We thank Dr. Evgeny Ivashkin for methodological advice. This work was supported financially by the Swedish Research Council (A.S.C), The Russian Scientific Foundation (grant #19-15-00241 to ASC), Karolinska Institute (A.S.C.), StratRegen KI and Stiftelsen Konung Gustaf V:s 80-&#229;rsfond (A.S.C.), Stiftelsen Frimurare Barnhuset and Sallskapet Barnavard (P.T.N.), Chinese Scholarship Council (B.Z.). The confocal microscope was funded by the Knut and Alice Wallenberg Foundation.

All mouse work was approved by the Ethical Committee on Animal Experiments (Stockholm North Committee/Norra Djurf&#246;rs&#246;ksetiska N&#228;md) and conducted in accordance with The Swedish Animal Agency&#180;s Provisions and Guidelines for Animal Experimentation.
1. Mouse breeding
<ol>
	<li>To generate the strain, cross the R26R-Confetti mouse with the Cre line of interest. Wean the pups at 21 days of age. 
	NOTE: Mice can be used for breeding from approximately 8 weeks of age, or according to the local guidelines. Genotyping (not described here) can be performed from biopsies collected at weaning. The age at weaning can be adjusted as per local guidelines.</li>
</ol>
2. Confetti labeling
NOTE: This labeling strategy is appropriate for tamoxifen inducible Cre strains.
<ol>
	<li>Prepare a solution of up to 2.5 mg/mL tamoxifen in corn oil in a glass bottle. Dissolve by heating to 37 &#176;C in an oven for up to 60 min, agitating every 5 min using a vortex until the powder has dissolved. 
	NOTE: For increased doses of tamoxifen, solutions up to 20 mg/mL can be prepared using this method.</li>
	<li>Store at 4 &#176;C for up to 3 months, protected from light.</li>
	<li>Induce recombination by administering 50 &#181;L of 2.5 mg/mL tamoxifen dissolved in corn oil intraperitoneally on the first postnatal day (P1). 
	NOTE: In principle, tamoxifen can be administered at any age, as early as the F1 generation. The dose of tamoxifen should be varied based on Cre expression, mouse age, and experimental application. Further information can be found in the discussion section. Cre negative mice treated in the same way can be used as a negative control for Confetti fluorescent signals. 
	CAUTION: Ensure animal handlers are warned of the tamoxifen use and ensure that cage materials are disposed of according to local environmental and safety guidelines.</li>
</ol>
3. Tissue collection
<ol>
	<li>Euthanize mice on postnatal day 27 (P27) by gradually filling the space containing the mice with carbon dioxide up to at least 80% by volume and maintaining this concentration for 3 min. Perform cervical dislocation. Keep mice on ice during the dissection of the other animals.</li>
	<li>Dissect out the tissue of interest. Outlined below is a protocol for collecting the proximal tibial and distal femoral growth plates and articular cartilage of the knee, suitable for postnatal mice up to the age of 6 months.
	<ol>
		<li>Remove the skin around the hind limbs as far as the foot, using sharp scissors and toothed forceps.</li>
		<li>Roughly trim the muscle and fat tissue from the legs with sharp scissors. 
		NOTE: Do not clean the bones perfectly, because the surrounding tissues provide some structural support when sectioning, but ensure all skin is removed.</li>
		<li>Using a scalpel, cut through the soft tissue where the top of the leg meets the body until the femoral head is visible, then cut the ligaments in the hip joint to remove the leg. 
		NOTE: Alternatively, if it is not possible to find the femoral head, cut through the femur close to the hip with strong scissors.</li>
		<li>Dissect the foot from the rest of the leg using a scalpel or sharp scissors.</li>
	</ol>
	</li>
</ol>
4. Tissue fixation and processing
NOTE: Based on the tissue of interest, follow one of the two protocols detailed below (4.1 for soft tissues or 4.2 for mineralized mouse tissues over the age of approximately 45 days, as detailed below). For both methods, store the dissected samples in the 3.7% formaldehyde/phosphate buffered saline (PBS) solution on ice while dissecting the remaining animals.
<ol>
	<li>For soft tissues and mineralized tibiae and femora up to the age of approximately P45, fix the tissue in precooled 3.7% formaldehyde/PBS for 6 h, rolling gently on a rotator at 4 &#176;C. Use a volume at least 10x greater than that of the tissue. Continue directly to step 4.3.</li>
	<li>For mineralized tissues, including the tibiae and femora over the age of approximately P45, a decalcification step is required.
	<ol>
		<li>Prepare 1 L of 10% EDTA/dH2O solution with the pH adjusted to 8.05 using sodium hydroxide. Next, dilute the formaldehyde to 3.7% using this solution. The working concentration of EDTA will be 9%.</li>
		<li>Fix the tissue by placing into precooled 3.7% formaldehyde/PBS overnight, gently rolling on a rotator at 4 &#176;C.</li>
		<li>Place the tissue into precooled 3.7% formaldehyde/9% EDTA/dH2O solution (pH 8.05), and gently rotate at 4 &#176;C in a volume at least 10x greater than that of the tissue. Repeat this step by placing the bones into fresh, precooled 3.7% formaldehyde/9% EDTA/dH2O for 3x over the next 48 h. 
