The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta–catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.
22 Related JoVE Articles!
In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin
Institutions: University of Notre Dame , Colorado State University , Wayne State University School of Medicine.
Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1
, spinal cord3
, optic nerve4
, sensory hair cells5
, and fins6
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 °C, this process occurs within six hours post-amputation (hpa, Figure 1B
. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D
. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E
. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt
, msx, retinoic acid, shh, notch, bmp, and activin-betaA
genes, is up-regulated during specific stages of fin regeneration9-16
. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13
, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12
. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus
, chick, and mouse development17-19
. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.
Developmental Biology, Issue 61, Electroporation, morpholino, zebrafish, fin, regeneration
Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development
Institutions: University of Cambridge, University of Cambridge.
Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis
oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro
matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.
Developmental Biology, Issue 96, Xenopus oocyte, oocyte maturation, Intracytoplasmic sperm injection, embryonic development, maternal factors, maternal depletion, micromanipulation, gene interference
Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization
Institutions: University of Notre Dame.
The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ
hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.
Developmental Biology, Issue 89, animals, vertebrates, fishes, zebrafish, growth and development, morphogenesis, embryonic and fetal development, organogenesis, natural science disciplines, embryo, whole mount in situ hybridization, flat mount, deyolking, imaging
Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Institutions: University of Notre Dame.
The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.
Cellular Biology, Issue 90,
zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis
Institutions: Center for Vision Research, SUNY Eye Institute, Upstate Medical University.
Measurement of the visual function in the tadpoles of the frog, Xenopus laevis
, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus
laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.
Neuroscience, Issue 88, eye, retina, vision, color preference, Xenopus laevis, behavior, light, guidance, visual assay
Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae
Institutions: Rensselaer Polytechnic Institute.
has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae
has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.
Microbiology, Issue 92, Aging, mutations, genome instability, Saccharomyces cerevisiae, fluctuation test, magnetic sorting, mother cell, replicative aging
Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq
Institutions: MRC National Institute for Medical Research.
The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and organ formation during development. Determining the locations and levels of such chromatin features in vivo
will provide valuable information about the spatio-temporal regulation of genomic elements, and will support aspirations to mimic embryonic tissue development in vitro
. The most commonly used method for genome-wide and high-resolution profiling is chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). This protocol outlines how yolk-rich embryos such as those of the frog Xenopus
can be processed for ChIP-Seq experiments, and it offers simple command lines for post-sequencing analysis. Because of the high efficiency with which the protocol extracts nuclei from formaldehyde-fixed tissue, the method allows easy upscaling to obtain enough ChIP material for genome-wide profiling. Our protocol has been used successfully to map various DNA-binding proteins such as transcription factors, signaling mediators, components of the transcription machinery, chromatin modifiers and post-translational histone modifications, and for this to be done at various stages of embryogenesis. Lastly, this protocol should be widely applicable to other model and non-model organisms as more and more genome assemblies become available.
Developmental Biology, Issue 96, Chromatin immunoprecipitation, next-generation sequencing, ChIP-Seq, developmental biology, Xenopus embryos, cross-linking, transcription factor, post-sequencing analysis, DNA occupancy, metagene, binding motif, GO term
Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons
Institutions: University of Iowa Carver College of Medicine, University of Iowa Carver College of Medicine, EZ BioResearch LLC.
High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g.,
in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (ΔE-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.
Neuroscience, Issue 95, AP2, genotyping, glial feeder layer, mouse tail, neuronal culture, nucleic-acid extraction, PCR, tattoo, torsinA
Quantification of Orofacial Phenotypes in Xenopus
Institutions: Virginia Commonwealth University.
has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial development will allow for more rigorous analysis of orofacial phenotypes upon abrogation with substances that can genetically or molecularly manipulate gene expression or protein function. Using two dimensional images of the embryonic heads, traditional size dimensions-such as orofacial width, height and area- are measured. In addition, a roundness measure of the embryonic mouth opening is used to describe the shape of the mouth. Geometric morphometrics of these two dimensional images is also performed to provide a more sophisticated view of changes in the shape of the orofacial region. Landmarks are assigned to specific points in the orofacial region and coordinates are created. A principle component analysis is used to reduce landmark coordinates to principle components that then discriminate the treatment groups. These results are displayed as a scatter plot in which individuals with similar orofacial shapes cluster together. It is also useful to perform a discriminant function analysis, which statistically compares the positions of the landmarks between two treatment groups. This analysis is displayed on a transformation grid where changes in landmark position are viewed as vectors. A grid is superimposed on these vectors so that a warping pattern is displayed to show where significant landmark positions have changed. Shape changes in the discriminant function analysis are based on a statistical measure, and therefore can be evaluated by a p-value. This analysis is simple and accessible, requiring only a stereoscope and freeware software, and thus will be a valuable research and teaching resource.
