Distinguishing the Contributions of the Perichondrium, Cartilage, and Vascular Endothelium to Skeletal Development

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Developmental Biology 269 (2004) 55–69 www.elsevier.com/locate/ydbio

Distinguishing the contributions of the perichondrium, cartilage, and vascular endothelium to skeletal development

Ce´line Colnot, Chuanyong Lu, Diane Hu, and Jill A. Helms*

Department of Orthopaedic Surgery, University of California, San Francisco, CA 94143-0514, USA Received for publication 10 November 2003, revised 8 January 2004, accepted 9 January 2004

Abstract

During the initiation of endochondral ossification three events occur that are inextricably linked in time and space: chondrocytes undergo terminal differentiation and cell death, the skeletal vascular endothelium invades the hypertrophic cartilage matrix, and osteoblasts differentiate and begin to deposit a bony matrix. These developmental programs implicate three tissues, the cartilage, the perichondrium, and the vascular endothelium. Due to their intimate associations, the interactions among these three tissues are exceedingly difficult to distinguish and elucidate. We developed an ex vivo system to unlink the processes initiating endochondral ossification and establish more precisely the cellular and molecular contributions of the three tissues involved. In this ex vivo system, the renal capsule of adult mice was used as a host environment to grow skeletal elements. We first used a genetic strategy to follow the fate of cells derived from the perichondrium and from the vasculature. We found that the perichondrium, but not the host vasculature, is the source of both trabecular and cortical osteoblasts. Endothelial cells residing within the perichondrium are the first cells to participate in the invasion of the hypertrophic cartilage matrix, followed by endothelial cells derived from the host environment. We then combined these lineage analyses with a series of tissue manipulations to address how the absence of the perichondrium or the vascular endothelium affected skeletal development. We show that although the perichondrium influences the rate of chondrocytes maturation and hypertrophy, it is not essential for chondrocytes to undergo late hypertrophy. The perichondrium is crucial for the proper invasion of blood vessels into the hypertrophic cartilage and both the perichondrium and the vasculature are essential for endochondral ossification. Collectively, these studies clarify further the contributions of the cartilage, perichondrium, and vascular endothelium to long bone development. D 2004 Elsevier Inc. All rights reserved.

Keywords: Perichondrium; Hypertrophic chondrocyte; Angiogenesis; Endochondral ossification; Osteoblast; Lineage analysis; Renal capsule transplantation

Introduction given set of instructive cues was constrained by its own developmental history. Fifty years of experimental embry- One of the fundamental principles of vertebrate devel- ology have provided overwhelming support that these opment is that organogenesis is the cumulative result of principles are applicable to all organ development, including tissue interactions. By the first part of the 20th century, Hans that of the skeleton. Spemann, along with their students and colleagues, eluci- Two tissues that give rise to the appendicular skeleton, dated two elemental tenets underlying these tissue interac- namely the cartilage and the perichondrium, are derived tions (Spemann and Mangold, 1924). The first principle was from the same population of mesenchymal cells. In response that one tissue acted in an instructive role and could to unknown cues, some mesenchymal cells begin to aggre- instigate an entire morphogenetic program, even if it had gate into a condensation, the shape of which presages the to do so with a naive partner tissue. The second tenet was future skeletal element. Shortly thereafter, mesenchymal that the extent to which a naive tissue could respond to a cells that surround this condensation begin to flatten, elongate, and form the perichondrium. Cells in the central condensation differentiate into chondrocytes and form the * Corresponding author. Department of Orthopedic Surgery, University of California at San Francisco, 533 Parnassus Avenue, U-453, San cartilage anlagen while those in the perichondrium differ- Francisco, CA 94143-0514. Fax: +1-415-476-1128. entiate into osteoblasts and give rise to the periosteum E-mail address: [email protected] (J.A. Helms). (Eames et al., 2003; Hall and Miyake, 2000).

