RESUMO
Tumors often secrete wasting factors associated with atrophy and the degeneration of host tissues. If tumors were to be affected by the wasting factors, mechanisms allowing tumors to evade the adverse effects of the wasting factors must exist, and impairing such mechanisms may attenuate tumors. We use Drosophila midgut tumor models to show that tumors up-regulate Wingless (Wg) to oppose the growth-impeding effects caused by the wasting factor, ImpL2 (insulin-like growth factor binding protein [IGFBP]-related protein). Growth of Yorkie (Yki)-induced tumors is dependent on Wg while either elimination of ImpL2 or elevation of insulin/insulin-like growth factor signaling in tumors revokes this dependency. Notably, Wg augmentation could be a general mechanism for supporting the growth of tumors with elevated ImpL2 and exploited to attenuate muscle degeneration during wasting. Our study elucidates the mechanism by which tumors negate the action of ImpL2 to uphold their growth during cachexia-like wasting and implies that targeting the Wnt/Wg pathway might be an efficient treatment strategy for cancers with elevated IGFBPs.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/metabolismo , Via de Sinalização Wnt , Proteína Wnt1/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Proteínas de Neoplasias/genética , Neoplasias Experimentais/genética , Proteína Wnt1/genéticaRESUMO
BACKGROUND: The gut visceral musculature plays essential roles in not only moving substances through the lumen but also maintaining the function and physiology of the gut. Although the development of the visceral musculature has been studied in multiple model organisms, how it degenerates is poorly understood. RESULTS: Here, we employ the Drosophila midgut as a model to demonstrate that the visceral musculature is disrupted by intrinsic and extrinsic factors, such as aging, feeding, chemical-induced tissue damage, and oncogenic transformation in the epithelium. Notably, we define four prominent visceral musculature disruption phenotypes, which we refer as "sprout," "discontinuity," "furcation," and "crossover" of the longitudinal muscle. Given that the occurrence of these phenotypes is increased during aging and under various stresses, we propose that these phenotypes can be used as quantitative readouts of deterioration of the visceral musculature. Intriguingly, administration of a tissue-damaging chemical dextran sulfate sodium (DSS) induced similar visceral musculature disruption phenotypes in zebrafish larvae, indicating that ingestion of a tissue-damaging chemical can disrupt the visceral musculature in a vertebrate as well. CONCLUSIONS: Our study provides insights into the deterioration of the gut visceral musculature and lays a groundwork for investigating the underlying mechanisms in Drosophila as well as other animals.
Assuntos
Proteínas de Drosophila , Peixe-Zebra , Animais , Drosophila/genética , Proteínas de Drosophila/genética , Endoderma , MúsculosRESUMO
Adhesive interactions are essential for tissue patterning and morphogenesis yet difficult to study owing to functional redundancies across genes and gene families. A useful system in which to dissect roles for cell adhesion and adhesion-dependent signaling is the pattern formed by pigment cells in skin of adult zebrafish, in which stripes represent the arrangement of neural crest derived melanophores, cells homologous to melanocytes. In a forward genetic screen for adult pattern defects, we isolated the pissarro (psr) mutant, having a variegated phenotype of spots, as well as defects in adult fin and lens. We show that psr corresponds to junctional adhesion protein 3b (jam3b) encoding a zebrafish orthologue of the two immunoglobulin-like domain receptor JAM3 (JAM-C), known for roles in adhesion and signaling in other developing tissues, and for promoting metastatic behavior of human and murine melanoma cells. We found that zebrafish jam3b is expressed post-embryonically in a variety of cells including melanophores, and that jam3b mutants have defects in melanophore survival. Jam3b supported aggregation of cells in vitro and was required autonomously by melanophores for an adherent phenotype in vivo. Genetic analyses further indicated both overlapping and non-overlapping functions with the related receptor, Immunoglobulin superfamily 11 (Igsf11) and Kit receptor tyrosine kinase. These findings suggest a model for Jam3b function in zebrafish melanophores and hint at the complexity of adhesive interactions underlying pattern formation.
