The cytochrome P450 Cyp6t3 is not required for ecdysone biosynthesis in Drosophila melanogaster

Abstract

The steroid hormone 20-hydroxyecdysone (20E) is essential for proper development and the timing of intermediary stage transitions in insects. As a result, there is intense interest in identifying and defining the roles of the enzymes and signaling pathways that regulate 20E production in the prothoracic gland (PG), the major endocrine organ of juvenile insect phases. Transcriptomics is one powerful tool that has been used to identify novel genes that are up- or down-regulated in the PG which may contribute to 20E regulation. Additional functional characterization of putative regulatory candidate genes typically involves qRT-PCR and/or RNAi mediated knockdown of the candidate mRNA in the PG to assess whether the gene's expression shows temporal regulation in the PG and whether its expression is essential for proper 20E production and the correct timing of developmental transitions. While these methods have proved fruitful for identifying novel regulators of 20E production, characterizing the null phenotype of putative regulatory genes is the gold standard for assigning gene function since RNAi is known to generate various types of "off target" effects. Here we describe the genetic null mutant phenotype of the Drosophila melanogaster Cyp6t3 gene . Cyp6t3 was originally identified as a differentially regulated gene in a PG microarray screen and assigned a place in the "Black Box" step of the E biosynthetic pathway based on RNAi mediated knockdown phenotypes and rescue experiments involving feeding of various intermediate compounds of the E biosynthetic pathway. In contrast, we find that Crispr generated null mutations in Cyp6t3 are viable and have normal developmental timing. Therefore, we conclude that Cyp6t3 is not required for E production under typical lab growth conditions and therefore is not an obligate enzymatic component of the Black Box.

Figure 1.

A. Ecdysone biosynthetic pathway. The feeding substrates for placement in the Black Box are shown in red. Number of arrows in the Black Box is for illustrative purpose only. B. Cartoon of the Cyp6t3 genomic region. Exons are shown in light blue and dashed lines denote the Cytochrome P450 domain. Sequences for the guide RNAs are shown in anti-sense because the targeted sequence is the negative DNA strand. Three new alleles are described below. C. Clustal alignment . Cyp6t3 aligned with Cyp6t3 mutant proteins. Pale green highlight indicates amino acid identity. Note that Cyp6t3[Cr1] and Cyp6t3[Cr3] are early truncations, while more than 80% of the protein is retained in Cyp6t3[Cr2]. D. Developmental timing. Cyp6t3 transheterozygous combinations and wild type (black line). Non-linear regression using Prism software with 50% pupariation indicated by dashed line.