RESUMEN
How complex 3D tissue shape emerges during animal development remains an important open question in biology and biophysics. Here, we discover a mechanism for 3D epithelial shape change based on active, in-plane cellular events that is analogous to inanimate "shape programmable" materials, which undergo blueprinted 3D shape transformations from in-plane gradients of spontaneous strains. We study eversion of the Drosophila wing disc pouch, when the epithelium transforms from a dome into a curved fold, quantifying 3D tissue shape changes and mapping spatial patterns of cellular behaviors on the evolving geometry using cellular topology. Using a physical model inspired by shape programming, we find that active cell rearrangements are the major contributor to pouch eversion and validate this conclusion using a knockdown of MyoVI, which reduces rearrangements and disrupts morphogenesis. This work shows that shape programming is a mechanism for animal tissue morphogenesis and suggests that patterns in nature could present design strategies for shape-programmable materials.
Asunto(s)
Morfogénesis , Alas de Animales , Animales , Alas de Animales/crecimiento & desarrollo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila , Modelos Biológicos , Discos Imaginales/metabolismo , Discos Imaginales/crecimiento & desarrolloRESUMEN
Histone acetylation regulates gene expression, cell function and cell fate1. Here we study the pattern of histone acetylation in the epithelial tissue of the Drosophila wing disc. H3K18ac, H4K8ac and total lysine acetylation are increased in the outer rim of the disc. This acetylation pattern is controlled by nuclear position, whereby nuclei continuously move from apical to basal locations within the epithelium and exhibit high levels of H3K18ac when they are in proximity to the tissue surface. These surface nuclei have increased levels of acetyl-CoA synthase, which generates the acetyl-CoA for histone acetylation. The carbon source for histone acetylation in the rim is fatty acid ß-oxidation, which is also increased in the rim. Inhibition of fatty acid ß-oxidation causes H3K18ac levels to decrease in the genomic proximity of genes involved in disc development. In summary, there is a physical mark of the outer rim of the wing and other imaginal epithelia in Drosophila that affects gene expression.
Asunto(s)
Acetilcoenzima A , Núcleo Celular , Cromatina , Drosophila melanogaster , Animales , Acetato CoA Ligasa/metabolismo , Acetilcoenzima A/metabolismo , Acetilación , Transporte Biológico , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , Cromatina/genética , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica , Histonas/química , Histonas/metabolismo , Discos Imaginales/citología , Discos Imaginales/crecimiento & desarrollo , Discos Imaginales/metabolismo , Lisina/metabolismo , Oxidación-Reducción , Alas de Animales/citología , Alas de Animales/crecimiento & desarrollo , Alas de Animales/metabolismoRESUMEN
Regeneration is a response mechanism aiming to reconstruct lost or damaged structures. To achieve this, the cells repopulating the lost tissue often have to change their original identity, a process that involves chromatin remodelling.We have analysed the issue of chromatin remodelling during regeneration in the wing disc of Drosophila . In this disc the ablation of the central region (the pouch) induces the regenerative response of the cells from the lateral region (the hinge), which reconstitute the wing pouch. We have examined euchromatin and heterochromatin histone marks during the process and find that heterochromatin marks disappear but are recovered when regeneration is complete. Euchromatin marks are not modified. We also describe the transcription of two retrotransposons, Roo and F-element in the regenerating cells. We have established a temporal correlation between the alterations of heterochromatin marks and the levels of transcription of two retrotransposons, Roo and F-element, both during embryonic development and in the regeneration process.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Drosophila melanogaster/embriología , Discos Imaginales/crecimiento & desarrollo , Regeneración/fisiología , Retroelementos/genética , Alas de Animales/embriología , Acetilación , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Eucromatina/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Elementos de Nucleótido Esparcido Largo/genética , Metilación , Alas de Animales/crecimiento & desarrolloRESUMEN
Cell competition induces the elimination of less-fit "loser" cells by fitter "winner" cells. In Drosophila, cells heterozygous mutant in ribosome genes, Rp/+, known as Minutes, are outcompeted by wild-type cells. Rp/+ cells display proteotoxic stress and the oxidative stress response, which drive the loser status. Minute cell competition also requires the transcription factors Irbp18 and Xrp1, but how these contribute to the loser status is partially understood. Here we provide evidence that initial proteotoxic stress in RpS3/+ cells is Xrp1-independent. However, Xrp1 is sufficient to induce proteotoxic stress in otherwise wild-type cells and is necessary for the high levels of proteotoxic stress found in RpS3/+ cells. Surprisingly, Xrp1 is also induced downstream of proteotoxic stress, and is required for the competitive elimination of cells suffering from proteotoxic stress or overexpressing Nrf2. Our data suggests that a feed-forward loop between Xrp1, proteotoxic stress, and Nrf2 drives Minute cells to become losers.
