克隆了罗氏沼虾(Macrobrachium rosenbergii)含DM结构域的Dsx基因(MroDsx)部分序列,DM结构域包含5个保守的半胱氨酸(Cysteine)和2个组氨酸(Histidine),且与中国对虾(Fenneropenaeus chinensis)Dsx基因DM结构域具有73%的相似性。荧光定量...克隆了罗氏沼虾(Macrobrachium rosenbergii)含DM结构域的Dsx基因(MroDsx)部分序列,DM结构域包含5个保守的半胱氨酸(Cysteine)和2个组氨酸(Histidine),且与中国对虾(Fenneropenaeus chinensis)Dsx基因DM结构域具有73%的相似性。荧光定量PCR(Quantitative real time PCR,qPCR)结果显示:MroDsx基因仅在性腺中特异性表达,且在精巢中的表达量显著高于卵巢;在精巢发育早期(主要是精原细胞)高表达,随着精巢的发育MroDsx基因的表达量逐渐降低。原位杂交(In situ hybridization,ISH)结果进一步显示:MroDsx转录本在精原细胞表达量高,在精母细胞以及精细胞中较弱,在成熟的精子中不表达,与荧光定量的结果一致。基于对MroDsx转录本的表达分析,推测MroDsx可能参与罗氏沼虾精巢的发育过程。展开更多
The highly conserved doublesex(dsx) and doublesex/mab-3 related(Dmrt) genes control sexually dimorphic traits across animals. The dsx gene encodes sex-specific transcription factors, Dsx^(M) in males and Dsx^(F) in fe...The highly conserved doublesex(dsx) and doublesex/mab-3 related(Dmrt) genes control sexually dimorphic traits across animals. The dsx gene encodes sex-specific transcription factors, Dsx^(M) in males and Dsx^(F) in females, which function differentially and often oppositely to establish sexual dimorphism. Here, we report that mutations in dsx, or overexpression of dsx, result in abnormal distribution of the basement membrane(BM) protein Collagen Ⅳ in the fat body. We find that Dsx isoforms regulate the expression of Collagen Ⅳ in the fat body and its secretion into the BM of other tissues. We identify the procollagen lysyl hydroxylase(dPlod) gene, which is involved in the biosynthesis of Collagen Ⅳ, as a direct target of Dsx. We further show that Dsx regulates Collagen Ⅳ through d Plod-dependent and independent pathways. These findings reveal how Dsx isoforms function in the secretory fat body to regulate Collagen Ⅳ and remotely establish sexual dimorphism.展开更多
Sex-specific neurons play pivotal roles in regulating sexually dimorphic behaviors.In insects,the sex determination gene doublesex(dsx)establishes major sexual dimorphism of the nervous system,in which male-specific d...Sex-specific neurons play pivotal roles in regulating sexually dimorphic behaviors.In insects,the sex determination gene doublesex(dsx)establishes major sexual dimorphism of the nervous system,in which male-specific dsx^(M)promotes neuronal development,while female-specific dsx^(F)inhibits neuronal development by promoting neuronal apoptosis.In this study,we find that dsx regulates the number of dsx-expressing central neurons in Drosophila in cell-specific manners.Although dsx^(M)overall promotes an increase in the number of dsx neurons,it inhibits the emergence of specific pC1 neurons.dsx^(F)reduces the number of different pC1/pC2 subtypes,but promotes the formation of pC1d.We also find that dsx^(M)and dsx^(F)barely affect the number of some pC2 neurons.Changes in the number of pC1/pC2 neurons alter their roles in regulating different behaviors,including courtship,aggression,and locomotion.Our results illustrate the multifaceted functions of dsx in sexually dimorphic neuronal development and behaviors.展开更多
Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of...Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit-fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The doublesex gene of S. litura (Sldsx) has male-specific SldsxM and female-specific SldsF isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/μL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at Go generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild-type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of doublesex on the male are similar to other insects.展开更多