A third tissue, the vascular endothelium, is also involved were used as either donor or host for lineage analyses. in the process of skeletogenesis. Initially, a vascular network Rosa26 mice were genotyped using whole mount X-gal occupies the entire limb field. In the region of the chondro- staining on tail tissue. Briefly, the tip of the tail was cut genic condensation, blood vessels are cleared just before and the tissue was fixed in 0.2% glutaraldehyde (0.2% mesenchymal cells begin to aggregate. Endothelial cells glutaraldehyde, 5 mM EDTA and 2 mM MgCl2 in 1 Â continue to occupy the surrounding area until a signal from PBS) for 30 min at room temperature or in 0.4% parafor- terminally differentiated chondrocytes induces them to once maldehyde at 4jC overnight. The tissue was then washed again invade the hypertrophic cartilage matrix (Kronenberg, with wash buffer (2 mM MgCl2, 0.01% sodium deoxy- 2003). Thus, the skeleton is generated as a result of cholate, 0.02% NP40 and 0.1 M PB in H2O) three times for interactions among three tissues, the cartilage, the perichon- 15 min and incubated in X-gal staining solution (1 mg/ml drium, and the vascular endothelium. X-Gal, 2.1 mg/ml potassium ferrocyanide, and 1.64 mg/ml As one might expect, disruptions to the development or potassium ferricyanide in wash buffer) overnight at 37jC. maturation of one tissue will adversely affect development Rosa26-positive mice exhibited blue staining. or maturation of any associated tissue (reviewed in de Mice used as host for renal capsule transplantations Crombrugghe et al., 2001; Karsenty and Wagner, 2002). were 3–6 months old. Embryos used as donor tissues The skeletal phenotypes of several null mutant mice exem- were obtained by precisely timed pregnancies. On day 0, plify this complex interplay between the cartilage, the a 3-h window was permitted for mating. To obtain e14.0 perichondrium, and the vasculature (Zelzer and Olsen, embryos, pregnant mice were sacrificed by cervical disloca- 2003). For example, the most obvious defect of Runx2À/À tion following anesthesia, 14 days after mating during the embryos is the absence of bone (Komori et al., 1997; Otto et same 3-h time period. al., 1997). However, long bones in Runx2À/À embryos also have a defect in cartilage differentiation (Inada et al., 1999; Renal capsule transplantations (ex vivo culture of skeletal Kim et al., 1999) and in vascular invasion (Zelzer et al., elements) 2001). This mouse phenotype highlights a conundrum: how does one effectively distinguish among the cell-autonomous Anlagen of the stylopod were removed from e14.0 and cell non-autonomous effects of delayed chondrocyte embryos and transillumination was used to identify the differentiation, faulty osteoblast differentiation, and defec- tissue-dense condensation of the humerus or the femur. tive angiogenesis? Cartilage elements were dissected free of the surrounding We used this question as a starting point and devised an connective tissues then transplanted directly, or after in vitro approach that allowed us to dissect apart the processes of manipulation (see below), underneath the renal capsule of cartilage differentiation, perichondrial maturation, and an- adult wild-type mice (Hom and Cunha, 2000). All proce- giogenic invasion at an early stage of fetal skeletogenesis. dures followed standard UCSF CAR/LARC protocols. Mice The renal capsule has been used to great advantage to were anesthetized, an incision was made in the back skin characterize tissue interactions that govern the development and the body wall and the kidney was exteriorized. A small of many organs (Ferguson et al., 2001; Norman et al., 1986; incision was made in the renal capsule and a small fire- Vu et al., 2003). We modified this ex vivo method to polished Pasteur pipette was used to open the capsule specifically investigate the contributions of the hypertrophic pocket. One or two samples (approximately 1.0 mm in cartilage, perichondrium, and the skeletal vascular endothe- length) were grafted into a host mouse, the kidney eased lium that are crucial to the program of bone formation. back into the body cavity, the body wall sutured and the Using this model system, we developed a unique approach back skin aligned and closed using wound clips. The to elucidate osteoblast and endothelial cell lineages during development and vascularization of cartilage elements were skeletogenesis. In addition, we were able to address the role assessed after 24, 48 h, 3 days to 8 weeks. of the perichondrium in vascular invasion and endochondral ossification and showed that direct interactions between the Lineage analyses perichondrium and the surrounding vasculature are required for endochondral ossification. Cartilage elements were isolated from e14.0 Rosa26 embryos and transplanted to wild-type host mice to follow cells derived from the graft, or the reverse, to follow cells Materials and methods derived from the host. To follow cells derived specifically from the perichondrium, cartilage elements from e14.0 wild- Mice generation and genotyping type embryos without perichondrium were recombined in vitro for 2–6 h with perichondrium dissected from e14.0 C57B6 mice were used as hosts and donors for the renal Rosa26 embryos; the chimeric element was then trans- capsule transplantations to avoid complications arising from planted into wild-type host mice. Samples were harvested immune rejection. Rosa26 mice (Jackson Laboratory, Maine, at 6, 10, 16 days and up to 8 weeks, processed for whole USA) carrying the LacZ transgene in a C57B6 background mount X-gal staining (see mice generation and genotyping), C. Colnot et al. / Developmental Biology 269 (2004) 55–69 57 dehydrated in a graded ethanol series, and embedded in Tissue processing and histology paraffin. Sections were counter-stained in eosin. At various time points following renal capsule transplan- Removal of the perichondrium tation or culture, tissue samples were dissected under RNase-free conditions and fixed in 4% paraformaldehyde Removal of the perichondrium was performed either to at 4jC overnight. Samples dissected from the renal capsule follow the development of cartilage anlagen alone or to more than 4 days post-transplantation were decalcified at recombine perichondrial tissue with cartilage anlagen. In the 4jC in 19% EDTA, pH 7.4, for 1–7 days, following which first case, fetal perichondrium was removed from e14.0 tissues were dehydrated and embedded in paraffin. Five cartilage elements using both enzymatic digestion and tissue micron-thick sections were cut and collected on TESPA- dissection. After removing loose connective tissues, the treated slides for histology using Safranin-O/Fast Green cartilage anlagen were treated with dispase (1 mg/ml in (SO/FG) and Trichrome (TC) staining. PBS, for 5 min at room temperature). During the dispase treatment, the perichondrium was partially removed using Immunohistochemistry and TUNEL thin forceps. The tissue was transferred in PBS to stop the reaction and the remaining perichondrium was carefully Tartrate resistant acid phosphatase (TRAP) staining was dissected in the central part of the cartilage element sur- performed using a leucocyte acid phosphatase kit (Sigma, rounding the proliferative, mature and hypertrophic chon- St. Louis, MO). Tissue sections were then counter-stained in drocytes, and around the articular surfaces. Total removal of 0.1% Fast Green or 5% Methyl Green. For platelet endo- the perichondrium was a difficult task to achieve; in some thelial cell adhesion molecule (PECAM) immunohisto- cases, residual perichondrium was observed on tissue sec- chemistry, tissue sections were immersed in 0.3% H2O2/ tions (see Figs. 4E and 5F). In the second case, the enzymatic methanol for 5 min, washed in PBS, treated with a ficin digestion was not applied to preserve the cells at the solution (Zymed, South San Francisco, CA) for 5 min at cartilage–perichondrium interface. Instead, the perichondri- 37jC, washed in PBS, then with 0.1 M glycine for 30 s, um was isolated using forceps only. Cartilage elements, with washed in PBS, and incubated in the following blocking or without perichondrium, were directly transplanted to the solutions and interspersed with intermediate PBS washes: renal capsule for 1 day to 1 month or were cultured for 24 to 5% powdered milk for 10 min, 1.0 mg/ml ovalbumin for 10 48 h in BGJ medium containing 10% FCS and penicillin- min, and 5% goat serum for 30 min. Sections were incu- streptavidin. Small variations in developmental stage exist bated overnight at 4jC in monoclonal rat anti-mouse among embryos of the same litter; therefore, the contra- PECAM-1 (1:100, BD Pharmingen, San Diego, CA). Sec- lateral skeletal element was always used as a control. tions were washed in PBS, blocked in 5% sheep serum for 30 min, and incubated for 1 h at room temperature in mFlt-IgG treatment biotinylated anti-rat IgG (1:100, BD Pharmingen). Slides were washed in PBS and incubated in horseradish peroxi- To prevent cartilage angiogenesis, we produced a tran- dase-conjugated streptavidin (Amersham, Cleveland, OH) sient, localized inhibition in VEGF signaling by using a and binding was revealed by incubation in a diaminobenzi- recombinant soluble receptor mFlt(1–3)-IgG (mFlt-IgG, N. dine solution containing < 1% CoCl2 and < 1% Ni3SO4 for Ferrara, Genentech, Gerber et al., 1999). Affigel blue beads PECAM immunostaining. Sections were counter-stained in were soaked in the soluble mFlt-IgG (10.9 mg/ml) for 1 h at 0.1% Fast Green or 5% Methyl Green. 37jC and implanted adjacent to the cartilage elements (4 TUNEL staining was performed to detect DNA fragmen- beads per sample) at the time of transplantation in the renal tation in apoptotic cells using a kit from Roche Diagnostics capsule. No bead or beads soaked in PBS were used as Corporation (Indianapolis, IN). Apoptotic cells were labeled controls. Alternatively, cartilage elements were cultured for by incubating the tissue sections with fluorescein-labeled 24 h in BGJ medium containing mFlt-IgG (100 Ag/ml) and nucleotides and terminal deoxynucleotidyl transferase. Sec- transplanted in the renal capsule. Samples were harvested at tions were then counterstained with Hoechst dye to visualize 4 and 7 days and assessed for vascular invasion and bone the nuclei. Using Adobe Photoshop, TUNEL staining (in formation. Equivalent results were obtained with both green) and Hoechst dye staining (colorized in red) were methods of treatment. superimposed, thus allowing the detection of TUNEL-positive nuclei (yellow). Blocking physically the host vasculature In situ hybridization To obtain a more efficient inhibition of vascular invasion, we used two filters (Millipore millex-GV 0.22 Am) that were In situ hybridization was performed using 35S-labelled interposed (1) between the graft and the kidney capsule and antisense riboprobes corresponding to cDNAs for Collagen (2) between the graft and the kidney (see Fig. 7). Nutrients type X (ColX), Collagen type I (Col1), Osteocalcin (Oc), were able to diffuse through and in between the filters. Osteopontin (Op), matrix metalloproteinase (MMP13), 58 C. Colnot et al. / Developmental Biology 269 (2004) 55–69

Indian hedgehog (Ihh) and vascular endothelial growth developed, ex vivo, for varying periods of time (Fig. 1). The factor (VEGF). Tissues were hybridized and signal was forelimb stylopod undergoes vascular invasion and ossifi- visualized as described previously (Ferguson et al., 1999). cation at e15.0 (Colnot and Helms, 2001); therefore, we isolated the stylopod anlagen at e14.0 in order that subsequent angiogenesis and bone formation would occur in the Results new environment. We next performed a variety of histological analyses and An ex vivo environment supports skeletogenesis confirmed that chondrocytes underwent normal hypertrophy, terminal differentiation, and removal in the ex vivo Three concurrent events are critical for long bone devel- environment (Figs. 1A–F). The hypertrophic cartilage ma- opment: chondrocytes must undergo terminal differentia- trix was invaded by the vasculature and a marrow cavity tion, the vasculature must invade the hypertrophic cartilage was established (Figs. 1E–H, black arrows). An ossification matrix, and osteoblasts must initiate their differentiation. In center formed in the central region of the anlagen and vivo, these events transpire within the space of 10–12 growth plates were evident at the bone ends (Figs. 1I–P). h (Colnot and Helms, 2001). Consequently, the nature of Within the course of 1 month, we noticed minor differences the tissue interactions between the cartilage, the perichon- between in vivo and ex vivo skeletal development: bone drium, and the vascular endothelium is not well understood. length was reduced by 20% ex vivo compared to in vivo We set out to devise a method to separate these events in (data not shown). Ex vivo, bones were wider and thinner time and space. We began by isolating the anlagen of the probably due to the mechanical influence of the kidney stylopod from donor embryos and transplanting them un- membrane. Moreover, the articular cartilage layer was thin derneath the renal capsule of syngenic host mice where they and irregular, suggesting that joint morphogenesis requires

