RESUMEN
Although well investigated, the importance of collagen fibers in supporting angiogenesis is not well understood. In this study, we demonstrate that extracellular collagen fibers provide guidance cues for endothelial cell migration during regenerative angiogenesis in the caudal zebrafish fin. Inhibition of collagen cross-linking by ß-Aminopropionitrile results in a 70% shorter regeneration area with 50% reduced vessel growth and disintegrated collagen fibers. The disrupted collagen scaffold impedes endothelial cell migration and induces formation of abnormal angioma-like blood vessels. Treatment of the Fli//colRN zebrafish line with the prodrug Nifurpirinol, which selectively damages the active collagen-producing 1α2 cells, reduced the regeneration area and vascular growth by 50% with wider, but less inter-connected, capillary segments. The regenerated area contained larger vessels partially covered by endothelial cells embedded in atypical extracellular matrix containing cell debris and apoptotic bodies, macrophages and granulocytes. Similar experiments performed in early embryonic zebrafish suggested that collagens are important also during embryonic angiogenesis. In vitro assays revealed that collagen I allows for the most efficient endothelial cell migration, followed by collagen IV relative to the complete absence of exogenous matrix support. Our data demonstrates severe vascular defects and restricted fin regeneration when collagens are impaired. Collagen I therefore, provides support and guidance for endothelial cell migration while collagen IV is responsible for proper lumen formation and vascular integrity.
Asunto(s)
Capilares , Colágenos Fibrilares/metabolismo , Neovascularización Fisiológica , Regeneración , Pez Cebra , Aletas de Animales/irrigación sanguínea , Aletas de Animales/metabolismo , Aletas de Animales/ultraestructura , Animales , Biomarcadores , Línea Celular , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Células Endoteliales , Colágenos Fibrilares/genética , Técnica del Anticuerpo Fluorescente , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Neovascularización Fisiológica/efectos de los fármacos , Cicatrización de Heridas/genéticaRESUMEN
Secreted growth factors can act as morphogens that form spatial concentration gradients in developing organs, thereby controlling growth and patterning. For some morphogens, adaptation of the gradients to tissue size allows morphological patterns to remain proportioned as the organs grow. In the zebrafish pectoral fin, we found that BMP signaling forms a two-dimensional gradient. The length of the gradient scales with tissue length and its amplitude increases with fin size according to a power-law. Gradient scaling and amplitude power-laws are signatures of growth control by time derivatives of morphogenetic signaling: cell division correlates with the fold change over time of the cellular signaling levels. We show that Smoc1 regulates BMP gradient scaling and growth in the fin. Smoc1 scales the gradient by means of a feedback loop: Smoc1 is a BMP agonist and BMP signaling represses Smoc1 expression. Our work uncovers a layer of morphogen regulation during vertebrate appendage development.
Asunto(s)
Aletas de Animales/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Transducción de Señal , Pez Cebra/metabolismo , Aletas de Animales/anatomía & histología , Aletas de Animales/crecimiento & desarrollo , Aletas de Animales/ultraestructura , Animales , Animales Modificados Genéticamente , Anisotropía , Larva/ultraestructura , Tamaño de los Órganos , Fenotipo , Proteínas de Pez Cebra/metabolismoRESUMEN
Tunas of the genus Thunnus are a group of high-performance pelagic fishes with many locomotor traits that are convergently shared with other high-performance fish groups. Because of their swimming abilities, tunas continue to be an inspiration for both comparative biomechanics and the design of biomimetic autonomous underwater vehicles (AUVs). Despite the strong history of studies in tuna physiology and current interest in tuna biomechanics and bio-inspired design, we lack quantitative data on the function of many features of tunas. Here we present data on the morphology, behavior, and function of tunas, focusing especially on experimentally examining the function of tuna lateral keels, finlets, and pectoral fins by using simple physical models. We find that both triangular lateral keels and flexible finlets decrease power requirements during swimming, likely by reducing lateral forces and yaw torques (compared to models either without keels or with rectangular keels, and models with stiff finlets or strip fins of equal area, respectively). However, both triangular keels and flexible finlets generate less thrust than other models either without these features or with modified keels or finlets, leading to a tradeoff between power consumption and thrust. In addition, we use micro computed tomography (µCT) to show that the flexible lateral keels possess a lateral line canal, suggesting these keels have a sensory function. The curved and fully-attached base of tuna pectoral fins provides high lift-to-drag ratio at low angles of attack, and generates the highest torques across speeds and angles of attack. Therefore, curved, fully-attached pectoral fins grant both better gliding and maneuvering performance compared to flat or curved, partially-attached designs. We provide both 3D models of tuna morphology derived from µCT scans and conclusions about the performance effects of tuna-like features as a resource for future biological and engineering work for next-generation tuna-inspired AUV designs.