Sexual development in insects is regulated by a complicated hierarchical cascade of sex determination.The primary signals are diverse,whereas the central nexus doublesex(dsx)gene is relatively conserved within the pat...Sexual development in insects is regulated by a complicated hierarchical cascade of sex determination.The primary signals are diverse,whereas the central nexus doublesex(dsx)gene is relatively conserved within the pathway.Aedes(Stegomyia)albopictus is an important vector with an extensive worldwide distribution.We previously reported that Ae.albopictus dsx(Aalbdsx)yields one male-(Aalbdsx^(M))and three female-specific isoforms(Aalbdsx^(F1-3));however,the spatiotemporal expression profiles and mechanisms regulating sex-specific alternative splicing require further investigation.In this study,we demonstrated that the Aalbdsx^(M) messenger RNA(mRNA)represents the default pattern when analyzed in human foreskin fibroblasts and HeLa cells.We combined reverse transcription polymerase chain reaction with RNA immunoprecipitation using specific antibodies against tagged Ae.albopictus male-determining factor AalNix and confirmed that AalNix indirectly regulates dsx pre-mRNA and regulates its alternative splicing.During the early embryo stage(0-2 and 4-8 h),maternal dsx^(F) and default splicing dsx^(M) were detected in both sexes;the expression of dsx^(M) then decreased until sufficient AalNix transcripts accumulated in male embryos at 20-24 h.These findings suggest that one or more potential dsx splicing enhancers can shift dsx^(M) to dsx^(F) in both sexes;however,the presence of Nix influences the function of this unknown splicing enhancer and ultimately leads to the formation of dsx^(M) in males.Finally,our results provide important insight into the regulatory mechanism of dsx alternative splicing in the mosquito.展开更多
文摘为了探索doublesex1(dsx1)基因是否在蚤状溞的生殖转换过程中发挥作用,利用蚤状溞滤食摄食的特点将体外合成的dsx1双链RNA(ds RNA)通过浸泡得方法分别摄入不同生殖状态的溞体中,实现dsx1基因的表达沉默。随后采用荧光定量PCR(q PCR)以及整体原位杂交分别检测不同生殖状态下蚤状溞在RNAi前后体内dsx1的表达水平变化,进而研究dsx1在蚤状溞生殖转换过程中的作用。结果表明:雄性溞、两性溞、孤雌溞在RNAi后均出现了dsx1 m RNA表达水平的显著下调,且下调量具有显著性差异(P<0.05)其中在孤雌溞中下调68%,在三种生殖状态中最为显著,其次是雄性溞(56%)和两性溞(20%)。同时发现,干扰前能够在溞的第一触角、第一胸肢和复眼上检测到明显的信号位点,而在RNAi后,仅在溞体的第一触角上发现少量dsx1基因表达位点,且第一胸肢及复眼上未检测到相关信号。结合未干扰前dsx1在不同生殖状态溞的表达定量以及定位结果,我们推测dsx1可能在蚤状溞的生殖转化基因调控过程中扮演关键角色,并且在维持雄性蚤状溞性别特征的机制中发挥重要作用。
文摘克隆了罗氏沼虾(Macrobrachium rosenbergii)含DM结构域的Dsx基因(MroDsx)部分序列,DM结构域包含5个保守的半胱氨酸(Cysteine)和2个组氨酸(Histidine),且与中国对虾(Fenneropenaeus chinensis)Dsx基因DM结构域具有73%的相似性。荧光定量PCR(Quantitative real time PCR,qPCR)结果显示:MroDsx基因仅在性腺中特异性表达,且在精巢中的表达量显著高于卵巢;在精巢发育早期(主要是精原细胞)高表达,随着精巢的发育MroDsx基因的表达量逐渐降低。原位杂交(In situ hybridization,ISH)结果进一步显示:MroDsx转录本在精原细胞表达量高,在精母细胞以及精细胞中较弱,在成熟的精子中不表达,与荧光定量的结果一致。基于对MroDsx转录本的表达分析,推测MroDsx可能参与罗氏沼虾精巢的发育过程。
基金supported by grants from National Key R&D Program of China (2019YFA0802400)the National Natural Science Foundation of China (31970943 and 31700905)the Jiangsu Innovation and Entrepreneurship Team Program
文摘The highly conserved doublesex(dsx) and doublesex/mab-3 related(Dmrt) genes control sexually dimorphic traits across animals. The dsx gene encodes sex-specific transcription factors, Dsx^(M) in males and Dsx^(F) in females, which function differentially and often oppositely to establish sexual dimorphism. Here, we report that mutations in dsx, or overexpression of dsx, result in abnormal distribution of the basement membrane(BM) protein Collagen Ⅳ in the fat body. We find that Dsx isoforms regulate the expression of Collagen Ⅳ in the fat body and its secretion into the BM of other tissues. We identify the procollagen lysyl hydroxylase(dPlod) gene, which is involved in the biosynthesis of Collagen Ⅳ, as a direct target of Dsx. We further show that Dsx regulates Collagen Ⅳ through d Plod-dependent and independent pathways. These findings reveal how Dsx isoforms function in the secretory fat body to regulate Collagen Ⅳ and remotely establish sexual dimorphism.