Fig. 1. Ex vivo skeletal development. Morphological and histological analyses using Safranin-O/Fast Green (SO-FG, second line) and Trichrome (TC, third and fourth lines) staining demonstrated that explanted e14.0 skeletal anlagen underwent proper development in the renal capsule. (A, B) At the time of transplant (e14.0, 0 h), the humerus was comprised of cartilage surrounded by perichondrium (pc); hypertrophic cartilage (hc, arrow and bracket) was present in the center of the cartilage anlage. (C, D) After 24 h ex vivo, the overall size of the cartilage anlage was increased by 40%. The hypertrophic zone was enlarged (arrow, bracket), and was separated by a zone of late hypertrophic chondrocytes (lhc) in the central region. (E, F) After 48 h ex vivo, the cartilage anlage had doubled in size, and vascular invasion (arrowhead) and cartilage degradation (black arrows) were detected in the center of the hypertrophic zone. (G, H) After 72hex vivo, the ossification center (oc) had developed and vascular invasion was progressing (arrowhead). (I–L) After 7 days ex vivo, the skeletal element was fully vascularized, the growth plates (gp) were established, the marrow cavity (mc) was expanding, and abundant new bone was detected in (K) the metaphysis and (L) the diaphysis. (M–P) After 16 days ex vivo, the overall size of the skeletal element had increased 5-fold, the secondary ossification center was established (arrow), the growth plate was well organized and bone remodeling was occurring. (K, O) correspond to boxed areas in (J, N), respectively. lhc: late hypertrophic chondrocytes, po: periosteum, rz: resting zone, pz: proliferating zone, hz: hypertrophic zone, tb: trabecular bone. Scale bars: (A, C,E,G,I,M)= 1 mm; (B, D, F, H) = 200 Am, (J, N) = 400 Am; (K, L, O, P) = 200 Am. C. Colnot et al. / Developmental Biology 269 (2004) 55–69 59 additional stimuli for proper development. These data indi- the adaptation of the graft tissue to the new environment and cated that although the ex vivo environment supported this phenomenon is usually observed in tissue explants. normal bone formation and bone remodeling, the shape and size of skeletal elements were slightly affected by the Osteoblasts are derived from the perichondrium absence of surrounding muscles, tendons, and other skeletal elements. In our ex vivo system, the host environment supported the growth of the grafted skeletal element and also appeared to The ex vivo environment prolongs maturation of the participate actively in vascular invasion and ossification of cartilage and the perichondrium the grafted cartilage element. To distinguish between the cellular contribution of the graft and the contribution of the Our next goal was to compare the time course of cartilage host to the process of vascular invasion and ossification, we differentiation, perichondrial maturation, and vascular inva- performed a series of lineage analyses that made use of sion in the ex vivo system and in vivo, by employing a Rosa26 mice, which carry the LacZ transgene under the variety of histological, cellular, and molecular assays. At control of a ubiquitous promoter (Friedrich and Soriano, e14.0, the time at which the stylopod anlagen were 1991). First, we focused on the tissue contributions to explanted, chondrocytes had entered late hypertrophy as ossification. determined by the presence of a few MMP13-expressing The Rosa26 mice first acted as hosts and their syngenic, chondrocytes within the ColX expression domain (Fig. 2A). wild-type counterparts served as the source for the stylopod PECAM-positive cells were present in the perichondrium explants. X-gal staining was used to follow the fate of cells (Fig. 2B) as were TRAP-positive osteoclasts (Fig. 2C).By derived from the host environment during the process of e14.5 in vivo, chondrocytes exhibited strong MMP13 ex- skeletogenesis. Since osteoclasts and blood cells are derived pression in the central region of the anlagen (Fig. 2D). from the hematopoietic lineage (reviewed in Chambers, Endothelial cells, which expressed PECAM and exhibited a 2000), we had a mechanism to confirm that the host Rosa26 characteristic morphology (Fig. 2E), accumulated at the site vasculature participated in endochondral ossification of the predestined for vascular invasion. TRAP-expressing osteo- graft. While most osteoclasts and blood cells in these clasts were found at the same location (Fig. 2F). By e15.0 in explants were X-gal positive (Figs. 3A–I), we found that vivo, terminally differentiated hypertrophic chondrocytes osteoblasts and osteocytes in the periosteum and in bone were in the process of being replaced by the marrow cavity. trabeculae were X-gal negative (n = 35; Figs. 3B and F, PECAM-positive endothelial cells had invaded the cartilage black arrows). These findings demonstrated that the host matrix. TRAP-positive osteoclasts and MMP13-positive vasculature contributed to the development of the explanted osteoblasts (Sasano et al., 2002) were in the center of the skeletal elements, and that osteoblasts were not delivered newly formed ossification center (Figs. 2G–I). via the Rosa26 vasculature. Instead the data suggested that The same molecular and cellular processes occurred ex the transplanted stylopod was the source of the osteoblasts. vivo, but instead of taking place within a 24-h window they Next, we reversed the identity of host and donor tissue so occurred over 72-h time period. Thus, 24 h after e14.0 that stylopod explants were derived from Rosa26 embryos stylopod elements were explanted to the renal capsule, and the host mice were wild-type. Osteoblasts and osteo- chondrocytes were still in the late hypertrophy stage as cytes were now X-gal positive (n = 12; Figs. 3J and K), indicated by MMP13 expression (Fig. 2J). PECAM- and confirming that they were derived from the skeletal anlagen. TRAP-positive cells were not yet localized to the site As expected, the majority of blood cells was X-gal negative presaged for vascular invasion (Figs. 2K and L). After 48 (Figs. 3J and M), demonstrating that they were derived from h ex vivo, terminally differentiated chondrocytes were be- the wild-type host vasculature. ginning to be removed, PECAM-positive cells had invaded To identify if the perichondrium was the primary source the cartilage matrix, and TRAP-positive cells had begun of osteoblasts, we isolated e14.0 wild-type cartilage anlagen degrading the hypertrophic cartilage matrix (Figs. 2M–O). and recombined them with e14.0 Rosa26 perichondrium, After 72 h ex vivo, the primary ossification center was then transplanted the recombinations into a wild-type host. established, which effectively separated the expression do- In these samples, osteoblasts and osteocytes within the bone main of ColX (Fig. 2P, brackets). PECAM staining delineat- trabeculae, as well as those in the periosteum, were X-gal ed the newly forming marrow cavity, and osteoclasts were positive (n =4;Figs. 3N and O). These findings demon- evident within the primary ossification center (Figs. 2Q and strated that both trabecular and periosteal osteoblasts orig- R). Thus, the programs of chondrocyte terminal differentia- inated from the perichondrium. tion, vascular invasion, and ossification were indistinguish- able between the in vivo and ex vivo settings, with one The skeletal vascular endothelium has a dual derivation exception. The entire process was prolonged ex vivo, which afforded us the opportunity to separate some of the molecular Using the same experimental strategy, we explored the and cellular events that led to the rapid initiation of endo- source of endothelial cells that invade the cartilage anlagen chondral ossification. This prolongation was certainly due to and lead to endochondral ossification. When Rosa26 mice 60 C. Colnot et al. / Developmental Biology 269 (2004) 55–69

Fig. 2. Terminal differentiation and vascular invasion of hypertrophic cartilage in the ex vivo skeletal elements was delayed by 48 h. In situ hybridization and immunohistochemical analyses of e14.0 humerus transplanted in the renal capsule for 24–72 h (J–R), compared to its in vivo development (A–I). (A) At e14.0, hypertrophic cartilage (hc) expressing collagen type X (colX, red) occupied the central region of the developing humerus. Few late hypertrophic chondrocytes expressing MMP13 were localized to the periphery of the hypertrophic zone (white arrows). (B) Endothelial cells labeled by PECAM antibody were detected in the perichondrium lining the cartilage anlage and in the surrounding soft tissue (black staining, arrowheads). (C) Very few TRAP-positive osteoclasts were found in the perichondrium (purple staining, arrow) and had not become localized to the future site of vascular invasion. (D) At e14.5, the colX-expressing hypertrophic cartilage (red) was enlarged and a characteristic constriction was seen in the center of the anlage, where (E) PECAM-positive endothelial cells (arrowheads) and (F) TRAP-positive osteoclasts (arrow) accumulated adjacent to late hypertrophic chondrocytes that expressed MMP13 (D, yellow). (G) By e15.0, the colX expressing (red) hypertrophic zone was separated by the area of vascular invasion (brackets) where MMP13-positive osteoblasts were detected (yellow), as well as (H) PECAM and (I) TRAP-positive cells. (J) Twenty-four hours after transplantation into the renal capsule, the center of the cartilage element contained late hypertrophic chondrocytes expressing MMP13 (yellow). (K) Very few PECAM (arrowhead) and (L) TRAP- positive cells were surrounding the hypertrophic cartilage. (M) After 48 h ex vivo, the hypertrophic zone started to be separated into two colX expressing domains (red). (N) PECAM (arrowheads) and (O) TRAP-positive (arrows) cells were found in the hypertrophic cartilage. (P) After 72 h ex vivo, the hypertrophic zone was completely separated into two distinct domains (red), and was invaded by MMP13-positive osteoblasts (yellow), (Q) PECAM-positive endothelial cells (arrowheads) and (R) TRAP-positive osteoclasts (arrows). oc: ossification center, Scale bars = 200 Am. C. Colnot et al. / Developmental Biology 269 (2004) 55–69 61