Assuntos
Padronização Corporal/genética , Molécula C de Adesão Juncional/genética , Molécula C de Adesão Juncional/metabolismo , Animais , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Melanóforos/metabolismo , Metamorfose Biológica/genética , Morfogênese , Mutação/genética , Crista Neural/citologia , Fenótipo , Pigmentação/genética , Transdução de Sinais/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The zebrafish adult pigment pattern has emerged as a useful model for understanding the development and evolution of adult form as well as pattern-forming mechanisms more generally. In this species, a series of horizontal melanophore stripes arises during the larval-to-adult transformation, but the genetic and cellular bases for stripe formation remain largely unknown. Here, we show that the seurat mutant phenotype, consisting of an irregular spotted pattern, arises from lesions in the gene encoding Immunoglobulin superfamily member 11 (Igsf11). We find that Igsf11 is expressed by melanophores and their precursors, and we demonstrate by cell transplantation and genetic rescue that igsf11 functions autonomously to this lineage in promoting adult stripe development. Further analyses of cell behaviors in vitro, in vivo, and in explant cultures ex vivo demonstrate that Igsf11 mediates adhesive interactions and that mutants for igsf11 exhibit defects in both the migration and survival of melanophores and their precursors. These findings identify the first in vivo requirements for igsf11 as well as the first instance of an immunoglobulin superfamily member functioning in pigment cell development and patterning. Our results provide new insights into adult pigment pattern morphogenesis and how cellular interactions mediate pattern formation.
Assuntos
Padronização Corporal/genética , Moléculas de Adesão Celular/genética , Proteínas de Peixes/genética , Imunoglobulinas/genética , Melanóforos/metabolismo , Pigmentação/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Evolução Biológica , Diferenciação Celular , Movimento Celular , Sobrevivência Celular , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Larva/genética , Melanóforos/citologia , Melanóforos/transplante , Mutação , FenótipoRESUMO
During neural tube closure, specialized regions called hinge points (HPs) display dynamic and polarized cell behaviors necessary for converting the neural plate into a neural tube. The molecular bases of such cell behaviors (e.g. apical constriction, basal nuclear migration) are poorly understood. We have identified a two-dimensional canonical BMP activity gradient in the chick neural plate that results in low and temporally pulsed BMP activity at the ventral midline/median hinge point (MHP). Using in vivo manipulations, high-resolution imaging and biochemical analyses, we show that BMP attenuation is necessary and sufficient for MHP formation. Conversely, BMP overexpression abolishes MHP formation and prevents neural tube closure. We provide evidence that BMP modulation directs neural tube closure via the regulation of apicobasal polarity. First, BMP blockade produces partially polarized neural cells, which retain contact with the apical and basal surfaces but where basolateral proteins (LGL) become apically localized and apical junctional proteins (PAR3, ZO1) become targeted to endosomes. Second, direct LGL misexpression induces ectopic HPs identical to those produced by noggin or dominant-negative BMPR1A. Third, BMP-dependent biochemical interactions occur between the PAR3-PAR6-aPKC polarity complex and phosphorylated SMAD5 at apical junctions. Finally, partially polarized cells normally occur at the MHP, their frequencies inversely correlated with the BMP activity gradient in the neural plate. We propose that spatiotemporal modulation of the two-dimensional BMP gradient transiently alters cell polarity in targeted neuronal cells. This ensures that the neural plate is flexible enough to be focally bent and shaped into a neural tube, while retaining overall epithelial integrity.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Polaridade Celular , Morfogênese/fisiologia , Tubo Neural/citologia , Tubo Neural/embriologia , Tubo Neural/fisiologia , Transdução de Sinais/fisiologia , Animais , Proteínas Morfogenéticas Ósseas/genética , Diferenciação Celular/fisiologia , Embrião de Galinha , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Smad/genética , Proteínas Smad/metabolismoRESUMO
Specialized cellular protrusions facilitate local intercellular communications in various species, including mammals. Among these, airinemes play a crucial role in pigment pattern formation in zebrafish by mediating long-distance Notch signaling between pigment cells. Remarkably, airinemes exhibit large vesicle-like structure at their tips, which are pulled by a macrophage subpopulation and delivered to target cells. The interaction between macrophages and Delta-ligand carrying airineme vesicles is essential for initiating airineme-mediated signaling, yet the molecular detail of this interaction remains elusive. Through high-resolution live imaging and genetic in vivo manipulations, we found that adhesive interactions via the extracellular domain of CD44, a class I transmembrane glycoprotein, between macrophages and airineme vesicles are critical for airineme signaling. Mutants lacking the extracellular domain of CD44 lose their adhesiveness, resulting in a significant reduction in airineme extension and pigment pattern defects. Our findings provide valuable insights into the role of adhesive interactions between signal-sending cells and macrophages in a long-range intercellular signaling.
