The insect exoskeleton is mainly composed of chitin filaments linked by cuticle proteins. When insects molt, the cuticle of the exoskeleton is renewed by degrading the old chitin and cuticle proteins and synthesizing ...The insect exoskeleton is mainly composed of chitin filaments linked by cuticle proteins. When insects molt, the cuticle of the exoskeleton is renewed by degrading the old chitin and cuticle proteins and synthesizing new ones. In this study, chitin-binding activity of the wing disc cuticle protein BmWCP4 in Bombyx mori was studied. Sequence analysis showed that the protein had a conservative hydrophilic ＂R＆R＂ chitin-binding domain （CBD）. Western blotting showed that BmWCP4 was predominately expressed in the wing disc-containing epidermis during the late wandering and early pupal stages. The immunohistochemistry result showed that the BmWCP4 was mainly present in the wing disc tissues containing wing bud and trachea blast during day 2 of wandering stage. Recombinant full-length BmWCP4 protein, ＂R＆R＂ CBD peptide （CBD）, non-CBD peptide （BmWCP4-CBD^-）, four single site-directed mutated peptides （M1, M2, M3 and M4） and four-sites-mutated peptide （MF） were generated and purified, respectively, for in vitro chitin-binding assay. The results indicated that both the full-length protein and the ＂R＆R＂ CBD peptide could bind with chitin, whereas the BmWCP4-CBD- could not bind with chitin. The single residue mutants M1, M2, M3 and M4 reduced but did not completely abolish the chitin-binding activity, while four-sites-mutated protein MF completely lost the chitin-binding activity. These data indicate that BmWCP4 protein plays a critical role by binding to the chitin filaments in the wing during larva-to-pupa transformation. The conserved aromatic amino acids are critical in the interaction between chitin and the cuticle protein.展开更多
Metazoan development requires coordination of signaling pathways to regulate patterns of gene expression.In Drosophila,the wing imaginal disc provides an excellent model for the study of how signaling pathways interac...Metazoan development requires coordination of signaling pathways to regulate patterns of gene expression.In Drosophila,the wing imaginal disc provides an excellent model for the study of how signaling pathways interact to regulate pattern formation.The determination of the dorsal-ventral（DV） boundary of the wing disc depends on the Notch pathway,which is activated along the DV boundary and induces the expression of the homeobox transcription factor,Cut.Here,we show that Broad（Br）,a zinc-finger transcription factor,is also involved in regulating Cut expression in the DV boundary region.However,Br expression is not regulated by Notch signaling in wing discs,while ecdysone signaling is the upstream signal that induces Br for Cut upregulation.Also,we find that the ecdysone-Br cascade upregulates cut-lacZ expression,a reporter containing a 2.7 kb cut enhancer region,implying that ecdysone signaling,similar to Notch,regulates cut at the transcriptional level.Collectively,our findings reveal that the Notch and ecdysone signaling pathways synergistically regulate Cut expression for proper DV boundary formation in the wing disc.Additionally,we show br promotes Delta,a Notch ligand,near the DV boundary to suppress aberrant high Notch activity,indicating further interaction between the two pathways for DV patterning of the wing disc.展开更多
The Decapentaplegic(Dpp)and Wingless(Wg)signal pathways play important roles in numerous biological processes in Drosophila.The Drosophila vestigial(vg)gene is selectively required for wing imaginal disc cell prolifer...The Decapentaplegic(Dpp)and Wingless(Wg)signal pathways play important roles in numerous biological processes in Drosophila.The Drosophila vestigial(vg)gene is selectively required for wing imaginal disc cell proliferation,which is essential for the formation of the adult wing and halter structures,and is regulated by Dpp and Wg signaling.Using a Drosophila invasion model of wing epithelium,we showed herein that inhibition of Dpp or Wg signaling promoted cells to migrate across the cell lineage restrictive anterior/posterior(A/P)compartment boundary.Being downstream of both Dpp and Wg signaling,vg can block cell migration induced by loss of either pathway.In addition,suppression of vg is sufficient to induce cell migration across the A/P boundary.Transcriptomic analysis revealed potential downstream genes involved in the cell migration after suppressing vg in the wing disc.We further demonstrated that the c-Jun N-terminal kinase(JNK)signaling promoted cell migration induced by vg suppression by upregulating Caspase activity.Taken together,our results revealed the requirement ofVg for suppressing cell migration and clarified how developmental signals collaborate to stabilize cells along the compartment boundary.展开更多