Fig. 3. Lineage analysis of osteoblast and endothelial cells during long bone development. (A–Q) X-gal staining (blue) and eosin counterstain on longitudinal sections of cartilage elements transplanted underneath the renal capsule and (B–D, F–I, K–M, O–Q) double staining with X-Gal and TRAP on adjacent sections shown at high magnification (right column). (A–I) E14.0 wild-type humerus transplanted in Rosa26 host kidney capsule. (A, C) At 6 days post- transplantation, X-gal-positive and TRAP-positive osteoclasts derived from the host were located at the chondrovascular junction and in the diaphysis (arrowheads). (B) Osteocytes and osteoblasts (black arrows) in the bone trabeculae and in the periosteum were X-gal-negative therefore derived from the graft. (D) Endothelial cells in the ossification center were also X-gal-negative (red arrow). (E–I) At 16 days, X-gal-positive osteoclasts (arrowheads) and blood cells (white arrowhead) derived from the host are detected in the marrow cavity. (F) X-gal-negative osteocytes and osteoblasts (black arrows) are derived from the graft. (H) Both X-gal-negative and (I) X-gal-positive endothelial cells (red arrows) are found in the metaphysis and diaphysis indicating that the host and the graft contribute as a source of endothelial cells. (J–M) E14.0 Rosa26 humerus transplanted in wild-type kidney capsule, harvested at 6 days post-transplant. (J) Hypertrophic chondrocytes, cells in the periosteum and in bone trabeculae derived from the graft were all X-gal-positive, except blood cells (white arrowhead), which were derived from the host. (K, L) Osteoblasts and osteocytes (black arrows) as well as (M) endothelial cells (red arrow) were X-gal-positive, confirming that they were derived from the graft. (L) Some TRAP-positive osteoclasts were also X-gal-positive (arrowhead). (N–Q) E14.0 wild-type humerus without perichondrium recombined with e14.0 Rosa26 perichondrium and transplanted into a wild-type kidney capsule. (N) At 10 days post-transplant, X-gal-positive cells were detected at the chondrovascular junction (arrowhead), in the periosteum and in bone trabeculae (black arrows). (O) Osteocytes (arrow) and (P) some TRAP-positive osteoclasts (arrowhead) were X-gal-positive, indicating that osteoblasts were derived from the perichondrium. (Q) X-gal-positive endothelial cells derived from the perichondrium were also found (red arrows). Schematics on the right indicated the origin of the graft (g) and the host (h) tissue (blue = Rosa26, white = wild-type). hc: hypertrophic cartilage; po: periosteum, Scale bar: (A, E, J, N) = 100 Am, (B–D, F–I, K–M, O–Q) = 10 Am. acted as hosts and the e14.0 anlagen were isolated from wild- time points (e.g., starting at 7 days ex vivo), we began to type embryos, the endothelial cells that invaded the hyper- detect X-gal-positive endothelial cells within the skeletal trophic cartilage matrix by day 6 were X-gal-negative (Fig. anlagen (Fig. 3I, 16 days ex vivo), indicating that endothelial 3D). These results suggest that endothelial cells, which reside cells from the host vasculature eventually participated in in the perichondrium, were the primary contributor to the vascular invasion and the formation of the marrow cavity. We vessels that had invaded the hypertrophic cartilage. At later confirmed these results by reversing the source of host and 62 C. Colnot et al. / Developmental Biology 269 (2004) 55–69 donor tissue. When wild-type mice acted as the host and the e14.0 anlagen were isolated from Rosa26 embryos, endothelial cells were X-gal-positive (Fig. 3M, day 6), once again indicating their primary derivation from the perichondrium. Using the strategy of recombination between Rosa26 perichondrium and wild-type cartilage anlagen, we were able to confirm that the endothelial cells that had invaded the hypertrophic cartilage matrix were derived from the perichondrium by day 10 (Fig. 3Q). Only at later stages did we observe endothelial cells from the host vasculature that began to participate in vascular invasion and the formation of the marrow cavity (data not shown and Fig. 3I). Alto- gether, these results indicate that the perichondrium is a source of cells that are required for vascular invasion and bone formation.

Chondrocyte differentiation occurs in the absence of perichondrial signals

If the perichondrium is the principle source of osteoblasts and endothelial cells, we reasoned that its removal would affect bone formation and vascular invasion during endochondral ossification. Therefore, we dissected e14.0 stylopod elements free of their perichondrium and explanted them to the renal capsule, where their development was compared to that of contralateral elements whose perichondrium remained intact. We first assessed the effects of perichondrial removal on chondrocyte hypertrophy and terminal differentiation, since chondrocyte differentiation is intimately linked with perichondrial maturation (Chung et al., 1998, 2001; Karp et al., 2000; Liu et al., 2002; Vortkamp et al., 1996). Fig. 4. Terminal differentiation of chondrocytes after perichondrium removal. (A, E) SO-FG, (B, C, F, G) in situ hybridization and (D, H) Within 24 h, the cartilage was not vascularized, and we noted TUNEL analyses on e14.0 humerus transplanted for 24 h in the renal that in the absence of a perichondrium, more chondrocytes capsule, (A–D) with or (E–H) without perichondrium. (A) After 24 h ex underwent hypertrophy as indicated by the increase in the vivo, hypertrophic cartilage (hc) was in the center of the cartilage anlage size of the ColX domain (n = 7; compare Figs. 4B with F). and (B) expressed the marker colX (red). (B) ColX expression was The removal of the perichondrium did not inhibit chondro- downregulated in late hypertrophic chondrocytes expressing MMP13 (yellow) and (C) Vegf (blue). (C) Ihh expression (purple) was detected in cytes from reaching the stage of late hypertrophy though, prehypertrophic chondrocytes. (D) TUNEL-positive nuclei were rarely since they expressed MMP13 and Vegf (Figs. 4B, C, F, G). found in the area of late hypertrophy (yellow, arrowheads); Hoechst Perichondrial removal also led to a slight expansion in the counterstain (red) showed all nuclei. (E–F) When the perichondrium was population of pre-hypertrophic chondrocytes as shown by an removed before transplantation in the renal capsule, the domains of ColX extended Ihh domain (Figs. 4C and G). To ensure that the and MMP13 expression were expanded (red and yellow, respectively) compared to samples with the perichondrium intact. (G) The Ihh expression enzymatic digestion, which facilitated the perichondrial domain (purple) was also enlarged indicating that perichondrium removal removal, did not have any untoward effect on chondrocyte prolonged the steps of chondrocyte maturation and hypertrophy. Expression viability, we examined the anlagen carefully for signs of of Vegf in late hypertrophic chondrocytes was observed but at lower level programmed cell death. We found that the number of compared to the cartilage anlage with perichondrium intact. (H) The apoptotic chondrocytes did not increase when the perichon- number of TUNEL-positive nuclei (yellow, arrowheads) was comparable to the number found in the control samples. Scale bars: (A–C, E–G) = drium was removed (Figs. 4D and H). Thus, perichondrial 200 Am, (D, H) = 50 Am. removal did not adversely affect chondrocyte viability nor did it prevent chondrocyte hypertrophy; the rate of chondrocyte hypertrophy, however, was altered. without a perichondrium were 18% smaller (n = 10, 2.4 mm) than their contralateral controls with an intact peri- Perichondrium removal delays vascular invasion and chondrium (n = 10, 2.9 mm; P < 0.01). In addition, the inhibits ossification samples without a perichondrium showed a severely constricted mid-diaphyseal region (compare Fig. 5E with Fig. The most notable consequence of perichondrial removal 5A). The cellular morphology within this constricted region at later time points was growth arrest. By 4 days, anlagen was neither consistent with hypertrophic cartilage, nor did C. Colnot et al. / Developmental Biology 269 (2004) 55–69 63