Asunto(s)
Aletas de Animales/ultraestructura , Vehículos a Motor , Natación/fisiología , Atún/anatomía & histología , Aletas de Animales/fisiología , Animales , Fenómenos Biomecánicos , Biomimética/instrumentación , Diseño de Equipo , Hidrodinámica , Atún/fisiología , Microtomografía por Rayos XRESUMEN
Present study highlighted the ultramicroscopic (SEM) alterations of the skin, eye, barbel, and fins of spawn of an air-breathing teleost (Clarias batrachus, Linn. 1758) induced by UV-B radiation (280-320 nm) at a dose (@4.07 × 10-20J/photon/m2) under the time-frame of 5, 10 and 15 min/d in the laboratory condition for the periods of 5 and 10 days. Limnological parameters revealed no significant changes throughout the period of experimentation which were measured by PCS Testr 35 Multi-Parameter. Morphometric analysis revealed that during the extended exposure period of 10 days the spawn size and weight were reduced as analysed through Specific Growth Rate (SGR). SGR values in terms of weight for 5 and 10 days under 3 time-frames were 17.12%, 12.52%, 11.46% and 9.09%, 6.43%, 6.09% respectively, which revealed a declined trend along with the exposure days. In the skin of C. batrachus, the compact regular orientation of the stratified epithelial cells and mucous cells became distorted and the microridges and double-ridged structures showed destruction and fragmentations. The body striations and microfolds became shrinked and swollen and finally degenerated to form a mass. The distribution of mucous cells throughout the epidermis was disorganised and releasing secretory contents on the surface through small pores. Appearance of huge quantity of biogenic semi-hexagonal plate like crystals (guanine platelets) on the skin surface of the body was the most significant observations during UV-B radiation. In the developmental phases the eyeball showed shrinkage loosing normal regular concave structure and to become a dome-shaped one. The supportive connective infoldings became loosened. The choroid coat displayed deformities and the iris deformed the pupil. The fibroblast on the epithelium and melanocytes depicted dispersed arrangement. The pairs of ventral barbels near the mouth depicted the presence of taste buds that became severely damaged exposing the sensory as well as neuroepithelial cells. Compact regular arrangement of the SECs was completely destroyed leaving long and deep channels inbetween them; the disintegrated concentric MRs also showed a mass.
Asunto(s)
Aletas de Animales/efectos de la radiación , Bagres , Ojo/efectos de la radiación , Piel/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Aletas de Animales/ultraestructura , Animales , Relación Dosis-Respuesta en la Radiación , Epidermis/efectos de la radiación , Epidermis/ultraestructura , Epitelio/efectos de la radiación , Epitelio/ultraestructura , Ojo/ultraestructura , Piel/ultraestructuraRESUMEN
Fish skin is in direct contact with water and forms the first line of defense against pathogens and toxicants present in the surrounding water. The effect of mercuric chloride (HgCl2) on surface architecture of the epidermis of caudal fin of an air breathing fish, Channa punctatus was examined by scanning electron microscopy (SEM) and revealed the presence of microridges that formed intricate, maze-like patterns. The exposed fish showed significant alterations including disorganization of microridge pattern and increase in number as well as enlargement of mucus cell openings. These findings exhibited concentration- and time- dependent alterations in fin epithelium. Data demonstrated that fin epithelium of fish may be successfully employed as a bioindicator of water pollution.