Insect wings are developed from the wing disc during metamorphosis.Bombyx mori,a model lepidopteran insect,loses flight ability after long-term domestication from the wild silkworm,Bombyx mandarina.The mw mutant(ul 1 ...Insect wings are developed from the wing disc during metamorphosis.Bombyx mori,a model lepidopteran insect,loses flight ability after long-term domestication from the wild silkworm,Bombyx mandarina.The mw mutant(ul 1 strain)shows minute wings compared to wild type(e.g.,p50 strain)wings.RNA sequencing analysis previously revealed differential Hippo-pathway-related gene expression between the ull and p50strains.The Hippo pathway is an evolutionarily conserved signaling cascade that controls organ size during development in animals.In this study,the function of BmSd which has been characterized as one of the Hippo-pathway-related genes was analyzed for silkworm wing development.We found that mats,warts,and hippo expression levels were higher in u11 compared to p50 wing discs.BmSd(scalloped)expression,which encodes a prominent transcriptional partner to Yorkie(Yki),gradually decreased during the wandering stage in ull,but exhibited the opposite expression pattern in p50.When BmSd was knocked down by small interfering RNA during the wandering stage in the p50 strain,57.9%of the individuals showed minute wings.Additionally,ex,kibras and wingless expression levels decreased in the BmSd knockdown mutant.Further,BmSd deletion mediated by clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9 induced 50%of individuals with minute wings,a phenotype similar to the mw mutant.This result demonstrates that BmSd plays pivotal roles in silkworm wing development.Our results show that the Hippo signaling pathway participates and plays crucial roles in the regulation of silkworm wing development,and our findings provide a basis for further research on B.mori wing development.展开更多
Manipulating an exogenous or endogenous gene of interest at a defined level is critical for a wide variety of experiments.The Gal4/UAS system has been widely used to direct gene expression for studying complex genetic...Manipulating an exogenous or endogenous gene of interest at a defined level is critical for a wide variety of experiments.The Gal4/UAS system has been widely used to direct gene expression for studying complex genetic and biological problems in Drosophila melanogaster and other model organisms.Driven by a given tissue-specific Gal4,expressing UAS-transgene or UAS-RNAi(RNA interference)could be used to up-or down-regulate target gene expression,respectively.However,the efficiency of the Gal4/UAS system is roughly predefined by properties of transposon vector constructs and the insertion site in the transgenic stock.Here,we describe a simple way to modulate optomotor blind(omb)expression levels in its endogenous expression region of the wing disc.We co-expressed UAS-omb and UAS-omb-RNAi together under the control of dpp-Gal4 driver which is expressed in the omb expression region of the wing pouch.The repression effect is more sensitive to temperature than that of overexpression.At low temperature,overexpression plays a dominant role but the efficiency is attenuated by UAS-omb-RNAi.In contrast,at high temperature RNAi predominates in gene expression regulation.By this strategy,we could manipulate omb expression levels at a moderate level.It allows us to manipulate omb expression levels in the same tissue between overexpression and repression at different stages by temperature control.展开更多
The highly conserved Notch signaling is precisely regulated at different steps in a series of developmental events. However, little is known about the regulation of Notch receptor at transcriptional level. Here, we de...The highly conserved Notch signaling is precisely regulated at different steps in a series of developmental events. However, little is known about the regulation of Notch receptor at transcriptional level. Here, we demonstrate that dBrmsl is involved in regulating Notch signaling in Drosophila wing. We show that knockdown of dBrmsl by RNA interference （RNAi） in wing disc suppresses the expression of Notch signaling target genes wingless （wg）, cut and Enhancer of split m8 [E（spl）m8]. Consistently, the levels of Wg and Cut are reduced in the dBrmsl mutant clones. Importantly, loss of dBrmsl leads to significant reduction of Notch proteins. Furthermore, depletion of dBrmsl results in apparent downregulation of Notch transcription in the wing disc. Moreover, we find that dBrmsl is functionally conserved with human Breast cancer metastasis suppressor 1 like （hBRMSIL） in the modulation of Notch signaling. Taken together, our data provide important insights into the biological function of dBrmsl in regulating Notch signaling.展开更多
基金This study was supported by research grants of the National Natural Science Foundation of China （31172265 31330071 31301918） and the National Basic Research Program of China （2012CB 114602）.