		NOTE: This short decalcification is enough to allow for sectioning of the femur and tibia bones of up to 6 months-old mice. For more densely mineralized tissues further decalcification may be required to improve histology.</li>
	</ol>
	</li>
	<li>Transfer the tissue into precooled 30% sucrose/dH2O. Make sure to fill the container to the brim, and gently rotate overnight at 4 &#176;C. Use a volume at least 10x greater than that of the tissue. The tissue will often float when first placed into this solution and sink to the bottom of the bottle by the morning.</li>
	<li>To remove the residual sucrose solution, briefly wash the tissue by removing it from the sucrose solution with forceps and completely covering the sample with approximately 5 ml of optimal cutting temperature compound (OCT) for several seconds.</li>
	<li>Fill a prelabeled cryomold with OCT and position the tissue at the bottom. Work quickly to avoid the samples sitting at room temperature for too long. 
	NOTE: It is important to place the sample in a convenient orientation for the sectioning step. To increase the chances of sectioning through entire growth plate columns after tracing with Col2CreERT:Confetti, place the medial side facing down using the femoral head as a guide, and position the sample as flat to the bottom of the mold as possible.</li>
	<li>Embed the sample by placing the mold onto dry ice and waiting for the OCT to entirely solidify. Place the cryomolds as flat as possible to avoid movement/sliding of the tissue in the mold. Once finished, store the samples at -20 &#176;C, if they are not used immediately. 
	NOTE: Use within 12 weeks, because with age the blocks become more difficult to section.</li>
</ol>
5. Sectioning
NOTE: Perform cryosectioning using a cryostat with disposable blades suitable for hard tissue. Any standard cryostat is suitable.
<ol>
	<li>Remove the block from the mold and secure it to the chuck by applying OCT between the top of the block and the chuck and cooling in the cryostat at -20 &#176;C for at least 5 min, so that the OCT has completely solidified.</li>
	<li>Precool both the sample holder and the blade holder to -20 &#176;C and then place the sample/chuck in the chuck holder and the blade in the blade holder to equilibrate for several min. 
	NOTE: The specified temperatures may be varied by several degrees depending on the cryostat and the tissue of interest.</li>
	<li>Use the cryostat to trim the block and cut tissue sections of a suitable thickness to allow for the visualization of the clones in the tissue of interest.
	<ol>
		<li>For postnatal growth plate analysis, prepare sections of 30&#8722;160 &#181;m and collect them on slides. Some cryostat models may only allow the collection of thick sections using the trim setting.</li>
	</ol>
	</li>
	<li>Air dry the slides at room temperature until they are completely dry. 
	NOTE: The duration of this step can vary based on tissue properties and section thickness. The temperature of the room is also likely to affect the speed of this step.</li>
	<li>Store the slides at -20 &#176;C for up to 36 months, or if processing immediately, proceed to step 6.2.</li>
</ol>
6. Slide preparation and mounting
<ol>
	<li>Remove the slide from the freezer and bring up to room temperature horizontally in a slide holder. Allow any condensation to evaporate.</li>
	<li>Remove the OCT by using a Pasteur pipette to gently apply PBS at room temperature onto the slide. The thicker the section, the longer time is required. For 160 &#181;m thick sections, perform two rinses: incubate once for 15 min and remove the liquid, and then apply fresh PBS for 5 min and remove the liquid. 
	NOTE: This step is to remove the OCT and can be varied based on tissue properties and section thickness, as appropriate. The temperature of the room is likely to affect the speed of this step.</li>
	<li>Mount the slides in a solution of 75% 2,2-thiodiethanol/dH2O at room temperature with 60 mm long cover slips. 
	NOTE: Slides can be prepared immediately before imaging, but the fluorescent signal is stable for 1&#8722;2 weeks if kept out of the light in a humidified chamber at 4 &#176;C.</li>
</ol>
7. Imaging by confocal microscopy
<ol>
	<li>Set up the microscope.
	<ol>
		<li>Turn on the microscope and open the software. Click the Start System button.</li>
		<li>Select the channels by first clicking Smart Setup under the Acquisition tab and then selecting the three channels corresponding to red fluorescent protein (RFP), yellow fluorescent protein (YFP), and cyan fluorescent protein (CFP) under the Dye heading. Click Best signal then Apply to select the lasers. 
		NOTE: The contents of the Acquisition tab are shown in Supplementary file 1A. If the required channels are not present, add them using the &#8216;+&#8217; button under the Dye heading. The necessary information regarding laser, excitation wavelengths, and the range of recorded emission wavelengths is shown for the three Confetti fluorescent proteins in Supplementary file 1B-D.</li>
