Name: quantification of orofacial phenotypes in xenopus
Uploaded: 2014-11-06T15:00:00.000+00:00
Duration: 9 min 26 s
Description: Watch this Scientific Journal Video about Quantification of Orofacial Phenotypes in Xenopus at JoVE.com

Fondos para la instalación de A. Dickinson de VCU apoyaron este trabajo. Los autores desean agradecer a Dan Nacu por su talento artístico en la creación de la ilustración esquemática.

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Wnt+antagonists+Frzb-1+and+Crescent+locally+regulate+basement+membrane+dissolution+in+the+developing+primary+mouth.">The Wnt antagonists Frzb-1 and Crescent locally regulate basement membrane dissolution in the developing primary mouth.</a> Development. 136, 1071-1081 (2009).</li><li>Barnett, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syndrome+Transcription+Factor+is+critical+for+neural+crest+cell+function+in+Xenopus+laevis.">Syndrome Transcription Factor is critical for neural crest cell function in Xenopus laevis.</a> Mech Dev. 129, 324-338 (2012).</li><li>Gonzales, B., Yang, H., Henning, D., Valdez, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+functional+characterization+of+the+Xenopus+orthologue+of+the+Treacher+Collins+syndrome+(TCOF1)+gene+product.">Cloning and functional characterization of the Xenopus orthologue of the Treacher Collins syndrome (TCOF1) gene product.</a> Gene. 359, 73-80 (2005).</li><li>Reisoli, E., De Lucchini, S., Nardi, I., Ori, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+2B+receptor+signaling+is+required+for+craniofacial+morphogenesis+and+jaw+joint+formation+in+Xenopus.">Serotonin 2B receptor signaling is required for craniofacial morphogenesis and jaw joint formation in Xenopus.</a> Development. 137, 2927-2937 (2010).</li><li>Schuff, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoxN3+is+required+for+craniofacial+and+eye+development+of+Xenopus+laevis.">FoxN3 is required for craniofacial and eye development of Xenopus laevis.</a> Dev Dyn. 236, 226-239 (2007).</li><li>Slater, B. J., Liu, K. J., Kwan, M. D., Quarto, N., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranial+osteogenesis+and+suture+morphology+in+Xenopus+laevis:+a+unique+model+system+for+studying+craniofacial+development.">Cranial osteogenesis and suture morphology in Xenopus laevis: a unique model system for studying craniofacial development.</a> PLoS One. 4, (2009).</li><li>Vandenberg, L. N., Adams, D. S., Levin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalized+shape+and+location+of+perturbed+craniofacial+structures+in+the+Xenopus+tadpole+reveal+an+innate+ability+to+achieve+correct+morphology.">Normalized shape and location of perturbed craniofacial structures in the Xenopus tadpole reveal an innate ability to achieve correct morphology.</a> Dev Dyn. 241, 863-878 (2012).</li><li>Bugaighis, I., Mattick, C. R., Tiddeman, B., Hobson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Facial+Morphometry+in+Children+with+Oral+Clefts.">3D Facial Morphometry in Children with Oral Clefts.</a> Cleft Palate Craniofac J. , (2013).</li><li>Farkas, L. G., Katic, M. J., Forrest, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+anatomy+of+the+face+in+Down's+syndrome:+anthropometric+proportion+indices+in+the+craniofacial+regions.">Surface anatomy of the face in Down's syndrome: anthropometric proportion indices in the craniofacial regions.