Fig. 5. Consequences of perichondrial removal on vascular invasion and endochondral ossification. Morphological and biomolecular analyses of e14.0 humerus with and without perichondrium, 4 days after transplantation in the renal capsule. (A, B) In the presence of the perichondrium, the cartilage anlage were fully vascularized, an ossification center was present (oc) and the growth plates were established. (C) PECAM-positive blood vessels (arrowheads) and (D) TRAP-positive matrix resorbing cells (arrows) were localized at the chondrovascular junction and in the ossification center. (E) In the absence of the perichondrium, a constriction was seen in the center of the cartilage element (brackets). (F) This area did not stain for SO (brackets), (G) very few PECAM- positive cells (arrowhead) and (H) no TRAP-positive cells were detected. (I) In the presence of the perichondrium, periosteum and bone trabeculae formed after 4 days in the renal capsule, as shown by TC (blue). (J) ColI (green) was strongly expressed in the periosteum and in bone trabeculae. (K) Few TUNEL-positive cells (yellow, white arrowheads) were localized near the chondrovascular junction. Hoechst counterstain (red) showed all other nuclei. (L) When the wild-type graft was transplanted in Rosa26 host, X-gal staining indicated the presence of osteoclasts and blood cells derived from the host vasculature (arrows). (M) In the absence of perichondrium, TC staining confirmed that a small amount of bone matrix had been produced. (N) Although vascular invasion did not occur in the absence of the perichondrium, cells in the center of the cartilage anlage downregulated colX (red) and upregulated colI expression (in brackets). (O) Very few TUNEL-positive cells (yellow, white arrowheads) were in the central area of the perichondrium-removed cartilage elements, similar to those with perichondrium intact (K). (P) The majority of the cells in the central area did not stain for X-gal indicating that they were not derived from the Rosa26 host. hc: hypertrophic chondrocytes, tb: trabecular bone, po: periosteum. (K, O) correspond to boxed area in (J, N), respectively, and the white dotted line indicates the limit of hypertrophic chondrocytes. Scale bars: (A, E) = 1 mm, (B–D, F–H) = 200 Am, (I, J, M, N) = 200 Am, (K, L, O, P) = 50 Am. the tissue stain positively for proteoglycans (Fig. 5F). The The vast majority of the cells in the central region of the region was poorly vascularized, as indicated by the presence graft was derived from the graft tissue (Figs. 5L and P), of only a few PECAM-positive endothelial cells at the which may indicate that they were hypertrophic chondro- periphery (compare Fig. 5G with C). The absence of cytes, either arrested at a stage of terminal differentiation, or TRAP-positive cells (Figs. 5D and H), suggested that matrix trans-differentiating into osteoblasts. As shown in Figs. 5F degradation and remodeling were suspended when the and 4E, residual perichondrial tissue could be observed in perichondrium was removed. The formation of trabecular some cases, however, the consequences on skeletal devel- and periosteal bone was also halted (compare control Figs. opment were still significant. Taken together, these molec- 5I–J with M–N). A marrow cavity did not develop either, ular and cellular analyses suggested that in the absence of and instead, a dense mass of cells occupied this central the perichondrium, the initiation of vascular invasion and region. Cells within this constricted domain expressed ColI endochondral ossification were inhibited. (Fig. 5N), MMP13, and Osteopontin but not Osteocalcin Even after 10 days ex vivo, anlagen in which the (data not shown). Perichondrial removal did not induce perichondrium had been removed formed very little trabec- excessive programmed cell death relative to samples in ular and periosteal bone (n = 28; Figs. 6D–F). Instead, the which the perichondrium was intact (Figs. 5K and O). central region became occupied by blood cells, indicating 64 C. Colnot et al. / Developmental Biology 269 (2004) 55–69

Fig. 6. Delayed vascular invasion in the absence of the perichondrium. Morphological analyses of e14.0 humerus with perichondrium intact (A–C), perichondrium removed (D–F), and perichondrium removed then recombined (G–I), 10 days after transplantation underneath the renal capsule. (A) The humerus with an intact perichondrium contained a large marrow cavity and (B, C) Trichrome (TC) staining indicated the presence of trabecular and periosteal (arrows) bone. (D–F) After perichondrium removal, although the presence of blood cells on histological sections indicated that some vascular invasion had occurred, the humerus lacked a large marrow cavity, and trabecular and periosteal (arrow) bone formation were minimal. (G) As a control for the surgery, perichondrium removal and recombination with perichondrial tissue restored the formation of a marrow cavity, (H–I) as well as trabecular and periosteal (arrows) bone. hc: hypertrophic chondrocytes, tb: trabecular bone, po: periosteum. Scale bars: (A, D, G) = 1 mm, (B, C, E, F, H, I) = 200 Am.

that vascular invasion could eventually occur even in the invasion of hypertrophic cartilage, while the perichondrium absence of a perichondrium (n = 28; Figs. 6E and F). One remained intact. obvious explanation for the observed phenotype was that VEGF is a known regulator of cartilage angiogenesis the removal of the perichondrium irreparably damaged the (Gerber et al., 1999; Maes et al., 2002; Zelzer et al., 2002). cartilage anlagen, thereby halting subsequent development To block vascular invasion of the hypertrophic cartilage, we of the skeletal anlagen. To control for such a possibility, we used a recombinant soluble receptor mFlt-IgG to produce a removed the perichondrium, then recombined the cartilage transient, localized inhibition in VEGF signaling. Four days anlagen with perichondrial graft obtained from another after mFlt-IgG treatment, we observed an inhibition of the donor. We found that these cartilage–perichondrium recom- initial vascularization of cartilage anlagen and endochondral bined samples were properly invaded by the vasculature and ossification (n =8;Figs. 7F–H). The treatment did not, that bone formation occurred normally (n =4;Figs. 6G–I). however, impair intramembranous ossification in the peri- The fact that their development paralleled that of intact osteum. Contralateral elements showed normal vasculariza- e14.0 anlagen indicated that the surgical manipulations were tion and ossification (n = 13; Figs. 7A–E). These results not traumatic to the point where ossification and vascular indicated that VEGF is required for many processes that invasion were impeded. normally occur during endochondral ossification, but does not appear to participate to the same extent in intramem- Blocking angiogenesis prevents ossification branous ossification within the periosteum. The effect of VEGF inhibition was not prolonged and was reversible Since perichondrial removal affected both vascular inva- since skeletal elements underwent normal vascular invasion sion and bone formation, we could not differentiate whether and ossification by day 7 (n =5;Figs. 7I–J). the ossification defect was simply the consequence of To assess the long-term effects of angiogenesis inhibi- removing the source of osteoblasts residing in the perichon- tion, we blocked physically the migration of blood vessels drium, or was a consequence of delayed vascular invasion. from the host kidney into the grafted tissue. e14.0 skeletal To separate the contribution of the vascular endothelium elements were placed in between two filters, which pre- from the contribution of the perichondrium during endo- vented direct contacts with the kidney and acted as physical chondral ossification, we used two approaches, one bio- barriers for blood vessels. In the presence of these filter chemical and the other mechanical, to prevent vascular sandwiches, vascular invasion of hypertrophic cartilage did C. Colnot et al. / Developmental Biology 269 (2004) 55–69 65