RESUMO
The skin, the largest organ, functions as a primary defense mechanism. Epidermal stem cells supply undifferentiated keratinocytes that differentiate as they migrate toward the outermost skin layer. Although such a replenishment process is disrupted in various human skin diseases, its underlying mechanisms remain elusive. With high-resolution live imaging and in vivo manipulations, we revealed that Notch signaling between keratinocytes is mediated by signaling filopodia called cytonemes and is essential for proper keratinocyte differentiation and proliferation. Inhibiting keratinocyte cytonemes reduced Notch expression within undifferentiated keratinocytes, leading to abnormal differentiation and hyperproliferation, resembling human skin disease phenotypes. Overproduction of Interleukin (IL)-17 signal, associated with skin diseases like psoriasis, induces psoriatic phenotypes via cytonemes in zebrafish. Our study suggests that intercellular signaling between keratinocytes through cytonemes is critical for epidermal maintenance, and its misregulation could be an origin of human skin diseases.
RESUMO
During development, a flat neural plate rolls up and closes to form a neural tube. This process, called neural tube closure, is complex and requires morphogenetic events to occur along multiple axes of the neural plate. Recent studies suggest that cell and tissue polarity play a major role in neural tube morphogenesis. While the planar cell polarity pathway is known to be involved in this process, a role for the apicobasal polarity pathway has only recently begun to be elucidated. These studies show that bone morphogenetic proteins can regulate the apicobasal polarity pathway in the neural plate in a cell cycle dependent manner. This dynamically modulates apical junctions in the neural plate, resulting in cell and tissue shape changes that help bend, shape and close the neural tube.
Assuntos
Polaridade Celular , Placa Neural/embriologia , Tubo Neural/embriologia , Animais , Fenômenos Biomecânicos , Padronização Corporal , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Ciclo Celular , Movimento Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Galinhas/genética , Galinhas/crescimento & desenvolvimento , Galinhas/fisiologia , Epitélio/metabolismo , Epitélio/fisiologia , Placa Neural/citologia , Placa Neural/fisiologia , Tubo Neural/citologia , Tubo Neural/fisiologia , OrganogêneseRESUMO
BACKGROUND: The amenability of the chick embryo to a variety of manipulations has made it an ideal experimental model organism for over 100 years. The ability to manipulate gene function via in ovo electroporations has further revolutionized its value as an experimental model in the last 15 years. Although in ovo electroporations are simple to conduct in embryos ≥ E2, in ovo electroporations at early E1 stages have proven to be technically challenging due to the tissue damage and embryonic lethality such electroporations produce. RESULTS AND CONCLUSIONS: Here we report our success with in vivo microelectroporations of E1 embryos as young as Hamburger-Hamilton Stage 4 (HH4). We provide evidence that such electroporations can be varied in size and can be spatially targeted. They cause minimal disruption of tissue-size, 3-dimensional morphology, cell survival, proliferation, and cell-fate specification. Our paradigm is easily adapted to a variety of experimental conditions since it does not depend upon the presence of a lumen to enclose the DNA solution during electroporation. It is thus compatible with the in vivo examination of E1 morphogenetic events (e.g., neural tube closure) where preservation of 3-dimensional morphology is critical.
Assuntos
Eletroporação/métodos , Técnicas de Transferência de Genes , Animais , Proliferação de Células , Embrião de Galinha , Mesencéfalo/anatomia & histologia , Mesencéfalo/fisiologia , Tamanho do ÓrgãoRESUMO
Dae Seok Eom spoke with Cell Reports about his scientific journey, inspirations to become a scientist, and his work on cellular protrusions called "airinemes" that are involved in long-range intercellular signal transmission in zebrafish; in particular, he discussed recent work regarding characterization of a macrophage population that plays a critical role in this process.