Asunto(s)
Aletas de Animales , Epitelio/efectos de los fármacos , Peces , Cloruro de Mercurio/toxicidad , Aletas de Animales/efectos de los fármacos , Aletas de Animales/ultraestructura , Animales , Epitelio/ultraestructura , Microscopía Electrónica de RastreoRESUMEN
How some animals regenerate missing body parts is not well understood. Taking advantage of the zebrafish caudal fin model, we performed a global unbiased time-course transcriptomic analysis of fin regeneration. Biostatistics analyses identified extracellular matrix (ECM) as the most enriched gene sets. Basement membranes (BMs) are specialized ECM structures that provide tissues with structural cohesion and serve as a major extracellular signaling platform. While the embryonic formation of BM has been extensively investigated, its regeneration in adults remains poorly studied. We therefore focused on BM gene expression kinetics and showed that it recapitulates many aspects of development. As such, the re-expression of the embryonic col14a1a gene indicated that col14a1a is part of the regeneration-specific program. We showed that laminins and col14a1a genes display similar kinetics and that the corresponding proteins are spatially and temporally controlled during regeneration. Analysis of our CRISPR/Cas9-mediated col14a1a knockout fish showed that collagen XIV-A contributes to timely deposition of laminins. As changes in ECM organization can affect tissue mechanical properties, we analyzed the biomechanics of col14a1a-/- regenerative BM using atomic force microscopy (AFM). Our data revealed a thinner BM accompanied by a substantial increase of the stiffness when compared to controls. Further AFM 3D-reconstructions showed that BM is organized as a checkerboard made of alternation of soft and rigid regions that is compromised in mutants leading to a more compact structure. We conclude that collagen XIV-A transiently acts as a molecular spacer responsible for BM structure and biomechanics possibly by helping laminins integration within regenerative BM.
Asunto(s)
Aletas de Animales/crecimiento & desarrollo , Membrana Basal/crecimiento & desarrollo , Colágeno/genética , Regeneración/genética , Proteínas de Pez Cebra/genética , Aletas de Animales/ultraestructura , Animales , Membrana Basal/ultraestructura , Sistemas CRISPR-Cas , Matriz Extracelular/genética , Matriz Extracelular/ultraestructura , Proteínas de la Matriz Extracelular/genética , Regulación del Desarrollo de la Expresión Génica/genética , Cinética , Transcriptoma/genética , Cicatrización de Heridas/genética , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
The embryonic fins and the tip of adult fins of teleost fish are supported by rows of straight, unmineralized fibrils called actinotrichia. The proximal ends of the actinotrichia are bundled and the mineralized bones called lepidotrichia are made along them. Since malformation in actinotrichia causes wavy fin bones, the correct configuration of actinotrichia is essential for the correct construction of the fin shape. Past studies suggested that two types of cells, basal keratinocytes, and mesenchymal cells involve in the formation of actinotrichia. However, the mechanism how these cells contribute is unknown. In this study, we elucidated the role of basal keratinocytes in actinotrichia formation. First, we developed the imaging tool that specifically visualizes the basal keratinocytes and actinotrichia. Then, we established the in vitro culture method of the basal keratinocytes and found that the keratinocytes developed fine needle-like structures in it. The TEM image of them showed the specific shadow pattern of actinotrichia, indicating that the fine needle-like structures are the newly made actinotrichia. Finally, we cultured the basal keratinocytes with mature actinotrichia and observed that the basal keratinocytes actively holded actinotrichia with their membrane, and often generated a linear array of cells holding a single actinotrichium. This behavior suggested a mechanism with which long actinotrichia are made by relatively small cells. Our results clarified the role of basal keratinocyte and provided a novel insight into understanding the mechanism of actinotrichia formation.