Fig. 7. Consequences of blocking angiogenesis on ossification. Histological and immunohistochemical analyses, at days 4 and 7 post-transplantation, in (A–E) control samples, (F–J) samples treated with the VEGF inhibitor mFlt-IgG and (K–O) samples placed between two filters separating the graft from the kidney. (A) SO-FG staining on control sample showed the growth plate, the chondrovascular junction and the adjacent periosteum. (B, D) PECAM immunostaining indicated the presence of blood vessels in the ossification center and in the periosteum (arrowheads). (C, E) TC staining revealed the presence of osteoid matrix in the periosteum (po) and in the bone trabeculae (tb). (F) After mFlt-IgG treatment, the domain of hypertrophic cartilage was enlarged, (G) and was not invaded by blood vessels by day 4. PECAM-positive cells were detected in the periosteum (arrowhead). (H) Periosteal bone was detected by TC staining, however, no trabecular bone was observed. (I, J) By day 7, vascular invasion of hypertrophic cartilage and endochondral ossification occurred, as seen in control samples. (K) When filters were interposed between the graft and the kidney, chondrocytes underwent hypertrophy as shown by SO-FG. The bended shape of the cartilage element was due to the shape of the kidney (L, N). Hypertrophic cartilage did not get vascularized as shown by the presence of few PECAM-positive cells at the periphery of the graft (arrowhead). (M, O) TC staining on adjacent sections shows that both intramembranous and endochondral ossification were impaired. Schematics on the top indicate how migration of blood vessels from the host environment was blocked. Black: graft; green: kidney; yellow: kidney capsule, pink: mFlt-IgG; blue: filters. Scale bars: A, F, K = 200 Am; B–E, G–J, L–O = 100 Am. not occur (n =5;Figs. 7K–O). PECAM-positive cells still impaired in the cases where host vessels did not reach residing in the perichondrium at the time of transplantation the graft (n =4;Figs. 7N–O). The viability of the tissue were still detected adjacent to the cartilage anlagen (Figs. 7L however was not affected, indicating that the phenotype was and N). In addition, the perichondrium did not differentiate not due to the lack of nutrients from the host environment. into periosteum and no trabecular bone formed (Fig. 7M). In the cases where host blood vessels were able to grow in These data indicated that both endochondral and intramem- between the filters and reached the graft, the skeletal branous ossification were halted. By day 7, ossification was elements became vascularized and ossified (n = 4; data 66 C. Colnot et al. / Developmental Biology 269 (2004) 55–69 not shown). These results demonstrated that vascular inva- anlagen. Endothelial cells and osteoblasts may share a sion of hypertrophic cartilage and a direct interaction common lineage (Qi et al., 2003), raising the possibility between the perichondrium and the surrounding vasculature that the endothelial cells, may actually trans-differentiate were essential for both endochondral and intramembranous into osteoblasts once they invade the hypertrophic cartilage ossification. matrix. There is also evidence for circulating skeletal stem cells (Kuznetsov et al., 2001) but the extent to which they contribute to trabecular bone is not yet clear. Previous Discussion observations suggest that cortical osteoblasts arise from the perichondrium (Bruder and Caplan, 1990; Nakahara et An impressive body of research has accumulated on the al., 1990), however, it is not clear whether cortical and molecular signals involved in chondrogenesis (Bi et al., trabecular osteoblasts have distinct or similar origins. 1999; Lanske et al., 1996; Vortkamp et al., 1996) and Our ex vivo system indicates that the source of both osteogenesis (Ducy et al., 1997; Komori et al., 1997; Otto cortical and trabecular osteoblasts is the perichondrium (Fig. et al., 1997). An equally abundant literature has documented 3). At this juncture, we cannot identify which cell type in the the importance of the vasculature during bone formation perichondrium gives rise to osteoblasts. Thus far, genetic (Gerber et al., 1999; Haigh et al., 2000; Vu et al., 1998; approaches have not been particularly useful in identifying Zelzer et al., 2001, 2002). Clearly, a dialogue exists among this elusive stem cell population, in large part because of the chondrocytes, osteoblasts, and endothelial cells, which con- lack of specific genetic markers for osteoblast precursor trols their synchronized differentiation (Kronenberg, 2003) cells. Even genes that are essential for bone formation, such but there is an inherent difficulty in studying the tissue as Runx2 (Ducy et al., 1997; Komori et al., 1997; Otto et al., interactions during endochondral ossification. These inter- 1997), are also expressed in chondrocytes (Inada et al., actions either occur simultaneously or in rapid succession, 1999; Kim et al., 1999). Nonetheless, our results exclude the therefore, it becomes difficult to distinguish primary defects vasculature surrounding skeletal elements as a source of from secondary ones. In a great number of skeletal defor- osteoblasts during development. The vasculature and the mities (Kronenberg, 2003; Zelzer and Olsen, 2003), all three soft tissue might, however, act as sources of skeletal stem tissues are adversely affected, which exemplifies the inter- cells during later stages of bone growth and remodeling and dependent nature of their development and maturation. during the process of bone repair in the adult. Another feature that complicates the separation of chondrogenesis and osteogenesis is that many molecules are The cartilage anlagen as a source of osteoblasts? expressed in both terminally differentiated chondrocytes and in newly minted osteoblasts (Colnot and Helms, Although osteoblasts appeared to be derived exclusively 2001; Jime´nez et al., 1999; Kim et al., 1999), which makes from the perichondrium during long bone development, we it a challenge to distinguish the origins of the two cell types. cannot completely exclude a participation of the cartilage The inherent difficulties in studying the tissue interac- anlagen as a source for osteoblasts. In the absence of the tions during skeletogenesis instigated us to adapt an ex vivo perichondrium, chondrocytes differentiate up to the late system to distinguish the contributions of the cartilage, the hypertrophic stage but do not undergo a normal transition perichondrium and the vascular endothelium. Using molec- to terminal differentiation and programmed cell death, even ular and cellular analyses, we found that all the steps of in an ex vivo environment that supports vascular invasion endochondral ossification were recapitulated ex vivo. More- (Fig. 5). A signal required for the terminal differentiation over, the initiation of cartilage angiogenesis and the onset of and chondrocyte death may originate from the perichondri- ossification occurred over a protracted time course in the um or from the vasculature (Maeda and Noda, 2003).In renal capsule, thus allowing us to more closely scrutinize absence of this death-inducing signal, hypertrophic chon- some of these developmental events. In addition, we could drocytes may remain viable (Fig. 5). Whether these remain- perform novel mapping experiments to address a long- ing cells represent terminally differentiated chondrocytes or standing question about the origins of osteoblasts during pre-osteoblasts is open to debate (Bianco et al., 1998; Kahn appendicular skeletogenesis. and Simmons, 1977; Roach et al., 1995). This cell population might participate to a small extent in the process of The perichondrium is the source of osteoblasts endochondral ossification.

