RSF has received funding from the European Union's - Seventh Framework Programme (FP7/2007-2013]) under grant agreement n&#176; 618432 &#8211; MC&#8212;Epi-Patho-Stem, as well as by the Israeli Science Foundation (218/14).

Ethics Statement: In this study animal care and use were conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All experiments were approved by the local ethical committee.
1. Choose a Suitable Cre-recombinase Expressing Mouse Strain
<ol>
	<li>Out of the great variety of Cre transgenic lines available for purchase26 and depending on the targeted tissue to be investigated, choose a Cre transgenic mouse strain in which the Cre recombinase gene is controlled by a promoter that is specific for the tissue of interest. In case temporal control is needed, choose an inducible Cre line. 
	Note: We chose the tamoxifen inducible K14-CreERT transgene to specifically label limbal and corneal epithelial stem/progenitor cells24. Note that there may be differences between strains, as Di Girolamo and coworkers reported limbal specificity with no corneal expression for their K14-CreERT22. An advantage to using inducible transgenic Cre mice is that it allows the induction of lineage tracing at versatile time frames.</li>
</ol>
2. Choose a Suitable Br Mouse Strain
<ol>
	<li>Choose the appropriate Br cassette depending on the research question and available Cre line16,18,20.
	<ol>
		<li>If a non-inducible Cre mouse strain is used, choose a Br cassette that does not allow continuous DNA inversions/excisions and thus give rise to dead-end products (for example Br1.0 or Br1.1)16.</li>
	</ol>
	</li>
</ol>
3. Cross the Transgenic Lines of Interest
<ol>
	<li>For the protocol shown here, cross the homozygous R26R-Confetti strain (Br2.1/Br2.1) with the homozygous CreERT strain (CreERT/CreERT) to obtain hemizygous offsprings (Br2.1/+;Cre/+)that are ready for mono-allelic lineage tracing as they all contain a single Br2.1 allele and a single Cre allele. 
	Note: To increase the efficiency of labeling, one could generate mice that contain two Cre alleles (Br2.1/+; Cre/Cre), while two Br2.1 alleles (Br2.1/Br2.1; Cre/+) would increase color combinations (as detailed in 22).</li>
</ol>
4. Cre-recombinase Induction
Note: The administration of Tamoxifen in these experiments was carried out by daily intraperitoneal injection, for the duration of 3-4 days, in order to induce the translocation of CreERT recombinase into the nucleus of limbal and corneal basal K14-positive stem/progenitor epithelial cells. The following protocol can be used for the injection of four mice. Alternatively, topical application of Tamoxifen to the ocular surface yields higher efficiency. The Tamoxifen solution can be kept in the dark at 4 &#176;C for up to one week. Warm the solution before use.
<ol>
	<li>Weigh 100 mg of Tamoxifen and dissolve it in 5 ml of corn oil to form a 20 mg/ml tamoxifen solution.</li>
	<li>Inject 200 &#181;l of Tamoxifen (4 mg) solution intraperitoneally to 8 weeks old mice for 3-4 consecutive days. 
	Note: The Tamoxifen solution can be kept in the dark at 4 &#176;C for up to one week. Warm the solution before use.</li>
	<li>For topical application: weigh 60 mg of Tamoxifen and dissolve it in 1 ml of corn oil to form a 60 mg/ml tamoxifen solution. Vortex and set the solution in a shaking water bath maintained at 65 &#176;C until it becomes clear leave it in the bath until use.&#160;&#160;
	<ol>
		<li>Carefully apply 100 &#956;l of the Tamoxifen solution to each ocular surface of the mouse.</li>
	</ol>
	</li>
</ol>
5. Tissue Preparation for Imaging
<ol>
	<li>Sacrifice animals using isoflurane (2% vaporized in oxygen) anesthesia (confirm proper anesthetization by the lack of reflexes) followed by CO2 inhalation at appropriate time points following Tamoxifen injection and collect the relevant tissue.
	<ol>
		<li>For these experiments, enucleate eyes at the following time points: 1, 4, 8, 12, and 16 weeks after Tamoxifen injection. Achieve enucleation by using curved forceps. Press the eyelids causing the eyeball to pop out of the eye socket. Place the forceps behind the eyeball holding the optic nerve. Gently pull the eyeball and detach it. 
		Note: At this stage, as described below, tissues can be prepared for wholemount analysis of labeled cells. Regular frozen tissue sections can also be prepared, fixed in PFA and imaged as described below (see section 6 and 22).</li>