Asunto(s)
Aletas de Animales/anatomía & histología , Aletas de Animales/embriología , Queratinocitos/citología , Pez Cebra/anatomía & histología , Aletas de Animales/ultraestructura , Animales , Células Cultivadas , Genes Reporteros , Queratinocitos/ultraestructura , Larva/ultraestructura , Pez Cebra/embriologíaRESUMEN
The skeleton of adult zebrafish fins comprises lepidotrichia, which are dermal bones of the rays, and actinotrichia, which are non-mineralized spicules at the distal margin of the appendage. Little is known about the regenerative dynamics of the actinotrichia-specific structural proteins called Actinodins. Here, we used immunofluorescence analysis to determine the contribution of two paralogous Actinodin proteins, And1/2, in regenerating fins. Both proteins were detected in the secretory organelles in the mesenchymal cells of the blastema, but only And1 was detected in the epithelial cells of the wound epithelium. The analysis of whole mount fins throughout the entire regenerative process and longitudinal sections revealed that And1-positive fibers are complementary to the lepidotrichia. The analysis of another longfin fish, a gain-of-function mutation in the potassium channel kcnk5b, revealed that the long-fin phenotype is associated with an extended size of actinotrichia during homeostasis and regeneration. Finally, we investigated the role of several signaling pathways in actinotrichia formation and maintenance. This revealed that the pulse-inhibition of either TGFß/Activin-ßA or FGF are sufficient to impair deposition of Actinodin during regeneration. Thus, the dynamic turnover of Actinodin during fin regeneration is regulated by multiple factors, including the osteoblasts, growth rate in a potassium channel mutant, and instructive signaling networks between the epithelium and the blastema of the regenerating fin.
Asunto(s)
Aletas de Animales/fisiología , Regeneración/fisiología , Proteínas de Pez Cebra/fisiología , Pez Cebra/fisiología , Aletas de Animales/ultraestructura , Estructuras Animales/metabolismo , Estructuras Animales/ultraestructura , Animales , Colágeno/metabolismo , Colágeno/ultraestructura , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Homeostasis , Mesodermo , Osteoblastos/metabolismo , Cicatrización de Heridas/fisiología , Pez Cebra/genética , Proteínas de Pez Cebra/biosíntesis , Proteínas de Pez Cebra/genéticaRESUMEN
Animal propulsion systems are believed to show high energy and mechanical efficiency in assisting movement compared to artificial designs. As an example, batoid fishes have very light cartilaginous skeletons that facilitate their elegant swimming via enlarged wing-like pectoral fins. The aim of this work is to illustrate the hierarchical structure of the pectoral fin of a representative batoid, the Longnose Skate (Raja rhina), and explain the mechanical implications of its structural design. At the macro level, the pectoral fins are comprised of radially oriented fin rays, formed by staggered mineralized skeletal elements stacked end-to-end. At the micro level, the midsection of each radial element is composed of three mineralized components, which consist of discrete segments (tesserae) that are mineralized cartilage and embedded in unmineralized cartilage. The radial elements are wrapped with aligned, unmineralized collagen fibers. This is the first report of the detailed structure of the ray elements, including the observation of a 3-chain mineralized tesserae. Structural analyses demonstrate that this configuration enhances stiffness in multiple directions. A two-dimensional numerical model based on the morphological analysis demonstrated that the tessera structure helps distributing shear, tensile and compressive stress more ideally, which can better support both lift and thrust forces when swimming without losing flexibility. STATEMENT OF SIGNIFICANCE: Batoid fishes have very light cartilaginous skeletons that facilitate their elegant swimming by applying their enlarged wing-like pectoral fins. Previous studies have shown structural features and mechanical properties of the mineralized cartilage skeleton in various batoid fishes. However, the details of the pectoral fin structure at different length scales, as well as the relationship between the mechanical properties and structural design remains unknown. The present work illustrates the hierarchical structure of the pectoral fin of the Longnose Skate (a representative batoid fish) and verifies the materials configuration and structural design increases the stiffness of fin skeleton without a loss in flexibility. These results have implications for the design of strong but flexible materials and bio-inspired autonomous underwater vehicles (AUVs).
Asunto(s)
Aletas de Animales/anatomía & histología , Aletas de Animales/fisiología , Huesos/anatomía & histología , Huesos/fisiología , Rajidae/fisiología , Aletas de Animales/ultraestructura , Animales , Fenómenos Biomecánicos , Huesos/diagnóstico por imagen , Huesos/ultraestructura , Calcificación Fisiológica , Espectrometría por Rayos X , Natación , Resistencia a la Tracción , Microtomografía por Rayos XRESUMEN
Severe infections by the protozoan parasite belonging to the genera Myxozoa are causing serious problems in ornamental fish reared in breeding farms. Histopathological study is being used for diagnosis of the severity of diseases. Myxozoan infections result in large scale histopathological damages in different fish tissues. No information is available regarding the histopathological changes of goldfish due to infection with myxozoans. The present study deals with the histopathological changes of the gill, fin, and skin of goldfish, infected with myxozoan parasites along with an ultrastructural study using scanning electron microscope. Several histological alterations have been observed in goldfish, like tissue damage, epithelial hyperplasia, necrosis, anoxia, localized lymphocytic infiltration, excess mucus, cellular necrosis, and epithelial proliferation. The present study revealed the invasion intensified by the occurrence of morphological lesions in the gill, skin, and fin exposed to Myxosporidia, which may lead to respiratory insufficiency in fish and even cause mass mortality.