The origins of osteoblasts during endochondral ossifica- The perichondrium regulates the initiation of vascular tion are somewhat controversial. One prevailing theory invasion proposes that trabecular osteoblasts arise from satellite cells resident in the surrounding soft tissues (Asakura et al., Cartilage angiogenesis is controlled by hypertrophic 2001) or from pericytes (Doherty et al., 1998; Schor et al., cartilage through the secretion of angiogenic factors such 1995), and that both cell types might use the invading as VEGF (Gerber et al., 1999; Maes et al., 2002; Zelzer et vasculature as a mechanism to gain entry into the cartilage al., 2002). Our results show that the perichondrium also C. Colnot et al. / Developmental Biology 269 (2004) 55–69 67 participates in the regulation of cartilage vascular invasion. differentiate. Additionally, the ossification defect might also Removing the perichondrium prevents the timely invasion arise from the lack of osteoclasts that would be brought by of blood vessels into the hypertrophic cartilage matrix. the vasculature. Altogether, our results indicate that vascular Since the perichondrium is the primary source for endothe- invasion and ossification are linked in time and space either lial cells (Fig. 3), the delay in vascular invasion seems because direct cell–cell contacts between osteoblast precur- intuitive. Removing the perichondrium, however, does not sors and endothelial cells are required for osteoblast differ- prevent vascular invasion at later time points (Fig. 6), which entiation, or because a factor from the blood stream is can be provided by the host environment. Therefore, the necessary for osteoblast differentiation. perichondrium appears to promote the initiation of cartilage angiogenesis. A model to elucidate skeletal phenotypes In a two-step process blood vessels become localized in the perichondrium, then invade the cartilage matrix (Zelzer Our findings have implications for understanding the et al., 2002). We had the opportunity to explore these two skeletal phenotypes of transgenic mice in which chondro- steps when we blocked invasion of host blood vessels. genesis, osteogenesis, and angiogenesis are all perturbed. A Treatment with mFlt-IgG initially blocked invasion of number of molecules, including Runx2 (Komori et al., 1997; hypertrophic cartilage and as a consequence, delayed the Otto et al., 1997),Ihh(St-Jacques et al., 1999),VEGF formation of trabecular bone. Ossification in the periosteum, (Gerber et al., 1999), TGFh (Alvarez et al., 2001, 2002), however, was not inhibited most likely because the perios- retinoic acid (Di Nino et al., 2001, 2002; Koyama et al., teum vasculature was able to connect to the host vascula- 1999), fibroblast growth factors (Liu et al., 2002; Vortkamp ture. When we left the perichondrium intact and placed a et al., 1996), Wnt signaling molecules (Hartmann and Tabin, physical barrier between the kidney and the skeletal graft, 2000), and matrix metalloproteinases (Jimenez et al., 2001; endothelial cells were in the perichondrium but because of Vu et al., 1998) synchronize the development of cartilage and the presence of the filter, the host blood vessels were unable perichondrium. In many cases, genetic inactivations produce to participate (Fig. 7). We observed that in the absence of an embryonic lethal phenotype, which until now has pre- this vascular contribution from the host, endothelial cells cluded analyses of later stages of skeletal development. residing in the perichondrium were unable to invade the Determining whether a molecule is required for osteogenesis, hypertrophic cartilage matrix despite the lack of any phys- chondrogenesis, or vascular invasion, or simply supports ical barrier. These results indicate that both intact host blood these processes, may be elucidated using an ex vivo system vessels and endothelial cells residing in the perichondrium that allows for the recombination of wild-type and mutant are required for vascular invasion of hypertrophic cartilage. skeletal tissues. Our analyses conducted with wild-type and We surmise that endothelial cells in the perichondrium need Rosa26 tissue recombinations have shed light on the origins to connect with an intact surrounding vascular network to of osteoblasts during long bone development, as well as initiate angiogenesis. These events might reflect what nor- elucidated the process by which endothelial cells invade the mally occurs in vivo. For example, endothelial cells might hypertrophic cartilage matrix and induce endochondral ossi- differentiate in situ from precursor cells localized in the fication. These data provide a framework in which to perichondrium. Following this process of vasculogenesis, interpret the skeletal phenotypes of mice in which genetic which takes place within the perichondrium, blood vessels inactivation perturbs the development of the cartilage, the may fuse with the surrounding vasculature and thus gain the perichondrium, and the skeletal vascular endothelium. capacity to invade the hypertrophic cartilage. Alternatively, blood vessels surrounding the cartilage anlagen may migrate into the perichondrium through an angiogenic process, Acknowledgments while maintaining their connection with the vascular network. In either case, the perichondrium promotes the The authors would like to thank S. Huang for expert initiation of vascular invasion by inducing a vasculogenic/ technical assistance and L. de la Fuente and B.F. Eames for angiogenic program, which positions blood vessels adjacent helpful discussions and comments on the manuscript. This to the site of chondrocyte terminal differentiation. work was supported by funds from R01 DE31497 and P60 The perichondrium therefore plays a dual role in the DE13058 (J.A.H.). CC was supported by R01 AR46238 process of endochondral ossification by providing osteo- (Z. Werb). blast precursors and regulating vascular invasion. Our system does not allow us to separate these two events however. Obstructing host blood vessels using a filter mimicked the effect of perichondrium removal by impeding References both vascular invasion and ossification. Unlike the peri- Alvarez, J., Horton, J., Sohn, P., Serra, R., 2001. The perichondrium plays chondrial removal, the filter did not disrupt the source of an important role in mediating the effects of TGF-beta1 on endochon- osteoblast precursors, indicating that if the perichondrium is dral bone formation. Dev. Dyn. 221, 311–321. not properly vascularized, osteoblasts lose their ability to Alvarez, J., Sohn, P., Zeng, X., Doetschman, T., Robbins, D.J., Serra, R., 68 C. Colnot et al. / Developmental Biology 269 (2004) 55–69