	</ol>
	</li>
	<li>Place each eye in a 60 mm dish filled with PBS.</li>
	<li>Under a stereo-microscope using forceps hold the eye near the optic nerve with the cornea facing down and use a scalpel in order to remove the optic nerve, muscle and connective tissue and make a small incision in the posterior part of the eye.</li>
	<li>Using spring scissors carefully enlarge the cut letting the lens pop out and remove the iris using forceps.</li>
	<li>Flatten the cornea by making 4-5 radial incisions starting from the center towards the periphery using a scalpel.</li>
	<li>Use a scalpel to cut excess peripheral tissue beyond the limbus.</li>
	<li>Fix corneas in PFA 4% for 10 min, then briefly wash them with PBS.</li>
	<li>Incubate samples with DAPI for 10 min and briefly wash with PBS.</li>
	<li>Mount corneas on glass slides with the epithelial side facing up. Place a drop of mounting media on top of the cornea and cover with a cover-glass. Let slides dry O/N in RT. Keep slides at 4 &#176;C.</li>
</ol>
6. Confocal Microscopy and Imaging Analysis
<ol>
	<li>Use a spectral confocal system which enables separation of YFP/GFP/CFP emissions. Set fluorescence excitation and emission wavelengths depending on the fluorescent proteins of the Br cassette. Choose the most suitable excitation wavelength for each protein aiming at minimizing cross excitation. Choose narrow emission ranges in order to minimize the leak of signals from the different proteins.
	<ol>
		<li>For the Br2.1 cassette, use the following set up of fluorescence excitation (ex) and emission (em) as a starting point: DAPI &#8212; ex 405 nm/em 420&#8211;480 nm, CFP &#8212; ex 458 nm/em 470&#8211;500 nm, GFP &#8212; ex 488 nm/em 497&#8211;508 nm, YFP &#8212; ex 514 nm/em 529&#8211;540 nm, and RFP &#8212; ex 561 nm/em 575&#8211;615 nm.</li>
	</ol>
	</li>
	<li>In order to visualize large tissue areas with high resolution, acquire tile imaging. For the results shown in Figure 2A-B, image the entire cornea by obtaining an array of 12 x 12 images, i.e., acquire 144 fields (512 x 512) using 4 fluorescent channels in each field. Altogether collect 576 images and then merge.</li>
	<li>To explore the localization of tagged cells in different depths and structures of the tissue, acquire Z-stack images of different regions of interest. In order to image the full depth of the cornea, acquire 8 optical sections at 3 &#181;m intervals. Orthogonal views of the sections collected as well as image projections based on maximum intensities can be constructed using conventional imaging softwares 24.</li>
</ol>
7. Corneal Injury Combined with Tamoxifen Induction
<ol>
	<li>Anesthetize mice with isoflurane (2% vaporized in oxygen) and administer analgesics (10 mg/kg Carpofen, injected subcutaneously). Confirm proper anesthetization by unresponsiveness to toe-pinch.</li>
	<li>Weigh 1 g of Tamoxifen powder and dissolve it in 5 ml of sterile DMSO to produce 200 mg/ml concentration solution.</li>
	<li>Warm the Tamoxifen solution to 65 &#176;C in a water bath until the solution becomes clear. Keep solution in the bath until use.</li>
	<li>Apply eye ointment to the untreated contralateral eye to prevent dehydration during anesthesia.&#160;Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return mice that had undergone eye wounding to the company of other mice until fully recovered. Mice should be closely monitored following the injury. In case they lose more than 10% of their body weight, suffer from corneal erosion,&#160;or discomfort,&#160;the experiment should be stopped.&#160;Repeat analgesics administration every 24 hr throughout the whole experiment.&#160;</li>
	<li>Using a sterile syringe, without a needle attached to it; apply 200 &#181;l Tamoxifen solution topically over the whole corneal surface of one eye of each mouse. 
	Note: The liquid solution solidifies quickly on the eye surface at RT.&#160;The effect of corneal injury depends on the severity of the wound. Following one application of Tamoxifen/DMSO solution no adverse effects are observed in the mice.&#160; In case of repeated applications corneal opacity can be observed.&#160;&#160;</li>
	<li>Continue with tissue preparation and imaging (see sections 5 and 6) at relevant time points. For the results shown here, sacrifice the mice (as detailed in stage 5.1) one week post wounding (Figure 2 and 24).</li>
</ol>