Asunto(s)
Competencia Celular , Proteínas de Unión al ADN , Proteínas de Drosophila , Proteínas Ribosómicas , Animales , Apoptosis/genética , Competencia Celular/genética , Proteínas de Unión al ADN/genética , Drosophila melanogaster/genética , Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica/genética , Discos Imaginales/crecimiento & desarrollo , Discos Imaginales/metabolismo , Estrés Oxidativo/genética , Proteínas Ribosómicas/genética , Ribosomas/genética , Transducción de Señal/genética , Factores de Transcripción/genéticaRESUMEN
Regeneration is a complex process that requires a coordinated genetic response to tissue loss. Signals from dying cells are crucial to this process and are best understood in the context of regeneration following programmed cell death, like apoptosis. Conversely, regeneration following unregulated forms of death, such as necrosis, have yet to be fully explored. Here, we have developed a method to investigate regeneration following necrosis using the Drosophila wing imaginal disc. We show that necrosis stimulates regeneration at an equivalent level to that of apoptosis-mediated cell death and activates a similar response at the wound edge involving localized JNK signaling. Unexpectedly, however, necrosis also results in significant apoptosis far from the site of ablation, which we have termed necrosis-induced apoptosis (NiA). This apoptosis occurs independent of changes at the wound edge and importantly does not rely on JNK signaling. Furthermore, we find that blocking NiA limits proliferation and subsequently inhibits regeneration, suggesting that tissues damaged by necrosis can activate programmed cell death at a distance from the injury to promote regeneration.
Asunto(s)
Drosophila melanogaster/fisiología , Regulación del Desarrollo de la Expresión Génica , Discos Imaginales/crecimiento & desarrollo , Regeneración/genética , Animales , Animales Modificados Genéticamente , Apoptosis/genética , Proliferación Celular/genética , Proteínas de Drosophila/metabolismo , Femenino , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Masculino , Necrosis/genética , Alas de Animales/crecimiento & desarrolloRESUMEN
How morphogen gradients control patterning and growth in developing tissues remains largely unknown due to lack of tools manipulating morphogen gradients. Here, we generate two membrane-tethered protein binders that manipulate different aspects of Decapentaplegic (Dpp), a morphogen required for overall patterning and growth of the Drosophila wing. One is "HA trap" based on a single-chain variable fragment (scFv) against the HA tag that traps HA-Dpp to mainly block its dispersal, the other is "Dpp trap" based on a Designed Ankyrin Repeat Protein (DARPin) against Dpp that traps Dpp to block both its dispersal and signaling. Using these tools, we found that, while posterior patterning and growth require Dpp dispersal, anterior patterning and growth largely proceed without Dpp dispersal. We show that dpp transcriptional refinement from an initially uniform to a localized expression and persistent signaling in transient dpp source cells render the anterior compartment robust against the absence of Dpp dispersal. Furthermore, despite a critical requirement of dpp for the overall wing growth, neither Dpp dispersal nor direct signaling is critical for lateral wing growth after wing pouch specification. These results challenge the long-standing dogma that Dpp dispersal is strictly required to control and coordinate overall wing patterning and growth.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Alas de Animales/metabolismo , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/genética , Proteínas Morfogenéticas Óseas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Discos Imaginales/crecimiento & desarrollo , Discos Imaginales/metabolismo , Microscopía Confocal , Mutación , Transducción de Señal/genética , Alas de Animales/crecimiento & desarrolloRESUMEN
Cell competition is a homeostatic process that eliminates by apoptosis unfit or undesirable cells from animal tissues, including tumor cells that appear during the life of the organism. In Drosophila there is evidence that many types of oncogenic cells are eliminated by cell competition. One exception is cells mutant for polyhomeotic (ph), a member of the Polycomb family of genes; most of the isolated mutant ph clones survive and develop tumorous overgrowths in imaginal discs. To characterize the tumorigenic effect of the lack of ph, we first studied the growth of different regions of the wing disc deficient in ph activity and found that the effect is restricted to the proximal appendage. Moreover, we found that ph-deficient tissue is partially refractory to apoptosis. Second, we analyzed the behavior of clones lacking ph function and found that many suffer cell competition but are not completely eliminated. Unexpectedly, we found that nonmutant cells also undergo cell competition when surrounded by ph-deficient cells, indicating that within the same tissue cell competition may operate in opposite directions. We suggest two reasons for the incompleteness of cell competition in ph mutant cells: 1) These cells are partially refractory to apoptosis, and 2) the loss of ph function alters the identity of imaginal cells and subsequently their cell affinities. It compromises the winner/loser interaction, a prerequisite for cell competition.
