View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Developmental Biology 252, 257–270 (2002) doi:10.1006/dbio.2002.0862 Dlx5 Is a Positive Regulator of Chondrocyte Differentiation during Endochondral Ossification Deborah Ferrari and Robert A. Kosher1 Department of BioStructure and Function, University of Connecticut Health Center, Farmington, CT 06030 The process of endochondral ossification in which the bones of the limb are formed after generation of cartilage models is dependent on a precisely regulated program of chondrocyte maturation. Here, we show that the homeobox-containing gene Dlx5 is expressed at the onset of chondrocyte maturation during the conversion of immature proliferating chondrocytes into postmitotic hypertrophying chondrocytes, a critical step in the maturation process. Moreover, retroviral misexpression of Dlx5 during differentiation of the skeletal elements of the chick limb in vivo results in the formation of severely shortened skeletal elements that contain excessive numbers of hypertrophying chondrocytes which extend into ectopic regions, including sites normally occupied by immature chondrocytes. The expansion in the extent of hypertrophic maturation detectable histologically is accompanied by expanded and upregulated domains of expression of molecular markers of chondrocyte maturation, particularly type X collagen and osteopontin, and by expansion of mineralized cartilage matrix, which is characteristic of terminal hypertrophic differentiation. Furthermore, Dlx5 misexpression markedly reduces chondrocyte proliferation concomitant with promoting hypertrophic maturation. Taken together, these results indicate that Dlx5 is a positive regulator of chondrocyte maturation and suggest that it regulates the process at least in part by promoting conversion of immature proliferating chondrocytes into hypertrophying chondrocytes. Retroviral misexpression of Dlx5 also enhances formation of periosteal bone, which is derived from the Dlx5-expressing perichondrium that surrounds the diaphyses of the cartilage models. This suggests that Dlx5 may be involved in regulating osteoblast differentiation, as well as chondrocyte maturation, during endochondral ossification. © 2002 Elsevier Science (USA) Key Words: Dlx5; chondrocyte differentiation; chondrocyte maturation; chondrocyte hypertrophy; endochondral ossifi- cation; osteoblast differentiation; bone development; limb. INTRODUCTION tions between the perichondrium and chondrocytes of the cartilage model play a major role in regulating various The skeletal elements of the limb develop by a process aspects of endochondral ossification (Long and Linsen- called endochondral ossification in which bone is formed mayer, 1998; Di Nino et al., 2001). after the establishment of a cartilaginous template (Olsen et Chondrocytes within the cartilage models progressively al., 2000; de Crombrugghe et al., 2001; Wagner and undergo a precisely regulated and coordinated program of Karsenty, 2001). At the onset of this process, mesenchymal maturation from proliferating to prehypertrophic to hyper- cells in discrete regions of the developing limb bud aggre- trophic chondrocytes (Olsen et al., 2000; de Crombrugghe gate into precartilage condensations which prefigure the et al., 2001; Wagner and Karsenty, 2001). Maturation com- future skeletal elements. The cartilage anlagen undergo mences in the central regions or diaphyses of the cartilage rapid longitudinal and appositional growth as a result of models where immature chondrocytes exit the cell cycle, proliferation and matrix deposition to form the cartilage enlarge, and are converted into prehypertrophic chondro- models of the bones of the limb. The developing cartilage cytes which are characterized by the expression of the models become surrounded by a longitudinally oriented secreted signaling molecule Indian hedgehog (Ihh) (Vort- group of flattened cells called the perichondrium. Interac- kamp et al., 1996). The prehypertrophic chondrocytes un- dergo further differentiation to hypertrophic chondrocytes 1 To whom correspondence should be addressed. Fax: (860) 679- during which they continue to enlarge, initiate expression 2910. E-mail: [email protected]. of type X collagen and other hypertrophic markers, includ- 0012-1606/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved. 257 258 Ferrari and Kosher ing osteopontin and bone sialoprotein, and deposit a min- Inada et al., 1999). Wnt-4 expressed by cells flanking the eralized matrix. In parallel with the differentiation of hy- epiphysis of the cartilage models has also been implicated pertrophying chondrocytes, the perichondrium flanking the in the positive control of maturation (Hartmann and Tabin, maturing chondrocytes becomes the