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1.
Science ; 372(6538)2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33833095

RESUMO

During multicellular development, spatial position and lineage history play powerful roles in controlling cell fate decisions. Using a serine integrase-based recording system, we engineered cells to record lineage information in a format that can be read out in situ. The system, termed integrase-editable memory by engineered mutagenesis with optical in situ readout (intMEMOIR), allowed in situ reconstruction of lineage relationships in cultured mouse cells and flies. intMEMOIR uses an array of independent three-state genetic memory elements that can recombine stochastically and irreversibly, allowing up to 59,049 distinct digital states. It reconstructed lineage trees in stem cells and enabled simultaneous analysis of single-cell clonal history, spatial position, and gene expression in Drosophila brain sections. These results establish a foundation for microscopy-readable lineage recording and analysis in diverse systems.


Assuntos
Linhagem da Célula , Expressão Gênica , Células-Tronco Embrionárias Murinas/citologia , Neurônios/citologia , Análise de Célula Única , Animais , Encéfalo/citologia , Linhagem Celular , Células Clonais/citologia , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Perfilação da Expressão Gênica , Resposta ao Choque Térmico , Hibridização in Situ Fluorescente , Integrases/metabolismo , Camundongos , Mutagênese , Análise Espacial , Imagem com Lapso de Tempo , Transcrição Genética
2.
Nat Commun ; 12(1): 2412, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33893303

RESUMO

The development of a widely adopted cryopreservation method remains a major challenge in Drosophila research. Here we report a robust and easily implemented cryopreservation protocol of Drosophila melanogaster embryos. We present innovations for embryo permeabilization, cryoprotectant agent loading, and rewarming. We show that the protocol is broadly applicable, successfully implemented in 25 distinct strains from different sources. We demonstrate that for most strains, >50% embryos hatch and >25% of the resulting larvae develop into adults after cryopreservation. We determine that survival can be significantly improved by outcrossing to mitigate the effect of genetic background for strains with low survival after cryopreservation. We show that flies retain normal sex ratio, fertility, and original mutation after successive cryopreservation of 5 generations and 6-month storage in liquid nitrogen. Lastly, we find that non-specialists are able to use this protocol to obtain consistent results, demonstrating potential for wide adoption.


Assuntos
Criopreservação/métodos , Drosophila melanogaster/embriologia , Embrião não Mamífero/embriologia , Reaquecimento/métodos , Vitrificação , Animais , Crioprotetores/farmacologia , Drosophila melanogaster/genética , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/ultraestrutura , Feminino , Fertilidade/genética , Larva/genética , Larva/metabolismo , Microscopia Eletrônica , Permeabilidade/efeitos dos fármacos , Temperatura , Fatores de Tempo
3.
J Vis Exp ; (167)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33554960

RESUMO

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and computational modeling to classify and compare biological processes. Although cell culture model systems are great for high content imaging, high throughput studies of cell morphology suggest that ex vivo cultures are limited in recapitulating the morphological complexity found in cells within living organisms. As such, there is a need for a scalable high throughput model system to image living cells within an intact organism. Described here is a protocol for using a high content image analyzer to simultaneously acquire multiple time-lapse videos of embryonic Drosophila melanogaster development during the syncytial blastoderm stage. The syncytial blastoderm has traditionally served as a great in vivo model for imaging biological events; however, obtaining a significant number of experimental replicates for quantitative and high-throughput approaches has been labor intensive and limited by the imaging of a single embryo per experimental repeat. Presented here is a method to adapt imaging and microinjection approaches to suit a high content imaging system, or any inverted microscope capable of automated multipoint acquisition. This approach enables the simultaneous acquisition of 6-12 embryos, depending on desired acquisition factors, within a single imaging session.


Assuntos
Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Processamento de Imagem Assistida por Computador , Microinjeções , Microscopia/métodos , Animais , Automação , Dessecação
5.
Dev Biol ; 469: 1-11, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-32950464

RESUMO

The regulation of formation of the Drosophila heart by the Nkx 2.5 homologue Tinman is a key event during embryonic development. In this study, we identify the highly conserved transcription cofactor Akirin as a key factor in the earliest induction of tinman by the Twist transcription cofactor. akirin mutant embryos display a variety of morphological defects in the heart, including abnormal spacing between rows of aortic cells and abnormal patterning of the aortic outflow tract. akirin mutant embryos have a greatly reduced level of tinman transcripts, together with a reduction of Tinman protein in the earliest stages of cardiac patterning. Further, akirin mutants have reduced numbers of Tinman-positive cardiomyoblasts, concomitant with disrupted patterning and organization of the heart. Finally, despite the apparent formation of the heart in akirin mutants, these mutant hearts exhibit fewer coordinated contractions in akirin mutants compared with wild-type hearts. These results indicate that Akirin is crucial for the first induction of tinman by the Twist transcription factor, and that the success of the cardiac patterning program is highly dependent upon establishing the proper level of tinman at the earliest steps of the cardiac developmental pathway.


