RESUMO
The current view of hematopoiesis considers leukocytes on a continuum with distinct developmental origins, and which exert non-overlapping functions. However, there is less known about the function and phenotype of ontogenetically distinct neutrophil populations. In this work, using a photoconvertible transgenic zebrafish line; Tg(mpx:Dendra2), we selectively label rostral blood island-derived and caudal hematopoietic tissue-derived neutrophils in vivo during steady state or upon injury. By comparing the migratory properties and single-cell expression profiles of both neutrophil populations at steady state we show that rostral neutrophils show higher csf3b expression and migration capacity than caudal neutrophils. Upon injury, both populations share a core transcriptional profile as well as subset-specific transcriptional signatures. Accordingly, both rostral and caudal neutrophils are recruited to the wound independently of their distance to the injury. While rostral neutrophils respond uniformly, caudal neutrophils respond heterogeneously. Collectively, our results reveal that co-existing neutrophils populations with ontogenically distinct origin display functional differences.
Assuntos
Neutrófilos , Peixe-Zebra , Animais , Peixe-Zebra/genética , Neutrófilos/metabolismo , Animais Geneticamente Modificados , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , HematopoeseRESUMO
Currently, inflammatory bowel disease (IBD) is a serious public health problem on the rise worldwide. In this work, we utilized the zebrafish to introduce a new model of intestinal inflammation triggered by food intake. Taking advantage of the translucency of the larvae and the availability of transgenic zebrafish lines with fluorescently labeled macrophages, neutrophils, or lymphocytes, we studied the behavior of these cell types in vivo during the course of inflammation. We established two feeding strategies, the first using fish that were not previously exposed to food (naïve strategy) and the second in which fish were initially exposed to normal food (developed strategy). In both strategies, we analyzed the effect of subsequent intake of a control or a soybean meal diet. Our results showed increased numbers of innate immune cells in the gut in both the naïve or developed protocols. Likewise, macrophages underwent drastic morphological changes after feeding, switching from a small and rounded contour to a larger and dendritic shape. Lymphocytes colonized the intestine as early as 5 days post fertilization and increased in numbers during the inflammatory process. Gene expression analysis indicated that lymphocytes present in the intestine correspond to T helper cells. Interestingly, control diet only induced a regulatory T cell profile in the developed model. On the contrary, soybean meal diet induced a Th17 response both in naïve and developed model. In addition, when feeding was performed in rag1-deficient fish, intestinal inflammation was not induced indicating that inflammation induced by soybean meal is T cell-dependent.
Assuntos
Ração Animal/efeitos adversos , Glycine max/efeitos adversos , Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/imunologia , Células Th17/imunologia , Peixe-Zebra/imunologia , Animais , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Doenças Inflamatórias Intestinais/induzido quimicamente , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Células Th17/patologia , Peixe-Zebra/genéticaRESUMO
Copper is an essential ion that forms part of the active sites of many proteins. At the same time, an excess of this metal produces free radicals that are toxic for cells and organisms. Fish have been used extensively to study the effects of metals, including copper, present in food or the environment. It has been shown that different metals induce different adaptive responses in adult fish. However, until now, scant information has been available about the responses that are induced by waterborne copper during early life stages of fish. Here, acute toxicity tests and LC50 curves have been generated for zebrafish larvae exposed to dissolved copper sulphate at different concentrations and for different treatment times. We determined that the larvae incorporate and accumulate copper present in the medium in a concentration-dependent manner, resulting in changes in gene expression. Using a transgenic fish line that expresses enhanced green fluorescent protein (EGFP) under the hsp70 promoter, we monitored tissue-specific stress responses to waterborne copper by following expression of the reporter. Furthermore, TUNEL assays revealed which tissues are more susceptible to cell death after exposure to copper. Our results establish a framework for the analysis of whole-organism management of excess external copper in developing aquatic animals.