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The authors have nothing to disclose.

Lineage tracing experiments have been conducted for many years. The recent technological improvements and the emergence of the Confetti mouse strains opened new avenues for research. Today, researchers can benefit from a large number of commercially available tissue-specific Cre lines, knockout mice and transgenic mice. This provides an opportunity for designing versatile experimental systems for addressing scientific questions with basic expertise, avoiding laborious practice, while providing robust and reliable data.
R26R-Confetti mice are an elegant tool for efficient tracking and studying the nature and behavior of cells in a living organism. The system is easy to establish and it allows non-invasive lineage tracing of single cells during embryogenesis, stem cell regeneration under homeostasis, or under different circumstances such as injury, inflammation and cancer. The obtainable data provides a robust description of the survival of targeted cells, clonal cell expansion and cell migration, in a quantifiable manner. A critical step would be to choose the appropriate genetic mouse strain that can reliably permit efficient tissue specific labeling at a precise developmental stage. One limitation of this system is that for monitoring a living organism, the tissue must be accessible and transparent. Similarly, tracking a thick tissue within a living animal may be only facilitated by two-photon or multi-photon microscopy. However, cell tracking is possible on postmortem tissue sections and perhaps the intact tissue may be studied after it has been transformed to become transparent by the CLARITY method32,33. Another limitation to be considered is that although adjacent cells that express the same fluorophore likely belong to the same clonal lineage, it is also possible that they may be derived from independent cell origin that randomly expressed the same fluorescent gene. This possibility becomes very unlikely when using multiple Br alleles to allow numerous color combinations.
In the future, combining the Confetti system with available genetic tools (i.e., knockout, knockin and transgenic mouse models) will permit the study of genes and their mutations in health and pathology. Likewise, lineage tracing under different system perturbations (i.e., stem cell niche destruction, laser mediated cell ablation, drug treatment) will bring new insights onto the involvement of signaling pathways in cell fate decisions. Ultimately, the combinatorial use of fluorescent and bioluminescence labeling, genetic reporters of signaling pathways and cutting edge microscopy will provide researchers a robust system to learn how single cells respond to their surrounding in their native environment.