Developmental Biology, Issue 93, Orofacial quantification, geometric morphometrics, Xenopus, orofacial development, orofacial defects, shape changes, facial dimensions
Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples
Institutions: San Diego State University, DOE Joint Genome Institute, University of Colorado, University of Colorado.
The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.
Molecular Biology, Issue 94, virome, microbiome, metagenomics, metatranscriptomics, cystic fibrosis, mucosal-surface
Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus
Institutions: Hospital for Sick Children, University of Western Ontario, University of Western Ontario, Hospital for Sick Children, University of Western Ontario.
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.
Developmental Biology, Issue 95, Xenopus, organogenesis, in situ hybridization, RNA methods, embryology, imaging, whole mount
Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages
Institutions: University of Alabama at Birmingham, INRA UR1067, INRA UR1037.
Due to the inherent difficulty and time involved with studying the myogenic program in vivo
, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata,
however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.
teleost fish) and full regeneration following appendage loss (i.e.
urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio
), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae
. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum
) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4
Basic Protocol, Issue 86, myogenesis, zebrafish, myoblast, cell culture, giant danio, moustached danio, myotubes, proliferation, differentiation, Danioninae, axolotl
Single Cell Electroporation in vivo within the Intact Developing Brain
Institutions: University of British Columbia - UBC, University of British Columbia - UBC.
Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.
Neuroscience, Issue 17, electroporation, gene delivery, transfection, fluorescence labeling, neuronal imaging, micropipette
Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species
Institutions: Uppsala University, Swedish University of Agricultural Sciences.
The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ
hybridization, a technique used to localize cell specific mRNA expression. The in situ
hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ
experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies
). Here we present a modified DIG in situ
hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies
. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana
and Brassica napus
. The protocol worked equally well for the species and genes studied. AtAP3
were observed in second and third whorl floral organs in A. thaliana
and B. napus
and DAL13 in microsporophylls of male cones from P. abies
. For P. abies
the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ
protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundström at Jens.Sundstrom@vbsg.slu.se
Plant Biology, Issue 26, RNA, expression analysis, Norway spruce, Arabidopsis, rapeseed, conifers
Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization
Institutions: Yale University.
Whole mount in situ
hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ
hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ
hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2
. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3
. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4
. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila
indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5
. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.
Developmental Biology, Issue 25, zebrafish, tyramide signal amplification, in situ hybridization, nuclear labeling
Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica
Institutions: University of Minnesota Medical School.
Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1
. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10
, represent an important system for studying evolution of the central nervous system11, 12
and have behavioral screening potential13, 14.
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo
RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15
. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21
. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo
RNAi by feeding) using the planarian Dugesia japonica
Developmental Biology, Issue 54, Stem Cells, Regeneration, Planarian, Flatworm, Dugesia japonica
Electroporation of Craniofacial Mesenchyme
Institutions: King's College London.
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.
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 (˜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
Developmental Biology, Issue 57, craniofacial, electroporation, Xenopus laevis, frog, cartilage, mesenchyme
Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure
Institutions: Children's Memorial Research Center, Northwestern University.
Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2
. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8
. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.
Medicine, Issue 61, Zebrafish, fetal alcohol exposure, Danio rerio, development, mRNA expression, morpholino, ethanol exposure
RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells
Institutions: Children's Mercy Hospital and Clinics, School of Medicine, University of Missouri-Kansas City.
The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g.
drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2
. RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4
in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.
Genetics, Issue 72, Molecular Biology, Immunology, Medicine, Genomics, Proteins, RNA-seq, Next Generation DNA Sequencing, Transcriptome, Transcription, Thrombin, Endothelial cells, high-throughput, DNA, genomic DNA, RT-PCR, PCR
Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture
Institutions: Max von Pettenkofer Institute, University of Cambridge, Ludwig-Maximilians-University Munich.
The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e.
total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila
, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.
Genetics, Issue 78, Cellular Biology, Molecular Biology, Microbiology, Biochemistry, Eukaryota, Investigative Techniques, Biological Phenomena, Gene expression profiling, RNA synthesis, RNA processing, RNA decay, 4-thiouridine, 4sU-tagging, microarray analysis, RNA-seq, RNA, DNA, PCR, sequencing
Isolation, Culture, and Transplantation of Muscle Satellite Cells
Institutions: University of Minnesota Medical School.
Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.
However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo
expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.
Cellular Biology, Issue 86, skeletal muscle, muscle stem cell, satellite cell, regeneration, myoblast transplantation, muscular dystrophy, self-renewal, differentiation, myogenesis
Isolation of Genomic DNA from Mouse Tails
Institutions: University of California, Irvine (UCI).
Basic Protocols, Issue 6, genomic, DNA, genotyping, mouse