Transcriptional Profiling Shows that BHR4 and E74A Are the Regulators of CPH33 and CPH34 Gene Expression

Abstract

Our endeavor is to identify with the basic system that regulates sequentially expressed cuticular protein genes around pupation. The present study was conducted to clarify the regulatory mechanism of a cuticular protein gene of Bombyx mori, BmorCPH33, whose expression in wing discs was atypical from others and peaked at wandering stage day 3 (W3) early to W3 late stage when ecdysteroid began to decrease before pupation. We compared the expression profiles of BmorCPH33 and BmorCPH34 to clarify their regulatory mechanism. Developmental profile of BmorCPH33 resembled BHR4, therefore we compared the expression profile of this transcription factor with that of BmorCPH33. BHR4 transcripts increased by the addition of 20E. BmorCPH33 expression was also induced by the addition of 20E, which was inhibited by cycloheximide. Both BHR4 and BmorCPH33 transcripts were induced after 6h 20E removal after exposure to 20E, but they were inhibited by the addition of cycloheximide. Thus, the results showed relatedness of BmorCPH33 and BHR4. Through this culture system it is suggested that BHR4 is a regulator of insect metamorphosis. The expression profiles of BmorCPH34 resembled those of E74A, therefore we compared the expression profiles of this transcription factor with BmorCPH34. BmorCPH34, E74A transcripts were up-regulated by the 20E pulse treatment. The results showed the relatedness of BmorCPH34 and E74A. The present findings showed different regulation of cuticular protein genes by ecdysone-responsive transcription factors at the pre-pupal stage of wing discs of B. mori.

Successively expressed cuticular protein genes at the prepupal stage in wing discs of Bombyx mori

“…Generally the epicuticle and the procuticle are the cuticular layers in the insect and later secreted to thick exo-cuticle and then endo-cuticle (Wigglesworth, 1972) and constructed by chitin and cuticle proteins with R&R consensus, and other types of cuticular protein constructed three cuticle layers. Correspondent with this, the clustering CP genes expressed around the molting stage were observed in B. mori (Okamoto, Futahashi, Kojima, Mita, & Fujiwara, 2008;Liang, Zhang, Xiang, & He, 2010;Ali, Iwanaga, & Kawasaki, 2013;Ali, Rahman, & Swapon, 2015;Ali, Mishra, Rahman, & Swapon, 2016;Ali, Hossain, & Mishra, 2018) and A. gambiae (Togawa et al 2008). Charles (2010) described about the regulation of expression of insect CP genes.…”

Transcriptional regulation of cuticular genes during insect metamorphosis

“…The peak expression of Group 2 in vivo is observed a little after the peak of Group 1 (44). Expression patterns of the "Group 3" genes BmorCPR34 and BmorCPH33 resemble the pattern of the ecdysone early late gene BHR4, the transcript levels of which increase early on the fourth day of the wandering stage (W3E) and show a broad expression peak around W3E and late in W3 (W3L) (44)(45)(46). Expression of "Group 4" genes (BmorCPG11, BmorCPG13, and BmorCPH30) increases from W0 and reaches a peak on W3L (44,47,48).…”

“…We challenged exposed wing discs with an ecdysone pulse in culture medium. Transcripts of Group 3 and Group 4 genes were induced by 6 h and reached their peak at 12h and 18h after the pulse, respectively (46,48). Group 5 genes were increased 12h after incubation, peaking at 18h (44,52).…”

“…The developmental expression pattern of BmorCPH5 indicates that it occurs after the expression peak of Group 1 and EcR, as shown in Figure 1. Developmental profiles and reporter assays also suggest that the expression of Group 3 CP genes BmorCPH33 and BmorCPR34 is regulated by BHR4 (44,59). Group 4 expression is believed to be regulated by BR-C, as the expression profiles of BmorCPG11, BmorCPG13, and BmorCPH30 are similar to the expression profile of BR-C, and a BR-C binding site is located upstream of BmorCPG11 and BmorCPG13 (47,48,60) (48 Ref3).…”