RESUMEN
The dynamics of the diffraction peak shape during pulsed heat load on mosaic single-crystal tungsten were measured at the `Plasma' scattering station on the eighth beamline of the VEPP-4 synchrotron radiation source at the Budker Institute of Nuclear Physics. The observed evolution of the diffraction peak shape agrees with theoretical predictions based on calculations of deformation caused by pulsed heating. Three clearly distinguishable stages of the diffraction-peak evolution were found, correlating with the evolution of temperature and deformation distributions. The residual plastic deformation increased with subsequent heating pulses.
RESUMEN
Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to "dial" the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.
Asunto(s)
Lesión Renal Aguda/etiología , Modelos Animales de Enfermedad , Terapia por Láser , Animales , Proteínas Fluorescentes Verdes/genética , Nefronas/fisiología , Pronefro/metabolismo , Pez Cebra/embriologíaRESUMEN
Mustn1 is a small nuclear protein that is involved in the development and regeneration of the musculoskeletal system. Previous work established a role for Mustn1 in myogenic and chondrogenic differentiation. In addition, recent evidence suggests a potential role for Mustn1 in cilia function in zebrafish. A detailed study of Mustn1 expression has yet to be conducted in zebrafish. As such, we report herein the cloning of the zebrafish Mustn1 orthologs, mustn1a and mustn1b, and their expression during zebrafish embryonic and larval development. Results indicate a 44% nucleotide identity between the two paralogs. Phylogenetic analysis further confirmed that the Mustn1a and 1b predicted proteins were highly related to other vertebrate members of the Mustn1 protein family. Whole mount in situ hybridization revealed expression of both mustn1a and 1b at the 7-somite stage through 72hpf in structures such as Kupffer's vesicle, segmental mesoderm, head structures, and otic vesicle. Additionally, in 5day old larva, mustn1a and 1b expression is detected in the neurocranium, otic capsule, and the gut. Although both were expressed in the neurocranium, mustn1a was localized in the hypophyseal fenestra whereas mustn1b was found near the posterior basicapsular commissure. mustn1b also displayed expression in the ceratohyal and ceratobranchial elements of the pharyngeal skeleton. These expression patterns were verified temporally by q-PCR analysis. Taken together, we conclude that Mustn1 expression is conserved in vertebrates and that the variations in expression of the two zebrafish paralogs suggest different modes of molecular regulation.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Nucleares/biosíntesis , Somitos/embriología , Proteínas de Pez Cebra/biosíntesis , Pez Cebra/embriología , Animales , Proteínas Nucleares/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
New nephron formation (nephrogenesis) ceases in mammals around birth and is completely absent in adults. In contrast, postembryonic nephrogenesis is well documented in the mesonephric kidneys of fishes and amphibians. The transient mesonephros in reptiles (including birds) and mammals is replaced by the metanephros during embryogenesis. Thus, one may speculate that postembryonic nephrogenesis is restricted to the mesonephric kidney. Previous reports have suggested the metanephros of non-avian reptiles (hereafter reptiles) may continually form nephrons throughout life. We investigated the presence of adult nephrogenesis in reptiles by examining adult kidneys from several species including Trachemys scripta, Chrysemys picta, Boa constrictor, Tupinambis tegu, Anolis carolinensis, and Alligator mississipiensis among others. We found that all major reptilian groups (Testudines, Crocodylia, and Squamates) showed the presence of adult nephrogenesis. The total amount of nephrogenesis varied greatly between species with turtles displaying the highest density of nephrogenesis. In contrast, we were unable to detect adult nephrogenesis in monotremes, and in the iguanid A. carolinensis. Nephron progenitor cells express the transcription factor Six2, which in mammals, becomes downregulated as the progenitor cell population is exhausted and nephrogenesis ends. Using the alligator as a model, we were able to detect Six2-positive cap mesenchyme cells in the adult kidney, which spatially correlated with areas of nephrogenesis. These results suggest that the metanephric kidney of reptiles has maintained the ability to continually grow new nephrons during postembryonic life, a process lost early in mammalian evolution, likely due to the persistence of a Six2-expressing progenitor cell population.