Asunto(s)
Carcinogénesis , Competencia Celular , Proteínas de Unión al ADN/fisiología , Proteínas de Drosophila/fisiología , Discos Imaginales/crecimiento & desarrollo , Complejo Represivo Polycomb 1/fisiología , Animales , Apoptosis , Drosophila , Sistema de Señalización de MAP QuinasasRESUMEN
The growth of imaginal discs in holometabolous insects is coordinated with larval growth to ensure the symmetrical and proportional development of the adult appendages. In ants, the differential growth of these discs generates distinct castes-the winged male and queen castes and the wingless worker caste. In the hyperdiverse ant genus Pheidole, the worker caste is composed of two morphologically distinct subcastes: small-headed minor workers and larger, big-headed, soldiers. Although these worker subcastes are completely wingless, soldier larvae develop rudimentary forewing discs that function in generating the disproportionate head-to-body scaling and size of soldiers. It remains unclear, however, how rudimentary forewing discs in soldier larvae are coordinated with other imaginal discs. Here we show, using quantitative nano-CT imaging and three-dimensional analyses, that the increase in the volume of the soldier rudimentary forewing discs is coordinated with larval size as well as with the increase in the volume of the leg and eye-antennal (head) discs. However, relative to larval size, we found that when the rudimentary forewing discs appear during the last larval instar, they are relatively smaller but increase in volume faster than that of the head (eye-antennal) and leg discs. These findings show that the rudimentary wing disc in soldier larvae has evolved novel patterns of inter-organ coordination as compared with other insects to generate the big-headed soldier caste in Pheidole. More generally, our study raises the possibility that novel patterns of inter-organ coordination are a general feature of rudimentary organs that acquire novel regulatory functions during development and evolution.
Asunto(s)
Hormigas , Discos Imaginales/crecimiento & desarrollo , Animales , Hormigas/anatomía & histología , Hormigas/crecimiento & desarrollo , Larva/anatomía & histología , Larva/crecimiento & desarrollo , Masculino , Morfogénesis , Nanotecnología , Tomografía Computarizada por Rayos X , Alas de AnimalesRESUMEN
Aneuploidy causes birth defects and miscarriages, occurs in nearly all cancers and is a hallmark of aging. Individual aneuploid cells can be eliminated from developing tissues by unknown mechanisms. Cells with ribosomal protein (Rp) gene mutations are also eliminated, by cell competition with normal cells. Because Rp genes are spread across the genome, their copy number is a potential marker for aneuploidy. We found that elimination of imaginal disc cells with irradiation-induced genome damage often required cell competition genes. Segmentally aneuploid cells derived from targeted chromosome excisions were eliminated by the RpS12-Xrp1 cell competition pathway if they differed from neighboring cells in Rp gene dose, whereas cells with normal doses of the Rp and eIF2γ genes survived and differentiated adult tissues. Thus, cell competition, triggered by differences in Rp gene dose between cells, is a significant mechanism for the elimination of aneuploid somatic cells, likely to contribute to preventing cancer.