During embryonic development, tissue and organ morphogenesis take place through a precise program that can be described in terms of cell proliferation, cell migration and lineage specification1-4. These biological processes are also involved in maintaining adult tissue homeostasis, which requires stem cell regeneration. Lineage tracing, the identification and tracking of the progeny of a specific type of cell population, provides an important tool for studying embryogenesis and stem cell homeostasis. Indeed, it is increasingly being used in many fields of research. Particularly, lineage tracing became an essential tool in identifying the origin and fate of stem cells in homeostasis and cancer development. This is because it may provide essential information about how the cell behaves in the context of the intact tissue or organism, with minimal experimental intervention, in contrast to cell isolation and in&#160;vitro culture or transplantation that may result in unwanted bias.
Traditionally, dyes such as lipid-soluble carbocyanine, which incorporate into the plasma membrane of cells, were used for lineage tracing. Although these types of experiments have successfully led to major discoveries and shaped seminal concepts in developmental biology5,6, they have some limitations, including the difficulty to control the specificity of targeted cells, the unwanted impact of the dye on cell behavior and the dilution of the label over time. Nucleotide analogue labeling approaches have enabled documenting the existence of slow-cycling &#8220;label retaining cells&#8221;&#160;that presumably correspond to quiescent stem cells7,8. However, while this technique could demonstrate the presence of slow cycling cells based on proliferation kinetics over a limited time period it could not provide substantial insights regarding the functionality of stem cells. An optimal lineage tracing methodology should minimize the effect of labeling on the properties of the marked cell, its progeny, or its neighbors. Additionally, it would be beneficial to have control on the labeling induction, namely, to have the ability to tag the cell population of interest at a stage and time dependent manner as well as in a single cell (clonal) manner. A stable and irreversible labeling method that is passed on to all progeny of the founder cell is important so that the label would be retained over time, and not transferred to neighboring cells.
More recently, genetic approaches of cell labeling were developed. In these systems, cell specific promoters can be used to trigger Cre recombinase expression in order to remove a loxP-flanked roadblock and allow the expression of reporter genes such as green fluorescent protein or &#914;-galactosidase reporter genes9-13. This approach enables not only the tracking of cells and their progeny but also permits cell isolation and molecular characterization. Cell tracking at the single cell level became possible by induction of the expression of a single fluorescent reporter gene. One important limitation of this system is the fact that only when very low percentage of cells is labeled it is possible to separate and trace individual clones. To overcome this limitation multicolor reporters can be used. When using multicolor labeling, the tracking of differential fate of neighboring cells in the same niche can be achieved efficiently14-17. Recently, a variety of Brainbow (Br) alleles were developed for lineage tracing experiments, enabling a stochastic expression of multiple fluorescent reporter genes utilizing the Cre/lox system. The Cre/lox system consists of the Cre recombinase enzyme, which recognizes and binds to specific DNA sequences such as loxP sites. Following the binding of Cre recombinase, site specific recombination occurs, which causes removal of loxP flanked sequences resulting in random expression of different fluorescent proteins (the mechanism is described below). A variety of Br lines are available for use (see reviews16,18,19). The major differences between the Br strains include (i) differences in the number of fluorescent genes included in the cassette, ranging from 2 (Br 2.0), 3 (Br1.0) to 4 (Br1.1, Br2.1) fluorescent genes, (ii) the presence of reciprocally orientated lox sites to allow gene inversion (Br2.0-2.1), (iii) the type of lox sites used, and (iv) the expression or silence of fluorescent gene expression prior to Cre activation. The recently established Br3 offers an improved method for multicolor imaging of neurons. One of the main features of this transcript is that it contains a new set of photostable farnesylated fluorescent proteins, which enable an even staining of the finest neuronal processes20.
Importantly, different versions of Br cassettes may contain the neuronal specific Thy1 promoter or the ubiquitously expressed CAG (CMV) promoter. Finally, while some of the Br transgenic mice may contain a single Br transgene, others may consist of multiple transgene copies, thereby allowing the production of an immense number of possibilities for color combinations to be expressed in each cell (for example see 16).
In our study, we used the R26R-Confetti mouse which contains the Br 2.1 cassette21. Br2.1 is uniquely designed to allow Cre-mediated DNA inversions and not only excisions because of the reciprocal orientation of the loxP sites (Figure 1). Thus, these inversion/excision events which lead to color change can continue as long as Cre is present16. For that reason, an inducible temporal Cre expression system is essential for reliable cell tracking using Br2.1. As shown in Figure. 1, the Br2.1 contains a ubiquitous CAG promoter followed by loxP-flanked NeoR-cassette, which acts as a transcriptional roadblock, that is followed by 4 fluorescent reporter genes, encoding for green (GFP), yellow (YFP), red (RFP) and cyan (CFP) fluorescent proteins, positioned in two tandems. No fluorescence is expressed prior to Cre activation. Induction of Cre recombinase causes the removal of the Neo-R cassette enabling the random expression of one of the 4 fluorescent proteins in a given cell. The first segment contains loxP-flanked GFP and a reversed YFP, while the second segment contains loxP-flanked RFP and a reversed CFP. These DNA segments may continuously be inverted (using loxP that is in reversed orientation), or excised, as long as Cre recombinase is present. Therefore, a transient and controlled Cre transgene should be used. The Br2.1 cassette provides an excellent system for distinguishing between overlapping emissions upon signal location: the expression of YFP and RFP is cytoplasmic, the GFP is nuclear and the CFP is bound to the cell membrane. GFP and RFP emissions are easily separated by conventional fluorescent microscopy. In order to separate YFP/GFP/CFP emissions, a spectral confocal imaging system is needed. Altogether, the Br2.1 cassette allows the random, inducible, and tissue specific expression of one out of four distinct fluorescent genes in each targeted cell. In fact, when a larger number of color combinations is needed, one can generate homozygous mice that contain 2 alleles of Br2.1, thus resulting in the expression of 2 color tags for each cell and in raising the number of possible color combinations to 10 22. Some of the Br mouse strains enable the expression of a much greater number of possible color combinations since multiple copies of the cassette were integrated into their genome16. Yet, in most cases, the four color combinations provided by a single Br2.1 allele are sufficient. Following the protocol provided here, one can establish this method using a relatively simple setup of spectral microscopy with little if any need for calibration.
Here we provide an adaptable protocol for the use of the R26R-Confetti mice that contain a single Br2.1 allele, for lineage tracing experiments of the corneal epithelium. This transgenic mouse strain has been used for studying the origin and fate of colon 21,23 and corneal epithelial stem cells22,24, the presence of&#160;stem cell&#160;activity&#160; in &#160;intestinal cancer25, and for characterizing kidney podocyte in focal segmental glomerulosclerosis (FSGS)17.

