RESUMEN
The study of cellular and developmental processes in physiologically relevant three-dimensional (3D) systems facilitates an understanding of mechanisms underlying cell fate, disease and injury. While cutting-edge microscopy technologies permit the routine acquisition of 3D datasets, there is currently a limited number of open-source software packages to analyse such images. Here, we describe General Image Analysis of Nuclei-based Images (GIANI; https://djpbarry.github.io/Giani), new software for the analysis of 3D images. The design primarily facilitates segmentation of nuclei and cells, followed by quantification of morphology and protein expression. GIANI enables routine and reproducible batch-processing of large numbers of images, and comes with scripting and command line tools. We demonstrate the utility of GIANI by quantifying cell morphology and protein expression in confocal images of mouse early embryos and by segmenting nuclei from light-sheet microscopy images of the flour beetle embryo. We also validate the performance of the software using simulated data. More generally, we anticipate that GIANI will be a useful tool for researchers in a variety of biomedical fields.
Asunto(s)
Imagenología Tridimensional , Microscopía , Algoritmos , Animales , Núcleo Celular , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Ratones , Programas InformáticosRESUMEN
The heart develops from 2 sources of mesoderm progenitors, the first and second heart field (FHF and SHF). Using a single-cell transcriptomic assay combined with genetic lineage tracing and live imaging, we find the FHF and SHF are subdivided into distinct pools of progenitors in gastrulating mouse embryos at earlier stages than previously thought. Each subpopulation has a distinct origin in the primitive streak. The first progenitors to leave the primitive streak contribute to the left ventricle, shortly after right ventricle progenitor emigrate, followed by the outflow tract and atrial progenitors. Moreover, a subset of atrial progenitors are gradually incorporated in posterior locations of the FHF. Although cells allocated to the outflow tract and atrium leave the primitive streak at a similar stage, they arise from different regions. Outflow tract cells originate from distal locations in the primitive streak while atrial progenitors are positioned more proximally. Moreover, single-cell RNA sequencing demonstrates that the primitive streak cells contributing to the ventricles have a distinct molecular signature from those forming the outflow tract and atrium. We conclude that cardiac progenitors are prepatterned within the primitive streak and this prefigures their allocation to distinct anatomical structures of the heart. Together, our data provide a new molecular and spatial map of mammalian cardiac progenitors that will support future studies of heart development, function, and disease.
Asunto(s)
Linaje de la Célula/genética , Corazón/embriología , Línea Primitiva/embriología , Animales , Linaje de la Célula/fisiología , Femenino , Gástrula , Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/genética , Corazón/fisiología , Atrios Cardíacos/embriología , Ventrículos Cardíacos/embriología , Masculino , Mesodermo , Ratones , Ratones Endogámicos C57BL , Morfogénesis , Línea Primitiva/fisiología , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodosRESUMEN
Perturbation of CaMKIIß expression has been associated with multiple neuropsychiatric diseases, highlighting CaMKIIß as a gene of interest. Yet, in contrast to CaMKIIα, the specific functions of CaMKIIß in the brain remain poorly explored. Here, we reveal a novel function for this CaMKII isoform in vivo during neuronal development. By using in utero electroporation, we show that CaMKIIß is an important regulator of radial migration of projection neurons during cerebral cortex development. Knockdown of CaMKIIß causes accelerated migration of nascent pyramidal neurons, whereas overexpression of CaMKIIß inhibits migration, demonstrating that precise regulation of CaMKIIß expression is required for correct neuronal migration. More precisely, CaMKIIß controls the multipolar-bipolar transition in the intermediate zone and locomotion in the cortical plate through its actin-binding and -bundling activities. In addition, our data indicate that a fine-tuned balance between CaMKIIß and cofilin activities is necessary to ensure proper migration of cortical neurons. Thus, our findings define a novel isoform-specific function for CaMKIIß, demonstrating that CaMKIIß has a major biological function in the developing brain.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/fisiología , Movimiento Celular/fisiología , Corteza Cerebral/fisiología , Neurogénesis/fisiología , Animales , Encéfalo/embriología , Encéfalo/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Corteza Cerebral/metabolismo , Embrión de Mamíferos/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Ratones , Proteínas de Microfilamentos/genética , Trastornos del Neurodesarrollo/genética , Neurogénesis/genética , Neuronas/metabolismo , Cultivo Primario de Células , Isoformas de Proteínas/metabolismo , Células Piramidales/metabolismoRESUMEN
Hirschsprung disease (HSCR) is characterized by absence of enteric neurons from the distal colon and severe intestinal dysmotility. To understand the pathophysiology and genetics of HSCR we developed a unique zebrafish model that allows combined genetic, developmental and in vivo physiological studies. We show that ret mutant zebrafish exhibit cellular, physiological and genetic features of HSCR, including absence of intestinal neurons, reduced peristalsis, and varying phenotype expressivity in the heterozygous state. We perform live imaging experiments using a UAS-GAL4 binary genetic system to drive fluorescent protein expression in ENS progenitors. We demonstrate that ENS progenitors migrate at reduced speed in ret heterozygous embryos, without changes in proliferation or survival, establishing this as a principal pathogenic mechanism for distal aganglionosis. We show, using live imaging of actual intestinal movements, that intestinal motility is severely compromised in ret mutants, and partially impaired in ret heterozygous larvae, and establish a clear correlation between neuron position and organised intestinal motility. We exploited the partially penetrant ret heterozygous phenotype as a sensitised background to test the influence of a candidate modifier gene. We generated mapk10 loss-of-function mutants, which show reduced numbers of enteric neurons. Significantly, we show that introduction of mapk10 mutations into ret heterozygotes enhanced the ENS deficit, supporting MAPK10 as a HSCR susceptibility locus. Our studies demonstrate that ret heterozygous zebrafish is a sensitized model, with many significant advantages over existing murine models, to explore the pathophysiology and complex genetics of HSCR.
Asunto(s)
Sistema Nervioso Entérico/metabolismo , Enfermedad de Hirschsprung/genética , Proteína Quinasa 10 Activada por Mitógenos/genética , Proteínas Proto-Oncogénicas c-ret/genética , Pez Cebra/genética , Animales , Colon/inervación , Colon/patología , Modelos Animales de Enfermedad , Sistema Nervioso Entérico/patología , Enfermedad de Hirschsprung/metabolismo , Enfermedad de Hirschsprung/patología , Humanos , Mutación , Neuronas/metabolismo , Neuronas/patología , Fenotipo , Proteínas Proto-Oncogénicas c-ret/metabolismoRESUMEN
The classical view of neural plate development held that it arises from the ectoderm, after its separation from the mesodermal and endodermal lineages. However, recent cell-lineage-tracing experiments indicate that the caudal neural plate and paraxial mesoderm are generated from common bipotential axial stem cells originating from the caudal lateral epiblast. Tbx6 null mutant mouse embryos which produce ectopic neural tubes at the expense of paraxial mesoderm must provide a clue to the regulatory mechanism underlying this neural versus mesodermal fate choice. Here we demonstrate that Tbx6-dependent regulation of Sox2 determines the fate of axial stem cells. In wild-type embryos, enhancer N1 of the neural primordial gene Sox2 is activated in the caudal lateral epiblast, and the cells staying in the superficial layer sustain N1 activity and activate Sox2 expression in the neural plate. In contrast, the cells destined to become mesoderm activate Tbx6 and turn off enhancer N1 before migrating into the paraxial mesoderm compartment. In Tbx6 mutant embryos, however, enhancer N1 activity persists in the paraxial mesoderm compartment, eliciting ectopic Sox2 activation and transforming the paraxial mesoderm into neural tubes. An enhancer-N1-specific deletion mutation introduced into Tbx6 mutant embryos prevented this Sox2 activation in the mesodermal compartment and subsequent development of ectopic neural tubes, indicating that Tbx6 regulates Sox2 via enhancer N1. Tbx6-dependent repression of Wnt3a in the paraxial mesodermal compartment is implicated in this regulatory process. Paraxial mesoderm-specific misexpression of a Sox2 transgene in wild-type embryos resulted in ectopic neural tube development. Thus, Tbx6 represses Sox2 by inactivating enhancer N1 to inhibit neural development, and this is an essential step for the specification of paraxial mesoderm from the axial stem cells.
Asunto(s)
Linaje de la Célula , Mesodermo/citología , Células-Madre Neurales/citología , Tubo Neural/citología , Factores de Transcripción SOXB1/metabolismo , Células Madre/citología , Factores de Transcripción/metabolismo , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Coristoma/embriología , Coristoma/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Datos de Secuencia Molecular , Placa Neural/citología , Placa Neural/embriología , Placa Neural/metabolismo , Tubo Neural/embriología , Tubo Neural/metabolismo , Factores de Transcripción SOXB1/genética , Proteínas de Dominio T Box , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Proteínas Wnt/antagonistas & inhibidores , Proteínas Wnt/metabolismo , Proteína Wnt3 , Proteína Wnt3ARESUMEN
To facilitate studies of neural network architecture and formation, we generated three Drosophila melanogaster variants of the mouse Brainbow-2 system, called Flybow. Sequences encoding different membrane-tethered fluorescent proteins were arranged in pairs within cassettes flanked by recombination sites. Flybow combines the Gal4-upstream activating sequence binary system to regulate transgene expression and an inducible modified Flp-FRT system to drive inversions and excisions of cassettes. This provides spatial and temporal control over the stochastic expression of one of two or four reporters within one sample. Using the visual system, the embryonic nervous system and the wing imaginal disc, we show that Flybow in conjunction with specific Gal4 drivers can be used to visualize cell morphology with high resolution. Finally, we demonstrate that this labeling approach is compatible with available Flp-FRT-based techniques, such as mosaic analysis with a repressible cell marker; this could further support the genetic analysis of neural circuit assembly and function.
Asunto(s)
Drosophila melanogaster/citología , Proteínas Luminiscentes/análisis , Red Nerviosa/citología , Neuronas/citología , Coloración y Etiquetado/métodos , Animales , Secuencia de Bases , Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de Unión al ADN/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Proteínas Luminiscentes/genética , Ratones , Datos de Secuencia Molecular , Red Nerviosa/embriología , Neuroglía/química , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/química , Neuronas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaRESUMEN
Nuclear architecture and chromatin reorganization have recently been shown to orchestrate gene expression and act as key players in developmental pathways. To investigate how regulatory elements in the mouse CD8 gene locus are arranged in space and in relation to each other, three-dimensional fluorescence in situ hybridization and chromosome conformation capture techniques were employed to monitor the repositioning of the locus in relation to its subchromosomal territory and to identify long-range interactions between the different elements during development. Our data demonstrate that CD8 gene expression in murine lymphocytes is accompanied by the relocation of the locus outside its subchromosomal territory. Similar observations in the CD4 locus point to a rather general phenomenon during T cell development. Furthermore, we show that this relocation of the CD8 gene locus is associated with a clustering of regulatory elements forming a tight active chromatin hub in CD8-expressing cells. In contrast, in nonexpressing cells, the gene remains close to the main body of its chromosomal domain and the regulatory elements appear not to interact with each other.
Asunto(s)
Antígenos CD8/genética , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Núcleo Celular/genética , Regulación del Desarrollo de la Expresión Génica/inmunología , Timo/inmunología , Timo/metabolismo , Animales , Antígenos CD4/genética , Antígenos CD8/biosíntesis , Posicionamiento de Cromosoma/genética , Sondas de ADN/genética , Femenino , Imagenología Tridimensional , Hibridación Fluorescente in Situ , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Tejido Linfoide/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Estructura Terciaria de Proteína/genética , Timo/citologíaRESUMEN
Sensory hair cells and their associated non-sensory supporting cells in the inner ear are fundamental for hearing and balance. They arise from a common progenitor, but little is known about the molecular events specifying this cell lineage. We recently identified two allelic mouse mutants, light coat and circling (Lcc) and yellow submarine (Ysb), that show hearing and balance impairment. Lcc/Lcc mice are completely deaf, whereas Ysb/Ysb mice are severely hearing impaired. We report here that inner ears of Lcc/Lcc mice fail to establish a prosensory domain and neither hair cells nor supporting cells differentiate, resulting in a severe inner ear malformation, whereas the sensory epithelium of Ysb/Ysb mice shows abnormal development with disorganized and fewer hair cells. These phenotypes are due to the absence (in Lcc mutants) or reduced expression (in Ysb mutants) of the transcription factor SOX2, specifically within the developing inner ear. SOX2 continues to be expressed in the inner ears of mice lacking Math1 (also known as Atoh1 and HATH1), a gene essential for hair cell differentiation, whereas Math1 expression is absent in Lcc mutants, suggesting that Sox2 acts upstream of Math1.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Oído Interno/embriología , Oído Interno/metabolismo , Transactivadores/metabolismo , Alelos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Diferenciación Celular , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Oído Interno/anomalías , Oído Interno/patología , Células Ciliadas Auditivas Internas/anomalías , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/patología , Ratones , Ratones Mutantes , Mutación/genética , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción SOXB1 , Transactivadores/deficiencia , Transactivadores/genética , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The hostile environment of the microscope stage poses numerous challenges to successful imaging of morphogenesis in live tissues. This review aims to highlight some of the main practical considerations to take into account when embarking on a project to image cell behaviour in the context of cells' normal surroundings. Scrutiny of these activities is likely to be the most informative approach to understanding mechanical morphogenesis but is often confounded by the substantial technical difficulties involved in imaging samples over extended periods of time. Repeated observation of cells in live tissue requires that strategies be adopted to prioritize the stability of the sample, ensuring that it remains viable and develops normally while being held in a manner accessible to microscopic examination. Key considerations when creating reliable protocols for time-lapse imaging may be broken down into three main criteria; labelling, mounting and image acquisition. Choices and compromises made here, however, will directly influence image quality, and even small refinements can substantially improve what information may be extracted from images. Live imaging of tissue is difficult but paying close attention to the basics along with a little innovation is likely to be well rewarded.This article is part of the themed issue 'Systems morphodynamics: understanding the development of tissue hardware'.
Asunto(s)
Microscopía/métodos , Morfogénesis , Imagen de Lapso de Tiempo/métodos , Microscopía/instrumentación , Imagen de Lapso de Tiempo/instrumentaciónRESUMEN
Despite great strides during the preceding 3 decades, the ability to consistently eliminate postoperative nausea and vomiting (PONV) continues to elude anesthesia practitioners. The occurrence of PONV related to anesthesia and surgery prolongs hospital stays and increases healthcare costs. Protracted recovery times place constraints on patients, healthcare systems, and healthcare financiers. Many pharmacological antiemetics have been developed and are in use in the attempt to alleviate PONV. Side effects and cost profiles of many of these interventions, however, reinforce the broadly held belief that there remains opportunity for improvement. Because the Western culture almost exclusively favors evidence-based scientific practice and interventions, the search continues for an ideal, cost-effective, safe, and efficacious pharmacological agent to prevent PONV. Eastern culture, on the other hand, relies heavily on naturopathic remedies whose successful use has spanned thousands of years. Increasing attention has been given to the potential benefits of nonpharmacological intervention for the prevention of PONV in association with anesthesia care. Therefore, the purpose of this AANA Journal course will be to focus attention on what is known and what is unknown in the literature regarding use of the nonallopathic remedy of acupressure as a nonpharmacological alternative to commonly utilized antiemetic prophylaxis.
Asunto(s)
Acupresión/instrumentación , Acupresión/métodos , Náusea y Vómito Posoperatorios/prevención & control , Antieméticos/efectos adversos , Humanos , Meridianos , Atención PerioperativaRESUMEN
The purpose of this study was to systematically review the instruments used to obtain anesthesia-specific patient satisfaction data and to determine the degree to which each instrument controlled for measurement error bias, such as poor survey design. By using an assessment and evaluation tool developed for the present study that held proven internal reliability and construct validity, we analyzed and scored each instrument according to the presence or absence of measurement error in survey design. We found that a paucity of anesthesia-specific patient satisfaction studies exists and that patient satisfaction studies dealing with anesthesia care were erratically defined, nonstandardized, and imprecise regarding intent and method. Moreover, the simple rating forms used in most of the reviewed studies were inadequate to achieve the goal of measuring the quality of anesthesia care. One instrument, the Iowa Satisfaction With Anesthesia Scale (ISAS), developed by Dexter et al (1997), was the first found to inculcate scientifically accepted psychometric item construction algorithms, an indicator of measurement reliability. Although the ISAS holds substantial potential for future application in this realm, we recommend that it be refined further and that the search for a superlative instrument to obtain anesthesia-specific patient satisfaction continue.
Asunto(s)
Enfermeras Anestesistas/normas , Satisfacción del Paciente , Atención Perioperativa/normas , Encuestas y Cuestionarios , Estudios de Evaluación como Asunto , HumanosRESUMEN
In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system (1-5). To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex (6-8). Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions. Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method( 9). However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice (10-11) (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA (12). These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C). Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.
Asunto(s)
Corteza Cerebral/fisiología , Dendritas/fisiología , Electroporación/métodos , Hipocampo/fisiología , Animales , Corteza Cerebral/química , Corteza Cerebral/ultraestructura , Dendritas/genética , Dendritas/ultraestructura , Embrión de Mamíferos , Hipocampo/química , Hipocampo/ultraestructura , RatonesRESUMEN
Little is known of the intracellular machinery that controls the motility of newborn neurons. We have previously shown that the proneural protein Neurog2 promotes the migration of nascent cortical neurons by inducing the expression of the atypical Rho GTPase Rnd2. Here, we show that another proneural factor, Ascl1, promotes neuronal migration in the cortex through direct regulation of a second Rnd family member, Rnd3. Both Rnd2 and Rnd3 promote neuronal migration by inhibiting RhoA signaling, but they control distinct steps of the migratory process, multipolar to bipolar transition in the intermediate zone and locomotion in the cortical plate, respectively. Interestingly, these divergent functions directly result from the distinct subcellular distributions of the two Rnd proteins. Because Rnd proteins also regulate progenitor divisions and neurite outgrowth, we propose that proneural factors, through spatiotemporal regulation of Rnd proteins, integrate the process of neuronal migration with other events in the neurogenic program.