Asunto(s)
Enfermedades de los Peces/parasitología , Carpa Dorada/parasitología , Myxozoa/fisiología , Enfermedades Parasitarias en Animales/parasitología , Aletas de Animales/parasitología , Aletas de Animales/ultraestructura , Animales , Enfermedades de los Peces/patología , Branquias/patología , Branquias/ultraestructura , India , Microscopía Electrónica de Rastreo , Myxozoa/aislamiento & purificación , Piel/parasitología , Piel/ultraestructuraRESUMEN
Many important biological questions can be addressed by studying in 3D large volumes of intact, cryo fixed hydrated tissues (⩾10,000µm3) at high resolution (5-20nm). This can be achieved using serial FIB milling and block face surface imaging under cryo conditions. Here we demonstrate the unique potential of the cryo-FIB-SEM approach using two extensively studied model systems; sea urchin embryos and the tail fin of zebrafish larvae. We focus in particular on the environment of mineral deposition sites. The cellular organelles, including mitochondria, Golgi, ER, nuclei and nuclear pores are made visible by the image contrast created by differences in surface potential of different biochemical components. Auto segmentation and/or volume rendering of the image stacks and 3D reconstruction of the skeleton and the cellular environment, provides a detailed view of the relative distribution in space of the tissue/cellular components, and thus of their interactions. Simultaneous acquisition of secondary and back-scattered electron images adds additional information. For example, a serial view of the zebrafish tail reveals the presence of electron dense mineral particles inside mitochondrial networks extending more than 20µm in depth in the block. Large volume imaging using cryo FIB SEM, as demonstrated here, can contribute significantly to the understanding of the structures and functions of diverse biological tissues.
Asunto(s)
Aletas de Animales/ultraestructura , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Aparato de Golgi/ultraestructura , Animales , Imagenología Tridimensional , Larva/ultraestructura , Erizos de Mar/embriología , Erizos de Mar/ultraestructura , Pez CebraRESUMEN
Our goal was the visualizing the vascular damage and acute inflammatory response to micro- and minibeam irradiation in vivo. Microbeam (MRT) and minibeam radiation therapies (MBRT) are tumor treatment approaches of potential clinical relevance, both consisting of parallel X-ray beams and allowing the delivery of thousands of Grays within tumors. We compared the effects of microbeams (25-100 µm wide) and minibeams (200-800 µm wide) on vasculature, inflammation and surrounding tissue changes during zebrafish caudal fin regeneration in vivo. Microbeam irradiation triggered an acute inflammatory response restricted to the regenerating tissue. Six hours post irradiation (6 hpi), it was infiltrated by neutrophils and fli1a(+) thrombocytes adhered to the cell wall locally in the beam path. The mature tissue was not affected by microbeam irradiation. In contrast, minibeam irradiation efficiently damaged the immature tissue at 6 hpi and damaged both the mature and immature tissue at 48 hpi. We demonstrate that vascular damage, inflammatory processes and cellular toxicity depend on the beam width and the stage of tissue maturation. Minibeam irradiation did not differentiate between mature and immature tissue. In contrast, all irradiation-induced effects of the microbeams were restricted to the rapidly growing immature tissue, indicating that microbeam irradiation could be a promising tumor treatment tool.
Asunto(s)
Plaquetas/efectos de la radiación , Vasos Sanguíneos/patología , Infiltración Neutrófila/efectos de la radiación , Adhesividad Plaquetaria/efectos de la radiación , Sincrotrones , Aletas de Animales/irrigación sanguínea , Aletas de Animales/efectos de la radiación , Aletas de Animales/ultraestructura , Animales , Tejido Conectivo/patología , Hemostasis , Inflamación/patología , Perfusión , Pez CebraRESUMEN
Zebrafish possess the remarkable ability to regenerate a vast variety of tissues, even as adults. However, direct imaging of regenerative processes in adult zebrafish remains challenging because of the lack of suitable anesthesia protocols. Here we present a description of an intubation-based anesthesia procedure that we developed to enable us to image regenerating zebrafish fins and which can be used to continuously anesthetize adult zebrafish for up to 2 d. Fish are immobilized in an imaging chamber followed by oral intubation. Subsequent delivery of anesthetic-containing water is achieved via a peristaltic pump. The setup of the system will take â¼90 min for two adult zebrafish, and it requires only a little previous experience of working with zebrafish. Our protocol will enable the imaging of regenerative processes in the fin and other tissues, and the investigation of processes that require long-term anesthesia, such as immune responses and surgical procedures.
Asunto(s)
Aletas de Animales/fisiología , Microscopía Confocal/métodos , Imagen de Lapso de Tiempo/métodos , Pez Cebra/fisiología , Anestesia/métodos , Anestesiología/instrumentación , Aletas de Animales/citología , Aletas de Animales/ultraestructura , Animales , Movimiento Celular , Diseño de Equipo , Femenino , Intubación/instrumentación , Intubación/métodos , Masculino , Microscopía Confocal/instrumentación , Regeneración , Imagen de Lapso de Tiempo/instrumentaciónRESUMEN
Cephalopods are famous for their ability to change color and pattern rapidly for signaling and camouflage. They have keen eyes and remarkable vision, made possible by photoreceptors in their retinas. External to the eyes, photoreceptors also exist in parolfactory vesicles and some light organs, where they function using a rhodopsin protein that is identical to that expressed in the retina. Furthermore, dermal chromatophore organs contain rhodopsin and other components of phototransduction (including retinochrome, a photoisomerase first found in the retina), suggesting that they are photoreceptive. In this study, we used a modified whole-mount immunohistochemical technique to explore rhodopsin and retinochrome expression in a number of tissues and organs in the longfin squid, Doryteuthis pealeii. We found that fin central muscles, hair cells (epithelial primary sensory neurons), arm axial ganglia, and sucker peduncle nerves all express rhodopsin and retinochrome proteins. Our findings indicate that these animals possess an unexpected diversity of extraocular photoreceptors and suggest that extraocular photoreception using visual opsins and visual phototransduction machinery is far more widespread throughout cephalopod tissues than previously recognized.
Asunto(s)
Decapodiformes/química , Decapodiformes/ultraestructura , Células Fotorreceptoras/química , Pigmentos Retinianos/análisis , Rodopsina/análisis , Aletas de Animales/química , Aletas de Animales/ultraestructura , Animales , Ganglios/química , Ganglios/ultraestructura , Inmunohistoquímica , Células Fotorreceptoras/ultraestructura , Retina/química , Retina/ultraestructuraRESUMEN
OBJECTIVES: This study aims at examining the ultrastructure of bone-derived biological apatite (BAp) from a series of small vertebrates and the effect of thermal treatment on its physiochemical properties. MATERIALS AND METHODS: Femurs/fin rays and vertebral bodies of 5 kinds of small vertebrates were firstly analyzed with X-ray microtomography. Subsequently, BAp was obtained with thermal treatment and low power plasma ashing, respectively. The properties of BAp, including morphology, functional groups, and crystal characteristics were then analyzed. RESULTS: The bones of grouper and hairtail were mainly composed of condensed bone. Spongy bone showed different distribution in the bones from frog, rat, and pigeon. No significant difference was found in bone mineral density of condensed bone and trabecular thickness of spongy bone. Only platelet-like crystals were observed for BAp obtained by plasma ashing, while rod-like and irregular crystals were both harvested from the bones treated by sintering. A much higher degree of crystallinity and larger crystal size but a lower content of carbonate were detected in the latter. CONCLUSION: Platelet-like BAp is the common inorganic component of vertebrate bones. BAp distributing in condensed and spongy bone may exhibit differing thermal reactivity. Thermal treatment may alter BAp's in vivo structure and composition.
Asunto(s)
Aletas de Animales/química , Aletas de Animales/ultraestructura , Apatitas/química , Fémur/química , Fémur/ultraestructura , Vértebras Torácicas/química , Vértebras Torácicas/ultraestructura , Animales , Anuros/fisiología , Densidad Ósea/fisiología , Columbidae/fisiología , Peces/fisiología , Ratas , Ratas Sprague-Dawley , Vertebrados/fisiología , Microtomografía por Rayos X/métodosRESUMEN
High-resolution imaging of autophagy has been used intensively in cell culture studies, but so far it has been difficult to visualize this process in detail in whole animal models. In this study we present a versatile method for high-resolution imaging of microbial infection in zebrafish larvae by injecting pathogens into the tail fin. This allows visualization of autophagic compartments by light and electron microscopy, which makes it possible to correlate images acquired by the 2 techniques. Using this method we have studied the autophagy response against Mycobacterium marinum infection. We show that mycobacteria during the progress of infection are frequently associated with GFP-Lc3-positive vesicles, and that 2 types of GFP-Lc3-positive vesicles were observed. The majority of these vesicles were approximately 1 µm in size and in close vicinity of bacteria, and a smaller number of GFP-Lc3-positive vesicles was larger in size and were observed to contain bacteria. Quantitative data showed that these larger vesicles occurred significantly more in leukocytes than in other cell types, and that approximately 70% of these vesicles were positive for a lysosomal marker. Using electron microscopy, it was found that approximately 5% of intracellular bacteria were present in autophagic vacuoles and that the remaining intracellular bacteria were present in phagosomes, lysosomes, free inside the cytoplasm or occurred as large aggregates. Based on correlation of light and electron microscopy images, it was shown that GFP-Lc3-positive vesicles displayed autophagic morphology. This study provides a new approach for injection of pathogens into the tail fin, which allows combined light and electron microscopy imaging in vivo and opens new research directions for studying autophagy process related to infectious diseases.
Asunto(s)
Autofagia , Microscopía Electrónica/métodos , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Infecciones por Mycobacterium no Tuberculosas/patología , Mycobacterium marinum/fisiología , Pez Cebra/microbiología , Aletas de Animales/microbiología , Aletas de Animales/patología , Aletas de Animales/ultraestructura , Animales , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes/metabolismo , Larva/ultraestructura , Microscopía Confocal , Proteínas Asociadas a Microtúbulos/metabolismo , Mycobacterium marinum/ultraestructura , Proteínas de Pez Cebra/metabolismoRESUMEN
The fitness of male guppies (Poecilia reticulata) highly depends on the size and number of their black, blue, and orange ornaments. Recently, progress has been made regarding the genetic mechanisms underlying male guppy pigment pattern formation, but we still know little about the pigment cell organization within these ornaments. Here, we investigate the pigment cell distribution within the black, blue, and orange trunk spots and selected fin color patterns of guppy males from three genetically divergent strains using transmission electron microscopy. We identified three types of pigment cells and found that at least two of these contribute to each color trait. Further, two pigment cell layers, one in the dermis and the other in the hypodermis, contribute to each trunk spot. The pigment cell organization within the black and orange trunk spots was similar between strains. The presence of iridophores in each of the investigated color traits is consistent with a key role for this pigment cell type in guppy color pattern formation.
Asunto(s)
Cromatóforos/fisiología , Color , Poecilia/fisiología , Pigmentación de la Piel/fisiología , Aletas de Animales/citología , Aletas de Animales/fisiología , Aletas de Animales/ultraestructura , Animales , Cromatóforos/citología , Cromatóforos/ultraestructura , Células Epidérmicas , Epidermis/fisiología , Epidermis/ultraestructura , Masculino , Melanóforos/citología , Melanóforos/fisiología , Melanóforos/ultraestructura , Microscopía Electrónica de Transmisión , Fenotipo , Poecilia/clasificación , Poecilia/genéticaRESUMEN
Regeneration is the ability of multicellular organisms to replace damaged tissues and regrow lost body parts. This process relies on cell fate transformation that involves changes in gene expression as well as in the composition of the cytoplasmic compartment, and exhibits a characteristic age-related decline. Here, we present evidence that genetic and pharmacological inhibition of autophagy - a lysosome-mediated self-degradation process of eukaryotic cells, which has been implicated in extensive cellular remodelling and aging - impairs the regeneration of amputated caudal fins in the zebrafish (Danio rerio). Thus, autophagy is required for injury-induced tissue renewal. We further show that upregulation of autophagy in the regeneration zone occurs downstream of mitogen-activated protein kinase/extracellular signal-regulated kinase signalling to protect cells from undergoing apoptosis and enable cytosolic restructuring underlying terminal cell fate determination. This novel cellular function of the autophagic process in regeneration implies that the role of cellular self-digestion in differentiation and tissue patterning is more fundamental than previously thought.