</a> J Craniofac Surg. 12, 519-524 (2001).</li><li>Scheuer, H. A., Holtje, W. J., Hasund, A., Pfeifer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognosis+of+facial+growth+in+patients+with+unilateral+complete+clefts+of+the+lip,+alveolus+and+palate.">Prognosis of facial growth in patients with unilateral complete clefts of the lip, alveolus and palate.</a> J Craniomaxillofac Surg. 29, 198-204 (2001).</li><li>Parsons, K. J., Andreeva, V., James Cooper, W., Yelick, P. C., Craig Albertson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+of+the+zebrafish+jaw:+development+beyond+the+embryo.">Morphogenesis of the zebrafish jaw: development beyond the embryo.</a> Methods Cell Biol. 101, 225-248 (2011).</li><li>Farkas, L. G., Katic, M. J., Forrest, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+anatomy+of+the+face+in+Down's+syndrome:+age-related+changes+of+anthropometric+proportion+indices+in+the+craniofacial+regions.">Surface anatomy of the face in Down's syndrome: age-related changes of anthropometric proportion indices in the craniofacial regions.</a> J Craniofac Surg. 13, 368-374 (2002).</li><li>Cooper, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bentho-pelagic+divergence+of+cichlid+feeding+architecture+was+prodigious+and+consistent+during+multiple+adaptive+radiations+within+African+rift-lakes.">Bentho-pelagic divergence of cichlid feeding architecture was prodigious and consistent during multiple adaptive radiations within African rift-lakes.</a> PLoS One. 5, (2010).</li><li>Klingenberg, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+alcohol+exposure+alters+the+patterns+of+facial+asymmetry.">Prenatal alcohol exposure alters the patterns of facial asymmetry.</a> Alcohol. 44, 649-657 (2010).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+cleft+palate+in+mice+with+a+targeted+mutation+of+the+LIM+homeobox+gene+lhx8.">Isolated cleft palate in mice with a targeted mutation of the LIM homeobox gene lhx8.</a> Proc Natl Acad Sci U S A. 96, 15002-15006 (1999).</li><li>Allam, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spectrum+of+median+craniofacial+dysplasia.">The spectrum of median craniofacial dysplasia.</a> Plast Reconstr Surg. , 812-821 (2011).</li><li>Sive, H. L., Grainger, R., Harlard, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Early development of Xenopus laevis: a laboratory manual. , (2000).</li><li>Cross, M. K., Powers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obtaining+eggs+from+Xenopus+laevis+females.">Obtaining eggs from Xenopus laevis females.</a> J Vis Exp. , (2008).</li><li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal Table of Xenopus Laevis (Daudin). , (1967).</li><li>Nieuwkoop, P. D. a. F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal Table of Xenopus laevis (Daudin): A Systematical and Chronological Survey of the Development from the Fertilized Egg till the End of Metamorphosis. , (1994).</li><li>Abdi, H., Williams, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principal+Component+Analysis.">Principal Component Analysis.</a> WIREs Computational Statistics. 2, (2010).</li><li>Hill, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> P. STATISTICS: Methods and Applications. , (2013).</li></ol>