RESUMO
Tissue-resident macrophages are heterogeneous and perform location-dependent functions. Skin resident macrophages play intriguing roles in long-distance intercellular signaling by mediating cellular protrusions called airinemes in zebrafish. These macrophages relay signaling molecules containing airineme vesicles between pigment cells, and their absence disrupts airineme-mediated signaling and pigment pattern formation. It is unknown if the same macrophages control both these signaling and typical immune functions or if a separate subpopulation functions in intercellular communication. With high-resolution imaging and genetic ablation approaches, we identify a macrophage subpopulation responsible for airineme-mediated signaling. These seem to be distinct from conventional skin-resident macrophages by their ameboid morphology and faster or expansive migratory behaviors. They resemble ectoderm-derived macrophages termed metaphocytes. Metaphocyte ablation markedly decreases airineme extension and signaling. In addition, these ameboid/metaphocytes require matrix metalloproteinase-9 for their migration and airineme-mediated signaling. These results reveal a macrophage subpopulation with specialized functions in airineme-mediated signaling, which may play roles in other aspects of intercellular communication.
Assuntos
Metaloproteinase 9 da Matriz , Peixe-Zebra , Animais , Peixe-Zebra/genética , Macrófagos , Comunicação Celular , Transdução de SinaisRESUMO
BACKGROUND: A critical event in neural tube closure is the formation of median hinge points (MHPs) and dorsolateral hinge points (DLHPs). Together, they buckle the ventral midline and elevate and juxtapose the neural folds for proper neural tube closure. Dynamic cell behaviors occur at hinge points (HPs), but their molecular regulation is largely unexplored. Bone morphogenetic proteins (BMPs) have been implicated in a variety of neural tube closure defects, although the underlying mechanisms are poorly understood. METHODS: In this study, we used in vivo electroporations, high-resolution microscopy, and biochemical analyses to explore the role of BMP signaling in chick midbrain neural tube closure. RESULTS: We identified a cell-cycle-dependent BMP gradient in the midbrain neural plate, which results in low-level BMP activity at the MHP. We show that although BMP signaling does not have a role in midbrain cell-fate specification, its attenuation is necessary and sufficient for MHP formation and midbrain closure. BMP blockade induces MHP formation by regulating apical constriction and basal nuclear migration. Furthermore, BMP signaling is critically important for maintaining epithelial organization by biochemically interacting with apicobasal polarity proteins (e.g., PAR3). As a result, prolonged BMP blockade disrupts apical junctions, desegregating the apical (PAR3(+), ZO1(+)) and basolateral (LGL(+)) compartments. Direct apical LGL-GFP misexpression in turn is sufficient to induce ectopic HPs. CONCLUSIONS: BMPs have a critical role in maintaining epithelial organization, a role that is conserved across species and tissue types. Its cell-cycle-dependent modulation in the neural plate dynamically regulates apicobasal polarity and helps to bend, shape, and close the neural tube.
Assuntos
Padronização Corporal/genética , Proteínas Morfogenéticas Ósseas/fisiologia , Crista Neural/embriologia , Tubo Neural/embriologia , Neurulação/genética , Animais , Animais Geneticamente Modificados , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Movimento Celular/genética , Movimento Celular/fisiologia , Polaridade Celular/genética , Polaridade Celular/fisiologia , Embrião de Galinha , Cinética , Morfogênese/genética , Morfogênese/fisiologia , Crista Neural/metabolismo , Placa Neural/citologia , Placa Neural/embriologia , Placa Neural/metabolismo , Tubo Neural/metabolismo , Defeitos do Tubo Neural/embriologia , Defeitos do Tubo Neural/genética , Neurulação/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
In addition to diffusive signals, cells in tissue also communicate via long, thin cellular protrusions, such as airinemes in zebrafish. Before establishing communication, cellular protrusions must find their target cell. Here, we demonstrate that the shapes of airinemes in zebrafish are consistent with a finite persistent random walk model. The probability of contacting the target cell is maximized for a balance between ballistic search (straight) and diffusive search (highly curved, random). We find that the curvature of airinemes in zebrafish, extracted from live-cell microscopy, is approximately the same value as the optimum in the simple persistent random walk model. We also explore the ability of the target cell to infer direction of the airineme's source, finding that there is a theoretical trade-off between search optimality and directional information. This provides a framework to characterize the shape, and performance objectives, of non-canonical cellular protrusions in general.