		<li>Select the 20x objective lens under the Objective heading in the Acquisition Mode tab.</li>
	</ol>
	</li>
	<li>Locate the growth plate or other tissue of interest.
	<ol>
		<li>To provide a distinct view, first select the RFP channel by clicking its name in the Channels tab. This causes details of the RFP channel to be displayed in the Light Path tab. Click the tick-box beside T-PMT. 
		NOTE: T-PMT channel shows nonreflected laser light, which is used to visualize the nonfluorescent parts of the tissue.</li>
		<li>Place the slide in the slide holder and position the growth plate or other tissue of interest directly between the light path and the objective lens.</li>
		<li>In order to simplify localizing the tissue, deselect the YFP and CFP channels using the tick-boxes under the Tracks heading of the Channels tab.</li>
		<li>Select the T-PMT channel by clicking its name in the Tracks heading. Click Live in the Acquisition tab and increase the Gain (Master) for the T-PMT channel in order to visualize the tissue section on the screen, which is shown in greyscale.</li>
		<li>Move the position of the slide using the joystick to locate the region of interest, then focus using the appropriate microscope knob.</li>
		<li>Click Stop in the Acquisition tab to turn off the laser exposure.</li>
	</ol>
	</li>
	<li>Define the imaging parameters.
	<ol>
		<li>Use the tick-boxes in the Channels tab to select one of the channels and click Live in the Acquisition tab to display the image of that channel on the screen. Then, using the left-click button on the mouse, adjust the slide-bars to set the range of emitted fluorescent signal that is collected, the laser power (sliding bar under Lasers heading), Gain (Master) and Digital Offset in the Channels tab to visualize the Confetti-labeled cells on the screen. Guideline values for all of these parameters are provided in Supplementary file 1B-D for RFP, YFP, and CFP. Complete this step for every channel, one by one. 
		NOTE: To ensure the visualized signal is not autofluorescence, a section from a Cre negative animal can be used as a negative control during this step. As T-PMT has been combined with the RFP laser (step 7.2.1) adjusting laser power for RFP will affect T-PMT signal.</li>
		<li>Adjust the Pinhole size to increase the fluorescence detection if necessary. 
		NOTE: The higher the pinhole size, the more fluorescent signal is detected in the Z-direction. A guideline for this is the resultant Airy Unit (AU), which is automatically indicated beneath the Pinhole indicator, where 1 AU is optimal for imaging quality. Increasing the AU too much will impair later analysis because it becomes more difficult to distinguish individual cells in the Z-direction).</li>
		<li>Check the setup to ensure that the recorded signals do not overlap.
		<ol>
			<li>Click on the tick-boxes in the Channels tab to select all three channels and press the Snap button in the Acquisition tab.</li>
			<li>In the resultant image, scroll between the displayed channels by clicking the blue-highlighted boxes above each listed Channel in the Dimensions tab. Manually check for the overlap between the channels on the screen. If the signals overlap (e.g., if every red cell is also yellow), return to the beginning of step 7.3 and repeat, adjusting the parameters for the overlapping channels until they can be distinguished from each other. Ensure that for each label (i.e., RFP, YFP, or CFP), at least one clone can be visualized that contains only one of the labels.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Acquire image.
	<ol>
		<li>Select the Acquisition Mode tab to specify image quality using the Frame Size, Speed, and Averaging Number values. 
		NOTE: The typical settings for these experiments are indicated in Supplementary file 1A. Reducing the Zoom under the Scan Area heading within the Acquisition tab can increase the field of view without altering the time required for image acquisition.</li>
		<li>In the Acquisition tab, under the Dimensional Acquisition heading, click the Z-Stack tick-box. Open the Z-stack tab to set the interval between images in the Z-direction to 2.5 &#181;m.</li>
		<li>To set the correct locations, select only the RFP/T-PMT in the Channels tab and click Live to visualize the red clones with a visible outline of the tissue. Set the first and last slice by clicking Set First and Set Last on the corresponding slice, adjusting the focus using the microscope knob.</li>
		<li>Click the Tile Scan tab within the Acquisition tab and enter the total number of tiles in the horizontal and vertical directions in the respective boxes.</li>
		<li>For image capture, select all the desired channels in the Channels tab, then click the Snap button to collect a single image, or the Start Experiment button to collect a Z stack/Tile scan.</li>
	</ol>
	</li>
	<li>Once the image has been acquired, click File then Save As and save the image in a file type that preserves as much information about the microscope settings as possible, to allow later review and reuse.</li>
	<li>Perform further analysis using image analysis software.</li>
</ol>

