<i>Drosophila</i>ventral furrow morphogenesis: a proteomic analysis

Gong, Lei; Puri, Mamta; Ünlü, Mustafa; Young, Margaret; Robertson, Katherine; Viswanathan, Surya; Krishnaswamy, Arun; Dowd, Susan R.; Minden, Jonathan S.

doi:10.1242/dev.00955

Cited by 68 publications

(66 citation statements)

References 45 publications

(39 reference statements)

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“…Wild-type oocytes were collected from hsFLP122;FRT82B͞FRT82B ovo D1 females treated in the same manner as above. Oocytes were collected, pooled in batches of 200, and frozen as described (20).…”

Section: Methodsmentioning

confidence: 99%

“…Difference Gel Electrophoresis (DIGE) and MS. Seventy to 100 g of protein homogenate from a particular genotype was conjugated with either Cy3 or Cy5 dye, mixed with equal protein from the other genotype that had been coupled with the opposite dye, and the mixture subjected to DIGE analysis, as described (20). Positive protein spots were excised from five gels, and proteins were extracted, digested with trypsin, and analyzed by a PerSpective Biosystems Voyager STR MALDI-TOF, as described (20).…”

Section: Methodsmentioning

confidence: 99%

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Targets of microRNA regulation in the Drosophila oocyte proteome

Nakahara

Kim

Sciulli

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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MicroRNAs (miRNAs) are a class of small RNAs that silence gene expression. In animal cells, miRNAs bind to the 3 untranslated regions of specific mRNAs and inhibit their translation. Although some targets of a handful of miRNAs are known, the number and identities of mRNA targets in the genome are uncertain, as are the developmental functions of miRNA regulation. To identify the global range of miRNA-regulated genes during oocyte maturation of Drosophila, we compared the proteome from wild-type oocytes with the proteome from oocytes lacking the dicer-1 gene, which is essential for biogenesis of miRNAs. Most identified proteins appeared to be subject to translation inhibition. Their transcripts contained putative binding sites in the 3 untranslated region for a subset of miRNAs, based on computer modeling. The fraction of genes subject to direct and indirect repression by miRNAs during oocyte maturation appears to be small (4%), and the genes tend to share a common functional relationship in protein biogenesis and turnover. The preponderance of genes that control global protein abundance suggests this process is under tight control by miRNAs at the onset of fertilization.translation control S mall RNAs, including microRNAs (miRNAs) and short interfering RNAs, are components of a RNA-based mechanism of gene silencing (1, 2). The miRNA branch of RNA-based gene regulation is found in plants and animals (3, 4). miRNAs have a specific size of Ϸ22 nt, and they are processed from hairpin-loop RNA precursors by endoribonucleases of the Dicer class. These precursors are transcribed from genes within plant and animal genomes, with the number of miRNA genes found in different species roughly corresponding to 0.5-1% of the total number of genes in their genomes (5). Most miRNA genes are conserved between related species, and Ϸ30% of miRNA genes are highly conserved, with orthologs found in vertebrate and invertebrate genomes. This suggests that a significant fraction of miRNAs have evolutionarily conserved biological functions.miRNAs specifically repress gene expression by negatively regulating complementary mRNAs. Plant miRNAs generally cause degradation of complementary mRNAs by near-perfect basepairing (5). Conversely, most characterized miRNAs from animals repress gene expression by blocking the translation of complementary mRNAs into protein. They interact with their targets by imperfectly basepairing to mRNA sequences within the 3Ј UTR. The exact mechanism of translation inhibition is unknown, although miRNAs have been found not to interfere with translation initiation (5).A question of great interest concerns the functions of miRNAs in animals. Although the functions of only a few miRNAs are known, available evidence suggests that miRNAs play diverse and important roles in development. This conclusion is based on the mutant phenotypes of individual miRNA genes and on the identification of genes that are direct targets of miRNA regulation. In Caenorhabditis elegans, the genes lin-14, lin-28, lin-41, and hbl-1 are trans...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Targets of microRNA regulation in the Drosophila oocyte proteome

Nakahara

Kim

Sciulli

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…Embryos to be injected were mounted on a coverslip as previously described (Gong et al, 2004). They were then dehydrated slightly, covered with halocarbon oil and injected with PBS (negative control) or a specific Rho Kinase inhibitor Y-27632 (Tocris, 50 mM).…”

Section: Embryo Injectionmentioning

confidence: 99%

“…To circumvent this lack of genetic tractability, researchers have begun injecting inhibitors and dsRNAs/siRNAs into early embryos (Gong et al, 2004;Royou et al, 2004;Martin et al, 2009). Typically, embryos are injected before or during cellularization to ensure the reagent can diffuse to all cells without membrane permeability issues.…”

Section: Introductionmentioning

confidence: 99%

Assessing the critical period for Rho kinase activity during Drosophila ventral furrow formation

Krajcovic

Minden

2012

Developmental Dynamics

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Background: Drosophila ventral furrow formation (VFF), which is the first morphogenetic event during embryo development, serves as a model for epithelial sheet folding. VFF can be subdivided into five cell shape changes: apical membrane flattening, apicobasal nuclear migration, apicobasal cell shortening, random apical constriction, and concerted apical constriction. These processes are generally believed to be driven by Rho kinase (Rok) activation of myosin II to stimulate the constriction of the apical actomyosin network. To test the role of Rok and its downstream target myosin II in VFF, timed injections of the Rok inhibitor, Y-27632, were performed. Results: Embryos injected with Y-27632 before the concerted apical constriction phase of VFF were able to execute apicobasal nuclear migration and random apical constriction, but were unable to enter the concerted apical constriction phase. Embryos injected with Y-27632 during concerted apical constriction reverted to the transition point between random apical constriction and concerted apical constriction. Finally, embryos injected with Y-27632 upon the initiation of furrow ingression were able to complete VFF. Conclusions: Together these results suggest a critical period for Rok activity and presumably myosin II activation during the initiation of the concerted apical constriction phase of VFF.

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“…2D-DIGE presents also the advantage that it is a quantitative approche since each protein spot has its own internal standard (IS), which ensure that the differences found are real and not due to a gel-to gel variation. Moreover, 2D-DIGE is a very sensitive technique with a detection threshold of around 1 femtomole of protein (Gong L et al, 2004). In the minimal labeling proteins are stained by cyanines, these dyes has a Nhydroxysuccinimidyl ester reactive group which forms a covalent bond with the epsilon amino group of the lysine in proteins via an amide connection.…”

Section: Quantitative Proteomics By Two-dimensional Differential Gel mentioning

confidence: 99%