2002. TGFbeta2 mediates the effects of hedgehog on hypertrophic dif- Inada, M., Yasui, T., Nomura, S., Miyake, S., Deguchi, K., Himeno, ferentiation and PTHrP expression. Development 129, 1913–1924. M., Sato, M., Yamagiwa, H., Kimura, T., Yasui, N., Ochi, T., Endo, Asakura, A., Komaki, M., Rudnicki, M., 2001. Muscle satellite cells are N., Kitamura, Y., Kishimoto, T., Komori, T., 1999. Maturational multipotential stem cells that exhibit myogenic, osteogenic, and adipo- disturbance of chondrocytes in Cbfa1-deficient mice. Dev. Dyn. genic differentiation. Differentiation 68, 245–253. 214, 279–290. Bi, W., Deng, J.M., Zhang, Z., Behringer, R.R., de Crombrugghe, B., 1999. Jimeńez, M.J., Balbıń, M., Lo´pez, J.M., Alvarez, J., Komori, T., Lo´pez- Sox9 is required for cartilage formation. Nat. Genet. 22, 85–89. Otıń, C., 1999. Collagenase 3 is a target of Cbfa1, a transcription factor Bianco, P., Cancedda, F.D., Riminucci, M., Cancedda, R., 1998. Bone of the runt gene family involved in bone formation. Mol. Cell. Biol. 19, formation via cartilage models: the ‘‘borderline’’ chondrocyte. Matrix 4431–4442. Biol. 17, 185–192. Jimenez, M.J., Balbin, M., Alvarez, J., Komori, T., Bianco, P., Holmbeck, Bruder, S.P., Caplan, A.I., 1990. Osteogenic cell lineage analysis is facil- K., Birkedal-Hansen, H., Lopez, J.M., Lopez-Otin, C., 2001. A regula- itated by organ cultures of embryonic chick periosteum. Dev. Biol. 141, tory cascade involving retinoic acid, Cbfa1, and matrix metalloprotei- 319–329. nases is coupled to the development of a process of perichondrial Chambers, T.J., 2000. Regulation of the differentiation and function of invasion and osteogenic differentiation during bone formation. J. Cell osteoclasts. J. Pathol. 192, 4–13. Biol. 155, 1333–1344. Chung, U.I., Lanske, B., Lee, K., Li, E., Kronenberg, H., 1998. The para- Kahn, A.J., Simmons, D.J., 1977. Chondrocyte-to-osteocyte transformation thyroid hormone/parathyroid hormone-related peptide receptor coordi- in grafts of perichondrium-free epiphyseal cartilage. Clin. Orthop. nates endochondral bone development by directly controlling chondro- Relat. Res. 65, 299–304. cyte differentiation. Proc. Natl. Acad. Sci. U. S. A. 95, 13030–13035. Karp, S.J., Schipani, E., St-Jacques, B., Hunzelman, J., Kronenberg, H., Chung, U.I., Schipani, E., McMahon, A.P., Kronenberg, H.M., 2001. In- McMahon, A.P., 2000. Indian hedgehog coordinates endochondral bone dian hedgehog couples chondrogenesis to osteogenesis in endochondral growth and morphogenesis via Parathyroid Hormone related-Protein- bone development. J. Clin. Invest. 107, 295–304. dependent and -independent pathways. Development 127, 543–548. Colnot, C.I., Helms, J.A., 2001. A molecular analysis of matrix remodeling Karsenty, G., Wagner, E.F., 2002. Reaching a genetic and molecular un- and angiogenesis during long bone development. Mech. Dev. 100, derstanding of skeletal development. Dev. Cell. 2, 389–406. 245–250. Kim, I.S., Otto, F., Zabel, B., Mundlos, S., 1999. Regulation of chondro- de Crombrugghe, B., Lefebvre, V., Nakashima, K., 2001. Regulatory mech- cyte differentiation by Cbfa1. Mech. Dev. 80, 159–170. anisms in the pathways of cartilage and bone formation. Curr. Opin. Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K., Deguchi, K., Cell Biol. 13, 721–727. Shimizu, Y., Bronson, R.T., Gao, Y.H., Inada, M., Sato, M., Okamoto, Di Nino, D.L., Long, F., Linsenmayer, T.F., 2001. Regulation of endochon- R., Kitamura, Y., Yoshiki, S., Kishimoto, T., 1997. Targeted disruption dral cartilage growth in the developing avian limb: cooperative involve- of Cbfa1 results in a complete lack of bone formation owing to matu- ment of perichondrium and periosteum. Dev. Biol. 240, 433–442. rational arrest of osteoblasts [see comments]. Cell 89, 755–764. Di Nino, D.L., Crochiere, M.L., Linsenmayer, T.F., 2002. Multiple mech- Koyama, E., Golden, E.B., Kirsch, T., Adams, S.L., Chandraratna, R.A., anisms of perichondrial regulation of cartilage growth. Dev. Dyn. 225, Michaille, J.J., Pacifici, M., 1999. Retinoid signaling is required for 250–259. chondrocyte maturation and endochondral bone formation during limb Doherty, M.J., Ashton, B.A., Walsh, S., Beresford, J.N., Grant, M.E., Can- skeletogenesis. Dev. Biol. 208, 375–391. field, A.E., 1998. Vascular pericytes express osteogenic potential in Kronenberg, H.M., 2003. Developmental regulation of the growth plate. vitro and in vivo. J. Bone Miner. Res. 13, 828–838. Nature 423, 332–336. Ducy, P., Zhang, R., Geoffroy, V., Ridall, A.L., Karsenty, G., 1997. Osf2/ Kuznetsov, S.A., Mankani, M.H., Gronthos, S., Satomura, K., Bianco, P., Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89, Robey, P.G., 2001. Circulating skeletal stem cells. J. Cell Biol. 153, 747–754. 1133–1139. Eames, B.F., de la Fuente, L., Helms, J.A., 2003. Molecular ontogeny of Lanske, B., Karaplis, A.C., Lee, K., Luz, A., Vortkamp, A., Pirro, A., the skeleton. Birth Defects Res., Part C Embryo Today 69, 93–101. Karperien, M., Defize, L.H.K., Ho, C., Mulligan, R.C., Abou-Samra, Ferguson, C., Alpern, E., Miclau, T., Helms, J.A., 1999. Does adult fracture A.B., Juppner, H., Segre, G.V., Kronenberg, H.M., 1996. PTH/PTHrP repair recapitulate embryonic skeletal formation? Mech. Dev. 87, 57–66. receptor in early development and Indian hedgehog-regulated bone Ferguson, C.A., Tucker, A.S., Heikinheimo, K., Nomura, M., Oh, P., Li, E., growth. Science 273, 663–666. Sharpe, P.T., 2001. The role of effectors of the activin signalling path- Liu, Z., Xu, J., Colvin, J.S., Ornitz, D.M., 2002. Coordination of chondro- way, activin receptors IIA and IIB, and Smad2, in patterning of tooth genesis and osteogenesis by fibroblast growth factor 18. Genes Dev. 16, development. Development 128, 4605–4613. 859–869. Friedrich, G., Soriano, P., 1991. Promoter traps in embryonic stem cells: a Maeda, Y., Noda, M., 2003. Coordinated development of embryonic long genetic screen to identify and mutate developmental genes in mice. bone on chorioallantoic membrane in ovo prevents perichondrium-de- Genes Dev. 5, 1513–1523. rived suppressive signals against cartilage growth. Bone 32, 27–34. Gerber, H.P., Vu, T.H., Ryan, A.M., Kowalski, J., Werb, Z., Ferrara, N., Maes, C., Carmeliet, P., Moermans, K., Stockmans, I., Smets, N., Collen, 1999. VEGF couples hypertrophic cartilage remodeling, ossification D., Bouillon, R., Carmeliet, G., 2002. Impaired angiogenesis and en- and angiogenesis during endochondral bone formation. Nat. Med. 5, dochondral bone formation in mice lacking the vascular endothelial 623–628. growth factor isoforms VEGF164 and VEGF188. Mech. Dev. 111, Haigh, J.J., Gerber, H.-P., Ferrara, N., Wagner, E.F., 2000. Conditional 61–73. inactivation of VEGF-A in areas of collagen2a1 expression results in Nakahara, H., Bruder, S.P., Goldberg, V.M., Caplan, A.I., 1990. In vivo embryonic lethality in the heterozygous state. Development 127, osteochondrogenic potential of cultured cells derived from the perios- 1445–1453. teum. Clin. Orthop., 223–232. Hall, B.K., Miyake, T., 2000. All for one and one for all: condensations and Norman, J.T., Cunha, G.R., Sugimura, Y., 1986. The induction of new the initiation of skeletal development. BioEssays 22, 138–147. ductal growth in adult prostatic epithelium in response to an embryonic Hartmann, C., Tabin, C.J., 2000. Dual roles of Wnt signaling during chon- prostatic inductor. Prostate 8, 209–220. drogenesis in the chicken limb. Development 127, 3141–3159. Otto, F., Thornell, A.P., Crompton, T., Denzel, A., Gilmour, K.C., Rose- Hom, Y.-K., Cunha, G.R., 2000. Transplantation and tissue recombination well, I.R., Stamp, G.W., Beddington, R.S., Mundlos, S., Olsen, B.R., techniques to study mammary gland biology. In: I. a. A. Kluwer (Ed.), Selby, P.B., Owen, M.J., 1997. Cbfa1, a candidate gene for cleidocranial Methods in mammary gland biology and breast cancer research: Aca- dysplasia syndrome, is essential for osteoblast differentiation and bone demic/Plenum, New York, pp. 289–306. development [see comments]. Cell 89, 765–771. C. Colnot et al. / Developmental Biology 269 (2004) 55–69 69

Qi, H., Aguiar, D.J., Williams, S.M., La Pean, A., Pan, W., Verfaillie, C.M., C.J., 1996. Regulation of rate of cartilage differentiation by Indian 2003. Identification of genes responsible for osteoblast differentiation hedgehog and PTH-related protein. Science 273, 613–622. from human mesodermal progenitor cells. Proc. Natl. Acad. Sci. U. S. A. Vu, T.H., Shipley, J.M., Bergers, G., Berger, J.E., Helms, J.A., Hanahan, 100, 3305–3310. D., Shapiro, S.D., Senior, R.M., Werb, Z., 1998. MMP-9/gelatinase B is Roach, H.I., Erenpreisa, J., Aigner, T., 1995. Osteogenic differentiation a key regulator of growth plate angiogenesis and apoptosis of hyper- of hypertrophic chondrocytes involves asymmetric cell divisions and trophic chondrocytes. Cell 93, 411–422. apoptosis. J. Cell Biol. 131, 483–494. Vu, T.H., Alemayehu, Y., Werb, Z., 2003. New insights into saccular Sasano, Y., Zhu, J.X., Tsubota, M., Takahashi, I., Onodera, K., Mizoguchi, development and vascular formation in lung allografts under the renal I., Kagayama, M., 2002. Gene expression of MMP8 and MMP13 capsule. Mech. Dev. 120, 305–313. during embryonic development of bone and cartilage in the rat man- Zelzer, E., Olsen, B.R., 2003. The genetic basis for skeletal diseases. Na- dible and hind limb. J. Histochem. Cytochem. 50, 325–332. ture 423, 343–348. Schor, A.M., Canfield, A.E., Sutton, A.B., Arciniegas, E., Allen, T.D., Zelzer, E., Glotzer, D.J., Hartmann, C., Thomas, D., Fukai, N., Soker, S., 1995. Pericyte differentiation. Clin. Orthop., 81–91. Olsen, B.R., 2001. Tissue specific regulation of VEGF expression Spemann, H., Mangold, H., 1924. Induction of Embryonic Primordia by during bone development requires Cbfa1/Runx2. Mech. Dev. 106, Implantation of Organizers from a Different Species. Hafner, New York. 97–106. St-Jacques, B., Hammerschmidt, M., McMahon, A.P., 1999. Indian hedge- Zelzer, E., McLean, W., Ng, Y.S., Fukai, N., Reginato, A.M., Lovejoy, S., hog signaling regulates proliferation and differentiation of chondrocytes D’Amore, P.A., Olsen, B.R., 2002. Skeletal defects in VEGF(120/120) and is essential for bone formation. Genes Dev. 13, 2072–2086. mice reveal multiple roles for VEGF in skeletogenesis. Development Vortkamp, A., Lee, K., Lanske, B., Segre, G.V., Kronenberg, H.M., Tabin, 129, 1893–1904.