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The authors have nothing to disclose.

The Confetti model was used extensively to study mineralized cartilage tissues12,13,14 and to describe the optimized protocol. Confetti mice2 with one copy or two copies of the R26R-Confetti allele can be used for breeding and experiments. Recombination in mice with one allele of the Brainbow 2.1 construct will give four potential labels: red fluorescent protein (RFP, cytoplasmic), yellow fluorescent protein (YFP, cytoplasmic), cyan fluorescent protein (CFP, membrane-localized), and green fluorescent protein (GFP, nucleus-localized), whereas recombination in the homozygous Confetti mice (i.e., containing two copies of the Brainbow 2.1 construct) will give 10 possible color outputs (RFP+RFP, RFP+YFP, RFP+CFP, RFP+GFP, YFP+YFP, YFP+CFP, YFP+GFP, CFP+CFP, CFP+GFP, GFP+GFP). However, because the DNA excision and inversion events occur in a stochastic manner, recombination to produce GFP occurs in only a small fraction of cells, as previously reported2, and seemingly differs by cell-type or Cre line used2,12,13. Hence, given the low occurrence of recombination events leading to the GFP expression, six color outputs are the most common in homozygous Confetti mice.
An important consideration when planning experiments with the Confetti mouse is to find a suitable Cre strain. First, since the Confetti allele has several loxP sites, one recombination event does not preclude a second4. This means that if a cell is labeled and divides to produce a clone of one color, a second recombination event in one of its daughter cells would generate a different color, thereby masking the true clonal expansion. Thus, noninducible Cre strains, where the enzyme is always active if the corresponding promoter is active, are not advisable. Second, in some strains the expression of Cre recombinase often comes at the expense of an endogenous protein, thereby creating a phenotype of its own (e.g., for the Prg4-GFPCreERT2 knock-in mouse strain in which mice harbor two Cre alleles15), so a single copy of the Cre allele is desirable. In all of the described experiments, a single copy of the Col2CreERT16 was carried by either the male or the female parent to generate Col2CreERT:Confetti mice, as described13. Next, the specificity of the Cre line to the cell type of interest is an important consideration. For example, Col2CreERT is chondrocyte-specific, but labels several distinct populations of chondrocytes in early postnatal cartilage17. Finally, the activity of different Cre strains can change considerably with animal age, as the endogenous activity of the promoter utilized for Cre expression changes, even in cells of the same type (e.g., Prg4-GFPCreERT2, which can require an increasing number of tamoxifen doses to label superficial zone cells as mice age15).
The number of labeled cells will be reflected by the amount of tamoxifen given, so it is not always desirable to give the maximum dose because distinguishing clones from each other may be difficult. Because Cre expression will vary under the regulation of different promoters as well as with age, the tamoxifen dosage will need to be adjusted to optimize labeling. It is important to note that cells are not immediately fluorescent upon triggering recombination because time is required for the recombination to occur and the resulting fluorescent proteins to accumulate sufficiently for imaging. A broad range of hours (between 24&minus;72 h) is needed, which appears to be influenced by the Cre line, cell-type, and animal age. Since tamoxifen can be biologically active long after administration18 it is always advisable to thoroughly check recombination efficiency in each model.
During the confocal microscopy step, excitation of the Confetti fluorescent proteins requires penetration of the tissue by laser light. Because different tissues have different properties, laser light might not penetrate 160 &micro;m thick sections of all tissues. However, such thick sections can be used for growth plate cartilage, as used in Figure 2, Figure 3. Note that every microscope is different, and it is unlikely that the described settings (Supplementary File 1) will work perfectly without minor adjustments. One of the major challenges is to distinguish the different fluorescent proteins to ensure that there is no contamination from one channel to another. For example, the signal in the YFP channel caused by the fluorescence coming from GFP overlaps between the YFP and RFP channels. YFP and CFP channels must also be carefully checked. This can be done in several ways. First, the emission range of fluorescent light wavelengths that are recorded for each fluorescent protein can be narrowed. Second, adjusting the laser power in combination with the digital gain can optimize the signal. In this study these steps were sufficient, but they rely on a strong fluorescent signal. This means that in theory, inefficient tissue processing can reduce the activity of the fluorescent proteins, or a weak laser light source can impair channel separation.
One of the advantages of the Confetti mouse is that it can be combined with a large number of genetically mutant strains that are available so that the impact of specific genes on clonal dynamics can be studied10,11,12,13,14,19,20, albeit with some limitations. For example, recombination of the Confetti alleles and the gene of interest cannot be separated when using Cre loxP-based mutants combined with clonal lineage tracing. Nevertheless, such coinciding recombination events can be effective10,14,20. For example, this possibility was used to explore the increased cell number in the resting zone of mice following postnatal chondrocyte-specific ablation of TSC1 in the growth plate21 and revealed the clonal relationship between these cells over time13. Additionally, the tissue-specific clonal analysis using Confetti mice can be used with non-Cre loxP-based mutant mice11, and it is also possible to combine these approaches with pharmacological8,9,13 and/or surgical models8 to visualize clonal responses to other functional perturbations. Further opportunities arise when combining Confetti labeled sections with immunodetection of endogenous proteins by immunofluorescence. Such analysis allows the identification of traced cells and their colocalization with a known marker. With the fixation method described herein, it is possible to conduct immunofluorescence and still visualize the fluorescence from the Confetti fluorescent proteins12,13. However, in the papers mentioned, antigen retrieval was not required. Harsh antigen retrieval methods, such as boiling in citrate buffer, leads to a complete loss of Confetti fluorescence. Although the Confetti fluorescent proteins can then be detected by using antibodies22, a loss of clonal identity can occur.
In summary, using the Confetti model to label cells in vivo is an informative tool to analyze the fate of those cells over time. The described method provides a fast and efficient way to directly visualize fluorescent protein expression in mineralized tissues.