Assuntos
Extensões da Superfície Celular , Peixe-Zebra , Animais , DifusãoRESUMO
Understanding the mechanisms of cell-to-cell communication is one of the fundamental questions in biology and medicine. In particular, long-range signalling where cells communicate over several cell diameters is vital during development and homeostasis. The major morphogens, their receptors and intracellular signalling cascades have largely been identified; however, there is a gap in our knowledge of how such signalling factors are propagated over a long distance. In addition to the diffusion-based propagation model, new modalities of disseminating signalling molecules have been identified. It has been shown that cells can communicate with direct contact through long, thin cellular protrusions between signal sending and receiving cells at a distance. Recent studies have revealed a type of cellular protrusion termed 'airinemes' in zebrafish pigment cell types. They share similarities with previously reported cellular protrusions; however, they also exhibit distinct morphology and features. Airinemes are indispensable for pigment pattern development by mediating long-distance Delta-Notch signalling between different pigment cell types. Notably, airineme-mediated signalling is dependent on skin-resident macrophages. Key findings of airineme-mediated intercellular signalling in pattern development, their interplay with macrophages and their implications for the understanding of cellular protrusion-mediated intercellular communication will be discussed.
Assuntos
Comunicação Celular , Extensões da Superfície Celular/fisiologia , Macrófagos/fisiologia , Transdução de Sinais , Animais , Transporte Biológico , Forma Celular , Vesículas Citoplasmáticas/metabolismo , Humanos , Especificidade de ÓrgãosRESUMO
Macrophages have diverse functions in immunity as well as in development and homeostasis. We identified a function for these cells in long-distance communication during postembryonic tissue remodeling. Ablation of macrophages in zebrafish prevented melanophores from coalescing into adult pigment stripes. Melanophore organization depends on signals provided by cells of the yellow xanthophore lineage via airinemes, long filamentous projections with vesicles at their tips. We show that airineme extension from originating cells, as well as vesicle deposition on target cells, depend on interactions with macrophages. These findings identify a role for macrophages in relaying long-range signals between nonimmune cells. This signaling modality may function in the remodeling and homeostasis of other tissues during normal development and disease.
Assuntos
Padronização Corporal , Comunicação Celular , Macrófagos/fisiologia , Melanóforos/fisiologia , Animais , Movimento Celular , Embrião não Mamífero/citologia , Transdução de Sinais , Pigmentação da Pele , Peixe-Zebra/embriologiaRESUMO
Recent studies indicate that activation of stress-activated protein kinases may be implicated in a broad range of biological activities including differentiation. To directly examine whether stress-activated protein kinases are involved in neuronal differentiation, we utilized retinoic acid-induced and spontaneous models of neurite outgrowth in dopaminergic neurons. Here, we show that retinoic acid-induced neurite outgrowth in MN9D dopaminergic neuronal cells was accompanied by activation of c-Jun N-terminal kinase but not p38. Consequently, cotreatment with a specific inhibitor of c-Jun N-terminal kinase or overexpression of c-Jun N-terminal kinase-binding domain of c-Jun N-terminal kinase-interacting protein-1 blocked retinoic acid-induced neurite outgrowth. In primary cultures of dopaminergic neurons, the extent of neurite outgrowth increased spontaneously in a time-dependent manner. When these cultures were treated with a specific inhibitor of c-Jun N-terminal kinase, the total extent of neurites, the primary neurite length and the number of neurites per cell were suppressed significantly. Thus, our data indicate that the c-Jun N-terminal kinase signal seems to play an important role during morphological differentiation in cultured dopaminergic neurons.