Aneuploid cells emerge when cellular division goes awry and a cell ends up with the wrong number of chromosomes, the tiny genetic structures carrying the instructions that control life's processes. Aneuploidy can lead to fatal conditions during development, and to cancer in an adult organism. A safety mechanism may exist that helps the body to detect and remove these cells. Yet, exactly this happens is still poorly understood: in particular, it is unclear how cells manage to 'count' their chromosomes. One way they could do so is through the ribosomes, the molecular 'factories' that create the building blocks required for life. In a cell, every chromosome carries genes that code for the proteins (known as Rps) forming ribosomes. Aneuploidy will alter the number of Rp genes, and in turn the amount and type of Rps the cell produces, so that ribosomes and the genes for Rps could act as a 'readout' of aneuploidy. Ji et al set out to test this theory in fruit flies. The first experiment used a genetic manipulation technique called site-specific recombination to remove parts of chromosomes from cells in the developing eye and wing. Cells which retained all their Rp genes survived, while those that were missing some usually died but only when the surrounding cells were normal. In this situation, healthy cells eliminated their damaged neighbours through a process known as cell competition. A second experiment, using radiation as an alternative method of damaging chromosomes, also gave similar results. The work by Ji et al. reveals how the body can detect and eliminate aneuploid cells, potentially before they can cause harm. If the same mechanism applies in humans, boosting cell competition may, one day, helps to combat diseases like cancer.
Asunto(s)
Aneuploidia , Competencia Celular , Drosophila melanogaster/fisiología , Dosificación de Gen , Proteínas Ribosómicas/metabolismo , Animales , Drosophila melanogaster/crecimiento & desarrollo , Humanos , Discos Imaginales/crecimiento & desarrollo , Discos Imaginales/fisiología , Neoplasias/genéticaRESUMEN
In both vertebrates and invertebrates, generating a functional appendage requires interactions between ectoderm-derived epithelia and mesoderm-derived cells. To investigate such interactions, we used single-cell transcriptomics to generate a temporal cell atlas of the Drosophila wing disc from two developmental time points. Using these data, we visualized gene expression using a multilayered model of the wing disc and cataloged ligand-receptor pairs that could mediate signaling between epithelial cells and adult muscle precursors (AMPs). We found that localized expression of the fibroblast growth factor ligands, Thisbe and Pyramus, in the disc epithelium regulates the number and location of the AMPs. In addition, Hedgehog ligand from the epithelium activates a specific transcriptional program within adjacent AMP cells, defined by AMP-specific targets Neurotactin and midline, that is critical for proper formation of direct flight muscles. More generally, our annotated temporal cell atlas provides an organ-wide view of potential cell-cell interactions between epithelial and myogenic cells.
Asunto(s)
Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Discos Imaginales/crecimiento & desarrollo , Transcriptoma , Animales , Epitelio/fisiología , Discos Imaginales/metabolismo , Larva/crecimiento & desarrollo , Larva/metabolismo , Mioblastos/fisiología , Análisis de la Célula Individual , Alas de Animales/crecimiento & desarrollo , Alas de Animales/metabolismoRESUMEN
Regeneration of Drosophila imaginal discs, larval precursors to adult tissues, activates a regeneration checkpoint that coordinates regenerative growth with developmental progression. This regeneration checkpoint results from the release of the relaxin-family peptide Dilp8 from regenerating imaginal tissues. Secreted Dilp8 protein is detected within the imaginal disc lumen, in which it is separated from its receptor target Lgr3, which is expressed in the brain and prothoracic gland, by the disc epithelial barrier. Here, we demonstrate that following damage the imaginal disc epithelial barrier limits Dilp8 signaling and the duration of regeneration checkpoint delay. We also find that the barrier becomes increasingly impermeable to the transepithelial diffusion of labeled dextran during the second half of the third instar. This change in barrier permeability is driven by the steroid hormone ecdysone and correlates with changes in localization of Coracle, a component of the septate junctions that is required for the late-larval impermeable epithelial barrier. Based on these observations, we propose that the imaginal disc epithelial barrier regulates the duration of the regenerative checkpoint, providing a mechanism by which tissue function can signal the completion of regeneration.
