RESUMO
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.
Assuntos
Bombyx/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Epóxido Hidrolases/metabolismo , Proteína Forkhead Box O1/metabolismo , Hormônios Juvenis/metabolismo , Metamorfose Biológica , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Animais Geneticamente Modificados , Bombyx/efeitos dos fármacos , Bombyx/crescimento & desenvolvimento , Sistemas CRISPR-Cas , Hidrolases de Éster Carboxílico/genética , Ecdisterona/farmacologia , Indução Enzimática/efeitos dos fármacos , Epóxido Hidrolases/genética , Feminino , Proteína Forkhead Box O1/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hidrólise/efeitos dos fármacos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Hormônios Juvenis/química , Masculino , Metamorfose Biológica/efeitos dos fármacos , Metoprene/farmacologia , Muda/efeitos dos fármacos , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Elementos de Resposta/efeitos dos fármacosRESUMO
Recently established, custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system provide attractive genome editing tools. Targeted gene mutagenesis using the CRISPR/Cas9 system has been achieved in several orders of insects. However, outside of studies on Drosophila melanogaster and the lepidopteron model insect Bombyx mori, little success has been reported, which is largely due to a lack of effective genetic manipulation tools that can be used in other insect orders. To create a simple and effective method of gene knockout analysis, especially for dissecting gene functioning during insect embryogenesis, we performed a functional analysis of the Bombyx Wnt1 (BmWnt1) gene using Cas9/sgRNA-mediated gene mutagenesis. The Wnt1 gene is required for embryonic patterning in various organisms, and its crucial roles during embryogenesis have been demonstrated in several insect orders. Direct injection of Cas9 mRNA and BmWnt1-specific sgRNA into Bombyx embryos induced a typical Wnt-deficient phenotype: injected embryos could not hatch and exhibited severe defects in body segmentation and pigmentation in a dose-dependent manner. Quantitative real-time PCR (qRT-PCR) analysis revealed that Hox genes were down-regulated after BmWnt1 depletion. Furthermore, large deletion, up to 18Kb, ware generated. The current study demonstrates that using the CRISPR/Cas9 system is a promising approach to achieve targeted gene mutagenesis during insect embryogenesis.
Assuntos
Bombyx/genética , Sistemas CRISPR-Cas , Proteínas de Insetos/genética , Proteína Wnt1/genética , Animais , Bombyx/embriologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Embrião não Mamífero , Técnicas de Inativação de Genes , Genoma de Inseto , Proteínas de Insetos/metabolismo , Pigmentação/genética , RNA Mensageiro/metabolismo , Proteína Wnt1/metabolismoRESUMO
Ras proteins play important roles in development especially for cell proliferation and differentiation in various organisms. However, their functions in the most insect species are still not clear. We identified three ras cDNAs from the silk worm, Bombyx mori. These sequences corresponded to three Ras of Drosophila melanogaster, but not to three mammalian Ras (H-Ras, K-Ras, N-Ras). Subsequently, the expression profiles of ras were investigated by quantitative real-time PCR using whole body of individuals from the embryonic to adult stages, and various tissues of 4th and 5th instar larvae. Each of three Bombyx ras showed different expression patterns. We also showed membrane localization of their products. These results indicate that the three Bombyx Ras are functional and have different roles.