Improved methods to monitor cell behavior are desirable to increase the experimental efficiency when using animal models. One of the most informative approaches to monitor cell behavior in vivo is clonal lineage tracing with multicolor reporter mouse strains1, including the widely used R26R-Confetti (Confetti) mouse2. By genetically labeling individual cells with fluorescent proteins and following those cells over time, researchers in many fields have used the Confetti mouse to reveal insights into a multitude of different biological systems.
The Confetti mouse is a loxP-based reporter system in which Cre dependent DNA recombination causes the permanent expression of one of several possible fluorescent proteins in a stochastic manner2. The R26R-Confetti allele includes the ubiquitously expressed CAGG promoter, which lies immediately upstream of a loxP-flanked NeoR-cassette2, whose polyadenylation sequence terminates transcription3, followed by the Brainbow 2.1 construct4. Hence, Cre-mediated recombination simultaneously excises the NeoR-cassette and leads to the production of certain fluorescent proteins depending on which parts of the Brainbow 2.1 construct are excised. This feature makes the Confetti mouse extremely adaptable because it can be used to target any cellular population in a mouse for which a specific Cre strain is available. The crossing of a tissue-specific Cre strain with the R26R-Confetti strain provides the specificity of labeling. However, the Cre strain should also be inducible (e.g., CreERT or CreERT2, which require tamoxifen binding to allow them to enter the nucleus and induce DNA recombination5) so that labeling can be achieved at specified time points. Thus, a cellular population can be labeled by injecting the resultant CreERT-positive/Confetti-positive offspring with tamoxifen at the desired time point and tracked to a certain age, when the tissues of interest can be collected for analysis. After tissue processing, the fluorescently labeled cells can be directly visualized by confocal microscopy so that the progeny of each initially-labeled cell can be separated from the progeny generated by any other labeled cell based on their specific fluorescent labels, thereby allowing clonality to be assessed (i.e., clonal genetic tracing).
Due to the flexibility of the Confetti model, it has been applied to study the development and maintenance of many different tissues in health and disease2,6,7,8,9,10,11. One common way to use the model is to label a certain population of cells and determine which tissues they (and/or their progeny) have contributed to. One such finding using this approach was that the adult tooth is comprised of cells with a glial origin6. Another application of the model is to study stem cell homeostasis. For example, the Confetti model revealed that stem cell niches in the intestinal crypts gradually become monoclonal because some stem cell clones are dominant during competition between stem cells2. The model can also be used to assess cell proliferation, which is particularly useful for studying slowly dividing cells. For example, clonal formation in the articular cartilage12 was assessed, and the short-term effects of a hedgehog antagonist on growth plate chondrocytes in five-week old mice was studied13.
Despite the relative simplicity of the Confetti model, the fluorescent signal can be technically challenging to preserve until image collection, particularly when analyzing mineralized tissues. Here, the protocol describes an optimized model to use the Confetti mouse with the postnatal bone (up to 6 months of age), enabling fluorescent proteins to be directly visualized by confocal microscopy without the need for immunodetection. This simplified protocol can be applied to nonmineralized tissues. Furthermore, included is a description of how to store the samples in order to preserve the fluorescent signal for a prolonged period of at least 3 years. An outline of the protocol is presented in Figure 1.

