Name: co-localization of cell lineage markers and the tomato signal
Uploaded: 2016-12-28T14:00:00
Description: Watch this Scientific Journal Video about Co-localization of Cell Lineage Markers and the Tomato Signal at JoVE.com

This study was supported by NIH grant DE025014 to JQF.

All protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Texas A&#38;M University College of Dentistry.
1. Animal Breeding
<ol>
	<li>Use three animal models in this study. To investigate the fate of the embryonic chondrocytes in condyle formation, first use Col10a1-Cre15 mice and cross them with Rosa26tdTomato (B6;129S6-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J) mice to obtain Col10a1-Cre<...

<ol>
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S., Freeman, T., Srinivas, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+the+hypertrophic+chondrocyte:+microenvironmental+perspectives+on+apoptosis+and+survival+in+the+epiphyseal+growth+plate.">Fate of the hypertrophic chondrocyte: microenvironmental perspectives on apoptosis and survival in the epiphyseal growth plate.</a> Birth Defects Res C Embryo Today. 75 (4), 330-339 (2005).</li><li>Kahn, A. J., Simmons, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrocyte-to-osteocyte+transformation+in+grafts+of+perichondrium-free+epiphyseal+cartilage.">Chondrocyte-to-osteocyte transformation in grafts of perichondrium-free epiphyseal cartilage.</a> Clin Orthop Relat Res. (129), 299-304 (1977).</li><li>Roach, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-differentiation+of+hypertrophic+chondrocytes+into+cells+capable+of+producing+a+mineralized+bone+matrix.">Trans-differentiation of hypertrophic chondrocytes into cells capable of producing a mineralized bone matrix.</a> Bone Miner. 19 (1), 1-20 (1992).</li><li>Park, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+pathways+to+endochondral+osteoblasts:+a+novel+chondrocyte-derived+osteoprogenitor+cell+identified+in+hypertrophic+cartilage.">Dual pathways to endochondral osteoblasts: a novel chondrocyte-derived osteoprogenitor cell identified in hypertrophic cartilage.</a> Biol Open. 4 (5), 608-621 (2015).</li><li>Kretzschmar, K., Watt, F. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage-tracing+methods+and+the+kidney.">Lineage-tracing methods and the kidney.</a> Kidney Int. 86 (3), 481-488 (2014).</li><li>Romagnani, P., Rinkevich, Y., Dekel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+lineage+tracing+to+study+kidney+injury+and+regeneration.">The use of lineage tracing to study kidney injury and regeneration.</a> Nat Rev Nephrol. 11 (7), 420-431 (2015).</li><li>Yang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenic+fate+of+hypertrophic+chondrocytes.">Osteogenic fate of hypertrophic chondrocytes.</a> Cell Res. 24 (10), 1266-1269 (2014).</li><li>Yang, L., Tsang, K. Y., Tang, H. C., Chan, D., Cheah, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertrophic+chondrocytes+can+become+osteoblasts+and+osteocytes+in+endochondral+bone+formation.">Hypertrophic chondrocytes can become osteoblasts and osteocytes in endochondral bone formation.</a> Proc Natl Acad Sci U S A. 111 (33), 12097-12102 (2014).</li><li>Jing, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrocytes+Directly+Transform+into+Bone+Cells+in+Mandibular+Condyle+Growth.">Chondrocytes Directly Transform into Bone Cells in Mandibular Condyle Growth.</a> J Dent Res. 94 (12), 1668-1675 (2015).</li><li>Zhou, X., von der Mark, K., Henry, S., Norton, W., Adams, H., de Crombrugghe, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrocytes+transdifferentiate+into+osteoblasts+in+endochondral+bone+during+development,+postnatal+growth+and+fracture+healing+in+mice.">Chondrocytes transdifferentiate into osteoblasts in endochondral bone during development, postnatal growth and fracture healing in mice.</a> PLoS Genet. 10 (12), (2014).</li><li>Purcell, P., Trainor, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mighty+Chondrocyte:+No+Bones+about+It.">The Mighty Chondrocyte: No Bones about It.</a> J Dent Res. 94 (12), 1625-1627 (2015).</li><li>Gebhard, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+expression+of+Cre+recombinase+in+hypertrophic+cartilage+under+the+control+of+a+BAC-Col10a1+promoter.">Specific expression of Cre recombinase in hypertrophic cartilage under the control of a BAC-Col10a1 promoter.</a> Matrix Biol. 27 (8), 693-699 (2008).</li><li>Kalajzic, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directing+the+expression+of+a+green+fluorescent+protein+transgene+in+differentiated+osteoblasts:+comparison+between+rat+type+I+collagen+and+rat+osteocalcin+promoters.">Directing the expression of a green fluorescent protein transgene in differentiated osteoblasts: comparison between rat type I collagen and rat osteocalcin promoters.</a> Bone. 31 (6), 654-660 (2002).</li><li>Akiyama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteo-chondroprogenitor+cells+are+derived+from+Sox9+expressing+precursors.">Osteo-chondroprogenitor cells are derived from Sox9 expressing precursors.</a> Proc Natl Acad Sci U S A. 102 (41), 14665-14670 (2005).</li><li>Henry, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+aggrecan-CreERT2+knockin+mice+for+inducible+Cre+activity+in+adult+cartilage.">Generation of aggrecan-CreERT2 knockin mice for inducible Cre activity in adult cartilage.</a> Genesis. 47 (12), 805-814 (2009).</li><li>Ren, Y., Lin, S., Jing, Y., Dechow, P. C., Feng, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+way+to+statistically+analyze+morphologic+changes+in+Dmp1-null+osteocytes.">A novel way to statistically analyze morphologic changes in Dmp1-null osteocytes.</a> Connect Tissue Res. 55, 129-133 (2014).</li><li>Pest, M. A., Beier, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+biology:+Is+there+such+a+thing+as+a+cartilage-specific+knockout+mouse?.">Developmental biology: Is there such a thing as a cartilage-specific knockout mouse?.</a> Nat Rev Rheumatol. 10 (12), 702-704 (2014).</li><li>Tsang, K. Y., Chan, D., Cheah, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+growth+plate+hypertrophic+chondrocytes:+death+or+lineage+extension?.">Fate of growth plate hypertrophic chondrocytes: death or lineage extension?.</a> Dev Growth Differ. 57 (2), 179-192 (2015).</li><li>Sugimoto, Y., Takimoto, A., Hiraki, Y., Shukunami, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+characterization+of+ScxCre+transgenic+mice.">Generation and characterization of ScxCre transgenic mice.</a> Genesis. 51 (4), 275-283 (2013).</li><li>Feng, J. 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Due to technological limitations, it is always difficult to investigate the behavior of cells in vivo. However, the cell lineage tracing technique is proving to be a powerful tool for studying cell biology7-9. In this study, we further improve this protocol by combining it with immunofluorescence. In this way, cell fate can be defined by multiple related markers, which broadens the application of lineage tracing. Moreover, this co-localization of immunofluorescence and tomato signal simultaneously dis...

