Co-localization of Cell Lineage Markers and the Tomato Signal

Summary

We developed two sets of tracing combinations in Rosa26^tdtomato (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.

Abstract

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-Cre^ERT2 (Agg-Cre^ERT2), crossed with Rosa26^tdTomato. 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.

Introduction

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 cells^1,2. 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 (Cre^ERT2)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 tracing^8,9. 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 Rosa26^tdTomato lineage tracing system, our group and other investigators have shown that HCs can change their phenotype into bone cells during development^10-14. To accomplish this, we developed two sets of tracing combinations with Rosa26^tdtomato (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.

Protocol

All protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Texas A&M University College of Dentistry.

1. Animal Breeding

1. 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 Rosa26^tdTomato (B6;129S6-Gt(ROSA)26Sor^{tm9(CAG-tdTomato)Hze}/J) mice to obtain Col10a1-Cre and Rosa26^tdTomato mice. Next, cross these mice with 2.3Col1a1-GFP mice¹⁶. Use Col10a1-Cre; Rosa26^tdTomato; 2.3Col1a1-GFP mice to perform the cell lineage tracing experiments (Figure 1B).
2. To study the fate of the postnatal chondrocytes, follow the same procedure as in step 1.1, but use the Aggrecan-cre^ERT2 (Agg-Cre^ERT2)^17,18 line and keep the Agg-Cre^ERT2; Rosa26^tdTomato; 2.3Col1a1-GFP mice to perform the cell lineage tracing experiments (Figure 1C). Inject tamoxifen on postnatal day 14.
3. To combine the cell lineage tracing technique with immunofluorescence, cross Agg-Cre^ERT2 mice and Rosa26^tdTomato mice. Use mice that carry both genes in the experiment and inject the tamoxifen on postnatal day 3 (Figure 1D).

2. Material Preparation

1. Dissolve the tamoxifen powder in 10% ethanol and 90% corn oil at a concentration of 10 mg/ml. The dosage for injection is 75 mg/kg.
2. Dissolve the paraformaldehyde (PFA) powder in PBS to a concentration of 4%, using 2 M sodium hydrate to adjust the pH value to 7.4. Use 4% PFA solution to fix the tissue (the mandibular condyle and hind leg are used here) after sacrifice. Because of its toxicity, handle PFA in the hood with gloves and a facemask.
3. Dissolve the EDTA powder in distilled water to a concentration of 10%, using 2 M sodium hydrate to adjust the pH value to 7.4. Use 10% EDTA to decalcify the mandibular condyle and hind leg.
4. Dissolve sucrose powder in PBS at concentrations of 15% and 30%. Use the sucrose solution to dehydrate the tissue after decalcification.
5. Dissolve the hyaluronidase powder in PBS at a concentration of 2 mg/ml, pH 5.0. This solution is for immunofluorescence antigen retrieval.
6. Use a 1.5-ml tube to prepare a blocking solution that contains 3% bovine serum albumin (BSA) and 20% goat serum in PBS for Runx2 or DMP1 immunofluorescent staining.
7. Use a 1.5-ml tube to prepare a primary antibody solution that contains 2% goat serum in PBS for Runx2 or DMP1 immunofluorescent staining. The concentration of the primary antibody is 1:400 for Runx2 and 1:100 for DMP1.
8. Use a 1.5-ml tube to prepare the rabbit IgG solution as the control for immunofluorescent staining to avoid false positive results. The solution contains 2% goat serum in PBS. The concentration of the rabbit IgG for Runx2 control is 1:400; for DMP1, 1:100. Perform the experiment and control staining simultaneously.
9. Use a 1.5 ml tube to prepare a secondary antibody solution that contains 2% goat serum in PBS for Runx2 or DMP1 immunofluorescent staining. Use the secondary antibody at a dilution of 1:500.

