We are grateful to S. Arber for the mGFP mice and to members of the Zervas lab who critically read the paper and provided technical assistance. A. Brown was supported by a Brain Science Kaplan Summer Graduate Research Award. E. Normand was supported by a Brain Science Program Graduate Research Award. This research was also funded by startup research funds (MZ).

Tamoxifen Preparation and Oral Gavage Procedure (E8.5)
<ul>
 <li> Prepare a 20 mg/ml stock solution of tamoxifen by dissolving tamoxifen in pre-warmed corn oil. </li>
 <li> Incubate the solution at 37&deg; C for 2 hours on a nutator and vortex intermittently.</li>
 <li> Protect the tamoxifen stock from light, store at 4&deg;C, and use for up to one month. </li>
 <li> For fate mapping experiments, establish a breeding pair consisting of a Swiss Webster female (wildtype; purchased from Taconic) and a male carrying both a gene specific CreERT allele and a reporter allele. For demonstration purposes, we are using Wnt1-CreERT;mGFP males (Ellisor 2009).</li>
 <li> Check Swiss Webster females each morning for the appearance of a vaginal plug. Designate the morning (0900) of the day a vaginal plug is seen as 0.5 days post-coitus and calculate the date of tamoxifen administration based on this starting point. (For this experiment, embryonic day 8.5 was used). </li>
 <li> Use a 1 ml syringe with an animal feeding needle (20G x 1-1/2) to draw up 200 &mu;l of the tamoxifen stock solution (4 mg tamoxifen).</li>
 <li> Firmly restrain the time-pregnant Swiss Webster female by grasping the nape of the neck and back to immobilize the head and turn over so the ventral side is facing up.</li>
 <li> Hold the tail between free fingers to keep the body in a straight line. </li>
 <li> Place the feeding needle into the corner of the mouth and gently guide the needle along the roof of the mouth.</li>
 <li> Rotate the needle so that it is parallel to the body, while simultaneously tilting the head back to keep the neck straight.</li>
 <li> Guide the needle down the esophagus, toward the stomach. Be careful not to enter the trachea. If there is resistance on the needle, the animal's gag reflex engages, or if the mouse struggles, immediately remove the needle and try the gavage again.</li>
 <li> Once the feeding needle is in the stomach, administer the tamoxifen into the stomach and return the female to her home cage until the date of dissection. </li>
</ul>
Craniotomy:
<ul>
 <li> Intracardially perfuse the adult fate mapped mouse with 4% PFA and remove the head with scissors by cutting through the spinal column just above the shoulders.</li>
 <li> Run a scalpel along the dorsal midline of the head (rostral to caudal) to cut through the scalp and expose the scull.</li>
 <li> Using the scalpel, scrape away any excess tissue or muscle from along the side and posterior of the cranium.</li>
 <li> With forceps, puncture the skull at the midline just rostral to the olfactory bulbs and create a small hole to accommodate the tips of fine scissors.</li>
 <li> Insert the fine scissors into this hole and make an incision medial to lateral approximately the length of the olfactory bulbs. This cut will break the skull at the intersection of the nasal bone and frontal bone and provide good access for the scissors.</li>
 <li> Cut along the sagittal sutures in the skull (dorsal midline) making sure to keep the scissor tips angled away from the brain to avoid damaging the underlying tissue. </li>
 <li> Gently grasp the skull with forceps and peel away the bone along the medial incision to expose the brain. The skull may chip off in small pieces or break away in larger sections. Continue using the forceps to remove all of the frontal, parietal, interparietal, and occipital bones.</li>
 <li> Crack the bones encasing the paraflocculi (located along the lateral edges of the brain at the level of the cerebellum) by pinching the spherical bones on each side. Gently pull away the bones.</li>
 <li> Turn the head upside down (dorsal side down) and use the forceps to sever the cranial nerves and release the brain from the skull.</li>
</ul>
Whole Mount Microscopy: Adult Brain
<ul>
 <li>Assess adult brain for GFP marking using a fluorescent dissecting scope.</li>
 <li>Transfer brain to a Petri dish containing PBS and photograph using PictureFrame or other appropriate software.</li>
</ul>
Whole Mount Microscopy: E12.5 Embryos
<ul>
 <li>Assess whole mount embryos for GFP marking using a fluorescent dissecting scope. </li>
 <li>Transfer those that are GFP positive by whole mount to a Petri dish containing PBS and photograph using PictureFrame or other appropriate software. </li>
 <li>Use forceps to pinch off a small piece of tail from each embryo, place each piece in a PCR tube and genotype the tissue using PCR (Ellisor 2009) for both alleles to confirm results seen via whole mount analysis. </li>
</ul>
Micro-dissection and explant preparation of the ventral mesencephalon from E12.5 embryos
<ul>
 <li> Following dissection from the uterine chain and identification of fate-mapped embryos by whole-mount fluorescence, transfer embryos to ice-cold sterile PBS in a cell culture dish.</li>
 <li> Using fine scissors, remove the head portion of the embryo cutting transversely, caudal to rhombomere 1.</li>
 <li> Next, remove the rostral portion of the head with a coronal cut through the diencephalon. This will expose a tube-like structure in which the 4th ventricle through the mesencephalic vesicle form a conduit between dorsal and ventral tissues.</li>
 <li> Carefully insert the scissor tips into the 4th ventricle, and snip along the dorsal midline, caudal to rostral, fully opening the tube, creating an "open-book" preparation. The ventral mesencephalon will now be exposed medially, while the two dorsal halves of the mesencephalon will reside laterally.</li>
