The FOXO transcription factor controls insect growth and development by regulating juvenile hormone degradation in the silkworm, Bombyx mori

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.

Keywords: Bombyx mori; CRISPR/Cas9; FOXO; JHE; development; insulin; juvenile hormone (JH).

Figure 1.

Developmental defects and reduced body size in FOXO mutants. A, the stages of larval development in WT and mutants (FOXO-M) (L1, first instar; L2, second instar; L3, third instar; L4, fourth instar; L5, fifth instar; P, pupae). L1-P stages are marked in red, yellow, green, blue, gray, and white sequentially through the developmental timeline. The dashed lines at time points 72, 120, 180, 252, and 336 h indicate the transition checkpoint of wild-type larvae. The curve between different stages shows the molting ratio. B, the size of wandering larvae of WT and FOXO-M. Two WT and FOXO-M larvae were photographed during the second day after larvae stopped feeding. The right graph show the male and female larval weight and body length at the wandering stage. C, adult body weight and wing area were reduced in mutants. The left graph shows the pictures of WT and FOXO-M adults (male on the top and female on the bottom). Both male and female adult weights were calculated (n = 20), the red line represents the average weight. The average areas of forewings and hindwings were shown as red and green bars, respectively. D, the mRNA levels of insulin signaling genes in the fat body of mutants. The relative InR, Akt, and 4EBP mRNA levels on the third day of fourth instar (L4D3) larvae were determined using RP49 as a control. The data are shown as the mean ± S.D. (n = 3). ***, p < 0.001 according to Student's t test.

Figure 2.

The ecdysteroid titers and 20E rescue of trimolter. A, measurement of relative 20E titers in the hemolymph on L4D2 and L4D3 of WT and FOXO-M. Hemolymph was collected from ∼10 larvae, and the pooled sample was used to determine 20E titers. **, p < 0.005 according to Student's t test. B, b, FOXO mutant larvae injected with 20E (left) or DMSO (right) on the sixth day after the third molt (n = 20). FOXO-M injected with 20E reached the fifth instar, whereas those injected with DMSO remained in the fourth instar; b′, one larva from the DMSO-injected group (fourth instar); b″, one larva from 20E-injected group (fifth instar); b‴, one larva from 20E-injected group, which partially recovered molting but failed to reach the next stage. C, the development of two groups in later stages. L4-P stages are marked in red, yellow, and green, respectively. The curve between different stages shows the transition ratio. The dark filled triangles indicate the time points when 20E or DMSO was injected. D, summary of 20E rescue. Twenty mutant larvae were treated in each group.

Figure 3.

Expression differences of ecdysone pathway genes in mutants. A–C, reduction in mRNA levels of E75, EcRA, and HR3 in the fat body. The bars with different colors in A-C represent the following: white bar, L4D2 in WT; pale gray bar, L4D2 in FOXO-M; dark gray bar, L4D3 in WT; and black bar, L4D3 in FOXO-M. D, the expression pattern of HR3 in fat body (FB) from the third instar to wandering stage. The x axis represents the time post second molt, and black lines below the time line show different larval developmental stages in WT and FOXO-M. E, EO expression in the fat body from WT and FOXO-M on L4D2 and L4D3. The relative mRNA levels were normalized using RP49 was as a control. The expression level of genes in L4D2 was set as 1. F, expression change of the genes involved in the ecdysone biosynthesis pathway in PG on L4D3. Six genes were investigated: Nvd, Spo, Phm, Dib, Sad, and Shd. **, p < 0.005; ***, p < 0.001 according to Student's t test.

Figure 4.