<table><tbody><tr><td>R26R-Confetti transgenic mice homozygous for the Brainbow 2.1 cassette</td><td>Jackson</td><td>strain #013731</td><td></td></tr><tr><td>K14-Cre&lt;sup&gt;ERT&lt;/sup&gt; mice</td><td>Jackson</td><td>strain #005107</td><td></td></tr><tr><td>tamoxifen</td><td>Sigma</td><td>T5648</td><td></td></tr><tr><td>isoflurane USP Terrell</td><td>Piramal Critical Care</td><td></td><td></td></tr><tr><td>Carprieve 30</td><td>b.wt</td><td></td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542</td><td>used at concentration of 4&amp;#160;&amp;micro;g/ml</td></tr><tr><td>Immu Mount</td><td>Thermo Scientific</td><td>9990402</td><td></td></tr><tr><td>LSM 510 Meta laser scanning confocal system hooked to an inverted motorized microscope (Axiovert 200M) equipped with a motorized stage</td><td>Zeiss</td><td></td><td>The spectral overlap between GFP, YFP and CFG requires the use of spectral imaging which allows the separation of the emission spectra of the fluorescent proteins. Here we use the Zeiss LSM 510 Meta spectral confocal microscope. The Meta detector included in this system has 32 detector elements each collecting emission from a 10-nanometer waveband. This technique termed &quot;spectral fingerprinting&quot; enables the user to set the most adequate limits for waveband filtering. There is a variety of spectral confocal systems that can be used, including Leica SP5(ref #21), SP8, Zeiss LSM 780 (ref #22).</td></tr><tr><td>Zen 2009 Imaging Software</td><td>Zeiss</td><td></td><td></td></tr><tr><td>Duratears-eye ointment</td><td>Alcon</td><td></td><td></td></tr></tbody></table>

a method for lineage tracing of corneal cells using multi-color fluorescent reporter mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