 <li> Remove any remaining non-neural tissue underneath the ventral mesencephalon in order for the tissue to lay more flatly. If need be, make notches in the rostral and caudal aspects such that the dorsal "wings" of the explant will lay flat as well. The explant should now resemble a "butterfly, in which the dorsal mesencephalon represents the wings, and rhombomere 1 the tail.</li>
 <li> To further isolate the ventral mesencephalon for FACS analysis or cell culture experiments, remove the lateral (dorsal aspects) of the tissue. </li>
</ul>
Representative Results:
In this GIFM experiment, Wnt1-expressing cells are permanently and heritably marked during embryogenesis by administering tamoxifen at E8.5 to Wnt1-CreERT;mGFP embryos. Subsequently, these marked cells are visualized via whole mount fluorescence and section immunohistochemistry to determine how Wnt1-derived cells contribute to neural structures across development. Whole mount fluorescence relies on the membrane bound GFP component of the mGFP reporter and therefore provides cellular information as well as an overarching view of Wnt1-derived neural projections. For example, with tamoxifen administration at E8.5, Wnt1-CreERTmGFP embryos at E12.5 exhibit GFP fluorescence primarily in the midbrain, posterior hindbrain and spinal cord (Figure 1A). At high magnification, fine neuronal projections are seen innervating the midbrain, body wall, limb, and craniofacial region. At this developmental stage, the embryo is translucent and internal GFP labeling is easily discerned. However, as the brain develops, the density of mature tissue obscures GFP fluorescence emanating from internal brain structures. Therefore, with tamoxifen administration at E8.5, only minor GFP labeling is observed in the superior colliculi of the adult by whole mount fluorescence (Figure 1C, inset). In addition, some axonal projections are seen coursing along the ventral surface of the hindbrain (not shown). In contrast, when tamoxifen is administered at E9.5, substantial GFP labeling occurs throughout the entire inferior colliculi (Figure 1B, inset). This increase in whole mount fluorescence may indicate that Wnt1-expressing cells at E9.5 contribute more significantly to superficial regions of the dorsal midbrain or extend more processes within this region than Wnt1 expressing cells from E8.5. Regardless, this difference in whole mount fluorescence reflects the dynamic nature of Wnt1 expression during development and demonstrates how GIFM can be used to follow distinct cell populations from the embryo into the mature adult.
With immunohistochemistry, fate mapped tissue sections from Wnt1-CreERT;mGFP animals are analyzed at the cellular level. Antibodies against GFP and &beta;-galactosidase (&beta;-gal) are used in conjunction with a variety of tissue specific biomarkers to determine the fine scale cellular contribution of the Wnt1 lineage to neural structures and cell types. With tamoxifen administration at E8.5 and tissue analysis at E12.5, double positive cells (GFP+ and &beta;-gal+) are observed throughout the midbrain and hindbrain with projections coursing through the ventral midbrain (Figure 1B). In the adult brain, the Wnt1-lineage marked at E8.5 gives rise to many midbrain structures including the superior and inferior colliculi as well as in the vicinity of dopaminergic cell populations of the ventral tegmental area and substantia nigra (Figure 1D) (see also Zervas 2004). In contrast, the Wnt1-lineage marked at E9.5 gives rise to inferior colliculi as well as to neurons in the vicinity of dopaminergic cell populations (Figure 1F).
<img src="/files/ftp_upload/1687/1687fig1.jpg" alt="Figure 1" /> Figure 1. Representative Results for Wnt1-fate mapping:
Examples of whole-mount and immunocytochemistry results in Wnt1-CreERT;mGFP embryos and adult brains fate mapped (marked) at E8.5 (A-D) and E9.5 (E-F). A. Wnt1-CreER;mGFP embryos at E12.5 exhibit GFP fluorescence primarily in the midbrain (Mb), posterior hindbrain (Hb) and spinal cord (Sp). B. Marked cells visualized and analyzed at the cellular level by immunohistochemistry as shown on this 1-&#956;m thick optical section (40x objective, z-series acquisition). Nuclear &beta;-galactosidase (n&beta;-gal) antibody labeling (red) and GFP antibody labeling (green) indicates fate-mapped cells shown in the ventral midbrain. Here, recombined cells are double positive (n&beta;-gal+/GFP+) because of the nature of the conditional mGFP reporter. C. Whole-mount florescence microscopy reveals faint GFP fluorescence in the superior colliculi (SC) of the adult (inset). Assessing the Wnt1 lineage marked at E8.5 on adult sections with low magnification (5X) microscopy reveals that the Wnt1 lineage gives rise to midbrain structures including the superior (SC) and inferior (IC) colliculi (C). D. A 40x, 1 mm optical section taken from the ventral midbrain in the vicinity of dopaminergic neurons with n&beta;-gal+ cells and a rich GFP+ axonal plexus. E. Marking at E9.5 results in substantial GFP labeling that is readily observed in the inferior colliculus of the midbrain by whole mount fluorescence (inset). The Wnt1 lineage marked at E9.5 on adult sections (&beta;-gal+, red) is concentrated in the inferior colliculus (dorsal-posterior midbrain) as seen with low magnification (5X) microscopy (E). F. A 40x, 1 mm optical section taken from the ventral midbrain in the vicinity of dopaminergic neurons with n&beta;-gal+ cells and a rich GFP+ axonal plexus. Wnt1-derived neurons marked at E8.5 versus E9.5 are progressively restricted from contributing to dorsal midbrain, while the Wnt1 lineage persists in contributing to ventral midbrain.

