The Occurrence of the Holometabolous Pupal Stage Requires the Interaction between E93, Krüppel-Homolog 1 and Broad-Complex

Abstract

Complete metamorphosis (Holometaboly) is a key innovation that underlies the spectacular success of holometabolous insects. Phylogenetic analyses indicate that Holometabola form a monophyletic group that evolved from ancestors exhibiting hemimetabolous development (Hemimetaboly). However, the nature of the changes underlying this crucial transition, including the occurrence of the holometabolan-specific pupal stage, is poorly understood. Using the holometabolous beetle Tribolium castaneum as a model insect, here we show that the transient up-regulation of the anti-metamorphic Krüppel-homolog 1 (TcKr-h1) gene at the end of the last larval instar is critical in the formation of the pupa. We find that depletion of this specific TcKr-h1 peak leads to the precocious up-regulation of the adult-specifier factor TcE93 and, hence, to a direct transformation of the larva into the adult form, bypassing the pupal stage. Moreover, we also find that the TcKr-h1-dependent repression of TcE93 is critical to allow the strong up-regulation of Broad-complex (TcBr-C), a key transcription factor that regulates the correct formation of the pupa in holometabolous insects. Notably, we show that the genetic interaction between Kr-h1 and E93 is also present in the penultimate nymphal instar of the hemimetabolous insect Blattella germanica, suggesting that the evolution of the pupa has been facilitated by the co-option of regulatory mechanisms present in hemimetabolan metamorphosis. Our findings, therefore, contribute to the molecular understanding of insect metamorphosis, and indicate the evolutionary conservation of the genetic circuitry that controls hemimetabolan and holometabolan metamorphosis, thereby shedding light on the evolution of complete metamorphosis.

Molecular mechanism underlying juvenile hormone-mediated repression of precocious larval–adult metamorphosis

“…E93 gene expression during the pupal stage functions as an adult specifier and plays a key role in inducing programmed cell death necessary for tissue remodeling (30)(31)(32)(33). Genetic studies have revealed that during the larval stage, JH suppresses E93 expression to prevent larvae from undergoing precocious metamorphosis to adults (34)(35)(36). In the present study, we clarified the molecular mechanism underlying the repression of E93 by JH.…”

“…These residues in Kr-h1 are highly conserved in other insect species (15), suggesting that Kr-h1 homologs in other insects may also recognize the consensus KBS. Previous genetic analyses in some insects demonstrated that Kr-h1 represses BR-C and/or E93 (25,35,36). Therefore, the function and mechanism by which Kr-h1 represses the transcription of E93 and/or BR-C are essentially shared by a wide variety of insects.…”

“…A recent study using RNAi in T. castaneum showed that a peak of Kr-h1 in the prepupal stage suppressed premature E93 induction (36). However, because all previous studies were performed using whole individuals, it remains unknown whether the suppression of E93 by JH-induced Kr-h1 is a cell-autonomous event.…”

“…The pupal specifier BR-C is differentially regulated by Kr-h1 in holometabolous insects; it is induced in response to JHmediated Kr-h1 expression at the prepupal and pupal stages, whereas Kr-h1 represses BR-C during the larval stage (17,18,24,25,28,36). The transcription factor CSL [CBF1/Su(H)/Lag-1] recruits a corepressor complex in the absence of Notch signaling to repress its target genes (51); however, Notch signaling converts the CSL repressor complex to an activator complex, resulting in transcriptional activation of the target genes (51).…”

“…Moreover, it was first reported in B. germanica that JH-inducible Kr-h1 suppressed E93 transcript levels (35), and this effect was also observed in D. melanogaster and T. castaneum (36); however, these findings were obtained mainly by RNAi-based genetic analyses using individual insects, and therefore the molecular mechanism underlying JH-mediated repression of E93 in the cells remains poorly understood. In particular, it is unknown whether Kr-h1 represses E93 transcription directly or indirectly, and how Kr-h1 represses the transcript of E93.…”