periosteum, which is 2000), and gain- and loss-of-function studies in vitro and in the source of osteogenic cells that deposit a bony collar vivo have shown that the transcription factor Cbfa1/Runx2 around the diaphyses of the cartilage rudiments (Pechak et expressed by prehypertrophic and hypertrophic chondro- al., 1986a). Blood vessels arising from the periosteum in- cytes is an important positive regulator of the maturation vade into the area of chondrocyte hypertrophy, a process process (Inada et al., 1999; Kim et al., 1999 Enomoto et al., dependent on vascular endothelial growth factor (VEGF) 2000; Takeda et al., 2001; Ueta et al., 2001; Leboy et al., expressed by hypertrophic chondrocytes (Gerber et al., 2001). 1999). Vascular invasion of the hypertrophic zone is accom- Identification of the regulatory genes and signaling mol- panied by degradation and resorption of the mineralized ecules that control the progression of chondrocyte matura- cartilage matrix, its replacement by trabecular bone depos- tion toward hypertrophy and the regulatory interactions ited by invading osteoblasts, and the concomitant excava- among them is crucial to understanding normal and abnor- tion of the bone marrow cavity. The continued maturation mal bone growth and development. In the present study, we of immature proliferating chondrocytes into hypertrophy- provide evidence indicating that the homeodomain tran- ing chondrocytes and their replacement by trabecular bone scription factor Dlx5 is an important positive regulator of and bone marrow results in the longitudinal growth of the chondrocyte maturation during endochondral ossification. long bones. Thus, a critical step in the longitudinal growth Dlx5 is one of six members of the Dlx family of homeobox- of long bones and endochondral ossification is the conver- containing genes that are homologs of the Drosophila sion of immature proliferating chondrocytes into hypertro- Distal-less gene, which is required for limb development in phying chondrocytes. Indeed, hypertrophic maturation ap- the fly embryo (Merlo et al., 2000; Zerucha and Ekker, pears to be the main determinant of longitudinal growth 2000). Dlx5 has been implicated in several aspects of early (Hunziker, 1994). vertebrate limb morphogenesis, including specification of The rate and progression of hypertrophic chondrocyte the limb territories of the lateral plate, activity of the apical maturation during endochondral ossification is regulated by ectodermal ridge which directs limb outgrowth, and pat- a coordinated balance between negative and positive sig- terning along the anteroposterior axis (Ferrari et al., 1995, nals. Ihh and parathyroid hormone-related peptide (PTHrP) 1999). Later in development, Dlx5 is expressed by differen- function in a regulatory loop to negatively control chondro- tiating osteoblasts during intramembranous and endochon- cyte maturation (Vortkamp et al., 1996; Kronenberg et al., dral ossification (Simeone et al., 1994; Zhao et al., 1994), 1997; St-Jacques et al., 1999). In particular, Ihh expressed by and several studies suggest that it may play a role in newly postmitotic prehypertrophic chondrocytes induces osteoblast differentiation (Newberry et al., 1998; Miyama et PTHrP expression in the periarticular perichondrium adja- al., 1999; Benson et al., 2000; Tadic et al., 2001, 2002). cent to the immature proliferating chondrocytes in the Here, we report that Dlx5 is expressed during the conver- epiphysis. In turn, PTHrP signals via its receptor PTH/ sion of immature proliferating chondrocytes into postmi- PTHrP-R to suppress the maturation of proliferating chon- totic hypertrophying chondrocytes, which is a critical step drocytes in the subjacent epiphysis, thus maintaining a in chondrocyte maturation during endochondral ossifica- zone of immature chondrocytes competent to undergo tion. Moreover, retroviral misexpression of Dlx5 during the proliferation (Vortkamp et al., 1996; Kronenberg et al., differentiation of the skeletal elements of the chick limb in 1997; St-Jacques et al., 1999). Members of the TGF-␤ family vivo promotes chondrocyte maturation, resulting in the produced by the perichondrium have also been implicated formation of severely shortened skeletal elements that as negative regulators of chondrocyte maturation (Serra et contain excessive numbers of hypertrophying chondro- al., 1997) and may act upstream of PTHrP to suppress the cytes. Our results indicate that Dlx5 positively regulates process (Serra et al., 1999). In addition, the transition from chondrocyte maturation, and may do so at least in part by prehypertrophic to hypertrophic chondrocytes is negatively promoting conversion of immature proliferating chondro- controlled by Wnt-5a produced