Col2-GFP reporter marks chondrocyte lineage and chondrogenesis during mouse skeletal development.

ABSTRACT Mice were generated in which a Col2-GFP transgene serves as a reporter for thechondrocyte lineage and for chondrogenesis inlive embryos and newborn pups. Cells activelyengaged in chondrogenesis were identiﬁed byconfocal optical sectioning within their nativeenvironments in embryos and in thick tissueslices. Chondrocytes exhibiting GFP ﬂuores-cence were puriﬁed from rib cages by high-speedcell sorting of crude cell suspensions. Intensity ofﬂuorescence correlated with biosynthesis of pro-collagen II in these cells. The use of these miceand their cells provides a novel approach forstudying chondrocyte differentiation and chon-drogenesis during skeletal development. Dev Dyn2000;218:394–400. © 2000 Wiley-Liss, Inc. Key words: transgenic mice; reporter mice; greenﬂuorescent protein; type II collagen;chondrocytes; chondrogenesis; skel-etal development; confocal micros-copy; cell sortingINTRODUCTION Chondrocytes play a key role in formation and main-tenance of the vertebrate skeleton (Tickle and Eichele,1994; Erlebacher et al., 1995; Johnson and Tabin, 1997;Beier et al., 1999; Neame et al., 1999; St-Jacques et al.,1999; Stevens and Williams, 1999). They differentiatein response to signals that determine where the futureskeleton will form. They proliferate, terminally differ-entiate and die in the growth plate in response tosignals that regulate linear bone growth. They synthe-size and maintain cartilage matrices that serve as tem-plates for endochondral ossiﬁcation, account for thefunctional properties of permanent cartilages and con-tribute to the repair of cartilage injuries. In short,chondrocytes exhibit a number of adaptive behaviors inresponse to changing environments in skeletal tissues.Despite their importance to skeletal developmentand homeostasis, chondrocyte behaviors are difﬁcult tostudy because the microenvironments responsible forthem are difﬁcult to reproduce experimentally. Indeed,loss of typical chondrocyte characteristics, i.e., dediffer-entiation, has been an obstacle for study of chondro-cytes, especially mammalian chondrocytes, in vitro(Benya et al., 1978; Benya and Shaffer, 1982; Ault-house et al., 1989; Ramdi et al., 1993). Even if chon-drocyte phenotypes can be produced in vitro, it is dif-ﬁcult to be certain that in vitro behaviors accuratelyreﬂect in vivo behaviors given the substantial differ-ences in microenvironment. Consequently, investiga-tors often turn to in situ approaches in which experi-ments are carried out in living animals or in organcultures that maintain native environments. These tis-sues are examined after ﬁxation with probes that canbe detected microscopically. Such analyses providesnapshots of relevant behaviors, but dynamic aspectsof chondrocyte behavior can not be studied directlybecause preparation of tissues for conventional micros-copy kills cells.We have developed an approach to study chondrocytebiology during skeletal development that allows anal-ysis of live cells within tissues with the potential forreal-time studies of adaptive behaviors relevant toskeletal biology. Transgenic mice were generated inwhich expression of enhanced Green Fluorescent Pro-tein (GFP) is controlled by the murine type II collagenpromoter/enhancer (