文摘The insect exoskeleton is mainly composed of chitin filaments linked by cuticle proteins. When insects molt, the cuticle of the exoskeleton is renewed by degrading the old chitin and cuticle proteins and synthesizing new ones. In this study, chitin-binding activity of the wing disc cuticle protein BmWCP4 in Bombyx mori was studied. Sequence analysis showed that the protein had a conservative hydrophilic ＂R＆R＂ chitin-binding domain （CBD）. Western blotting showed that BmWCP4 was predominately expressed in the wing disc-containing epidermis during the late wandering and early pupal stages. The immunohistochemistry result showed that the BmWCP4 was mainly present in the wing disc tissues containing wing bud and trachea blast during day 2 of wandering stage. Recombinant full-length BmWCP4 protein, ＂R＆R＂ CBD peptide （CBD）, non-CBD peptide （BmWCP4-CBD^-）, four single site-directed mutated peptides （M1, M2, M3 and M4） and four-sites-mutated peptide （MF） were generated and purified, respectively, for in vitro chitin-binding assay. The results indicated that both the full-length protein and the ＂R＆R＂ CBD peptide could bind with chitin, whereas the BmWCP4-CBD- could not bind with chitin. The single residue mutants M1, M2, M3 and M4 reduced but did not completely abolish the chitin-binding activity, while four-sites-mutated protein MF completely lost the chitin-binding activity. These data indicate that BmWCP4 protein plays a critical role by binding to the chitin filaments in the wing during larva-to-pupa transformation. The conserved aromatic amino acids are critical in the interaction between chitin and the cuticle protein.
基金supported by Dissertation Research Grant Award from Florida State Universitysupported by the National Institutes of Health grant(R01GM072562)the National Science Foundation(IOS-1052333)
文摘Metazoan development requires coordination of signaling pathways to regulate patterns of gene expression.In Drosophila,the wing imaginal disc provides an excellent model for the study of how signaling pathways interact to regulate pattern formation.The determination of the dorsal-ventral（DV） boundary of the wing disc depends on the Notch pathway,which is activated along the DV boundary and induces the expression of the homeobox transcription factor,Cut.Here,we show that Broad（Br）,a zinc-finger transcription factor,is also involved in regulating Cut expression in the DV boundary region.However,Br expression is not regulated by Notch signaling in wing discs,while ecdysone signaling is the upstream signal that induces Br for Cut upregulation.Also,we find that the ecdysone-Br cascade upregulates cut-lacZ expression,a reporter containing a 2.7 kb cut enhancer region,implying that ecdysone signaling,similar to Notch,regulates cut at the transcriptional level.Collectively,our findings reveal that the Notch and ecdysone signaling pathways synergistically regulate Cut expression for proper DV boundary formation in the wing disc.Additionally,we show br promotes Delta,a Notch ligand,near the DV boundary to suppress aberrant high Notch activity,indicating further interaction between the two pathways for DV patterning of the wing disc.
基金by the National Natural Science Foundation of China(NSFC31872293 and 31872295).
文摘The Decapentaplegic(Dpp)and Wingless(Wg)signal pathways play important roles in numerous biological processes in Drosophila.The Drosophila vestigial(vg)gene is selectively required for wing imaginal disc cell proliferation,which is essential for the formation of the adult wing and halter structures,and is regulated by Dpp and Wg signaling.Using a Drosophila invasion model of wing epithelium,we showed herein that inhibition of Dpp or Wg signaling promoted cells to migrate across the cell lineage restrictive anterior/posterior(A/P)compartment boundary.Being downstream of both Dpp and Wg signaling,vg can block cell migration induced by loss of either pathway.In addition,suppression of vg is sufficient to induce cell migration across the A/P boundary.Transcriptomic analysis revealed potential downstream genes involved in the cell migration after suppressing vg in the wing disc.We further demonstrated that the c-Jun N-terminal kinase(JNK)signaling promoted cell migration induced by vg suppression by upregulating Caspase activity.Taken together,our results revealed the requirement ofVg for suppressing cell migration and clarified how developmental signals collaborate to stabilize cells along the compartment boundary.