Asunto(s)
Nefronas/crecimiento & desarrollo , Reptiles , Células Madre/citología , Transactivadores/metabolismo , AnimalesRESUMEN
OBJECTIVES: Innate immune dysfunction after major burn injuries increases the susceptibility to organ failure. Lipid mediators of inflammation resolution, e.g., resolvin D2, have been shown recently to restore neutrophil functionality and reduce mortality rate in a rat model of major burn injury. However, the physiological mechanisms responsible for the benefic activity of resolvin D2 are not well understood. DESIGN: Prospective randomized animal investigation. SETTING: Academic research setting. SUBJECTS: Wistar male rats. INTERVENTIONS: Animals were subjected to a full-thickness burn of 30% total body surface area. Two hours after burn, 25 ng/kg resolvin D2 was administered IV and repeated every day, for 8 days. At day 10 post burn, 2 mg/kg of lipopolysaccharide was administered IV, and the presence of renal and hepatic injuries was evaluated at day 11 post burn by histology, immunohistochemistry, and relevant blood chemistry. MEASUREMENTS AND MAIN RESULTS: In untreated animals, we found significant tissue damage in the kidneys and liver, consistent with acute tubular necrosis and multifocal necrosis, and changes in blood chemistry, reflecting the deterioration of renal and hepatic functions. We detected less tissue damage and significantly lower values of blood urea nitrogen (26.4 ± 2.1 vs 36.0 ± 9.3 mg/dL; p ≤ 0.001), alanine aminotransferase (266.5 ± 295.2 vs 861.8 ± 813.7 U/L; p ≤ 0.01), and total bilirubin (0.13 ± 0.05 vs 0.30 ± 0.14 mg/dL; p ≤ 0.01) in resolvin D2-treated rats than in untreated animals. The mean blood pressure of all animals was above 65 mm Hg, indicating adequate tissue perfusion throughout the experiments. We measured significantly larger amounts of chromatin in the circulation of untreated than of resolvin D2-treated rats (575.1 ± 331.0 vs 264.1 ± 122.4 ng/mL; p ≤ 0.05) and identified neutrophil extracellular traps in kidney and liver tissues from untreated rats, consistent with the tissue damage. CONCLUSIONS: Pathologic changes in kidney and liver tissues in a rat model of major burn and endotoxin insults are ameliorated by resolvin D2.
Asunto(s)
Quemaduras/complicaciones , Ácidos Docosahexaenoicos/farmacología , Insuficiencia Hepática/tratamiento farmacológico , Insuficiencia Hepática/etiología , Insuficiencia Renal/tratamiento farmacológico , Insuficiencia Renal/etiología , Animales , Análisis Químico de la Sangre , Peso Corporal , Modelos Animales de Enfermedad , Hemodinámica , Insuficiencia Hepática/patología , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Pruebas de Función Renal , Lipopolisacáridos/farmacología , Pruebas de Función Hepática , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/biosíntesis , Masculino , Estudios Prospectivos , Distribución Aleatoria , Ratas , Ratas Wistar , Insuficiencia Renal/patologíaRESUMEN
Several evolutionary theories have been proposed to explain the adaptation of the long giraffe neck; however, few studies examine the fossil cervical vertebrae. We incorporate extinct giraffids, and the okapi and giraffe cervical vertebral specimens in a comprehensive analysis of the anatomy and elongation of the neck. We establish and evaluate 20 character states that relate to general, cranial and caudal vertebral lengthening, and calculate a length-to-width ratio to measure the relative slenderness of the vertebrae. Our sample includes cervical vertebrae (n=71) of 11 taxa representing all seven subfamilies. We also perform a computational comparison of the C3 of Samotherium and Giraffa camelopardalis, which demonstrates that cervical elongation occurs disproportionately along the cranial-caudal vertebral axis. Using the morphological characters and calculated ratios, we propose stages in cervical lengthening, which are supported by the mathematical transformations using fossil and extant specimens. We find that cervical elongation is anisometric and unexpectedly precedes Giraffidae. Within the family, cranial vertebral elongation is the first lengthening stage observed followed by caudal vertebral elongation, which accounts for the extremely long neck of the giraffe.