Asunto(s)
Proteínas de Drosophila/genética , Discos Imaginales/crecimiento & desarrollo , Péptidos y Proteínas de Señalización Intercelular/genética , Receptores Acoplados a Proteínas G/genética , Regeneración/genética , Animales , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Ecdisona/genética , Regulación del Desarrollo de la Expresión Génica , Discos Imaginales/metabolismo , Larva/genética , Larva/crecimiento & desarrollo , Neuronas/metabolismo , Transducción de Señal/genéticaRESUMEN
Highly reproducible tissue development is achieved by robust, time-dependent coordination of cell proliferation and cell death. To study the mechanisms underlying robust tissue growth, we analyzed the developmental process of wing imaginal discs in Drosophila Minute mutants, a series of heterozygous mutants for a ribosomal protein gene. Minute animals show significant developmental delay during the larval period but develop into essentially normal flies, suggesting there exists a mechanism ensuring robust tissue growth during abnormally prolonged developmental time. Surprisingly, we found that both cell death and compensatory cell proliferation were dramatically increased in developing wing pouches of Minute animals. Blocking the cell-turnover by inhibiting cell death resulted in morphological defects, indicating the essential role of cell-turnover in Minute wing morphogenesis. Our analyses showed that Minute wing discs elevate Wg expression and JNK-mediated Dilp8 expression that causes developmental delay, both of which are necessary for the induction of cell-turnover. Furthermore, forced increase in Wg expression together with developmental delay caused by ecdysone depletion induced cell-turnover in the wing pouches of non-Minute animals. Our findings suggest a novel paradigm for robust coordination of tissue growth by cell-turnover, which is induced when developmental time axis is distorted.
Asunto(s)
Proteínas de Drosophila/genética , Discos Imaginales/crecimiento & desarrollo , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas Ribosómicas/genética , Proteína Wnt1/genética , Animales , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Ecdisona/genética , Células Epiteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Discos Imaginales/metabolismo , Larva/genética , Larva/crecimiento & desarrollo , Metamorfosis Biológica/genética , Organogénesis/genética , Transducción de Señal/genética , Factores de Transcripción/genética , Alas de Animales/crecimiento & desarrollo , Alas de Animales/metabolismoRESUMEN
Regulation of microtubule stability is crucial for the maintenance of cell structure and function. While the acetylation of α-tubulin lysine 40 by acetylase has been implicated in the regulation of microtubule stability, the in vivo functions of N-terminal acetyltransferases (NATs) involved in the acetylation of N-terminal amino acids are not well known. Here, we identify an N-terminal acetyltransferase, Mnat9, that regulates cell signaling and microtubule stability in Drosophila Loss of Mnat9 causes severe developmental defects in multiple tissues. In the wing imaginal disc, Mnat9 RNAi leads to the ectopic activation of c-Jun N-terminal kinase (JNK) signaling and apoptotic cell death. These defects are suppressed by reducing the level of JNK signaling. Overexpression of Mnat9 can also inhibit JNK signaling. Mnat9 colocalizes with mitotic spindles, and its loss results in various spindle defects during mitosis in the syncytial embryo. Furthermore, overexpression of Mnat9 enhances microtubule stability. Mnat9 is physically associated with microtubules and shows a catalytic activity in acetylating N-terminal peptides of α- and ß-tubulin in vitro. Cell death and tissue loss in Mnat9-depleted wing discs are restored by reducing the severing protein Spastin, suggesting that Mnat9 protects microtubules from its severing activity. Remarkably, Mnat9 mutated in the acetyl-CoA binding site is as functional as its wild-type form. We also find that human NAT9 can rescue Mnat9 RNAi phenotypes in flies, indicating their functional conservation. Taken together, we propose that Mnat9 is required for microtubule stability and regulation of JNK signaling to promote cell survival in developing Drosophila organs.
Asunto(s)
Drosophila melanogaster/genética , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Acetiltransferasas N-Terminal/genética , Animales , Apoptosis/genética , Drosophila melanogaster/crecimiento & desarrollo , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica/genética , Discos Imaginales/crecimiento & desarrollo , Discos Imaginales/metabolismo , Microtúbulos/genética , Mitosis/genética , Transducción de Señal/genética , Alas de Animales/crecimiento & desarrollo , Alas de Animales/metabolismoRESUMEN
The Drosophila melanogaster wing imaginal disc is an epithelial sac that exhibits dramatic tissue growth during the larval stage. With its simple morphology and accessibility of genetic tools, studies using the wing disc have contributed to the understanding of the mechanisms of epithelial homeostasis including the control of mitotic spindle orientation. This chapter describes a detailed protocol for analyzing epithelial architecture and planar orientation of the mitotic spindle in the wing disc epithelium. The rapid dissection method, effective immunostaining, and mounting tips described here facilitate genetic and cell biological studies of the wing disc and can be applied to a wide array of studies using various Drosophila tissues.
