Holometabolous (complete-metamorphosis) insects alone possess a pupal stage, which Ureña et al. (2016) showed requires a specific crosstalk between three transcription factors: the juvenile-hormone-induced anti-metamorphic factor Krüppel-homolog 1 (Kr-h1), the pupal specifier Broad-Complex (Br-C), and the adult specifier E93. That paper left three mechanistic questions explicitly open: which proximal signal re-induces the prepupal Kr-h1 pulse; how Br-C escapes Kr-h1-mediated repression precisely at the prepupal stage; and what the molecular (cis-regulatory) basis of E93 induction is across hemimetabolous and holometabolous insects. Work published between 2016 and 2026 shows substantial but species-limited progress: in Drosophila, the prepupal Kr-h1 surge is directly driven by 20-hydroxyecdysone (20E) through an ecdysone response element, and two independent 2025 studies explained the Br-C escape (20E-opened positive Kr-h1 binding enhancers, and a JH-versus-20E-controlled SUMOylation switch on Kr-h1), while the Kr-h1-dependent repression arm of E93 regulation was mechanistically resolved in Bombyx and Locusta. The surviving open core is the in vivo signal for the prepupal Kr-h1 pulse in holometabolans other than Drosophila (notably Tribolium and Bombyx), the conservation of the Drosophila escape mechanisms across orders, and the enhancer-level logic of the activating inputs to E93, especially in hemimetabolous insects.

Background

Insects develop through two main modes. Hemimetabolous species (e.g. cockroaches, locusts) grow through nymphal instars that resemble the adult and molt directly into the adult; holometabolous species (e.g. beetles, moths, flies) pass through a larva, then a pupa, then the adult. The pupa is the defining innovation of complete metamorphosis, and its evolutionary origin from a hemimetabolan-like ancestor is a classic evo-devo question. Post-embryonic development in both modes is orchestrated by two hormones: juvenile hormone (JH), which maintains juvenile identity, and the steroid 20-hydroxyecdysone (20E), which triggers each molt and, at the end of juvenile life, metamorphosis. Downstream of these hormones operates a conserved transcription-factor circuit (the MEKRE93 pathway and the wider Metamorphic Gene Network): JH, through its receptor Methoprene-tolerant (Met) with its partner Taiman (Tai), induces Krüppel-homolog 1 (Kr-h1), a zinc-finger anti-metamorphic factor; 20E, through its nuclear receptor complex formed by the ecdysone receptor (EcR) and Ultraspiracle (USP), induces early-response genes including the pupal specifier Broad-Complex (Br-C, a BTB-zinc-finger transcription factor required for pupal morphogenesis) and E93 (Ecdysone-induced protein 93F, the adult specifier that triggers adult differentiation). A fourth BTB-zinc-finger factor, Chinmo (Chronologically inappropriate morphogenesis), acts as a larval specifier. Ureña et al. (2016), working mainly in the red flour beetle Tribolium castaneum with RNA interference (RNAi, double-stranded-RNA-mediated gene knockdown), established the stage-specific wiring that underlies the pupa: midway through the last larval instar, holometabolans transiently re-induce Kr-h1 (the prepupal Kr-h1 pulse, absent in hemimetabolous nymphs); this prepupal Kr-h1 represses E93, and the resulting low E93 permits the strong prepupal burst of Br-C (E93 being a potent Br-C repressor); the Br-C pulse then coordinates pupal morphogenesis. After pupal ecdysis, high E93 represses both Kr-h1 and Br-C to complete metamorphosis. The model implies that the pupa arose in evolution by redeploying this conserved hemimetabolan circuit through the transient Kr-h1 re-induction. Three mechanistic gaps were left open by that paper. First, the signal for the prepupal Kr-h1 pulse is unestablished: JH is the canonical Kr-h1 inducer, yet in Bombyx mori the prepupal Kr-h1 pulse persists after surgical removal of the JH-producing glands (corpora allata), and in Tribolium simultaneous knockdown of the two final JH-biosynthetic enzymes (JHAMT and CYP15A1) does not cause premature adult differentiation, so the proximal trigger (JH from other tissues, 20E, or another factor) remains unidentified. Second, a paradox: Kr-h1 directly represses Br-C in young larvae (Kr-h1 binds Kr-h1 binding sites, KBS, in the Br-C locus), yet at the prepupal stage Br-C is strongly induced despite the coincident Kr-h1 pulse, and forced Kr-h1 overexpression in prepupal Bombyx cannot block the Br-C pulse; the molecular basis of this stage-specific switch in Kr-h1's regulatory effect was unknown. Third, the signals that activate E93 expression were characterized only in Drosophila (direct induction by the prepupal 20E pulse; Baehrecke and Thummel 1995) and Bombyx, and the molecular basis of E93 regulation across other hemimetabolous and holometabolous insects was unexplored. Between 2016 and 2025, parts of these gaps were filled in Drosophila alone: the prepupal Kr-h1 surge in the wing disc was shown to be directly driven by 20E through an ecdysone response element, and the prepupal Br-C escape was explained by two mechanisms — 20E-induced H3K27 acetylation (an activating histone mark that renders enhancer chromatin accessible) at Br-C enhancers carrying positive Kr-h1-binding sites, and a JH/20E-controlled switch in the SUMOylation state of Kr-h1 (SUMOylation being the attachment of the small ubiquitin-like modifier SUMO to a protein, which changes its interaction partners). Whether these mechanisms operate outside Drosophila remains untested, and this non-drosophilid, cis-regulatory residue is what the present problem targets.

Problem Statement

Identify, at the molecular and endocrine level, the mechanisms that establish the prepupal state of the metamorphic gene network in holometabolous insects — the network whose Kr-h1-Br-C-E93 crosstalk Ureña et al. (2016) showed to be required for the pupal stage. Specifically: (1) determine the proximal signal(s) — endocrine (juvenile hormone, 20-hydroxyecdysone, or another hormone) or tissue-autonomous — and their in vivo timing that induce the transient prepupal re-expression of Kr-h1 in holometabolous insects beyond the Drosophila wing-disc case, in particular resolving why the prepupal Kr-h1 pulse in Bombyx mori is independent of the corpora allata and why blocking juvenile-hormone synthesis in Tribolium castaneum does not phenocopy Kr-h1 loss; (2) establish the molecular mechanism by which Broad-Complex is strongly induced at the prepupal stage despite the coincident Kr-h1 pulse — testing whether the two mechanisms demonstrated in Drosophila (20E-driven H3K27-acetylation-dependent opening of positive Kr-h1-binding enhancers of Br-C, and the JH/20E-controlled SUMOylation switch on Kr-h1) operate, alone or jointly, in other holometabolous insects, and if not, what replaces them; and (3) delineate the cis-regulatory basis of the activating inputs to E93 (which enhancers, hormone-response elements, and transcription factors drive its stage-specific surge at the onset of metamorphosis) and determine how these activating mechanisms, and the Kr-h1-dependent repression mechanism already characterized in Bombyx and Locusta, are conserved or diverged across hemimetabolous and holometabolous insects.

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