<table><tbody><tr><td>2,2-thiodiethanol</td><td>Sigma</td><td>MKCF9328</td><td></td></tr><tr><td>60 mm-long cover slips</td><td>Mendel-Gl&amp;auml;zer</td><td>220588</td><td></td></tr><tr><td>Confocal laser scanning microscope</td><td>Zeiss</td><td>LSM 710</td><td></td></tr><tr><td>Corn oil</td><td>Sigma</td><td>C8267</td><td></td></tr><tr><td>Cryomold (standard)</td><td>Sakura</td><td>4557</td><td></td></tr><tr><td>Cryostat</td><td>Thermo</td><td>Model: NX70</td><td></td></tr><tr><td>Disposable cryostat blades</td><td>Histolab</td><td>207500014</td><td>Specifically for use with hard tissue</td></tr><tr><td>EDTA</td><td>Scharlan</td><td>AC0960005P</td><td></td></tr><tr><td>Formaldehyde concentrate</td><td>Merck</td><td>8.18708.1000</td><td></td></tr><tr><td>Glass bottles</td><td>Sigma</td><td>V7130</td><td>Can be used for tamoxifen dilution and for tissue fixation and processing</td></tr><tr><td>Imaris software</td><td>Oxford Instruments</td><td>N/A</td><td>Image analysis software for 3D reconstruction</td></tr><tr><td>Isoflurane</td><td>Zoetis</td><td>11-8162</td><td></td></tr><tr><td>OCT</td><td>Sakura</td><td>102094-104</td><td></td></tr><tr><td>Pasteur pipette</td><td>Sarstedt</td><td>86.1171</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>14200075</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Merck</td><td>1.06498.1000</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S0389</td><td></td></tr><tr><td>Superfrost UltraPlus slides</td><td>Mendel-Gl&amp;auml;zer</td><td>J3800AMNZ</td><td></td></tr><tr><td>Tamoxifen</td><td>Sigma</td><td>T5648</td><td></td></tr><tr><td>Zen2 software</td><td>Zeiss</td><td>N/A</td><td>Freeware for Confocal laser scanning microscopy</td></tr></tbody></table>