<ol>
	<li>Ellisor, D., Koveal, D., Hagan, N., Brown, A., Zervas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+conditional+reporter+alleles+in+the+developing+embryo+and+embryonic+nervous+system.">Comparative analysis of conditional reporter alleles in the developing embryo and embryonic nervous system.</a> Gene Expr Patterns. 9, 475-489 (2009).</li><li>Zervas, M., Millet, S., Ahn, S., Joyner, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+behaviors+and+genetic+lineages+of+the+mesencephalon+and+rhombomere+1.">Cell behaviors and genetic lineages of the mesencephalon and rhombomere 1.</a> Neuron. 43, 345-357 (2004).</li></ol>

All authors contributed equally to this work and are listed alphabetically. The contribution of each individual is apparent by his or her participation in the video article.

The GIFM system that we have demonstrated can be used to answer a wide range of questions in a variety of cellular processes. For example, mice engineered with different Cre drivers can be used to target any cell type, tissue or genetic lineage of interest. In addition, because tamoxifen can be administered at any embryonic or postnatal time point, recombination can be targeted to any relevant developmental stage. The spatial and temporal control of this system are ideal for investigating how cells expressing particular...

a practical approach to genetic inducible fate mapping: a visual guide to mark and track cells in vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

Watch this Scientific Journal Video about A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo at JoVE.com

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

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16.6K Views.  Brown University. Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

Video: A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Also, the recent deluge of research on the neurophysiology and neurobehavioral genetics of olfaction in Drosophila has motivated ever more technically sophisticated and quantitative behavioral assays. Here, we modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. A fly is glued to a small steel pin and suspended in a magnetic field that enables it to yaw freely. Small diameter food odor plumes are directed downward over the fly s head, eliciting stable tracking by a hungry fly. Here we focus on the critical mechanics of tethering, aligning the magnets, devising the odor plume, and confirming stable odor tracking.

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...