Assuntos
Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/embriologia , Proteínas Nucleares/fisiologia , Proteínas Repressoras/biossíntese , Transativadores/biossíntese , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/fisiologia , Coração/embriologia , Mutação , Contração Miocárdica , Miocárdio/metabolismo , Miocárdio/patologia , Proteínas Nucleares/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Transativadores/genética , Proteína 1 Relacionada a Twist/metabolismo
6.
Elife ; 92020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33320085

RESUMO

Phosphoinositides (PI) are key regulators of cellular organization in eukaryotes and genes that tune PI signaling are implicated in human disease mechanisms. Biochemical analyses and studies in cultured cells have identified a large number of proteins that can mediate PI signaling. However, the role of such proteins in regulating cellular processes in vivo and development in metazoans remains to be understood. Here, we describe a set of CRISPR-based genome engineering tools that allow the manipulation of each of these proteins with spatial and temporal control during metazoan development. We demonstrate the use of these reagents to deplete a set of 103 proteins individually in the Drosophila eye and identify several new molecules that control eye development. Our work demonstrates the power of this resource in uncovering the molecular basis of tissue homeostasis during normal development and in human disease biology.


Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Drosophila melanogaster/genética , Olho/embriologia , Engenharia Genética/métodos , Fosfatidilinositóis/metabolismo , Animais , Drosophila melanogaster/embriologia , Olho/metabolismo , Edição de Genes/métodos , Técnicas de Inativação de Genes , Genoma de Inseto/genética , Metabolismo dos Lipídeos , RNA Guia/biossíntese , RNA Guia/genética , Deleção de Sequência/genética , Transdução de Sinais/fisiologia
7.
J Vis Exp ; (163)2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32986033

RESUMO

Light sheet-based fluorescence microscopy offers efficient solutions to study complex processes on multiple biologically relevant scales. Sample chamber-based setups, which are specifically designed to preserve the three-dimensional integrity of the specimen and usually feature sample rotation, are the best choice in developmental biology. For instance, they have been used to document the entire embryonic morphogenesis of the fruit fly Drosophila melanogaster and the red flour beetle Tribolium castaneum. However, many available live imaging protocols provide only experimental frameworks for single embryos. Especially for comparative studies, such approaches are inconvenient, since sequentially imaged specimens are affected by ambient variance. Further, this limits the number of specimens that can be assayed within a given time. We provide an experimental framework for simultaneous live imaging that increases the throughput in sample chamber-based setups and thus ensures similar ambient conditions for all specimens. Firstly, we provide a calibration guideline for light sheet fluorescence microscopes. Secondly, we propose a mounting method for multiple embryos that is compatible with sample rotation. Thirdly, we provide exemplary three-dimensional live imaging datasets of Drosophila, for which we juxtapose three transgenic lines with fluorescently labeled nuclei, as well as of Tribolium, for which we compare the performance of three transgenic sublines that carry the same transgene, but at different genomic locations. Our protocol is specifically designed for comparative studies as it pro-actively addresses ambient variance, which is always present in sequential live imaging. This is especially important for quantitative analyses and characterization of aberrational phenotypes, which result e.g., from knockout experiments. Further, it increases the overall throughput, which is highly convenient when access to light sheet fluorescence microscopes is limited. Finally, the proposed mounting method can be adapted for other insect species and further model organisms, e.g., zebrafish, with basically no optimization effort.