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.

To label chondrocytes in the epiphyseal cartilage and visualize their clonal expansion with age using tamoxifen administration on P1, Col2CreERT:Confetti mice were labeled, and their hind-limbs were collected on P27. The central section was determined by visualizing the cruciate ligament (Figure 2A) and clones in the proximal tibial growth plate were visualized (Figure 2B). These data show that individual cells that were Col2-positive on P1 and became labeled with Confetti fluorescent proteins when tamoxifen was administered, underwent clonal expansion, and subsequently remained in the growth plate until P27. The initial changes in clonal expansion from a similar developmental time point can be seen in Figure 1 of a recent publication13.
To give an example of the fluorescent signal after long-term storage (step 5.5), the section collected was stored immediately after (see Figure 2A,B) for more than 3 years before it was prepared and imaged (Figure 2C, Video 1).
To demonstrate some common problems with the protocol, a section broken during the cryosectioning step is shown in Figure 3A. The region of the section (between the growth plate and articular cartilage) expanded in Figure 3B shows that this dose of tamoxifen leads to such a high level of recombination in this area that it would preclude clonal analysis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60424/60424fig01.jpg" /> 
Figure 1: Experimental overview.&#160;Flow chart summarizing the key stages of the method. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60424/60424fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60424/60424fig02.jpg" /> 
Figure 2: Confetti images from a Col2CreERT:Confetti mouse.&#160;(A) Samples of Col2CreERT:Confetti mice were processed without decalcification or the long-term storage step. The central section was located using the cruciate ligament as a marker. This image was visualized using a tile scan after detecting RFP with T-PMT (Scale bar = 300 &#181;m). (B) Clonal columns are shown within the proximal tibia of the growth plate visible in (A), following 3D reconstruction. (C) An adjacent slide to that shown in (A) and (B) held in long-term storage for more than 3 years was processed for imaging and 3D reconstruction. RFP, YFP, and CFP are shown in (B) and (C) (Scale bars = 50 &#181;m). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60424/60424fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60424/60424fig03.jpg" /> 
Figure 3: Challenges when using the method.&#160;(A) The white dashed lines outline a split in the section through the growth plate of Col2CreERT:Confetti mice (Scale bar = 50 &#181;m). The area within the white box is shown in (B) (Scale bar = 25 &#181;m). RFP, YFP, and CFP are shown in both panels, and the nonreflected laser light (T-PMT) channel is also shown in (A) to visualize the tissue. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60424/60424fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Three-dimensional reconstruction of a growth plate from Col2CreERT: Confetti mouse. A slide prepared from a Col2CreERT: Confetti mouse was held in long-term storage after the sectioning for more than 3 years, and then processed for imaging. Three-dimensional reconstruction was conducted automatically with image analysis software and exported as a video. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60424/video_1.mov" target="_blank">Please click here to download this video.</a>
Supplementary file 1: Typical set-up of the confocal microscope. (A) The main controls present in the Acquisition tab. (B&#8722;D) The laser parameters, excitation wavelength, and the range of recorded emission wavelengths are shown for three Confetti fluorescent proteins. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60424/60424supfig01.jpg" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues at JoVE.com