Recently, we aimed at identifying the origin, survival and migration of stem cells and progenitor cells of the corneal epithelium24. Accumulating evidence supports the dogma that the corneal epithelium is regenerated by stem cells located exclusively in the limbal niche, a ring shaped zone at the corneal periphery. This theory is supported by in vitro and in vivo data, and by clinical evidence that provided the basis for successful pioneering limbal stem cell therapy27-29. However, this topic remained highly debated in the recent years due to a study based on limbal grafting experiments that suggested that the mouse limbus does not participate in corneal renewal30. To test this interesting hypothesis, lineage tracing experiments using R26R-Confetti mice were performed, to follow the fate of limbal and corneal epithelial stem/progenitor cells under steady-state conditions for up to 120 days24. Eyes were enucleated and flat-mount corneas were analyzed by spectral confocal laser scanning fluorescence microscopy 10 and 120 days post Tamoxifen injection. As expected, small sporadic clusters of fluorescent cells were observed throughout the ocular surface 10 days after injection, while 1-4 months later (Figure 2B and 24), elongated streaks that extend from the limbus towards the corneal center slowly developed. This result suggests that the cornea is regenerated by cells that migrate from the limbus toward the center of the cornea. Next, corneal regeneration under wounding conditions was examined. In order to test the fate of K14-positive limbal/corneal cells under wounding conditions while inducing efficient cell tracking, we dissolved the Tamoxifen in dimethyl sulfoxide (DMSO), which causes corneal injury upon topical application. This way we managed to induce mild wounding if a single topical application was applied (Figure 2C), moderate wounding if additional DMSO alone treatment was applied in the day before Tamoxifen induction or severe wound when DMSO alone was topically applied in 2 sequential days prior to Tamoxifen induction.
In contrast to the thin limbal stripes that slowly developed and reached the corneal center within 4 months under steady-state conditions, cell migration following injury was rapid. Remarkably, long and thick limbal stripes were already developed under these conditions within 7 days. Interestingly, color diversity was sometimes minimal (Figure 2D). In our hands, occasionally, mainly red cells were identified in the whole cornea, indicating that color diversity can be biased towards specific fluorophores. This unwanted bias can be calibrated by dose response tests as reported previously in Br1.0L31 and in Br2.121.
<img alt="Figure 1" src="/files/ftp_upload/53370/53370fig1.jpg" /> 
Figure 1. Schematic illustration of the R26R-Confetti construct. (A) The R26R-confetti construct contains a CAG promoter for high expression levels, a loxP (black triangle)-flanked NeoR-roadblock and the Brainbow2.1 cassette at the Rosa26 locus16,21. The Brainbow2.1 cassette includes two head-to-tail loxP-flanked dimers. One dimer consists of nuclear-localized green fluorescent protein (GFP) and a reverse-oriented cytoplasmic yellow fluorescent protein (YFP, the inversed orientation is designated - PFY). The second dimer contains cytoplasmic RFP and a reverse-oriented membrane-tethered cyan fluorescent protein (CFP, the inversed orientation is designated - PFC)16. (B-E) Upon Cre-recombinase activation, different excision and inversion events may occur; possible outcomes are exemplified in B-E. The Cre activity induces stochastic rearrangement of the cassette leading to gene removals and/or inversions and thus results in the random expression of one of the four fluorescent proteins. <a href="https://www.jove.com/files/ftp_upload/53370/53370fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53370/53370fig2.jpg" /> 
Figure 2. Corneal lineage tracing under homeostasis and injury. R26R-confetti/K14-Cre mice were injected with 4 mg Tamoxifen as detailed in the protocol. In the indicated time points post induction flattened corneas were imaged (A-B). Corneal wounding coupled with Cre induction was achieved by topical application of Tamoxifen dissolved in DMSO (C-D). Mosaic images obtained by multichannel tiling spectral confocal microscopy are shown (A-D). In homeostasis, radial limbal stripes of slow migrating cells extended between the limbus and the corneal center. These stripes developed slowly, reaching the corneal center within 4-5 months (B). Clusters of corneal cells were evident as well (white arrows, B). In contrast, large limbal streaks appeared within a single week under wounding conditions (C). An example for bias of color diversity towards red cells (D). The limbal-corneal border is annotated by a dashed line. Scale bar is 500 &#181;m. Abbreviations: CFP, cyan fluorescent protein; GFP, green fluorescent protein; RFP, red fluorescent protein; YFP, yellow fluorescent protein. This figure has been modified from 24. <a href="https://www.jove.com/files/ftp_upload/53370/53370fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice at JoVE.com

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

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JoVE Journal

Developmental Biology

17.3K Views.  Israel Institute of Technology. In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

Video: A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. Patterning of the Meckel&#39;s cartilage, a first pharyngeal arch derivative, involves the migration of cranial neural crest (CNC) cells and the progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described CNC migration during lower jaw&#160;morphogenesis, but the details of how the chondrocytes achieve organization in the growth and extension of Meckel&#8217;s cartilage remains unclear. The sox10 restricted and chemically induced Cre recombinase-mediated recombination generates permutations of distinct fluorescent proteins (RFP, YFP and CFP), thereby creating a multi-spectral labeling of progenitor cells and their progeny, reflecting distinct clonal populations. Using confocal time-lapse photography, it is possible to observe the chondrocytes behavior during the development of the zebrafish Meckel&#8217;s cartilage.
Multispectral cell labeling enables scientists to demonstrate extension of the Meckel&#8217;s chondrocytes.&#160;During extension phase of the Meckel&#8217;s cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single-cell layered row. Failure of this organized intercalating process to mediate cell extension provides the cellular mechanistic explanation for hypoplastic mandible that we observe in mandibular malformations.

Cell organization of craniofacial bones has long been hypothesized but never directly visualized. Multi-spectral cell labeling and in vivo live imaging allows visualization of dynamic cell behavior in zebrafish lower jaw. Here, we detail the protocol to manipulate Zebrabow transgenic fish and directly observe cell intercalation and morphological changes of chondrocytes in the Meckel&#8217;s cartilage.

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Neuroscience

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its longevity can be a powerful way to reveal mechanisms of tissue development and maintenance. One of the most important tools currently available to monitor cells in vivo is the Confetti mouse model. The Confetti model can be used to genetically label individual cells in living mice with various fluorescent proteins in a cell type-specific manner and monitor their fate, as well as the fate of their progeny over time, in a process called clonal genetic tracing or clonal lineage tracing. This model was generated almost a decade ago and has contributed to an improved understanding of many biological processes, particularly related to stem cell biology, development, and renewal of adult tissues. However, preserving the fluorescent signal until image collection and simultaneous capturing of various fluorescent signals is technically challenging, particularly for mineralized tissue. This publication describes a step-by-step protocol for using the Confetti model to analyze growth plate cartilage that can be applied to any mineralized or nonmineralized tissue.

This method describes the use of the R26R-Confetti (Confetti) mouse model to study mineralized tissues, covering all steps from the breeding strategy to the image acquirements. Included is a general protocol that can be applied to all soft tissues and a modified protocol that can be applied to mineralized tissues.

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its ...

Clonal Analysis of the Neonatal Mouse Heart using Nearest Neighbor Modeling

By replacing lost or dysfunctional myocardium, tissue regeneration is a promising approach to treat heart failure. However, the challenge of detecting bona fide heart regeneration limits the validation of potential regenerative factors. One method to detect new cardiomyocytes is multicolor lineage tracing with clonal analysis. Clonal analysis experiments can be difficult to undertake, because labeling conditions that are too sparse lack sensitivity for rare events such as cardiomyocyte proliferation, and diffuse labeling limits the ability to resolve clones. Presented here is a protocol to undertake clonal analysis of the neonatal mouse heart by using statistical modeling of nearest neighbor distributions to resolve cardiomyocyte clones. This approach enables resolution of clones over a range of labeling conditions and provides a robust analytical approach for quantifying cardiomyocyte proliferation and regeneration. This protocol can be adapted to other tissues and can be broadly used to study tissue regeneration.

Presented here is a protocol for using multicolor lineage tracing and nearest-neighbor modeling to identify clonally derived cardiomyocytes during growth and regeneration in mice. This approach is objective, works across different labeling conditions, and can be adapted to incorporate a variety of image analysis pipelines.