Asunto(s)
Células Epiteliales/citología , Discos Imaginales/citología , Huso Acromático/genética , Alas de Animales/citología , Animales , Drosophila melanogaster , Discos Imaginales/crecimiento & desarrollo , Alas de Animales/crecimiento & desarrolloRESUMEN
Dis3L2 is a highly conserved 3'-5' exoribonuclease which is mutated in the human overgrowth disorders Perlman syndrome and Wilms' tumour of the kidney. Using Drosophila melanogaster as a model system, we have generated a new dis3L2 null mutant together with wild-type and nuclease-dead genetic lines in Drosophila to demonstrate that the catalytic activity of Dis3L2 is required to control cell proliferation. To understand the cellular pathways regulated by Dis3L2 to control proliferation, we used RNA-seq on dis3L2 mutant wing discs to show that the imaginal disc growth factor Idgf2 is responsible for driving the wing overgrowth. IDGFs are conserved proteins homologous to human chitinase-like proteins such as CHI3L1/YKL-40 which are implicated in tissue regeneration as well as cancers including colon cancer and non-small cell lung cancer. We also demonstrate that loss of DIS3L2 in human kidney HEK-293T cells results in cell proliferation, illustrating the conservation of this important cell proliferation pathway. Using these human cells, we show that loss of DIS3L2 results in an increase in the PI3-Kinase/AKT signalling pathway, which we subsequently show to contribute towards the proliferation phenotype in Drosophila. Our work therefore provides the first mechanistic explanation for DIS3L2-induced overgrowth in humans and flies and identifies an ancient proliferation pathway controlled by Dis3L2 to regulate cell proliferation and tissue growth.
Asunto(s)
Proliferación Celular , Discos Imaginales/metabolismo , Animales , Proteína 1 Similar a Quitinasa-3/química , Proteína 1 Similar a Quitinasa-3/metabolismo , Secuencia Conservada , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Glicoproteínas/metabolismo , Células HEK293 , Humanos , Discos Imaginales/crecimiento & desarrollo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
Imaginal disc morphogenesis during metamorphosis in Drosophila melanogaster provides an excellent model to uncover molecular mechanisms by which hormonal signals effect physical changes during development. The broad (br) Z2 isoform encodes a transcription factor required for disc morphogenesis in response to 20-hydroxyecdysone, yet how it accomplishes this remains largely unknown. Here, we use functional studies of amorphic br5 mutants and a transcriptional target approach to identify processes driven by br and its regulatory targets in leg imaginal discs. br5 mutants fail to properly remodel their basal extracellular matrix (ECM) between 4 and 7 hr after puparium formation. Additionally, br5 mutant discs do not undergo the cell shape changes necessary for leg elongation and fail to elongate normally when exposed to the protease trypsin. RNA-sequencing of wild-type and br5 mutant leg discs identified 717 genes differentially regulated by br, including a large number of genes involved in glycolysis, and genes that encode proteins that interact with the ECM. RNA interference-based functional studies reveal that several of these genes are required for adult leg formation, particularly those involved in remodeling the ECM. Additionally, brZ2 expression is abruptly shut down at the onset of metamorphosis, and expressing it beyond this time results in failure of leg development during the late prepupal and pupal stages. Taken together, our results suggest that brZ2 is required to drive ECM remodeling, change cell shape, and maintain metabolic activity through the midprepupal stage, but must be switched off to allow expression of pupation genes.
Asunto(s)
Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Discos Imaginales/crecimiento & desarrollo , Hormonas de Insectos/metabolismo , Morfogénesis , Factores de Transcripción/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Matriz Extracelular/metabolismo , Glucólisis , Discos Imaginales/metabolismo , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
Morphogen gradients provide positional information during development. To uncover the minimal requirements for morphogen gradient formation, we have engineered a synthetic morphogen in Drosophila wing primordia. We show that an inert protein, green fluorescent protein (GFP), can form a detectable diffusion-based gradient in the presence of surface-associated anti-GFP nanobodies, which modulate the gradient by trapping the ligand and limiting leakage from the tissue. We next fused anti-GFP nanobodies to the receptors of Dpp, a natural morphogen, to render them responsive to extracellular GFP. In the presence of these engineered receptors, GFP could replace Dpp to organize patterning and growth in vivo. Concomitant expression of glycosylphosphatidylinositol (GPI)-anchored nonsignaling receptors further improved patterning, to near-wild-type quality. Theoretical arguments suggest that GPI anchorage could be important for these receptors to expand the gradient length scale while at the same time reducing leakage.
Asunto(s)
Tipificación del Cuerpo , Drosophila melanogaster/crecimiento & desarrollo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Proteínas Fluorescentes Verdes/genética , Discos Imaginales/crecimiento & desarrollo , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/genética , Alas de Animales/crecimiento & desarrolloRESUMEN
Promoter proximal pausing (PPP) of RNA polymerase II has emerged as a crucial rate-limiting step in the regulation of gene expression. Regulation of PPP is brought about by complexes 7SK snRNP, P-TEFb (Cdk9/cycT), and the negative elongation factor (NELF), which are highly conserved from Drosophila to humans. Here, we show that RNAi-mediated depletion of bin3 or Hexim of the 7SK snRNP complex or depletion of individual components of the NELF complex enhances Yki-driven growth, leading to neoplastic transformation of Drosophila wing imaginal discs. We also show that increased CDK9 expression cooperates with Yki in driving neoplastic growth. Interestingly, overexpression of CDK9 on its own or in the background of depletion of one of the components of 7SK snRNP or the NELF complex necessarily, and specifically, needed Yki overexpression to cause tumorous growth. Genome-wide gene expression analyses suggested that deregulation of protein homeostasis is associated with tumorous growth of wing imaginal discs. As both Fat/Hippo/Yki pathway and PPP are highly conserved, our observations may provide insights into mechanisms of oncogenic function of YAP-the ortholog of Yki in humans.
Asunto(s)
Carcinogénesis/genética , Proteínas de Drosophila/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Transactivadores/metabolismo , Animales , Quinasa 9 Dependiente de la Ciclina/genética , Quinasa 9 Dependiente de la Ciclina/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Discos Imaginales/crecimiento & desarrollo , Discos Imaginales/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismo , Proteínas Nucleares/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transactivadores/genética , Alas de Animales/crecimiento & desarrollo , Alas de Animales/metabolismo , Proteínas Señalizadoras YAPRESUMEN
The regulation of growth and the determination of organ-size in animals is an area of research that has received much attention during the past two and a half decades. Classic regeneration and cell-competition studies performed during the last century suggested that for size to be determined, organ-size is sensed and this sense of size feeds back into the growth control mechanism such that growth stops at the "correct" size. Recent work using Drosophila imaginal discs as a system has provided a particularly detailed cellular and molecular understanding of growth. Yet, a clear mechanistic basis for size-sensing has not emerged. I re-examine these studies from a different perspective and ask whether there is scope for alternate modes of size control in which size does not need to be sensed.
Asunto(s)
Drosophila/crecimiento & desarrollo , Discos Imaginales/crecimiento & desarrollo , Modelos Biológicos , Transducción de Señal/fisiología , Alas de Animales/crecimiento & desarrollo , Animales , Muerte Celular/fisiología , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Discos Imaginales/citología , Tamaño de los Órganos , Alas de Animales/citologíaRESUMEN
Organ growth and size are finely tuned by intrinsic and extrinsic signaling molecules. In Drosophila, the BMP family member Dpp is produced in a limited set of imaginal disc cells and functions as a classic morphogen to regulate pattern and growth by diffusing throughout imaginal discs. However, the role of TGFß/Activin-like ligands in disc growth control remains ill-defined. Here, we demonstrate that Myoglianin (Myo), an Activin family member, and a close homolog of mammalian Myostatin (Mstn), is a muscle-derived extrinsic factor that uses canonical dSmad2-mediated signaling to regulate wing size. We propose that Myo is a myokine that helps mediate an allometric relationship between muscles and their associated appendages.