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

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-genetic-tracing-using-confetti-mouse-to-study-mineralized

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

14.2K Views.  Karolinska Institutet. 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.

Video: Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

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. ...

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.

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 ...

Co-localization of Cell Lineage Markers and the Tomato Signal

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the reporter in a specific cell line and all progeny. Here, we used the cell lineage tracing technique to demonstrate that chondrocytes directly transform into osteoblasts and osteocytes during long bone and mandibular condyle development using two kinds of Cre, Col10a1-Cre and Aggrecan-CreERT2 (Agg-CreERT2), crossed with Rosa26tdTomato. Both Col10 and aggrecan are well-recognized markers for chondrocytes.
On this basis, we developed a new method-cell lineage tracing in conjunction with fluorescent immunohistochemistry-to define cell fate by analyzing the expression of specific cell markers. Runx2 (a marker for early-stage osteogenic cells) and Dentin matrix protein1 (DMP1; a marker for late-stage osteogenic cells) were used to identify chondrocyte-derived bone cells and their differentiation status. This combination not only broadens the application of cell lineage tracing, but also simplifies the generation of compound mice. More importantly, the number, location, and differentiation statuses of parent cell progeny are displayed simultaneously, providing more information than cell lineage tracing alone. In conclusion, the co-application of cell lineage tracing techniques and immunofluorescence is a powerful tool for investigating cell biology in vivo.

We developed two sets of tracing combinations in Rosa26tdtomato (ubiquitously expressed in all cells)/Cre (specifically expressed in chondrocytes) mice: one with 2.3Col1a1-GFP (specific to osteoblasts) and one with immunofluorescence (specific to bone cells). The data demonstrate the direct transformation of chondrocytes into bone cells.

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the ...

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 ...

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Neuroscience

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

Biology

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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 ...

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 ...

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their native environment was described more than 10 years ago and sheds new light on the process of platelet formation. Megakaryocytes extend elongated protrusions, called proplatelets, through the endothelial lining of sinusoid vessels. This paper presents a protocol to simultaneously image in real time fluorescently labeled megakaryocytes in the skull bone marrow and sinusoid vessels. This technique relies on a minor surgery that keeps the skull intact to limit inflammatory reactions. The mouse head is immobilized with a ring glued to the skull to prevent movements from breathing.
Using two-photon microscopy, megakaryocytes can be visualized for up to a few hours, enabling the observation of cell protrusions and proplatelets in the process of elongation inside sinusoid vessels. This allows the quantification of several parameters related to the morphology of the protrusions (width, length, presence of constriction areas) and their elongation behavior (velocity, regularity, or presence of pauses or retraction phases). This technique also allows simultaneous recording of circulating platelets in sinusoid vessels to determine platelet velocity and blood flow direction. This method is particularly useful to study the role of genes of interest in platelet formation using genetically modified mice and is also amenable to pharmacological testing (study the mechanisms, evaluating drugs in the treatment of platelet production disorders). It has become an invaluable tool, especially to complement in vitro studies as it is now known that in vivo and in vitro proplatelet formation rely on different mechanisms. It has been shown, for example, that in vitro microtubules are required for proplatelet elongation per se. However, in vivo, they rather serve as a scaffold, elongation being mainly promoted by blood flow forces.

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

We describe here the method for imaging megakaryocytes and proplatelets in the marrow of the skull bone of living mice using two-photon microscopy.

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their ...

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In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow