Description

During the course of the Drosophila development, metabolism readily adapts to meet the energetic and biosynthetic demands of each life stage (Gillette et al., 2021). This relationship between growth and metabolism is particularly apparent when examining changes in glycolytic gene expression. Approximately midway through embryonic development, transcripts representing nearly every enzyme involved in glycolysis are coordinately up-regulated (Abu-Shumays and Fristrom, 1997; Currie and Sullivan, 1994a, b; Madhavan et al., 1972; Roselli-Rehfuss et al., 1992; Shaw-Lee et al., 1992; Shaw-Lee et al., 1991; Sun et al., 1988; Tennessen et al., 2011; Tennessen et al., 2014; Tixier et al., 2013; Wright and Shaw, 1970). The resulting glycolytic program is maintained throughout the larval growth period and subsequently down-regulated prior to the onset of metamorphosis (White et al., 1999). These metabolic transitions are highly predictable and provide an opportunity to understand how metabolism adapts to meet the energetic demands of insect development.

Activation of glycolytic metabolism in Drosophila embryos depends on the Drosophila Estrogen-Related Receptor (dERR; FBgn0035849), which represents the sole fly ortholog of the orphan class of ERR nuclear receptors (Ostberg et al., 2003). In embryos cultured at 25ºC, dERR becomes transcriptionally active ~10-12 hours after oviposition, resulting in the coordinate up-regulation of genes involved in carbohydrate metabolism (Tennessen et al., 2011). The mechanisms that temporally regulate dERR activity during this metabolic switch, however, remain unknown and additional factors could be controlling embryonic glycolytic metabolism independent of dERR. In this regard, both dERR activation and onset of glycolytic gene expression correlate with the embryonic pulse of the steroid hormone 20-hydroxyecdysone (20E), which triggers dorsal closure, cuticle deposition, head involution, and a variety of other developmental events (Chavez et al., 2000; Kozlova and Thummel, 2003; Maróy et al., 1988; Warren et al., 2002). Moreover, not only is 20E known to induce Lactate Dehydrogenase (Ldh; FBgn0001258) expression in imaginal discs (Abu-Shumays and Fristrom, 1997), but the Ecdysone Receptor (EcR; FBgn0000546) and dERR are also reported to cooperatively regulate expression of glycolytic genes in insect cell culture (Kovalenko et al., 2019), thus raising the possibility that 20E-signaling is necessary for the up-regulation of embryonic carbohydrate metabolism.

To test the hypothesis that 20E transcriptionally regulates glycolysis in Drosophila embryos, I used developmental northern blots to analyze expression of key glycolytic genes in shadow (sad; FBgn0003312) mutants, which are unable to synthesize either ecdysone or 20E (Warren et al., 2002). As a positive control, I first analyzed expression of DHR3 (FBgn0000448), which is up-regulated in response to embryonic 20E production (Ruaud et al., 2010). Unlike w¹¹¹⁸ control samples that expressed DHR3 during mid-embryogenesis, DHR3 transcripts were undetectable in sad mutant samples – a result consistent with lack of 20E production in these embryos. In contrast, sad mutants still exhibit the coordinate up-regulation of Phosphoglucoisomerase (Pgi; FBgn0003074), Phosphofructokinase (Pfk; FBgn0003071), and Phosphoglyceromutase 78 (Pglym78; FBgn0014869) transcripts during mid-embryogenesis, indicating that 20E is not necessary to activate glycolytic gene expression during mid-embryogenesis. I would note, however, that Ldh transcripts levels were noticeably decreased at the 10-12 hour AEL timepoint in sad mutant samples when compared with controls, suggesting that 20E partially regulates Ldh expression at this early timepoint. Such a possibility is consistent with previous observations that 20E activates Ldh expression in imaginal discs and that embryonic Ldh mRNA transcript levels are elevated at an earlier timepoint when compared with other glycolytic genes (Abu-Shumays and Fristrom, 1997; Tennessen et al., 2011).

Overall, my findings suggest that that 20E signaling is not required for the coordinate up-regulation of glycolytic gene expression observed midway through embryonic development. All glycolytic genes examined in this study exhibited normal up-regulation in sad mutants, with the only exception being Ldh, which exhibited slightly delayed onset of expression when compared with the control strain. My finding, however, does not exclude the possibility that ecdysone or 20E controls glycolytic metabolism within individual cells or tissues that would be overlooked using northern blot analysis. Finally, my study further highlights the role of dERR as a central regulator of carbohydrate metabolism during mid-embryogenesis and again raises the question as to what developmental signals induce dERR activity during this metabolic transition.