Abstract
Forkhead box O (FOXO) functions as the terminal transcription factor of the insulin signaling pathway and regulates multiple physiological processes in many organisms, including lifespan in insects. However, how FOXO interacts with hormone signaling to modulate insect growth and development is largely unknown. Here, using the transgene-based CRISPR/Cas9 system, we generated and characterized mutants of the silkworm Bombyx mori FOXO (BmFOXO) to elucidate its physiological functions during development of this lepidopteran insect. The BmFOXO mutant (FOXO-M) exhibited growth delays from the first larval stage and showed precocious metamorphosis, pupating at the end of the fourth instar (trimolter) rather than at the end of the fifth instar as in the wild-type (WT) animals. However, different from previous reports on precocious metamorphosis caused by juvenile hormone (JH) deficiency in silkworm mutants, the total developmental time of the larval period in the FOXO-M was comparable with that of the WT. Exogenous application of 20-hydroxyecdysone (20E) or of the JH analog rescued the trimolter phenotype. RNA-seq and gene expression analyses indicated that genes involved in JH degradation but not in JH biosynthesis were up-regulated in the FOXO-M compared with the WT animals. Moreover, we identified several FOXO-binding sites in the promoter of genes coding for JH-degradation enzymes. These results suggest that FOXO regulates JH degradation rather than its biosynthesis, which further modulates hormone homeostasis to control growth and development in B. mori In conclusion, we have uncovered a pivotal role for FOXO in regulating JH signaling to control insect development.
Highlights
Forkhead box O (FOXO) functions as the terminal transcription factor of the insulin signaling pathway and regulates multiple physiological processes in many organisms, including lifespan in insects
The results showed that the expression of the insulin receptor (InR) gene was reduced by 86%, whereas the serine/threonine kinase (Akt) and 4E-binding protein (4EBP) were up-regulated in mutants when compared with their expression in WT (Fig. 1D and supplemental S2B)
In the present study, using a transgenic CRISPR/Cas9-derived somatic mutagenesis system, we showed that FOXO depletion affected growth and development as well as ecdysteroid and juvenile hormone (JH) signaling in B. mori
Summary
Forkhead box O (FOXO) functions as the terminal transcription factor of the insulin signaling pathway and regulates multiple physiological processes in many organisms, including lifespan in insects. How FOXO interacts with hormone signaling to modulate insect growth and development is largely unknown. We identified several FOXObinding sites in the promoter of genes coding for JH-degradation enzymes These results suggest that FOXO regulates JH degradation rather than its biosynthesis, which further modulates hormone homeostasis to control growth and development in B. mori. We elucidated how FOXO interacts with hormone signaling to regulate growth and metamorphosis in B. mori by knocking out BmFOXO using the transgene-based CRISPR/ Cas system. BmFOXO mutants showed a significant growth delay from the second larval instar and skipped the last molt, resulting in precocious metamorphosis after only four larval instars (trimolter), the entire developmental time of the larval instar was comparable with that of WT animals. Our results revealed a novel mechanism by which FOXO interacts with the JH degradation pathway to regulate growth and development in silkworms
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