Assuntos
Drosophila melanogaster/embriologia , Embrião não Mamífero/diagnóstico por imagem , Imageamento Tridimensional , Microscopia de Fluorescência/métodos , Tribolium/embriologia , Animais , Animais Geneticamente Modificados , Calibragem , Análise de Dados , Desenvolvimento Embrionário , Hipoclorito de Sódio
8.
Nat Commun ; 11(1): 4477, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901019

RESUMO

Individual cells detach from cohesive ensembles during development and can inappropriately separate in disease. Although much is known about how cells separate from epithelia, it remains unclear how cells disperse from clusters lacking apical-basal polarity, a hallmark of advanced epithelial cancers. Here, using live imaging of the developmental migration program of Drosophila primordial germ cells (PGCs), we show that cluster dispersal is accomplished by stabilizing and orienting migratory forces. PGCs utilize a G protein coupled receptor (GPCR), Tre1, to guide front-back migratory polarity radially from the cluster toward the endoderm. Posteriorly positioned myosin-dependent contractile forces pull on cell-cell contacts until cells release. Tre1 mutant cells migrate randomly with transient enrichment of the force machinery but fail to separate, indicating a temporal contractile force threshold for detachment. E-cadherin is retained on the cell surface during cell separation and augmenting cell-cell adhesion does not impede detachment. Notably, coordinated migration improves cluster dispersal efficiency by stabilizing cell-cell interfaces and facilitating symmetric pulling. We demonstrate that guidance of inherent migratory forces is sufficient to disperse cell clusters under physiological settings and present a paradigm for how such events could occur across development and disease.


Assuntos
Drosophila melanogaster/embriologia , Células Germinativas Embrionárias/fisiologia , Animais , Animais Geneticamente Modificados , Fenômenos Biomecânicos , Padronização Corporal/fisiologia , Caderinas/metabolismo , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/fisiologia , Células Germinativas Embrionárias/citologia , Microscopia de Fluorescência por Excitação Multifotônica , Miosina Tipo II/metabolismo , Transdução de Sinais , Análise de Célula Única , Proteínas rho de Ligação ao GTP/metabolismo
9.
Proc Natl Acad Sci U S A ; 117(26): 15096-15103, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541043

RESUMO

The regulatory specificity of a gene is determined by the structure of its enhancers, which contain multiple transcription factor binding sites. A unique combination of transcription factor binding sites in an enhancer determines the boundary of target gene expression, and their disruption often leads to developmental defects. Despite extensive characterization of binding motifs in an enhancer, it is still unclear how each binding site contributes to overall transcriptional activity. Using live imaging, quantitative analysis, and mathematical modeling, we measured the contribution of individual binding sites in transcriptional regulation. We show that binding site arrangement within the Rho-GTPase component t48 enhancer mediates the expression boundary by mainly regulating the timing of transcriptional activation along the dorsoventral axis of Drosophila embryos. By tuning the binding affinity of the Dorsal (Dl) and Zelda (Zld) sites, we show that single site modulations are sufficient to induce significant changes in transcription. Yet, no one site seems to have a dominant role; rather, multiple sites synergistically drive increases in transcriptional activity. Interestingly, Dl and Zld demonstrate distinct roles in transcriptional regulation. Dl site modulations change spatial boundaries of t48, mostly by affecting the timing of activation and bursting frequency rather than transcriptional amplitude or bursting duration. However, modulating the binding site for the pioneer factor Zld affects both the timing of activation and amplitude, suggesting that Zld may potentiate higher Dl recruitment to target DNAs. We propose that such fine-tuning of dynamic gene control via enhancer structure may play an important role in ensuring normal development.


Assuntos
Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análise Espaço-Temporal
10.
Proc Natl Acad Sci U S A ; 117(21): 11444-11449, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32381735

RESUMO

Morphogenetic flows in developmental biology are characterized by the coordinated motion of thousands of cells that organize into tissues, naturally raising the question of how this collective organization arises. Using only the kinematics of tissue deformation, which naturally integrates local and global mechanisms along cell paths, we identify the dynamic morphoskeletons behind morphogenesis, i.e., the evolving centerpieces of multicellular trajectory patterns. These features are model- and parameter-free, frame-invariant, and robust to measurement errors and can be computed from unfiltered cell-velocity data. We reveal the spatial attractors and repellers of the embryo by quantifying its Lagrangian deformation, information that is inaccessible to simple trajectory inspection or Eulerian methods that are local and typically frame-dependent. Computing these dynamic morphoskeletons in wild-type and mutant chick and fly embryos, we find that they capture the early footprint of known morphogenetic features, reveal new ones, and quantitatively distinguish between different phenotypes.