Assuntos
Morte Celular/efeitos dos fármacos , Sulfato de Cobre/toxicidade , Estresse Fisiológico/efeitos dos fármacos , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Embrião não Mamífero/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Imuno-Histoquímica , Larva/efeitos dos fármacos , Dose Letal Mediana , Fatores de Tempo , Testes de Toxicidade Aguda , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/embriologiaRESUMO
Copper is an essential ion that forms part of the active sites of many proteins. At the same time, an excess of this metal produces free radicals that are toxic for cells and organisms. Fish have been used extensively to study the effects of metals, including copper, present in food or the environment. It has been shown that different metals induce different adaptive responses in adult fish. However, until now, scant information has been available about the responses that are induced by waterborne copper during early life stages of fish. Here, acute toxicity tests and LC50 curves have been generated for zebrafish larvae exposed to dissolved copper sulphate at different concentrations and for different treatment times. We determined that the larvae incorporate and accumulate copper present in the medium in a concentration-dependent manner, resulting in changes in gene expression. Using a transgenic fish line that expresses enhanced green fluorescent protein (EGFP) under the hsp70 promoter, we monitored tissue-specific stress responses to waterborne copper by following expression of the reporter. Furthermore, TUNEL assays revealed which tissues are more susceptible to cell death after exposure to copper. Our results establish a framework for the analysis of whole-organism management of excess external copper in developing aquatic animals.
Assuntos
Animais , Morte Celular/efeitos dos fármacos , Sulfato de Cobre/toxicidade , Estresse Fisiológico/efeitos dos fármacos , Peixe-Zebra , Animais Geneticamente Modificados , Embrião não Mamífero/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , /metabolismo , Imuno-Histoquímica , Larva/efeitos dos fármacos , Fatores de Tempo , Testes de Toxicidade Aguda , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/embriologiaRESUMO
Numerous physical and chemical agents can destroy mechanosensory hair cells in the inner ear of vertebrates, a process that is irreversible in mammals. Few experimental systems allow the observation of hair cell death mechanisms in vivo, in the intact animal, one of these being the lateral line system in the zebrafish. In this work we characterize the behavior of dying lateral line hair cells in fish exposed to low doses of copper in the water. The concentration of copper used in our study kills hair cells in a few hours, but removal of the metal is followed by robust regeneration of new hair cells. We use a combination of membrane and nuclear live stains, ultrastructural analysis and measurement of reactive oxygen species to characterize the events leading to the death of hair cells under these conditions. Our results show that a combination of necrotic cell death, accompanied by apoptotic features such as rapid DNA fragmentation, lead to the loss of these cells. We also show that hair cells exposed to copper undergo oxidative stress and that antioxidants can protect these cells from the effects of the metal. The study of this process in the zebrafish lateral line allows rapid morphological analysis of hair cell death and may be used as an efficient end point for molecule screens aimed at preventing these effects.
Assuntos
Apoptose/efeitos dos fármacos , Cobre/toxicidade , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Sistema da Linha Lateral/citologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Apoptose/fisiologia , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Corantes Fluorescentes/química , Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Auditivas Internas/ultraestrutura , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Larva/citologia , Larva/efeitos dos fármacos , Larva/metabolismo , Microscopia Eletrônica de Transmissão , Estresse Oxidativo/fisiologia , Peixe-ZebraRESUMO
Unicellular eukaryotes and cultured cells from several animal species were invaluable in discovering the mechanisms that govern incorporation, handling, and excretion of copper at the cellular level. However, understanding the systemic regulation of copper availability and distribution among the different tissues of an intact multicellular organism has proven to be more challenging. This analysis is made even more difficult if the genetic variability among organisms is taken into account. The zebrafish has long been considered a powerful animal model because of its tractable genetics and embryology, but it has more recently become a player in environmental studies, pharmaceutical screening, and physiologic analysis. In particular, the use of the larvae, small enough to fit into a microtiter well, but developed enough to have full organ functionality, represents a convenient alternative for studies that aim to establish effects of environmental agents on the intact, living organism. Studies by our group and others have characterized absorption and tissue distribution of copper and have described the acute effects of the metal on larvae in terms of survival, organ stress, and functionality of sensory organs. A large body of work has shown that there is strong conservation in mechanisms and genes between fish and mammals, opening the possibility for genetic or small molecule screens or for generating fish models of human diseases related to copper metabolism. The variability within humans in terms of tolerance to copper excess or deficiency requires a genetic approach to be taken to understand the behavior of populations, because markers and vulnerabilities need to be identified. The zebrafish could represent a unique tool to move in this direction.