RESUMEN
The zebrafish pronephric tubule consists of proximal and distal segments and a collecting duct. The proximal segment is subdivided into the neck, proximal convoluted tubule (PCT) and proximal straight tubule (PST) segments. The distal segment consists of the distal-early (DE) and distal-late (DL) segments. How the proximal and distal segments develop along the anteroposterior axis is poorly understood. Here we show that knockdown of taz in zebrafish caused shortening and a significant reduction in the number of principal cells of the PST-DE segment, and proximalization of the pronephric tubule in 24 hpf embryos. RA treatment expanded the pronephric proximal domain in normal embryos as in taz morphants, an effect that was further enhanced upon exposure of taz morphants to RA. The early pronephric defects in 24 hpf taz morphants led to the failure of anterior pronephric tubule migration and convolution, and to PCT dilation and cyst formation in older embryos. In situ hybridization showed weak and transient expression of taz at the bud stage in the intermediate mesoderm, the source of pronephric progenitors. The present findings show that Taz is required in the anteroposterior patterning of the pronephric progenitor domain in the intermediate mesoderm, acting in part by regulating RA signaling in the pronephric progenitor field in the intermediate mesoderm.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/fisiología , Túbulos Renales/embriología , Pronefro/embriología , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/efectos de los fármacos , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Recuento de Células , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Túbulos Renales/citología , Túbulos Renales/metabolismo , Mesodermo/citología , Mesodermo/embriología , Mesodermo/metabolismo , Pronefro/citología , Pronefro/metabolismo , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Tretinoina/metabolismo , Tretinoina/farmacología , Pez Cebra/genética , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genéticaRESUMEN
Dysregulation of the actin cytoskeleton in podocytes represents a common pathway in the pathogenesis of proteinuria across a spectrum of chronic kidney diseases (CKD). The GTPase dynamin has been implicated in the maintenance of cellular architecture in podocytes through its direct interaction with actin. Furthermore, the propensity of dynamin to oligomerize into higher-order structures in an actin-dependent manner and to cross-link actin microfilaments into higher-order structures has been correlated with increased actin polymerization and global organization of the actin cytoskeleton in the cell. We found that use of the small molecule Bis-T-23, which promotes actin-dependent dynamin oligomerization and thus increased actin polymerization in injured podocytes, was sufficient to improve renal health in diverse models of both transient kidney disease and CKD. In particular, administration of Bis-T-23 in these renal disease models restored the normal ultrastructure of podocyte foot processes, lowered proteinuria, lowered collagen IV deposits in the mesangial matrix, diminished mesangial matrix expansion and extended lifespan. These results further establish that alterations in the actin cytoskeleton of kidney podocytes is a common hallmark of CKD, while also underscoring the substantial regenerative potential of injured glomeruli and identifying the oligomerization cycle of dynamin as an attractive potential therapeutic target to treat CKD.
Asunto(s)
Ácidos Cumáricos/administración & dosificación , Cianoacrilatos/administración & dosificación , Dinaminas/metabolismo , Podocitos/efectos de los fármacos , Proteinuria/tratamiento farmacológico , Insuficiencia Renal Crónica/tratamiento farmacológico , Acrilamida/administración & dosificación , Citoesqueleto de Actina/efectos de los fármacos , Animales , Dinaminas/química , Dinaminas/efectos de los fármacos , Humanos , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/patología , Glomérulos Renales/ultraestructura , Ratones , Modelos Animales , Podocitos/patología , Podocitos/ultraestructura , Proteinuria/metabolismo , Proteinuria/patología , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Pez CebraRESUMEN
Acute kidney injury is common and has a high mortality rate, and no effective treatment exists other than supportive care. Using cell culture models, we previously demonstrated that exocyst Sec10 overexpression reduced damage to renal tubule cells and speeded recovery and that the protective effect was mediated by higher basal levels of mitogen-activated protein kinase (MAPK) signaling. The exocyst, a highly-conserved eight-protein complex, is known for regulating protein trafficking. Here we show that the exocyst biochemically interacts with the epidermal growth factor receptor (EGFR), which is upstream of MAPK, and Sec10-overexpressing cells express greater levels of phosphorylated (active) ERK, the final step in the MAPK pathway, in response to EGF stimulation. EGFR endocytosis, which has been linked to activation of the MAPK pathway, increases in Sec10-overexpressing cells, and gefitinib, a specific EGFR inhibitor, and Dynasore, a dynamin inhibitor, both reduce EGFR endocytosis. In turn, inhibition of the MAPK pathway reduces ligand-mediated EGFR endocytosis, suggesting a potential feedback of elevated ERK activity on EGFR endocytosis. Gefitinib also decreases MAPK signaling in Sec10-overexpressing cells to levels seen in control cells and, demonstrating a causal role for EGFR, reverses the protective effect of Sec10 overexpression following cell injury in vitro. Finally, using an in vivo zebrafish model of acute kidney injury, morpholino-induced knockdown of sec10 increases renal tubule cell susceptibility to injury. Taken together, these results suggest that the exocyst, acting through EGFR, endocytosis, and the MAPK pathway is a candidate therapeutic target for acute kidney injury.