Assuntos
Embrião de Galinha/citologia , Embrião de Galinha/crescimento & desenvolvimento , Drosophila melanogaster/embriologia , Modelos Biológicos , Animais , Animais Geneticamente Modificados , Fenômenos Biomecânicos , Embrião de Galinha/efeitos dos fármacos , Simulação por Computador , Proteínas de Drosophila/genética , Embrião não Mamífero/citologia , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/metabolismo , Gástrula/crescimento & desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Indazóis/farmacologia , Microscopia/métodos , Morfogênese , Mutação , Proteína 1 Relacionada a Twist/genética
11.
Sci Rep ; 10(1): 8485, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444687

RESUMO

Despite their essential function in terminating translation, readthrough of stop codons occurs more frequently than previously supposed. However, little is known about the regulation of stop codon readthrough by anatomical site and over the life cycle of animals. Here, we developed a set of reporters to measure readthrough in Drosophila melanogaster. A focused RNAi screen in whole animals identified upf1 as a mediator of readthrough, suggesting that the stop codons in the reporters were recognized as premature termination codons (PTCs). We found readthrough rates of PTCs varied significantly throughout the life cycle of flies, being highest in older adult flies. Furthermore, readthrough rates varied dramatically by tissue and, intriguingly, were highest in fly brains, specifically neurons and not glia. This was not due to differences in reporter abundance or nonsense-mediated mRNA decay (NMD) surveillance between these tissues. Readthrough rates also varied within neurons, with cholinergic neurons having highest readthrough compared with lowest readthrough rates in dopaminergic neurons. Overall, our data reveal temporal and spatial variation of PTC-mediated readthrough in animals, and suggest that readthrough may be a potential rescue mechanism for PTC-harboring transcripts when the NMD surveillance pathway is inhibited.


Assuntos
Códon de Terminação , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Biossíntese de Proteínas , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Embrião não Mamífero/citologia , Feminino , Perfilação da Expressão Gênica , Masculino , Especificidade de Órgãos
12.
Genes Dev ; 34(13-14): 965-972, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32467225

RESUMO

Graded transcription factors are pivotal regulators of embryonic patterning, but whether their role changes over time is unclear. A light-regulated protein degradation system was used to assay temporal dependence of the transcription factor Dorsal in dorsal-ventral axis patterning of Drosophila embryos. Surprisingly, the high-threshold target gene snail only requires Dorsal input early but not late when Dorsal levels peak. Instead, late snail expression can be supported by action of the Twist transcription factor, specifically, through one enhancer, sna.distal This study demonstrates that continuous input is not required for some Dorsal targets and downstream responses, such as twist, function as molecular ratchets.


Assuntos
Padronização Corporal/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Animais , Padronização Corporal/efeitos da radiação , Proteínas de Drosophila/genética , Embrião não Mamífero , Luz , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteólise/efeitos da radiação , Fatores de Transcrição da Família Snail/metabolismo , Fatores de Transcrição/genética , Proteína 1 Relacionada a Twist/genética
13.
Am J Physiol Cell Physiol ; 318(6): C1107-C1122, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32267718

RESUMO

Tetraspanin-2A (Tsp2A) is an integral membrane protein of smooth septate junctions in Drosophila melanogaster. To elucidate its structural and functional roles in Malpighian tubules, we used the c42-GAL4/UAS system to selectively knock down Tsp2A in principal cells of the tubule. Tsp2A localizes to smooth septate junctions (sSJ) in Malpighian tubules in a complex shared with partner proteins Snakeskin (Ssk), Mesh, and Discs large (Dlg). Knockdown of Tsp2A led to the intracellular retention of Tsp2A, Ssk, Mesh, and Dlg, gaps and widening spaces in remaining sSJ, and tumorous and cystic tubules. Elevated protein levels together with diminished V-type H+-ATPase activity in Tsp2A knockdown tubules are consistent with cell proliferation and reduced transport activity. Indeed, Malpighian tubules isolated from Tsp2A knockdown flies failed to secrete fluid in vitro. The absence of significant transepithelial voltages and resistances manifests an extremely leaky epithelium that allows secreted solutes and water to leak back to the peritubular side. The tubular failure to excrete fluid leads to extracellular volume expansion in the fly and to death within the first week of adult life. Expression of the c42-GAL4 driver begins in Malpighian tubules in the late embryo and progresses upstream to distal tubules in third instar larvae, which can explain why larvae survive Tsp2A knockdown and adults do not. Uncontrolled cell proliferation upon Tsp2A knockdown confirms the role of Tsp2A as tumor suppressor in addition to its role in sSJ structure and transepithelial transport.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Epiteliais/metabolismo , Túbulos de Malpighi/metabolismo , Tetraspaninas/metabolismo , Junções Íntimas/metabolismo , Animais , Animais Geneticamente Modificados , Proliferação de Células , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/ultraestrutura , Impedância Elétrica , Células Epiteliais/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Larva/genética , Larva/metabolismo , Larva/ultraestrutura , Túbulos de Malpighi/embriologia , Túbulos de Malpighi/ultraestrutura , Via Secretória , Transdução de Sinais , Tetraspaninas/genética , Junções Íntimas/genética , Junções Íntimas/ultraestrutura , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo
14.
Development ; 147(5)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32122911