During development, endochondral bone formation accounts for over 80% of the skeletal volume. It is widely believed that it begins with the apoptosis of hypertrophic chondrocytes. Subsequently, the cells from the underlying bone marrow invade and initiate angiogenesis, followed by new bone deposition by bone marrow- and periosteum-derived cells1,2. The cell fate of hypertrophic chondrocytes (HCs), however, has been an issue of debate for decades3. Initially, HCs were considered to be the end of the chondrocyte differentiation pathway, and apoptosis was generally thought to be the ultimate fate of HCs. Now, some researchers suggest that at least some HCs could survive and contribute to endochondral bone formation. Although they proposed that growth plate chondrocytes had the ability to transdifferentiate into osteoblasts based on ultrastructure, immunohistochemical staining, and in vitro studies46, none of these methods were definitive in demonstrating chondrocyte contribution to the osteoblast lineage.
The cell lineage tracing technique provides a more rigorous way to study cell fate. Briefly speaking, a recombinase enzyme, which is only expressed in a specific type of cell, stimulates the expression of the reporter gene. In this way, this type of cell and their descendants are permanently labeled7. The Cre-loxP system is commonly used in lineage tracing. Cre (the recombinase enzyme) will excise the STOP sequence between the two loxP sites and activate the reporter in a specific cell line (Figure 1A). In some cases, the investigator can choose a favorable time point to activate Cre by using a drug, such as tamoxifen, causing Cre to fuse to a modified form of the estrogen receptor (CreERT2)8. Fluorescent reporters have become the standard in lineage tracing experiments because they dramatically reduce the complexity and improve the accuracy and efficiency of cell fate tracing8,9. tdTomato is becoming the best choice among fluorescent reporters since it has the brightest fluorescent protein and the strongest epifluorescence, making it easily visualized7 (Figure 1A).
By using the Rosa26tdTomato lineage tracing system, our group and other investigators have shown that HCs can change their phenotype into bone cells during development10-14. To accomplish this, we developed two sets of tracing combinations with Rosa26tdtomato (ubiquitous expression in all cells)/Cre (specific to chondrocytes) mice: 2.3Col1a1-GFP (specific to osteoblasts) and immunofluorescence (specific to bone cells). The data demonstrate that both methods are viable ways to study cell fate in vivo.