Asunto(s)
Lesión Renal Aguda/prevención & control , Endocitosis , Receptores ErbB/metabolismo , Túbulos Renales/enzimología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Pez Cebra/metabolismo , Lesión Renal Aguda/enzimología , Lesión Renal Aguda/genética , Lesión Renal Aguda/patología , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Perros , Endocitosis/efectos de los fármacos , Activación Enzimática , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Túbulos Renales/efectos de los fármacos , Túbulos Renales/patología , Células de Riñón Canino Madin Darby , Estrés Oxidativo , Fosforilación , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Factores de Tiempo , Transfección , Proteínas de Transporte Vesicular/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Acute kidney injury (AKI) is a common and significant medical problem. Despite the kidney's remarkable regenerative capacity, the mortality rate for the AKI patients is high. Thus, there remains a need to better understand the cellular mechanisms of nephron repair in order to develop new strategies that would enhance the intrinsic ability of kidney tissue to regenerate. Here, using a novel, laser ablation-based, zebrafish model of AKI, we show that collective migration of kidney epithelial cells is a primary early response to acute injury. We also show that cell proliferation is a late response of regenerating kidney epithelia that follows cell migration during kidney repair. We propose a computational model that predicts this temporal relationship and suggests that cell stretch is a mechanical link between migration and proliferation, and present experimental evidence in support of this hypothesis. Overall, this study advances our understanding of kidney repair mechanisms by highlighting a primary role for collective cell migration, laying a foundation for new approaches to treatment of AKI.
Asunto(s)
Lesión Renal Aguda/patología , Lesión Renal Aguda/fisiopatología , Movimiento Celular , Células Epiteliales/patología , Riñón/patología , Riñón/fisiopatología , Animales , Proliferación Celular , Transición Epitelial-Mesenquimal , Rayos Láser , Modelos Biológicos , Pez CebraRESUMEN
Glutamylation is a functionally important tubulin posttranslational modification enriched on stable microtubules of neuronal axons, mitotic spindles, centrioles, and cilia. In vertebrates, balanced activities of tubulin glutamyl ligase and cytoplasmic carboxypeptidase deglutamylase enzymes maintain organelle- and cell type-specific tubulin glutamylation patterns. Tubulin glutamylation in cilia is regulated via restricted subcellular localization or expression of tubulin glutamyl ligases (ttlls) and nonenzymatic proteins, including the zebrafish TPR repeat protein Fleer/Ift70. Here we analyze the expression patterns of ccp deglutamylase genes during zebrafish development and the effects of ccp gene knockdown on cilia formation, morphology, and tubulin glutamylation. The deglutamylases ccp2, ccp5, and ccp6 are expressed in ciliated cells, whereas ccp1 expression is restricted to the nervous system. Only ccp5 knockdown increases cilia tubulin glutamylation, induces ciliopathy phenotypes, including axis curvature, hydrocephalus, and pronephric cysts, and disrupts multicilia motility, suggesting that Ccp5 is the principal tubulin deglutamylase that maintains functional levels of cilia tubulin glutamylation. The ability of ccp5 knockdown to restore cilia tubulin glutamylation in fleer/ift70 mutants and rescue pronephric multicilia formation in both fleer- and ift88-deficient zebrafish indicates that tubulin glutamylation is a key driver of ciliogenesis.