RESUMO

Mutations in the Ultrabithorax (Ubx) gene cause homeotic transformation of the normally two-winged Drosophila into a four-winged mutant fly. Ubx encodes a HOX family transcription factor that specifies segment identity, including transformation of the second set of wings into rudimentary halteres. Ubx is known to control the expression of many genes that regulate tissue growth and patterning, but how it regulates tissue morphogenesis to reshape the wing into a haltere is still unclear. Here, we show that Ubx acts by repressing the expression of two genes in the haltere, Stubble and Notopleural, both of which encode transmembrane proteases that remodel the apical extracellular matrix to promote wing morphogenesis. In addition, Ubx induces expression of the Tissue inhibitor of metalloproteases in the haltere, which prevents the basal extracellular matrix remodelling necessary for wing morphogenesis. Our results provide a long-awaited explanation for how Ubx controls morphogenetic transformation.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Proteínas de Homeodomínio/genética , Morfogênese/genética , Fatores de Transcrição/genética , Asas de Animais/embriologia , Animais , Sistemas CRISPR-Cas , Drosophila melanogaster/genética , Inibidores de Metaloproteinases de Matriz/metabolismo , Proteínas de Membrana/genética , Serina Endopeptidases/genética
15.
Development ; 147(5)2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32161062

RESUMO

The modulation of mechanical tension is important for sculpturing tissues during animal development, yet how mechanical tension is controlled remains poorly understood. In Drosophila wing discs, the local reduction of mechanical tension at basal cell edges results in basal relaxation and the formation of an epithelial fold. Here, we show that Wingless, which is expressed next to this fold, promotes basal cell edge tension to suppress the formation of this fold. Ectopic expression of Wingless blocks fold formation, whereas the depletion of Wingless increases fold depth. Moreover, local depletion of Wingless in a region where Wingless signal transduction is normally high results in ectopic fold formation. The depletion of Wingless also results in decreased basal cell edge tension and basal cell area relaxation. Conversely, the activation of Wingless signal transduction leads to increased basal cell edge tension and basal cell area constriction. Our results identify the Wingless signal transduction pathway as a crucial modulator of mechanical tension that is important for proper wing disc morphogenesis.


Assuntos
Padronização Corporal/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Morfogênese/genética , Asas de Animais/embriologia , Proteína Wnt1/genética , Animais , Padronização Corporal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Transdução de Sinais/genética , Estresse Mecânico
16.
Nat Commun ; 11(1): 1495, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198383

RESUMO

Imaging of complex (biological) samples in the near-infrared (NIR) is beneficial due to reduced light scattering, absorption, phototoxicity, and autofluorescence. However, there are few NIR fluorescent materials known and suitable for biomedical applications. Here we exfoliate the layered pigment CaCuSi4O10 (Egyptian Blue, EB) via ball milling and facile tip sonication into NIR fluorescent nanosheets (EB-NS). The size of EB-NS can be tailored to diameters <20 nm and heights down to 1 nm. EB-NS fluoresce at 910 nm and the fluorescence intensity correlates with the number of Cu2+ ions. Furthermore, EB-NS display no bleaching and high brightness compared with other NIR fluorophores. The versatility of EB-NS is demonstrated by in-vivo single-particle tracking and microrheology measurements in Drosophila melanogaster embryos. EB-NS can be uptaken by plants and remotely detected in a low-cost stand-off detection setup. In summary, EB-NS have the potential for a wide range of bioimaging applications.