3. Slide Preparation for Confocal Microscopy (Cell Lineage Tracing Only, No Combination with Immunofluorescence)

1. Inject tamoxifen in the Agg-cre^ERT2 mice at a favorable time point.
   1. First, remove a mouse 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. Use the right hand to hold the syringe. The optimal entry point for injection is on the left or right side of hypogastrium, avoiding the liver and bladder.
   2. Keep the syringe parallel to the hind legs of the mouse and inject intraperitoneally. The dosage for injection is 75 mg/kg. The weights of the mice at the ages of 2 weeks, 3 weeks, and 4 weeks old are approximately 7 - 9 g, 11 - 13 g, and 16 - 18 g, respectively.
2. Anesthetize the mice with a Xylazine/Ketaset combination.
   1. To prepare the working solution, first dilute the Xylazine and Ketaset with distilled water to a concentration of 1 mg/ml and 5 mg/ml, respectively. Next, mix the Xylazine and Ketaset in a 1:2 ratio. The dosage for injection is 30 µl/g.
   2. Inject as in step 3.1. Confirm the anesthetization by pinching the mouse's ankle. The mouse is unconscious if it has no reaction.
3. Perfuse the mice with 4% PFA after anesthetization.
   1. After the mouse loses consciousness, fix the four legs of the mouse on a board to entirely expose the abdomen.
   2. Saturate the abdomen with 70% ethanol and make an excision from the lower abdomen to the neck along the middle line. Pinch and simultaneously pull the skin to the lateral sides to reveal the peritoneal membrane. Use dissection scissors to make a longitudinal excision.
   3. Cut off and remove the front ribs to expose the heart. Puncture the heart from the left ventricle with a 22 G syringe, hold the syringe, and simultaneously cut a slot in the right auricle.
   4. Slowly inject the 4% PFA, which is perfused along the cardiovascular system while the blood flushes out of the cut from the right auricle. The volume of the PFA for perfusion is 1 ml/gram. Perform this step in a Class I biosafety cabinet that is hard-ducted to the building exhaust system. 
4. Peel off the mouse's skin and put the whole body into a 50-ml polypropylene centrifuge tube that contains 40 ml of 4% PFA to fix overnight at 4 °C.
5. Use dissection scissors and # 3 and # 5 forceps to carefully remove the mandible and hind leg from the body and to remove the muscles and tendons on the surface. Perform this step in a Class I biosafety cabinet that is hard-ducted to the building exhaust system.
6. Cut the mandible into two pieces at the distal region of the third molar. Similarly, cut the femur and tibia in the midshaft to expose the bone marrow cavity in order to accelerate decalcification. Put the part that includes the condyle and condylar process and along with the hind leg into 40 ml of 10% EDTA to decalcify at 4 °C for 2 - 4 days in a 50-ml polypropylene centrifuge tube.
7. Use 50 ml of 15% sucrose to dehydrate the condyle and hind leg overnight at 4 °C in a 50-ml polypropylene centrifuge tube.
8. Use 30% sucrose to dehydrate the condyle and hind leg overnight at 4 °C in a 50-ml polypropylene centrifuge tube.
9. Along the sagittal plane, embed the sample with OCT on the cutting plate in the cryosection machine.
   1. Horizontally lay the condyle or the hind leg in the mounting mold. Submerge the tissue in OCT and leave it in the cryosection machine until the OCT freezes.
   2. Mount the OCT block on the cutting plate. Wait for approximately 15 min before cutting to ensure that the OCT is completely frozen.
10. Cut the condyle and hind leg into 10-µm sections. Collect the sections on slides and store at -20 °C.
11. Incubate the slide in a 37 °C chamber to remove the water before staining.
12. Wash the slides twice with distilled water for 5 min.
13. Wipe off the water around each section. Use a hydrophobic barrier pen to circle the sections and drop DAPI or non-fluorescing anti-fade mounting solution into the circle. Carefully lay down the cover slip.

4. Immunohistochemical Staining for Runx2 and DMP1

NOTE: The IgG control is necessary for immunohistochemical staining to avoid false-positive signals. The staining for the experimental and control groups needs to be performed simultaneously.

1. Incubate the slides in a 37 °C chamber to remove the water before staining.
2. Wash the slides twice with distilled water.
3. Use the hydrophobic barrier pen to circle all the sections on the slide. From this step, add all of the prepared solution into the circle to completely cover the sections.
4. Treat the sections with hyaluronidase in a humid chamber at 37 °C for 30 min. The volume of the solution (in steps 4.5 - 4.8) depends on the size of the section. Use 50 µl of solution for the condyle and 100 µl for the long bone. Wash with PBST (PBS that contains 0.1% Tween 20) three times.
5. Prepare and apply the blocking solution to each section and incubate them in a humid chamber for 1 hr at room temperature.
6. Incubate the sections with primary antibody solution (rabbit anti-mouse Runx2, or rabbit anti-mouse DMP1) at 4 °C overnight. Wash with PBS three times.
7. Incubate the sections with secondary antibody solution (goat anti-rabbit, Alexa Fluor 488) for 2 hr at room temperature. Wash with PBS three times.
8. Wipe off the water around the section and drop DAPI into the circle to cover the section on the slide. Carefully lay down the cover slip.

5. Confocal Microscopy

1. Capture fluorescent cell images using a confocal microscope at wavelengths ranging from 488 µm (green) to 561 µm (red). Take multiple stacked images at 200 Hz (dimensions of 1,024 × 1,024)¹⁹ using 10X, 20X, and 63X lenses.

Results

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-Cre^ERT2; Rosa26^tdTomato mice activated the red-tomato reporter in all ...

Discussion

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 biology^7-9. 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...

Materials

Name	Company	Catalog Number	Comments
Tamoxifen	Sigma	T5648	activate the Cre event
Paraformaldehyde	Sigma	P6148	fix the sample
Ethylenediaminetetraacetic acid	Alfa Aesar	A10713	decalcify the hard tissue
Sucrose	Sigma	S0389	dehydrate the tissue
Hyaluronidase from bovine testes	Sigma	H4272	retrieve antigen for immunochemical staining
Bovine serum albumin	Sigma	A3059	blocking solution
primary antibody for Runx2	Cell Signal	D1L7F	primary antibody for immunochemical staining
primary antibody for DMP1	provided by Dr. Chunlin Qin		primary antibody for immunochemical staining
anti-rabbit IgG	Sigma	18140	control for immunochemical staining
secondary antibody	Invitrogen	A11008	second antibody for immunochemical staining
OCT	Tissue-Tek	4583	embed the sample for frozen section
Tween 20	Fisher Scientific	BP337	PBST
non-fluorescing antifade mountant	Life technologies	P36934	mounting slides
DAPI	Life technologies	P36931	nuclear staining
Hydrophobic Barrier Pen	Vector Laboratories		circle the section on the slide for for immunochemical staining
Xylazine	AnaSed		anesthetization
Ketaset	Zoetis		anesthetization
cryosection machine	Leica	CM1860 UV
confocal microscope	Leica	DM6000 CFS