Assuntos
Dopamina/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Neuritos/fisiologia , Neurônios/citologia , Análise de Variância , Animais , Antracenos/farmacologia , Western Blotting/métodos , Células Cultivadas , Relação Dose-Resposta a Droga , Interações Medicamentosas , Embrião de Mamíferos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Hibridomas , Imuno-Histoquímica/métodos , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Mesencéfalo/citologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neuritos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Fatores de Tempo , Transfecção/métodos , Tretinoína/farmacologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Changes in gene activity are essential for evolutionary diversification. Yet, elucidating the cellular behaviors that underlie modifications to adult form remains a profound challenge. We use neural crest-derived adult pigmentation of zebrafish and pearl danio to uncover cellular bases for alternative pattern states. We show that stripes in zebrafish require a novel class of thin, fast cellular projection to promote Delta-Notch signaling over long distances from cells of the xanthophore lineage to melanophores. Projections depended on microfilaments and microtubules, exhibited meandering trajectories, and stabilized on target cells to which they delivered membraneous vesicles. By contrast, the uniformly patterned pearl danio lacked such projections, concomitant with Colony stimulating factor 1-dependent changes in xanthophore differentiation that likely curtail signaling available to melanophores. Our study reveals a novel mechanism of cellular communication, roles for differentiation state heterogeneity in pigment cell interactions, and an unanticipated morphogenetic behavior contributing to a striking difference in adult form.
Assuntos
Comunicação Celular , Cyprinidae/fisiologia , Regulação da Expressão Gênica , Melanóforos/fisiologia , Pigmentos Biológicos/metabolismo , Vesículas Secretórias/metabolismo , Transdução de Sinais , Animais , Cyprinidae/genéticaRESUMO
Pigment patterns are useful for elucidating fundamental mechanisms of pattern formation and how these mechanisms evolve. In zebrafish, several pigment cell classes interact to generate stripes, yet the developmental requirements and origins of these cells remain poorly understood. Using zebrafish and a related species, we identified roles for thyroid hormone (TH) in pigment cell development and patterning, and in postembryonic development more generally. We show that adult pigment cells arise from distinct lineages having distinct requirements for TH and that differential TH dependence can evolve within lineages. Our findings demonstrate critical functions for TH in determining pigment pattern phenotype and highlight the potential for evolutionary diversification at the intersection of developmental and endocrine mechanisms.
Assuntos
Padronização Corporal , Diferenciação Celular , Linhagem da Célula , Melanóforos/fisiologia , Pigmentação da Pele/fisiologia , Hormônios Tireóideos/fisiologia , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero/citologia , Melanóforos/citologia , Melanóforos/efeitos dos fármacos , Pigmentação da Pele/genética , Hormônios Tireóideos/genética , Hormônios Tireóideos/farmacologiaRESUMO
In ovo electroporation of chick embryos at ages ≥ E2 is simple to conduct and widely used to manipulate gene function. However, in ovo electroporation at early E1 stages has so far been unsuccessful because of unacceptable levels of tissue damage and embryonic lethality. Early E1 manipulations in the chick have therefore relied on in vitro electroporation, posing problems for morphogenetic studies in which the long-term preservation (>24 h) of three-dimensional tissue organization is critical. This article describes a simple technique for in vivo electroporation of E1 embryos as young as Hamburger-Hamilton stage 4 (HH4). It uses thin microelectrodes and low voltages, which permit precise localization of gene misexpression while causing minimal tissue damage and embryonic lethality. Critically, it does not depend on the presence of a lumen for DNA injections and can easily be adapted for a wide variety of tissues.
Assuntos
Embrião de Galinha , Eletroporação/métodos , Animais , Análise de SobrevidaRESUMO
Recent studies suggest that Bcl-2 may play an active role in neuronal differentiation. Here, we showed a marked neurite extension in MN9D dopaminergic neuronal cells overexpressing Bcl-2 (MN9D/Bcl-2) or Bcl-X(L) (MN9D/Bcl-X(L)). We found a specific increase in phosphorylation of c-Jun N-terminal kinase (JNK) accompanied by neurite extension in MN9D/Bcl-2 but not in MN9D/Bcl-X(L) cells. Consequently, neurite extension in MN9D/Bcl-2 but not in MN9D/Bcl-X(L) cells was suppressed by treatment with SP600125, a specific inhibitor of JNK. Inhibition of other mitogen-activated protein kinases-including p38 and extracellular signal-regulated kinase-did not affect Bcl-2-mediated neurite extension in MN9D cells. While the expression levels of such protein markers of maturation as SNAP-25, phosphorylated NF-H, and neuron-specific enolase were increased in MN9D/Bcl-2 cells, only upregulation of SNAP-25 was inhibited after treatment with SP600125. Thus, the JNK signal activated by Bcl-2 seems to play an important role during morphological and certain biochemical differentiation in cultured dopaminergic neurons.