By replacing lost or dysfunctional myocardium, tissue regeneration is a promising approach to treat heart failure. However, the challenge of detecting ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Biology

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Bioengineering

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...

Corneal Sensory Fiber Damage Protocols for Axon Regeneration Studies

Three Strategies to Induce Neurotrophic Keratitis and Nerve Regeneration in Murine Cornea

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation provides sensation and trophic function to the eye and contributes to preserving corneal integrity. The pathological disruption of this innervation is termed neurotrophic keratitis. This can be triggered by injury to the eye, surgery, or disease. In this study, we propose three different protocols for inflicting damage on the innervation in ways that recapitulate the three types of cases generally encountered in the clinic.
The first method consists in making an abrasion of the epithelium with an ophthalmic burr. This involves the removal of the epithelial layer, the free nerve endings, and the subbasal plexus in a manner similar to the photorefractive keratectomy surgery performed in the clinic. The second method&#160;only targets the innervation by sectioning it at the periphery with a biopsy punch, maintaining the integrity of the epithelium. This method is similar to the first steps of lamellar keratoplasty and leads to a degeneration of the innervation followed by regrowth of the axons in the central cornea. The last method damages the innervation of a transgenic mouse model using a multiphoton microscope, which specifically localizes the site of cauterization of the fluorescent nerve fibers. This method inflicts the same damage as photokeratitis, an overexposure to UV light.
This study describes different options for investigating the physiopathology of corneal innervation, particularly the degeneration and regeneration of the axons. Promoting regeneration is crucial for avoiding such complications as epithelium defects or even perforation of the cornea. The proposed models can help test new pharmacological molecules or gene therapy that enhance nerve regeneration and limit disease progression.

Author Spotlight: Advancing Corneal Innervation Research Through Innovative Models 

Here, we propose three different methods of damaging the sensory fibres innervating the cornea. These methods facilitate the study of axon regeneration in mice. These three methods, which are adaptable to other animal models, are ideal for the study of corneal innervation physiology and regeneration.

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation ...

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying cellular and molecular mechanisms regulating lineage transitions of neural stem cells (NSCs) and eventual production of neurons and glia in the developing neuroepithelium remains unclear. Methods to trace NSC division patterns and map the lineage of clonally related cells have advanced dramatically. However, many contemporary lineage tracing techniques suffer from the lack of cellular resolution of progeny cell fate, which is essential for deciphering progenitor cell division patterns. Presented is a protocol using mosaic analysis with double markers (MADM) to perform in vivo clonal analysis. MADM concomitantly manipulates individual progenitor cells and visualizes precise division patterns and lineage progression at unprecedented single cell resolution. MADM-based interchromosomal recombination events during the G2-X phase of mitosis, together with temporally inducible CreERT2, provide exact information on the birth dates of clones and their division patterns. Thus, MADM lineage tracing provides unprecedented qualitative and quantitative optical readouts of the proliferation mode of stem cell progenitors at the single cell level. MADM also allows for examination of the mechanisms and functional requirements of candidate genes in NSC lineage progression. This method is unique in that comparative analysis of control and mutant subclones can be performed in the same tissue environment in vivo. Here, the protocol is described in detail, and experimental paradigms to employ MADM for clonal analysis and lineage tracing in the developing cerebral cortex are demonstrated. Importantly, this protocol can be adapted to perform MADM clonal analysis in any murine stem cell niche, as long as the CreERT2 driver is present.

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers MADM

A protocol to perform lineage tracing and functional genetic analysis of candidate genes at a single cell level using mosaic analysis with double markers (MADM) is presented. MADM clonal analysis provides a quantitative framework to measure the proliferative behavior, cellular output, and lineage relationship of individual progenitors and their daughter cells.

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying ...

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Summary

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Chapters in this video

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel’s Cartilage

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

Clonal Analysis of the Neonatal Mouse Heart using Nearest Neighbor Modeling

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

Three Strategies to Induce Neurotrophic Keratitis and Nerve Regeneration in Murine Cornea

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM)