ReviewBR-C Z4 and FoxJ interact to regulate expression of a chitin synthase gene CHSA-2b in the pupal wing discs of the silkworm, Bombyx mori
Graphical abstract
Introduction
The stage- and tissue-specific expression of genes is important for insect morphogenesis. Ecdysteroids can regulate the stage- and cell-specific nuclear receptors and transcription factors to mediate insect development, such as midgut remodeling (Harker et al., 2013; Parthasarathy and Palli, 2007). In the Bombyx mori wing disc, there are 58 cuticle proteins specifically expressed during the prepupal stage (Ou et al., 2014). Many genes that are involved in chitin degradation and synthesis pathways are significantly up-regulated during the larva-pupa transition (Ou et al., 2014). Chitin is a polymer of N-acetylglucosamine and a component of insect epidermis, wings, trachea and the midgut peritrophic membrane (Merzendorfer, 2006; Moussian, 2010; Rezende et al., 2008; Tellam and Eisemann, 2000). Chitin synthase (CHS), including epidermal and midgut chitin synthase (CHSA and CHSB), catalyzes the synthesis of chitin. The RNA interference (RNAi) of CHSA in Drosophila melanogaster, Tribolium castaneum and Ostrinia furnacalis results in abnormal embryonic development (Ostrowski et al., 2002) and molting difficulty (Arakane et al., 2005; Qu and Yang, 2012). In some Lepidoptera, an extra alternative splicing in CHSA occurs in the second exon (Qu and Yang, 2012). CHSA-2a is expressed throughout growth and development, while CHSA-2b is only expressed in the wing disc at the mid-pupal stage (Xu et al., 2017). B. mori CHSA-2b RNAi resulted in the malfunction of wing development (Xu et al., 2017). These results indicate that stage- and tissue-specific genes are involved in successful development of insect tissues and organs.
During the pupal stage, insect wings composed of chitin and cuticle proteins develop along with extracellular matrix remodeling, cellular morphological changes and cuticle deposition (Zhang et al., 2013). Pupal development is regulated by the molting hormone 20-hydroxyecdysone (20E), which binds to a heterodimeric receptor complex consisting of Ecdysone Receptor (EcR) and Ultraspiracle (USP). The 20E/EcR/USP complex up-regulates ecdysis-associated primary response genes, such as Broad-Complex (BR-C), E75 and E74 (Burtis et al., 1990; DiBello et al., 1991; Segraves and Hogness, 1990). These early expressed transcription factors then activate the expression of secondary response genes, such as Ftz-F1, DHR3, DHR39 and E78B (Thummel, 2001), and enhance the expression of later expressed genes that are directly associated with molting and metamorphosis events. BR-C is a key member of the 20E regulatory pathway (Bayer et al., 1996). It encodes a Broad-Complex-Tramtrack-Bric-a-brac (BTB) domain protein with one of four alternatively spliced C2H2 zinc-finger motifs Z1-Z4 (DiBello et al., 1991; Konopova and Jindra, 2008). BR-C proteins have important regulatory roles during the larva-pupa transition and in sex determination in insects (Emery et al., 1994; Huet et al., 1993; Ito et al., 2013). In B. mori, many pupa-specific Wing Cuticle Proteins (WCPs) have been identified (Takeda et al., 2001), and 20E up-regulates the expression of WCP10 (Noji et al., 2003), WCP2 (Nita et al., 2009), cuticle protein GlyGlyTyr-repeat 1 (CPG1) (Suzuki et al., 2002) and WCP4 (Deng et al., 2012). The 20E-EcR/USP-BRC Z4-POUM2 is the pathway that regulates WCP4 expression during the prepupal stage (Deng et al., 2012, 2015). This indicates that pupal genes are up-regulated by 20E cascade signals. In addition to pupa-specific genes, non-stage-specific genes contribute to wing development, but their regulatory mechanism may be different. For example, in the B. mori pupa, two alternative splicing variants of epidermal chitin synthase, CHSA-2a and CHSA-2b, are expressed at different stages in response to a 20E pulse or 20E to ensure wing development (Xu et al., 2017). CHSA-2a is expressed in the epidermis and wing of larvae, pupae and adults, while CHSA-2b is expressed only in the wing during the mid-pupal stage.
How are these genes expressed at the pupal stage? In B. mori, the mechanism of stage- and tissue-specific expression of CHSA-2b shows that different rates of DNA methylation can result in different expression levels of CHSA-2b in the epidermis and wing disc. The down-regulated DNA methyltransferase (Dnmt1) allows the pupa-specific transcription factor Deaf1 to bind the same Dnmt1-bound position in the CHSA-2b promoter (Xu et al., 2018). In addition, the transcription factor forkhead box A (Fox A) inhibits pupa-specific WCP4 expression before the pupal stage by binding to the WCP4 promoter. Its down-regulation at the pupal stage indirectly enhances pupa-specific expression of WCP4 (Hu et al., 2019). The down-regulation of Dnmt1 and Fox A at pupal stage explains why CHSA-2b and WCP4 are expressed at the pupal stage of B. mori but not at the larval stage. However, it is unclear why these two 20E-induced genes are not expressed earlier when the 20E titer is high.
We studied the regulation of 20E-induced expression of CHSA-2b as an example of a pupal gene. We found that 20E-responsive FoxJ was inhibited by BR-C Z4 before the early pupal stage, but it was then up-regulated at the mid-pupal stage, thereby ensuring CHSA-2b expression. Our results show how a combination of 20E-pathway factors regulates pupa-specific gene expression.
Section snippets
Insects and cell line
B. mori strain Dazao was obtained from the Research and Development Center of the Sericultural Research Institute of the Academy of Agricultural Sciences of Guangdong Province, China. Larvae were reared on fresh mulberry leaves at 25 °C and a 12:12 h (L:D) photoperiod in an incubator located in a clean and temperature-controlled chamber.
The B. mori cell line (Bm12), originally developed from ovarian tissues (Khurad et al., 2009) was maintained at 28 °C in Grace medium (Invitrogen, Carlsbad, CA,
BR-C Z or Fox cis-regulatory element in the −734 to −716 nt region of the promoter enhancs CHSA-2b gene transcription
To investigate the 20E-related pathways regulating CHSA-2b expression in B. mori pupae, we analyzed the 20E-related cis-regulatory elements in the CHSA-2b promoter. Three known 20E-related cis-regulatory elements were predicated, including BR-C Z1 at −732 to −714 nt, BR-C Z4 or Fox at −734 to −716 nt, and E74A at −1658 to −1644 nt of the promoter. The sequence of −734 to −716 nt includes potential BR-C Z4 (taaaTAAAcaaa) and Fox (aaaTAAAcaaaca) cis-regulatory elements, where both cis-regulatory
Discussion
Wing disc development is a significant process in the holometabolous insect pupa. The wing disc consists of undifferentiated and proliferating cells. The disc undergoes dramatic morphological changes and eventually evaginates from the body to form the pupal wing disc (Klein, 2001). Pupal development is regulated by an ecdysteroid cascade, including 20E signaling pathway genes such as E74, E75, BR-C, Ftz and HR3 and other transcription factors (Burtis et al., 1990; DiBello et al., 1991; Segraves
Acknowledgments
This work was supported by the National High Technology Research and Development Program of China (863 Program) (No. 2011AA10A204-3).
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These authors equally contributed to the study.