At the early 5(th) larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E.At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues.In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level.

View Article: PubMed Central - PubMed

Affiliation: 1] Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding/Guangdong Provincial Sericulture and Mulberry Engineering Research Center, College of Animal Sciences, South China Agricultural University, Guangzhou, 510642, China [2] Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.

ABSTRACT
Resulted from alternative splicing of the 5' exons, the nuclear receptor gene E75 in the silkworm, Bombyx mori, processes three mRNA isoforms, BmE75A, BmE75B and BmE75C. From the early 5(th) larval instar to the prepupal stages, BmE75A mRNA and protein levels in the prothoracic glands display developmental profiles similar to ecdysteroid titer. In the fat body, mRNA levels but not protein levels of all three BmE75 isoforms correlate with ecdysteroid titer; moreover, proteins of all three BmE75 isoforms disappear at the prepupal stages, and a modified BmE75 protein with smaller molecular weight and cytoplasm localization occurs. At the early 5(th) larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E. At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues. In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level.

No MeSH data available.