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 s....

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

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

List of materials used in this article

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