<table border="0" cellpadding="0" cellspacing="0" width="1099">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Tamoxifen&#160;</td>
			<td style="width:220px;">Sigma</td>
			<td style="width:136px;">T5648</td>
			<td style="width:496px;">activate the Cre event&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma&#160;</td>
			<td>P6148</td>
			<td>fix the sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediaminetetraacetic acid</td>
			<td>Alfa Aesar</td>
			<td>A10713</td>
			<td>decalcify the hard tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose&#160;</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td>dehydrate the tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyaluronidase from bovine testes&#160;</td>
			<td>Sigma</td>
			<td>H4272</td>
			<td>retrieve antigen for immunochemical staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Bovine serum albumin</td>
			<td>Sigma&#160;</td>
			<td>A3059</td>
			<td>blocking solution&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">primary antibody for Runx2&#160;</td>
			<td>Cell Signal</td>
			<td>D1L7F</td>
			<td>primary antibody for immunochemical staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">primary antibody for DMP1</td>
			<td style="width:220px;">provided by Dr. Chunlin Qin</td>
			<td style="width:136px;"></td>
			<td>primary antibody for immunochemical staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-rabbit IgG</td>
			<td style="width:220px;">Sigma</td>
			<td style="width:136px;">18140</td>
			<td>control for immunochemical staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">secondary antibody&#160;</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>second antibody for immunochemical staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OCT</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td>embed the sample for frozen section</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337</td>
			<td>PBST</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">non-fluorescing antifade mountant</td>
			<td>Life technologies</td>
			<td>P36934</td>
			<td>mounting slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Life technologies</td>
			<td>P36931</td>
			<td>nuclear staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrophobic Barrier Pen</td>
			<td>Vector Laboratories</td>
			<td></td>
			<td>circle the section on the slide for for immunochemical staining&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine</td>
			<td style="width:220px;">AnaSed</td>
			<td></td>
			<td>anesthetization&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketaset&#160;</td>
			<td>Zoetis</td>
			<td></td>
			<td>anesthetization&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">cryosection machine</td>
			<td style="width:220px;">Leica</td>
			<td style="width:136px;">CM1860 UV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">confocal microscope</td>
			<td style="width:220px;">Leica</td>
			<td>DM6000 CFS&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Chondrocytes Directly Transform into Bone Cells (Osteoblasts and Osteocytes) in the Mandibular Condyle and Long Bone.
Aggrecan, a critical gene for chondrogenesis, is mainly expressed in early and mature chondrocytes18. As a result, the injection of tamoxifen at 2 weeks of age in Agg-CreERT2; Rosa26tdTomato mice activated the red-tomato reporter in all ...

Watch this Scientific Journal Video about Co-localization of Cell Lineage Markers and the Tomato Signal at JoVE.com

Co-localization of Cell Lineage Markers and the Tomato Signal

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 ...