Asunto(s)
Carboxipeptidasas/fisiología , Cilios/fisiología , Ácido Glutámico/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas de Pez Cebra/fisiología , Animales , Técnicas de Silenciamiento del Gen , Larva/citología , Larva/enzimología , Microtúbulos/metabolismo , Pronefro/citología , Pronefro/enzimología , Procesamiento Proteico-Postraduccional , Pez CebraRESUMEN
Inflammatory responses to infection and injury must be restrained and negatively regulated to minimize damage to host tissue. One proposed mechanism involves enzymatic inactivation of the pro-inflammatory mediator leukotriene B4, but it is difficult to dissect the roles of various metabolic enzymes and pathways. A primary candidate for a regulatory pathway is omega oxidation of leukotriene B4 in neutrophils, presumptively by CYP4F3A in humans and CYP4F18 in mice. This pathway generates ω, ω-1, and ω-2 hydroxylated products of leukotriene B4, depending on species. We created mouse models targeting exons 8 and 9 of the Cyp4f18 allele that allows both conventional and conditional knockouts of Cyp4f18. Neutrophils from wild-type mice convert leukotriene B4 to 19-hydroxy leukotriene B4, and to a lesser extent 18-hydroxy leukotriene B4, whereas these products were not detected in neutrophils from conventional Cyp4f18 knockouts. A mouse model of renal ischemia-reperfusion injury was used to investigate the consequences of loss of CYP4F18 in vivo. There were no significant changes in infiltration of neutrophils and other leukocytes into kidney tissue as determined by flow cytometry and immunohistochemistry, or renal injury as assessed by histological scoring and measurement of blood urea nitrogen. It is concluded that CYP4F18 is necessary for omega oxidation of leukotriene B4 in neutrophils, and is not compensated by other CYP enzymes, but loss of this metabolic pathway is not sufficient to impact inflammation and injury following renal ischemia-reperfusion in mice.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Inflamación/genética , Riñón/metabolismo , Leucotrieno B4/metabolismo , Animales , Sistema Enzimático del Citocromo P-450/metabolismo , Familia 4 del Citocromo P450 , Modelos Animales de Enfermedad , Humanos , Inflamación/metabolismo , Riñón/patología , Leucotrieno B4/genética , Ratones , Ratones Noqueados , Neutrófilos/metabolismo , Daño por Reperfusión/genética , Daño por Reperfusión/patologíaRESUMEN
Chordoma is a malignant tumor thought to arise from remnants of the embryonic notochord, with its origin in the bones of the axial skeleton. Surgical resection is the standard treatment, usually in combination with radiation therapy, but neither chemotherapeutic nor targeted therapeutic approaches have demonstrated success. No animal model and only few chordoma cell lines are available for preclinical drug testing, and, although no druggable genetic drivers have been identified, activation of EGFR and downstream AKT-PI3K pathways have been described. Here, we report a zebrafish model of chordoma, based on stable transgene-driven expression of HRASV12 in notochord cells during development. Extensive intra-notochordal tumor formation is evident within days of transgene expression, ultimately leading to larval death. The zebrafish tumors share characteristics of human chordoma as demonstrated by immunohistochemistry and electron microscopy. The mTORC1 inhibitor rapamycin, which has some demonstrated activity in a chordoma cell line, delays the onset of tumor formation in our zebrafish model, and improves survival of tumor-bearing fish. Consequently, the HRASV12-driven zebrafish model of chordoma could enable high-throughput screening of potential therapeutic agents for the treatment of this refractory cancer.
Asunto(s)
Cordoma/embriología , Cordoma/patología , Modelos Animales de Enfermedad , Mutación/genética , Notocorda/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Pez Cebra , Animales , Animales Modificados Genéticamente , Carcinogénesis/patología , Línea Celular Tumoral , Progresión de la Enfermedad , Humanos , Inmunohistoquímica , Notocorda/efectos de los fármacos , Notocorda/patología , Notocorda/ultraestructura , Especificidad de Órganos/efectos de los fármacos , Sirolimus/farmacología , Análisis de Supervivencia , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
Formation of a functional renal network requires the interconnection of two epithelial tubes: the nephron, which arises from kidney mesenchyme, and the collecting system, which originates from the branching ureteric epithelium. How this connection occurs, however, is incompletely understood. Here, we used high-resolution image analysis in conjunction with genetic labeling of epithelia to visualize and characterize this process. Although the focal absence of basal lamina from renal vesicle stages ensures that both epithelial networks are closely apposed, we found that a patent luminal interconnection is not established until S-shaped body stages. Precursor cells of the distal nephron in the interconnection zone exhibit a characteristic morphology consisting of ill-defined epithelial junctional complexes but without expression of mesenchymal markers such as vimentin and Snai2. Live-cell imaging revealed that before luminal interconnection, distal cells break into the lumen of the collecting duct epithelium, suggesting that an invasive behavior is a key step in the interconnection process. Furthermore, loss of distal cell identity, which we induced by activating the Notch pathway, prevented luminal interconnection. Taken together, these data support a model in which establishing the distal identity of nephron precursor cells closest to the nascent collecting duct epithelium leads to an active cell invasion, which in turn contributes to a patent tubular interconnection between the nephron and collecting duct epithelia.