Assuntos
Corantes Fluorescentes/efeitos da radiação , Raios Infravermelhos , Imagem Óptica/métodos , Óptica e Fotônica/métodos , Silicatos/efeitos da radiação , Animais , Cobre , Drosophila melanogaster/embriologia , Fluorescência , Íons , Modelos Teóricos , Nanopartículas
17.
Development ; 147(5)2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32098765

RESUMO

The dramatic changes in gene expression required for development necessitate the establishment of cis-regulatory modules defined by regions of accessible chromatin. Pioneer transcription factors have the unique property of binding closed chromatin and facilitating the establishment of these accessible regions. Nonetheless, much of how pioneer transcription factors coordinate changes in chromatin accessibility during development remains unknown. To determine whether pioneer-factor function is intrinsic to the protein or whether pioneering activity is developmentally modulated, we studied the highly conserved, essential transcription factor Grainy head (Grh). Prior work established that Grh is expressed throughout Drosophila development and is a pioneer factor in the larva. We demonstrated that Grh remains bound to mitotic chromosomes, a property shared with other pioneer factors. By assaying chromatin accessibility in embryos lacking maternal and/or zygotic Grh at three stages of development, we discovered that Grh is not required for chromatin accessibility in early embryogenesis, in contrast to its essential functions later in development. Our data reveal that the pioneering activity of Grh is temporally regulated and likely influenced by additional factors expressed at a given developmental stage.


Assuntos
Cromatina/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Fatores de Transcrição/genética , Animais , Drosophila melanogaster/genética , Desenvolvimento Embrionário/genética , Mitose/genética
18.
Development ; 147(5)2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32051172

RESUMO

Neural remodeling is essential for the development of a functional nervous system and has been extensively studied in the metamorphosis of Drosophila Despite the crucial roles of glial cells in brain functions, including learning and behavior, little is known of how adult glial cells develop in the context of neural remodeling. Here, we show that the architecture of neuropil-glia in the adult Drosophila brain, which is composed of astrocyte-like glia (ALG) and ensheathing glia (EG), robustly develops from two different populations in the larva: the larval EG and glial cell missing-positive (gcm+ ) cells. Whereas gcm + cells proliferate and generate adult ALG and EG, larval EG dedifferentiate, proliferate and redifferentiate into the same glial subtypes. Each glial lineage occupies a certain brain area complementary to the other, and together they form the adult neuropil-glia architecture. Both lineages require the FGF receptor Heartless to proliferate, and the homeoprotein Prospero to differentiate into ALG. Lineage-specific inhibition of gliogenesis revealed that each lineage compensates for deficiency in the proliferation of the other. Together, the lineages ensure the robust development of adult neuropil-glia, thereby ensuring a functional brain.


Assuntos
Astrócitos/citologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Neurópilo/citologia , Proteínas Nucleares/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Encéfalo/citologia , Encéfalo/embriologia , Linhagem da Célula/fisiologia , Proliferação de Células/fisiologia , Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/metabolismo , Metamorfose Biológica/genética , Metamorfose Biológica/fisiologia , Neurogênese/genética
19.
Adv Exp Med Biol ; 1218: 1-7, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32060868

RESUMO

The attention of science first turned to the gene that later earned the name Notch over a century ago, when the American scientist John S. Dexter discovered in his laboratory at Olivet College the characteristic notched-wing phenotype (a nick or notch in the wingtip) in mutant fruit flies Drosophila melanogaster. At present, it is generally accepted that the Notch pathway governs tissue patterning and many key cell fate decisions and other core processes during embryonic development and in adult tissues. Not surprisingly, a broad variety of independent inherited diseases (including CADASIL, Alagille, Adams-Oliver, and Hajdu-Cheney syndromes) have now convincingly been linked to defective Notch signaling. In the second edition of the book entitled Notch Signaling in Embryology and Cancer, leading researchers provide a comprehensive, highly readable overview on molecular mechanisms of Notch signaling (Volume I), and notch's roles in embryology (Vol. II) and cancer (Vol. III). In these introductory pages of Vol. II, we give a short overview on its individual chapters, which are intended to provide both basic scientists and clinicians who seek today's clearest understanding of the broad role of Notch signaling in embryology with an authoritative day-to-day source.


Assuntos
Padronização Corporal , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Humanos
20.
Adv Exp Med Biol ; 1218: 9-37, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32060869

RESUMO

The evolutionary highly conserved Notch pathway, which first developed during evolution in metazoans and was first discovered in fruit flies (Drosophila melanogaster), governs many core processes including cell fate decisions during embryonic development. A huge mountain of scientific evidence convincingly demonstrates that Notch signaling represents one of the most important pathways that regulate embryogenesis from sponges, roundworms, Drosophila melanogaster, and mice to humans. In this review, we give a brief introduction on how Notch orchestrates the embryonic development of several selected tissues, summarizing some of the most relevant findings in the central nervous system, skin, kidneys, liver, pancreas, inner ear, eye, skeleton, heart, and vascular system.


Assuntos
Desenvolvimento Embrionário , Receptores Notch/metabolismo , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Humanos , Especificidade de Órgãos
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