Trimolter larvae were fully rescued using the JH analog. A, measurement of the JHI titer in the hemolymph collected from larvae on the second day of the fourth instar (L4D2) of WT and FOXO-M. Hemolymph was collected from ∼10 larvae, and the pooled samples were analyzed. JHI was undetected in the hemolymph of FOXO-M. B, FOXO-M larvae on the sixth day (∼144 h) after application of methoprene or acetone (as a control). b, five individuals from different experimental groups. The group treated with methoprene (FOXO-M + Methoprene) molted into the fifth instar, whereas the other group treated with acetone (FOXO-M + Acetone) remained in the fourth instar. b′, one larva from the methoprene-treated group transformed into the fifth instar after 144 h. b″, one larva from the acetone-treated group remained in the fourth instar after 144 h. b‴, one larva in the acetone-treated group reached the wandering stage after 144 h. C, ratio of individuals reaching the fifth instar in two groups (n = 20). The white section of the bar represents individuals that reached the fifth instar (tetramolter), and the black section shows trimolter larvae. All individuals from the methoprene-treated group (100%) transitioned to the fifth instar, whereas only 20% in the acetone-treated group transitioned to this stage. D, the later stages of larval development in the two groups. L4-P stages are indicated in red, yellow, and green. The curve between different stages shows the transition ratio. The dark-filled triangle shows the time points when acetone or methoprene was applied.

Figure 5.

Activation of JH degradation rather than biosynthesis genes results in JH titer reduction. A, JHE enzymatic activity in the hemolymph was measured in the WT and FOXO-M during the fourth instar. The JHE activity in mutants (241.227 μm/min/ml) was 3-fold higher than that in the WT (61.495 μm/min/ml). B, relative expression of genes coding for JH degradation and synthesis enzymes in the fat body (FB). The expression changes of three significant genes in JH degradation: JHE, JHEH, and JHDK in L4D2 and L4D3 are shown. White bar, FB of WT on L4D2; pale gray bar, FB of FOXO-M on L4D2; dark gray bar, FB of WT on L4D3; black bar, FB of FOXO-M on L4D3. C–E, the expression patterns of JHE, Kr-h1α, and Kr-h1β in FB from third instar to wandering stage. The x axis represents the time post second molt, and black lines below the time line show different larval developmental stages in WT and FOXO-M. F, expression differences of several important genes coding for enzymes in JH biosynthesis, FPPS, FPPS-2, FPPS-3, IPPI, CYP15C1, and JHAMT in CA were investigated. The expression of all genes was normalized using RP49 as a control. The bars in C represent the following: white bar, CA of WT on L4D3; gray bar, CA of FOXO-M on L4D3. *, p < 0.05; **, p < 0.005; ***, p < 0.001 according to Student's t test.

Figure 6.

FOXO binds to conserved sequence in the promoters of genes coding for JH degradation enzymes. A, consensus FOXO-binding site sequence and potential binding sites in the promoter of BmJHE, BmJHDK, and BmJHEH. The black line indicates the genomic sequences of the three genes, and the white box and arrow show the first exon and transcription direction, respectively. The filled black triangle and numbers below (JHE, −1740 bp and −1390 bp; JHDK, −1404 bp and −228 bp; JHEH, −1036 bp) represent the distance between candidate FOXO-binding sites and TSS (transcription starting site). The sequence under the dashed line show the corresponding sequence (including the binding sequence), and the underlined red letters highlight the FOXO binding sequence. B–D, EMSA to test for binding of FOXO to the binding sites in the promoter regions of JHE (B), JHDK (C), and JHEH (D). Cy5-labeled oligos corresponding to the five sites (200–300 bp) were incubated with different concentrations (0, 0.2, 0.4, and 0.8 μg/μl) of purified FOXO protein. For competitive experiments, unlabeled or nonspecific DNA was added to the reaction mixture.

Figure 7.

A model for the FOXO regulatory network for modulation of growth and development. Loss of function of FOXO activates JH degradation genes (JHE, JHDK, and JHEH) resulting in a reduction in JH titer. Lower levels of JH and/or FOXO further perturb ecdysone biosynthesis and action during the late larval stages (L3–L4) and induce the loss of fourth larval molt (trimolter). In FOXO knock-out silkworms, insulin signaling is also reduced, causing growth delay and reduced body size. Taken together, these findings show that FOXO plays a pivotal role in regulating growth and development through interactions with hormone signaling.