Los autores no tienen nada que revelar.

Xenopus laevis se ha convertido en una herramienta útil para la disección de los mecanismos de desarrollo de desarrollo orofacial subyacente; sin embargo, actualmente no existen protocolos que describen los cambios de tamaño y forma de esta región en las ranas. El método aquí descrito contribuirá significativamente a la esfera del desarrollo orofacial al permitir una cuantificación más rigurosa de fenotipos orofaciales en Xenopus y otros vertebrados. El primer aspe...

Entre los tipos más frecuentes y devastadores de defectos de nacimiento en humanos son los que afectan a la boca y la cara, como hendiduras orofaciales 1. Los niños con estructuras orofaciales malformados se someten a múltiples cirugías durante toda su vida y luchan con desfiguraciones faciales, el habla, la audición y problemas de alimentación. Por lo tanto, lo que facilita una nueva investigación en el desarrollo de craneo y orofacial es de suma importancia para la prevención y el tratamiento de estos tipos de defectos de nacimiento en seres humanos. Xenopus laevis ha surgido como una nueva herramienta para la disección de los mecanismos que rigen el desarrollo craneofacial (algunos ejemplos incluyen 2,3,4 -11). Por lo tanto, un método cuantitativo para analizar los cambios de tamaño y forma durante el desarrollo de la cabeza y la cara de esta especie podría ser muy poderosa 3. A continuación, presentamos un método de este tipo; la combinación de mediciones de tamaño tradicional con morfometría geométrica adaptados de un estudio de Xenopus 12 </sup&gt; y una gran cantidad de estudios que analizan la forma facial humana 13-15. El objetivo de este protocolo es permitir a los investigadores para cuantificar el tamaño de la cara y las formas de distinguir entre los diferentes fenotipos orofaciales durante el desarrollo normal y anormal. Este análisis permitirá una mejor diferenciación entre los defectos craneofaciales sutiles tales como las derivadas de los efectos sinérgicos de los genes y / o factores ambientales. Además, este método de cuantificación también podría revelar incluso leve mejora o rescate de un defecto orofacial. Por lo tanto, esto hace que sea una guía útil en el análisis de productos terapéuticos potenciales. La combinación de medidas faciales y morfometría geométrica que aquí presentamos permite un análisis estadístico más exhaustivo del tamaño y forma de la región orofacial que los protocolos actuales que utilizan en gran medida sólo uno o el otro 15-18. Además, se presenta una forma sencilla de evaluar tanto el medial y planos laterales dela cara sin necesidad de equipos de imagen tridimensional sofisticada utilizada en los estudios actuales 13,19. Demostramos este protocolo en Xenopus laevis embriones tratados con un inhibidor del receptor del ácido retinoico que induce el desarrollo orofacial anormal y una mediana de 2,3 paladar hendido. La cuantificación de las dimensiones y la forma de la región orofacial en estos embriones han revelado cambios en el tercio medio facial que es análogo a los seres humanos con hendiduras palatinas similares y modelos de ratón 20,21. Sin embargo, este protocolo puede utilizarse para evaluar los efectos de otros compuestos sobre el desarrollo orofacial tales como sustancias naturales, herbicidas, o proteínas tales como factores de crecimiento. Además, el tamaño y forma cambia orofaciales derivados de la perturbación de la expresión génica a través de la pérdida o ganancia de experimentos de función (utilizando morfolinos antisentido o Crispers / Talens) también pueden ser cuantificados usando este protocolo. Por último, hemos desarrollado este método specificaLLY para evaluar la morfología de Xenopus; sin embargo, se puede modificar fácilmente para el análisis de cualquier vertebrado. Otras aplicaciones también podrían incluir el uso de este protocolo para la comparación de especies estrechamente relacionadas para los estudios evolutivos o ecológicos. Aunque el ejemplo que ofrecemos aquí utiliza este protocolo para describir el análisis de la región orofacial, que fácilmente podría ser modificado para el análisis de otras regiones, órganos o estructuras. Este protocolo cuantificación orofacial se convertirá en un recurso valioso para la comunidad de investigación, así como una excelente herramienta de enseñanza para los estudiantes de pregrado como una demostración en video.

<table><tbody><tr><td>Dissecting microscope</td><td>Zeiss</td><td></td><td>fitted with AxioCamICC1 camera</td></tr><tr><td>Dumont #5 Inox forceps</td><td>Fine Science Tools</td><td>11251-10</td><td></td></tr><tr><td>Sterile, disposable scalpel</td><td>Sklar</td><td>06-2015</td><td></td></tr><tr><td>24-well plate</td><td>Fisher Scientific</td><td>087721</td><td></td></tr><tr><td>Standard Disposable transfer pipettes</td><td>Fisher Scientific</td><td>13-711-7M</td><td></td></tr><tr><td>150 mm x 15 mm Petri dishes</td><td>Falcon</td><td>351058</td><td></td></tr><tr><td>Incubators</td><td>Ectotherm</td><td></td><td>set to 15 &amp;deg;C or 20 &amp;deg;C</td></tr><tr><td>Modeling Clay</td><td>Premo, or other non-toxic modeling clay</td><td></td><td>in black or white</td></tr><tr><td>Straight teasing needle</td><td>Thermo Scientific</td><td>19010</td><td></td></tr><tr><td>Capillary Tubing (for needles)</td><td>FHC</td><td>30-30-1</td><td>Borosil 1.0 mm OD x 0.5 mm ID/Fiber, 100 mm each</td></tr><tr><td>Needle Puller, Model P-97</td><td>Sutter Instrument Co.</td><td>Needle Puller: P-97 Flaming/ Bown micropipette puller Filament: FB300B</td><td>For filaments, use Sutter 3.00 mm square box filaments, 3.0 mm wide.</td></tr><tr><td>Pipettemen</td><td>Gilson</td><td>F144802, F123600, F123602</td><td></td></tr><tr><td>BMS-453</td><td>Tocris</td><td>3409</td><td></td></tr><tr><td>DMSO</td><td>American Bioanalytical</td><td>AB00435-01000</td><td></td></tr><tr><td>Cysteine</td><td>Sigma-Aldrich</td><td>52-90-4</td><td></td></tr><tr><td>Paraformaldehyde powder</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Petri dishes</td><td>Falcom</td><td>353003, 351058</td><td>100 mm diameter and 150 mm in diameter</td></tr><tr><td>100% Ethanol</td><td>VWR</td><td>89125-170</td><td></td></tr></tbody></table>