The overall goal of this cell lineage tracing technique and subsequent immunofluorescence is to investigate cell biology and cell fate in vivo. This method can help answer key questions in the cell biology and tissue development field regarding the origin of cells that form bone during endochondrogenesis. The main advantage of this technique is to display the number, location and differentiation status of the progeny of the founder cell simultaneously, thus broadening the application of lineage tracing.Though this method provides insight into the transformation from chondrocytes into osteogenic cells during normal development, it can also be applied to other tissues and disease models such as cell transformation in a tumor. Generally, individuals new to this method will struggle because frozen sectioning is difficult and the section can easily peel off during staining. We first had the idea for this method when we intended to demonstrate the differentiation status of osteogenic cells transformed from chrondocytes in trabecular bone.In order to trace the lineage of hypertrophic chrondocytes in the mouse, first breed the animals as described in the accompanying text protocol. This allows for cell lineage tracing in conjunction with fluorescent immunohistochemistry to define the chrondocyte's fate. Once the desired mouse has been bred, remove it from its cage.Then use the left thumb and index finger to grab the skin on the back of the mouse and turn it over exposing the abdomen. With the right hand, pick up a 0.5 milliliter syringe equipped with a 28.5 gauge needle and loaded with Tamoxifen. Place the syringe parallel to the hind legs and insert it into the peritoneum of the mouse.Inject the solution into the side of the hypogastrium avoiding the liver and bladder in order to activate the Cre system at a favorable time point. After two weeks, prepare the mouse for fixation by properly anesthetizing it and checking the mouse's pedal reflex. Then use tape to fix the four legs of the mouse on a board and expose the abdomen.Next, saturate the mouse's abdomen with 70%ethanol to sterilize the area and then make an incision from the lower abdomen to the neck along the middle line. Now, pinch and simultaneously pull the skin to the lateral sides to reveal the peritoneal membrane and use dissection scissors to make a longitudinal excision. Cut off and remove the front ribs to expose the heart.Then in a class I biosafety cabinet, use a syringe loaded with 4%Paraformaldehyde and equipped with a 22 gauge needle to puncture the wall of the heart and enter the left ventricle. While holding the syringe, cut a slot in the right auricle. Then slowly inject the solution into the left ventricle in order to perfuse the cardiovascular system while the blood flushes out of the cut from the right auricle.After removing the mouse's skin and organs, place the whole body into a 50 milliliter polypropylene centrifuge tube containing 40 milliliters of 4%Paraformaldehyde and fix the animal overnight at 4 degrees Celsius. The next day, transfer the mouse to a Petri dish in the class I biosafety. Use dissection scissors and number 3 and number 5 forceps to carefully remove a mandible and a hind leg from the body.Next, remove the muscles and tendons on the surface of the hind leg and mandible. To speed the decalcification, cut the mandible into two pieces at the distal region of the third molar. Put the parts that include the condyle and condylar process into 40 milliliters of 10%EDTA to decalcify.Similarly, cut the femur and tibia in the midshaft to expose the bone marrow cavity in order to accelerate decalcification. Once decalcified, transfer the samples into a 50 milliliter tube containing a 15%sucrose solution to dehydrate the condyle and hind leg. Further dehydrate the samples by transferring them into a fresh 50 milliliter tube containing a 30%sucrose solution.Then embed the condyle and hind leg in OCT compound along the sagittal plane respectively. Leave the samples in the cryosection machine until the OCT freezes. Next, use a drop of fresh OCT compound to mount the tissue block on the cutting plate.Wait for approximately 15 minutes before starting to cut the sample in order to ensure that the connection is stable. When ready to begin cutting sections, set the cut depth to 10 microns and section the condyle and hind leg, collecting the sections on slides and storing them at negative 20 degrees Celsius. Just before staining, incubate the slide in a dry 37 degrees Celsius chamber for 10 minutes to remove the water and adhere the section to the slide.Then wash the slides twice with distilled water for five minutes. Wipe off the water around each section and use a hydrophobic barrier pen to circle the sections. Next, add a drop of DAPI mounting solution into the circle and carefully lay down the coverslip.For Runx2 and DMP1 immunohistochemistry, prepare the slides as previously shown, washing them twice with distilled water for five minutes. However, instead of adding DAPI mounting solution right away, first treat the sections with hyaluronidase in a humid chamber at 37 degrees Celsius for 30 minutes. Next, wash the sections with PBST three times and incubate them for one hour at room temperature in an appropriate blocking solution.Then remove the blocking solution and add either rabbit anti-mouse Runx2 or rabbit anti-mouse DMP1 to each slide. Incubate the slides and their primary antibiodies at four degrees Celsius overnight. The next day, wash the sections three times with PBS and then add the secondary antibody solution.Incubate the slides in the dark at room temperature for two hours. Following incubation, wash the sections three times in PBS and then add a drop of DAPI mounting solution before adding the coverslip. Capture fluorescent cell images using a confocal microscope at wavelengths ranging from 488 micrometers to 561 micrometers.Take multiple stacked images at 200 hertz using 10X, 20X and 63X lenses. A Tamoxifen injection at two weeks of age activates the red tomato reporter in all chondrocytes and their daughter cells. In addition, all collagen 1 expressing bone cells are green fluorescing.Yellow cells indicate the presence of chondrocyte-derived bone cells. These results provided strong evidence that these bone cells were derived from chondrocytes. Similar results were also observed during long bone development where the majority of bone cells were derived from chondrocytes in both the epiphysis and the metaphysis.In the trabeculae, near the cartilage bone interface of three-week-old mice, red and some yellow fluorescing cells were predominant, while green fluorescing cells were scarce. In a slightly more inferior area, the majority of the cells were fluorescing yellow with slightly fewer fluorescing red. Green fluorescing cells appear to dominate only in the most inferior area of the condylar process.This trend indicates that condylar growth is mostly contributed to transformed bone cells from hypertrophic chondrocytes. The distribution of the red, yellow and green fluorescing cells in the condylar process is also consistent with the inferior to superior direction of condylar growth. In addition to crossing alone, the co-application of immunofluorescent staining and cell lineage tracing enables the tracking of cell differentiation.Shown here are Runx2 and DMP1 labeled sections showing the presence of immature osteoblasts and mature chondrocyte-derived osteocytes. While attempting this procedure, it is important to remember to select appropriate Cre lines in order to verify cell fate. In the future, investigators can attempt to perform immunofluorescence with two different antibodies over the tomato signal background to obtain more information in one section or use in situ immunofluorescence to decrease nonspecific staining.After its development, this technique paved the way for researchers in the field of cell biology and tissue development to explore the unknown differentiation capacity of cells. After watching this video, you should have a good understanding of how to combine the cell lineage tracing technique with immunofluorescence to investigate the cell fate and cell biology in vivo.

Research

JoVE Journal

Developmental Biology

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human ...

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

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 ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...