Examining the role of Zfrp8 with respect to iron/heme homeostasis and ecdysone production

• Author / Creator

  Dey, Dipanjan

• Ecdysone, the primary steroid hormone in insects produced primarily in the prothoracic gland during larval development, is responsible for coordinating developmental transitions such as larval molts and metamorphosis during the Drosophila life-cycle. The ecdysone biosynthesis pathway, which involves the conversion of dietary cholesterol to the biologically active form of ecdysone, requires cytochrome P450 enzymes to synthesize its steps. The cytochrome P450 enzymes require heme as a co-factor in order to function properly, which results in high demand for heme during the massive increase in cytochrome P450 enzyme production throughout the larval development and especially during the late larval ecdysone peak. Since free heme is highly toxic, it is produced when required through the heme biosynthesis pathway. Whenever the heme biosynthesis pathway is disrupted as a result of mutations in the latter stages of the pathway, it results in the accumulation of heme precursors in the prothoracic gland, which displays visible red autofluorescence on exposure to UV light. Since only two other tissues that are known to highly express cytochrome P450 enzymes also display red autofluorescence signifying the accumulation of heme precursors, this establishes the relationship between cytochrome P450 enzymes and heme by proving the importance of heme for the cytochrome P450 enzymes. Genetic screens conducted by King-Jones lab in collaboration with two other labs by knocking down genes of the entire Drosophila genome specifically in the prothoracic gland investigated for ecdysone-deficient phenotypes such as the arrest of larval molts, delay in metamorphosis which resulted in ~1,906 genes that were screened further in the King-Jones lab to classify ring glands based on their size and ability to display red autofluorescence under UV exposure. This resulted in 34 genes, which also included Zfrp8, the gene that I have investigated in my thesis. I hypothesized that Zfrp8 plays a role in iron/heme homeostasis and ecdysone production, for which I needed to observe the developmental effects
  ii
  when Zfrp8 is knocked down specifically in the prothoracic gland. This resulted in significant third instar larval arrest and big red ring gland under UV exposure, which resulted from the accumulated heme precursors. In order to determine the exact mechanism as to how Zfrp8 is regulating iron/heme homeostasis as well as ecdysone production, I decided to rescue the developmental defects by supplementing the diet of the PG>Zfrp8-RNAi animals with components of the ecdysone biosynthetic pathway as well as iron and heme. Interestingly, sterol supplements, namely 7-dC, 20E, and cholesterol, were able to rescue the developmental defects of the PG>Zfrp8-RNAi animals by a significant degree, but not completely. At the same time, dietary iron and exogenous heme failed to rescue the developmental defects of the animals mentioned above. Knocking down Zfrp8, specifically in the prothoracic gland, also resulted in the downregulation of the ecdysteroidogenic genes as determined by qPCR. Based on the results, I propose that Zfrp8 participates in the process of heme production not by the conventional heme biosynthesis pathway but by a yet-to-be-identified pathway or mechanism which allows the cytochrome P450 enzymes to function properly and produce the biologically active form of ecdysone. The significant rescue demonstrated by 7-dehydrocholesterol supplementation suggests that Zfrp8 possibly branches off to a separate pathway which is responsible for regulating iron or heme metabolism instead of feeding into the conventional ecdysone biosynthesis pathway.

• Subjects / Keywords

  • Ecdysone
  • Zfrp8
  • Heme
  • Iron

• Graduation date

  Fall 2020

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-0jk9-8c90

• License

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