quantification of orofacial phenotypes in xenopus

Xenopus se ha convertido en una herramienta importante para la disección de los mecanismos que rigen el desarrollo y defectos craneofaciales. Un método para cuantificar el desarrollo orofacial permitirá análisis más riguroso de fenotipos orofaciales en derogación con sustancias que genéticamente o molecularmente pueden manipular la expresión génica o la función de proteínas. El uso de dos imágenes dimensionales de las cabezas de embriones, se miden las dimensiones tradicionales de tamaño-tales como orofacial anchura, altura y zona-. Además, una medida de la redondez de la abertura de la boca embrionario se utiliza para describir la forma de la boca. Morfometría geométrica de estas dos imágenes dimensionales también se realiza para proporcionar una visión más sofisticada de los cambios en la forma de la región orofacial. Puntos de referencia se asignan a puntos específicos en la región orofacial y coordenadas se crean. Un análisis de componentes principio se utiliza para reducir coordenadas señal a los componentes principales que discriminan entonces el tratamientogrupos. Estos resultados se muestran como un gráfico de dispersión en el que los individuos con formas orofaciales similares se agrupan. También es útil para llevar a cabo un análisis de función discriminante, que compara estadísticamente las posiciones de los puntos de referencia entre dos grupos de tratamiento. Este análisis se muestra en una rejilla de transformación donde los cambios en la posición de punto de interés se consideran como vectores. Una rejilla se superpone a estos vectores a fin de que se muestra un patrón de deformación para mostrar dónde posiciones hitos significativos han cambiado. Cambios en la forma en el análisis de la función discriminante se basan en una medida estadística, y por lo tanto pueden ser evaluados por un valor de p. Este análisis es sencillo y accesible, que sólo requiere un estereoscopio y software freeware, y por lo tanto será una investigación y la enseñanza valiosa de recursos.

Aquí, se demostró un análisis cuantitativo de tamaño y forma orofacial para comparar los embriones tratados con un inhibidor de receptor de ácido retinoico (RAR inhibidor) a los controles no tratados. Los embriones se trataron con una concentración 1 M de este inhibidor químico de la etapa 24 a 30 (26-35 HPF), eliminado, y se fijaron en la etapa 42 (82 HPF). Luego fueron procesados ​​y analizados como se describe en el protocolo. Los resultados son los datos originales, pero consistente con las observaciones ...

Watch this Scientific Journal Video about Quantification of Orofacial Phenotypes in Xenopus at JoVE.com

Quantification of Orofacial Phenotypes in Xenopus

Un método para cuantificar el tamaño orofacial y la forma de los embriones de Xenopus laevis ha sido desarrollado. En este protocolo, las mediciones de tamaño tradicionales se combinan con morfometría geométrica para permitir análisis más sofisticados de desarrollo y defectos orofacial.

La cuantificación de fenotipos en Orofacial<em&gt; Xenopus</em

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial ...

quantification-of-orofacial-phenotypes-in-xenopus

Research

JoVE Journal

Biology

Quantification of Orofacial Phenotypes in Xenopus

9.7K vistas.  Virginia Commonwealth University.  Un método para cuantificar el tamaño orofacial y la forma de los embriones de Xenopus laevis ha sido desarrollado. En este protocolo, las mediciones de tamaño tradicionales se combinan con morfometría geométrica para permitir análisis más sofisticados de desarrollo y defectos orofacial. 

Video: Quantification of Orofacial Phenotypes in Xenopus

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

La comprensión temprana Organogenesis Usando una simplificado<em&gt; In Situ</em&gt; Protocolo de hibridación en<em&gt; Xenopus</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Investigación

Biología del Desarrollo

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Visualización y análisis cuantitativo de la angiogénesis embrionaria<em&gt; Xenopus tropicalis</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

Microinyección de ADN en los oculares en embriones de Xenopus laevis e imágenes de gFP que expresan los árboles axonales ópticos en los renacuajos de Xenopus vivos

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Células vivas, imágenes y análisis cuantitativo de las células epiteliales embrionarias en Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Biología

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

La neurogénesis en la evaluación primaria<em&gt; Xenopus</em&gt; Los embriones Usando Inmunoticción

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

Neurociencias

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

Electroporación de mesénquima craneofacial

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as accessibility to numerous imaging approaches, electrophysiological techniques and behavioral assays. The Xenopus tadpole olfactory system is particularly well suited to investigate the function of synapses established during normal development or reformed after injury. Here, we describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae. We outline a combination of in vivo measurements of presynaptic calcium responses in glomeruli of the olfactory bulb with olfactory-guided behavior assays. Methods can be combined with the transection of olfactory nerves to study the rewiring of synaptic connectivity. Experiments are presented using both wild-type and genetically modified animals expressing GFP reporters in central nervous system cells. Application of the approaches described to genetically modified tadpoles can be useful for unraveling the molecular bases that define vertebrate behavior.

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system in vivo. We describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae in normal rearing conditions or after injury.

Evaluación funcional de las vías olfatorias en los renacuajos de Xenopus de vida

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Visualización de Desarrollo Craneofacial en los SOX10: Kaede pez cebra transgénico Línea Usando Time-lapse microscopía confocal

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

Los trasplantes faciales en<em&gt; Xenopus laevis</em&gt; Los embriones

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from harmful ultraviolet radiations. Perturbations in melanocyte functioning lead to pigmentary disorders such as piebaldism, albinism, vitiligo, melasma, and melanoma. Zebrafish is an excellent model system to understand melanocyte functions. The presence of conspicuous pigmented melanocytes, ease of genetic manipulation, and availability of transgenic fluorescent lines facilitate the study of pigmentation. This study employs the use of wild-type and transgenic zebrafish lines that drive green fluorescent protein (GFP) expression under mitfa and tyrp1 promoters that mark various stages of melanocytes. 
Morpholino-based silencing of candidate genes is achieved to evaluate the phenotypic outcome on larval pigmentation and is applicable to screen for regulators of pigmentation. This protocol demonstrates the method from microinjection to imaging and fluorescence-activated cell sorting (FACS)-based dissection of phenotypes using two candidate genes, carbonic anhydrase 14 (Ca14) and a histone variant (H2afv), to comprehensively assess the pigmentation outcome. Further, this protocol demonstrates segregating candidate genes into melanocyte specifiers and differentiators that selectively alter melanocyte numbers and melanin content per cell, respectively.

Regulators of melanocyte functions govern visible differences in the pigmentation outcome. Deciphering the molecular function of the candidate pigmentation gene poses a challenge. Herein, we demonstrate the use of a zebrafish model system to identify candidates and classify them into regulators of melanin content and melanocyte number.

Enfoque genético inverso para identificar reguladores de pigmentación usando pez cebra

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from ...

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