Assuntos
Cobre/toxicidade , Peixe-Zebra/genética , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Cobre/deficiência , Cobre/metabolismo , Cobre/farmacologia , Marcadores Genéticos , Larva/metabolismo , Modelos Animais , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismoRESUMO
Mechanosensory hair cells are essential for audition in vertebrates, and in many species, have the capacity for regeneration when damaged. Regeneration is robust in the fish lateral line system as new hair cells can reappear after damage induced by waterborne aminoglycoside antibiotics, platinum-based drugs, and heavy metals. Here, we characterize the loss and reappearance of lateral line hair cells induced in zebrafish larvae treated with copper sulfate using diverse molecular markers. Transgenic fish that express green fluorescent protein in different cell types in the lateral line system have allowed us to follow the regeneration of hair cells after different damage protocols. We show that conditions that damage only differentiated hair cells lead to reappearance of new hair cells within 24 h from nondividing precursors, whereas harsher conditions are followed by a longer recovery period that is accompanied by extensive cell division. In order to characterize the cell population that gives rise to new hair cells, we describe the expression of a neural stem cell marker in neuromasts. The zebrafish sox2 gene is strongly expressed in neuromast progenitor cells, including those of the migrating lateral line primordium, the accessory cells that underlie the hair cells in neuromasts, and in interneuromastic cells that give rise to new neuromasts. Moreover, we find that most of the cells that proliferate within the neuromast during regeneration express this marker. Thus, our results describe the dynamics of hair cell regeneration in zebrafish and suggest the existence of at least two mechanisms for recovery of these cells in neuromasts.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Proteínas HMGB/biossíntese , Células Ciliadas Auditivas/fisiologia , Sistema da Linha Lateral/fisiologia , Neurônios/fisiologia , Células-Tronco/fisiologia , Fatores de Transcrição/biossíntese , Animais , Animais Geneticamente Modificados , Antimetabólitos , Bromodesoxiuridina , Proliferação de Células/efeitos dos fármacos , Cobre/toxicidade , Proteínas de Ligação a DNA/genética , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Proteínas HMGB/genética , Células Ciliadas Auditivas/efeitos dos fármacos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Hibridização In Situ , Mitose/fisiologia , Regeneração Nervosa/fisiologia , Fatores de Transcrição SOXB1 , Células-Tronco/efeitos dos fármacos , Fatores de Transcrição/genética , Peixe-ZebraRESUMO
In teleosts, the lateral line system is composed of neuromasts containing hair cells that are analogous to those present in the inner ear of all vertebrates. In the zebrafish embryo and early larva, this system is composed of the anterior lateral line (ALL), which covers the head, and the posterior lateral line (PLL), present in the trunk and tail. The mechanosensory hair cells found in neuromasts can be labeled in vivo using fluorescent dyes such as 4-di-2-Asp (DiAsp) or FM1-43. We have studied the effects of water-borne copper exposure on the function of the lateral line system in zebrafish larvae. Our results show that transient incubation of post-hatching larvae for 2h with non-lethal concentrations of copper (1-50 microM CuSO4) induces cellular damage localized to neuromasts, apoptosis, and loss of hair cell markers. This effect is specific to copper, as other metals did not show these effects. Since hair cells in fish can regenerate, we followed the reappearance of viable hair cells in neuromasts after copper removal. In the PLL, we determined that there is a threshold concentration of copper above which regeneration does not occur, whereas, at lower concentrations, the length of time it takes for viable hair cells to reappear is dependent on the amount of copper used during the treatment. The ALL behaves differently though, as regeneration can occur even after treatments with concentrations of copper an order of magnitude higher than the one that irreversibly affects the PLL. Regeneration of hair cells is dependent on cell division within the neuromasts as damage that precludes proliferation prevents reappearance of this cell type.