基金This project was supported by the National Natural Science Foundation of China(grant No.31572320)the Postgraduate Research&Practice Innovation Program of Jiangsu Province(KYCX181881).
文摘Insect wings are developed from the wing disc during metamorphosis.Bombyx mori,a model lepidopteran insect,loses flight ability after long-term domestication from the wild silkworm,Bombyx mandarina.The mw mutant(ul 1 strain)shows minute wings compared to wild type(e.g.,p50 strain)wings.RNA sequencing analysis previously revealed differential Hippo-pathway-related gene expression between the ull and p50strains.The Hippo pathway is an evolutionarily conserved signaling cascade that controls organ size during development in animals.In this study,the function of BmSd which has been characterized as one of the Hippo-pathway-related genes was analyzed for silkworm wing development.We found that mats,warts,and hippo expression levels were higher in u11 compared to p50 wing discs.BmSd(scalloped)expression,which encodes a prominent transcriptional partner to Yorkie(Yki),gradually decreased during the wandering stage in ull,but exhibited the opposite expression pattern in p50.When BmSd was knocked down by small interfering RNA during the wandering stage in the p50 strain,57.9%of the individuals showed minute wings.Additionally,ex,kibras and wingless expression levels decreased in the BmSd knockdown mutant.Further,BmSd deletion mediated by clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9 induced 50%of individuals with minute wings,a phenotype similar to the mw mutant.This result demonstrates that BmSd plays pivotal roles in silkworm wing development.Our results show that the Hippo signaling pathway participates and plays crucial roles in the regulation of silkworm wing development,and our findings provide a basis for further research on B.mori wing development.
基金supported by National Natural Science Foundation of China(NSFC31402021and NSFC31672364)2018 Special Talents Projects in Shanxi Province,China(201805D211019).
文摘Manipulating an exogenous or endogenous gene of interest at a defined level is critical for a wide variety of experiments.The Gal4/UAS system has been widely used to direct gene expression for studying complex genetic and biological problems in Drosophila melanogaster and other model organisms.Driven by a given tissue-specific Gal4,expressing UAS-transgene or UAS-RNAi(RNA interference)could be used to up-or down-regulate target gene expression,respectively.However,the efficiency of the Gal4/UAS system is roughly predefined by properties of transposon vector constructs and the insertion site in the transgenic stock.Here,we describe a simple way to modulate optomotor blind(omb)expression levels in its endogenous expression region of the wing disc.We co-expressed UAS-omb and UAS-omb-RNAi together under the control of dpp-Gal4 driver which is expressed in the omb expression region of the wing pouch.The repression effect is more sensitive to temperature than that of overexpression.At low temperature,overexpression plays a dominant role but the efficiency is attenuated by UAS-omb-RNAi.In contrast,at high temperature RNAi predominates in gene expression regulation.By this strategy,we could manipulate omb expression levels at a moderate level.It allows us to manipulate omb expression levels in the same tissue between overexpression and repression at different stages by temperature control.
基金supported by the grants from National Basic Research Program of China (Nos.2011CB943901, 2011CB943902 and 2011CB943802)the National Natural Science Foundation of China (Nos.31030049, 31271582 and 31071284)the Strategic Priority Research Program of the Chinese Academy of Sciences(No. XDA01010101)
文摘The highly conserved Notch signaling is precisely regulated at different steps in a series of developmental events. However, little is known about the regulation of Notch receptor at transcriptional level. Here, we demonstrate that dBrmsl is involved in regulating Notch signaling in Drosophila wing. We show that knockdown of dBrmsl by RNA interference （RNAi） in wing disc suppresses the expression of Notch signaling target genes wingless （wg）, cut and Enhancer of split m8 [E（spl）m8]. Consistently, the levels of Wg and Cut are reduced in the dBrmsl mutant clones. Importantly, loss of dBrmsl leads to significant reduction of Notch proteins. Furthermore, depletion of dBrmsl results in apparent downregulation of Notch transcription in the wing disc. Moreover, we find that dBrmsl is functionally conserved with human Breast cancer metastasis suppressor 1 like （hBRMSIL） in the modulation of Notch signaling. Taken together, our data provide important insights into the biological function of dBrmsl in regulating Notch signaling.