Asunto(s)
Aletas de Animales/fisiología , Autofagia , Regeneración , Pez Cebra/metabolismo , Aletas de Animales/citología , Aletas de Animales/ultraestructura , Animales , Autofagia/efectos de los fármacos , Proteína 5 Relacionada con la Autofagia , Butadienos/farmacología , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Macrólidos/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Nitrilos/farmacología , Oligonucleótidos Antisentido/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Adipose fins are enigmatic appendages found between the dorsal and caudal fins of some teleostean fishes. Long thought to be vestigial, degenerate second dorsal fins, remnants of the primitive gnathostome condition, adipose fins have since been recognized as novel morphologies. Unique among the fins of extant fishes, adipose fins have uniformly been described as passive structures, with no associated musculature. Here we provide the first description of a musculoskeletal linkage in an adipose fin, identified in the sun catfish Horabagrus brachysoma. Modified supracarinalis posterior muscles insert from the dorsal midline anterior to the adipose fin by tendons onto the fin base. An additional pair of posterior adipose-fin muscles also inserts upon the fin base and lay posterolateral to the fin, superficial to the axial muscle. This musculoskeletal linkage is an evolutionary innovation, a novel mechanism for controlling adipose-fin movement. These muscles appear to exemplify two approaches by which fins evolve to be actively controlled. We hypothesize that the anterior muscles arose through co-option of an existing fin linkage, while the posterior muscles originated as de novo fin muscles. These findings present adipose fins as a rich system within which to explore the evolution of novel vertebrate appendages.
Asunto(s)
Aletas de Animales/anatomía & histología , Bagres/anatomía & histología , Músculo Esquelético/anatomía & histología , Aletas de Animales/fisiología , Aletas de Animales/ultraestructura , Animales , Evolución Biológica , Fenómenos Biomecánicos , Bagres/fisiología , Músculo Esquelético/fisiología , NataciónRESUMEN
Hemicentin 1 (Hmcn1) and Hemicentin 2 (Hmcn2) belong to the fibulin family of extracellular matrix (ECM) proteins that play pivotal roles during development and homeostasis of a variety of vertebrate tissues. Recently, we have shown that mutations in zebrafish Hmcn1, also called Fibulin 6, lead to massive fin blistering, similar to the defects caused by the Fraser syndrome gene Fras1. In contrast, the role of Hmcn2 during vertebrate development has thus far been uncharacterized. In zebrafish, hmcn2, like fibulin 1 (fbln1), another member of the fibulin family, is predominantly expressed in fin mesenchymal cells and developing somites, contrasting the strict epithelial expression of hmcn1. While antisense morpholino oligonucleotide (MO)-based knockdown of hmcn2 did not yield any discernable defects, hmcn2/fbln1 double knockdown fish displayed blistering in the trunk, pointing to an essential contribution of these proteins from mesodermal sources for proper epidermal-dermal junction formation. In contrast, and unlike hmcn1 mutants, epidermal-dermal junctions in the fin folds of hmcn2/fbln1 double knockdown fish were only moderately affected. Instead, they displayed impaired migration of fin mesenchymal cells into the fin folds, pointing to a crucial role of Hmcn2 and Fbln1 to remodel the ECM of the fin fold interepidermal space, which is a prerequisite for fibroblast ingrowth. TEM analyses suggest that this ECM remodeling occurs at the level of actinotrichia, the collageneous migration substrate of mesenchymal cells, and at the level of cross fibers, which resemble mammalian microfibers. This work provides first insights into the role of Hmcn2 during vertebrate development, identifying it as an evolutionary conserved protein that acts in functional redundancy with Fbln1C and/or Fbln1D isoforms to regulate tissue adhesion and cell migration, while extending the current knowledge of the functions of vertebrate Fbln1.