基金supported by the National Key R&D Program of China(2021YFA1101300)the National Natural Science Foundation of China(32371067 to Y.P.)+1 种基金the Natural Science Foundation from Jiangsu Province(BK20231418 to Q.P.)the Fundamental Research Funds for the Central Universities(2242023R40054 to Q.P.).
文摘Sex-specific neurons play pivotal roles in regulating sexually dimorphic behaviors.In insects,the sex determination gene doublesex(dsx)establishes major sexual dimorphism of the nervous system,in which male-specific dsx^(M)promotes neuronal development,while female-specific dsx^(F)inhibits neuronal development by promoting neuronal apoptosis.In this study,we find that dsx regulates the number of dsx-expressing central neurons in Drosophila in cell-specific manners.Although dsx^(M)overall promotes an increase in the number of dsx neurons,it inhibits the emergence of specific pC1 neurons.dsx^(F)reduces the number of different pC1/pC2 subtypes,but promotes the formation of pC1d.We also find that dsx^(M)and dsx^(F)barely affect the number of some pC2 neurons.Changes in the number of pC1/pC2 neurons alter their roles in regulating different behaviors,including courtship,aggression,and locomotion.Our results illustrate the multifaceted functions of dsx in sexually dimorphic neuronal development and behaviors.
基金the National Natural Science Foundation of China (Grant No. 31330071,31772519)Natural Science Foundation of Guangdong Province (Grant No. S2012010010185,2017A030313210).
文摘Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit-fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The doublesex gene of S. litura (Sldsx) has male-specific SldsxM and female-specific SldsF isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/μL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at Go generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild-type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of doublesex on the male are similar to other insects.
基金supported by the National Natural Science Foundation of China(81672054,81871688,and 31830087)the National Institutes of Health,USA(All36850)the Natural Science Foundation of Guangdong Province(2017A030313120).
文摘Sexual development in insects is regulated by a complicated hierarchical cascade of sex determination.The primary signals are diverse,whereas the central nexus doublesex(dsx)gene is relatively conserved within the pathway.Aedes(Stegomyia)albopictus is an important vector with an extensive worldwide distribution.We previously reported that Ae.albopictus dsx(Aalbdsx)yields one male-(Aalbdsx^(M))and three female-specific isoforms(Aalbdsx^(F1-3));however,the spatiotemporal expression profiles and mechanisms regulating sex-specific alternative splicing require further investigation.In this study,we demonstrated that the Aalbdsx^(M) messenger RNA(mRNA)represents the default pattern when analyzed in human foreskin fibroblasts and HeLa cells.We combined reverse transcription polymerase chain reaction with RNA immunoprecipitation using specific antibodies against tagged Ae.albopictus male-determining factor AalNix and confirmed that AalNix indirectly regulates dsx pre-mRNA and regulates its alternative splicing.During the early embryo stage(0-2 and 4-8 h),maternal dsx^(F) and default splicing dsx^(M) were detected in both sexes;the expression of dsx^(M) then decreased until sufficient AalNix transcripts accumulated in male embryos at 20-24 h.These findings suggest that one or more potential dsx splicing enhancers can shift dsx^(M) to dsx^(F) in both sexes;however,the presence of Nix influences the function of this unknown splicing enhancer and ultimately leads to the formation of dsx^(M) in males.Finally,our results provide important insight into the regulatory mechanism of dsx alternative splicing in the mosquito.