Asunto(s)
Riñón/embriología , Animales , Movimiento Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Imagenología Tridimensional , Riñón/metabolismo , Túbulos Renales Distales/embriología , Túbulos Renales Distales/metabolismo , Masculino , Ratones , Ratones Transgénicos , Microscopía Confocal , Nefronas/embriología , Nefronas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
Organ development leads to the emergence of organ function, which in turn can impact developmental processes. Here we show that fluid flow-induced collective epithelial migration during kidney nephron morphogenesis induces cell stretch that in turn signals epithelial proliferation. Increased cell proliferation was dependent on PI3K signaling. Inhibiting epithelial proliferation by blocking PI3K or CDK4/Cyclin D1 activity arrested cell migration prematurely and caused a marked overstretching of the distal nephron tubule. Computational modeling of the involved cell processes predicted major morphological and kinetic outcomes observed experimentally under a variety of conditions. Overall, our findings suggest that kidney development is a recursive process where emerging organ function "feeds back" to the developmental program to influence fundamental cellular events such as cell migration and proliferation, thus defining final organ morphology.
Asunto(s)
Células Epiteliales/citología , Túbulos Renales/citología , Fenómenos Mecánicos , Morfogénesis , Fosfatidilinositol 3-Quinasas/metabolismo , Pronefro/embriología , Pez Cebra/embriología , Animales , Fenómenos Biomecánicos , Movimiento Celular , Proliferación Celular , Túbulos Renales/embriología , Modelos Biológicos , Pronefro/citología , Transducción de SeñalRESUMEN
The zebrafish has emerged as a powerful model to study organ development and regeneration. It has a number of advantages over other vertebrate model systems. The embryo can be kept transparent throughout embryonic development, which allows direct visualization of the developing organs. In addition, embryos can be easily manipulated surgically, genetically, or chemically. Furthermore, because nephron shape and function are remarkably conserved among vertebrates, zebrafish findings can directly inform human studies. Here, we describe a simple procedure that can be used by laboratories to investigate the development of zebrafish kidney and other organs by time-lapse microscopy.
Asunto(s)
Embrión no Mamífero/ultraestructura , Riñón/embriología , Microscopía Confocal/métodos , Pez Cebra/embriología , Animales , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Femenino , Procesamiento de Imagen Asistido por Computador/métodos , Morfolinos/metabolismoRESUMEN
Collective cell migration is emerging as a significant component of many biological processes including metazoan development, tissue maintenance and repair and tumor progression. Different contexts dictate different mechanisms by which migration is guided and maintained. In vascular endothelia subjected to significant shear stress, fluid flow is utilized to properly orient a migrating group of cells. Recently, we discovered that the developing zebrafish pronephric epithelium undergoes a similar response to luminal fluid flow, which guides pronephric epithelial migration towards the glomerulus. Intratubular migration leads to significant changes in kidney morphology. This novel process provides a powerful in vivo model for further exploration of the mechanisms underlying mechanotransduction and collective migration.
Asunto(s)
Movimiento Celular , Reología , Animales , Células Epiteliales/citología , Riñón/citología , Riñón/fisiología , Modelos Biológicos , Pez CebraRESUMEN
Mutations in polycystin1 (PKD1) account for the majority of autosomal dominant polycystic kidney disease (ADPKD). PKD1 mutations are also associated with vascular aneurysm and abdominal wall hernia, suggesting a role for polycystin1 in extracellular matrix (ECM) integrity. In zebrafish, combined knockdown of the PKD1 paralogs pkd1a and pkd1b resulted in dorsal axis curvature, hydrocephalus, cartilage and craniofacial defects, and pronephric cyst formation at low frequency (10-15%). Dorsal axis curvature was identical to the axis defects observed in pkd2 knockdown embryos. Combined pkd1a/b, pkd2 knockdown demonstrated that these genes interact in axial morphogenesis. Dorsal axis curvature was linked to notochord collagen overexpression and could be reversed by knockdown of col2a1 mRNA or chemical inhibition of collagen crosslinking. pkd1a/b- and pkd2-deficient embryos exhibited ectopic, persistent expression of multiple collagen mRNAs, suggesting a loss of negative feedback signaling that normally limits collagen gene expression. Knockdown of pkd1a/b also dramatically sensitized embryos to low doses of collagen-crosslinking inhibitors, implicating polycystins directly in the modulation of collagen expression or assembly. Embryos treated with wortmannin or LY-29400 also exhibited dysregulation of col2a1 expression, implicating phosphoinositide 3-kinase (PI3K) in the negative feedback signaling pathway controlling matrix gene expression. Our results suggest that pkd1a/b and pkd2 interact to regulate ECM secretion or assembly, and that altered matrix integrity may be a primary defect underlying ADPKD tissue pathologies.
Asunto(s)
Matriz Extracelular/metabolismo , Riñón Poliquístico Autosómico Dominante/genética , Canales Catiónicos TRPP/genética , Tipificación del Cuerpo/genética , Calcio/metabolismo , Condrogénesis/genética , Clonación Molecular , Colágeno/genética , Colágeno/metabolismo , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/patología , Reactivos de Enlaces Cruzados/metabolismo , Retículo Endoplásmico/enzimología , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Riñón/anomalías , Riñón/patología , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Canales Catiónicos TRPP/deficiencia , Canales Catiónicos TRPP/metabolismoRESUMEN
Tissue organization in epithelial organs is achieved during development by the combined processes of cell differentiation and morphogenetic cell movements. In the kidney, the nephron is the functional organ unit. Each nephron is an epithelial tubule that is subdivided into discrete segments with specific transport functions. Little is known about how nephron segments are defined or how segments acquire their distinctive morphology and cell shape. Using live, in vivo cell imaging of the forming zebrafish pronephric nephron, we found that the migration of fully differentiated epithelial cells accounts for both the final position of nephron segment boundaries and the characteristic convolution of the proximal tubule. Pronephric cells maintain adherens junctions and polarized apical brush border membranes while they migrate collectively. Individual tubule cells exhibit basal membrane protrusions in the direction of movement and appear to establish transient, phosphorylated Focal Adhesion Kinase-positive adhesions to the basement membrane. Cell migration continued in the presence of camptothecin, indicating that cell division does not drive migration. Lengthening of the nephron was, however, accompanied by an increase in tubule cell number, specifically in the most distal, ret1-positive nephron segment. The initiation of cell migration coincided with the onset of fluid flow in the pronephros. Complete blockade of pronephric fluid flow prevented cell migration and proximal nephron convolution. Selective blockade of proximal, filtration-driven fluid flow shifted the position of tubule convolution distally and revealed a role for cilia-driven fluid flow in persistent migration of distal nephron cells. We conclude that nephron morphogenesis is driven by fluid flow-dependent, collective epithelial cell migration within the confines of the tubule basement membrane. Our results establish intimate links between nephron function, fluid flow, and morphogenesis.
Asunto(s)
Movimiento Celular , Morfogénesis , Nefronas/citología , Nefronas/embriología , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Cartilla de ADN , Inmunohistoquímica , Hibridación in Situ , Pez CebraRESUMEN
The kidney and vasculature are intimately linked both functionally and during development, when nephric and blood/vascular progenitor cells occupy adjacent bands of mesoderm in zebrafish and frog embryos. Developmental mechanisms that underlie the differentiation of kidney versus blood/vascular lineages remain unknown. The odd skipped related1 (osr1) gene encodes a zinc-finger transcription factor that is expressed in the germ ring mesendoderm and subsequently in the endoderm and intermediate mesoderm, prior to the expression of definitive kidney or blood/vascular markers. Knockdown of osr1 in zebrafish embryos resulted in a complete, segment-specific loss of anterior kidney progenitors and a compensatory increase in the number of angioblast cells in the same trunk region. Histology revealed a subsequent absence of kidney tubules, an enlarged cardinal vein and expansion of the posterior venous plexus. Altered kidney versus vascular development correlated with expanded endoderm development in osr1 knockdowns. Combined osr1 loss of function and blockade of endoderm development by knockdown of sox32/casanova rescued anterior kidney development. The results indicate that osr1 activity is required to limit endoderm differentiation from mesendoderm; in the absence of osr1, excess endoderm alters mesoderm differentiation, shifting the balance from kidney towards vascular development.