162 Pacifici et al. of Patched in perichondrial cells surrounding IHH-expressing prehypertrophic chondrocytes seen in long bone anlagen in vivo (5,6,10). That is, IHH produced by the prehypertrophic chondrocytes, may diffuse into the perichondrium where it would trigger Patched gene expression as well as osteogenic cell differentiation and bone collar formation (as our model shown in Fig. 6 prescribes). As pointed out above, our data and conclusions are supported by the very recent report that in IHH-null mice there is no ossification in the limb (10). NEED FOR RETINOID SIGNALING IN ENDOCHONDRAL OSSIFICATION In a series of previous studies from our laboratory (ref. 16 and refs. therein), we had provided evi- dence that retinoic signaling promotes the development of immature chondrocytes into hypertrophic chondrocytes in vitro. We found that upon induction by retinoids, the cells progress to the terminal stage of maturation and closely resemble the posthypertrophic cells present at the chondro-osseous border in the growth plate in vivo. The phenotypic traits expressed by the retinoid-induced chon- drocytes include a very large cell diameter, production of mineralization-competent matrix vesicles, ability to deposit apatitic crystals and high alkaline phosphatase activity. Because all these traits are actually needed for the transition from mineralized hypertrophic cartilage to endochondral bone, our data suggested that by inducing such traits, retinoid signaling may be required for cartilage-to-bone transition in vivo. Thus, we conducted additional studies to obtain evidence in support of this impor- tant conclusion; these studies have been reported (8) and only key findings are shown here. In a first set of experiments, we asked whether expression of retinoid nuclear receptors is upregu- lated in prehypertrophic and/or hypertrophic chondrocytes. We reasoned that such upregulation may be necessary for retinoids to act on those cells and promote terminal maturation into mineralizing post- hypertrophic chondrocytes ready for replacement by bone cells. Thus, we used in situ hybridization to monitor expression of RAR_, RAR`, and RARa during long bone development in the embryonic limb. As above, we first examined newly emerged cartilaginous anlagen in young day 5.5 chick embryo limb; these anlagen are composed entirely of immature chondrocytes, display a still primitive mor- phological organization, and do not contain growth plates. We found that in these anlagen, the expres- sion of RAR_ and RARa was quite broad and diffuse throughout the cartilaginous tissue and that RAR` expression was strong in incipient perichondrial cells. We then examined older day 9 through day 18 skeletal elements that display typical elongated morphologies, well-defined diaphysis and epi- physes, and obvious growth plates (Fig. 2A). At these stages, RAR_ expression remained uniform, broad, and relatively low throughout the cartilaginous tissue (Fig. 2B), whereas RAR` remained con- fined to perichondrial tissue. Interestingly, expression of RARa was sharply and selectively upreg- ulated in hypertrophic chondrocytes (Fig. 2C). Identity of the hypertrophic cells was based on their large cell size and location as well as strong expression of a typical marker, type X collagen (Fig. 2D). Equally interesting was the finding that there was a sharp boundary and minimal overlap between RARa expression in hypertrophic chondrocytes and IHH expression in the preceding prehypertrophic chondrocyte zone (cfr. Fig. 2C,E). Type II collagen was uniformly strong from epiphysis to early hypertropic zone (Fig. 2F). Having shown that there is a selective upregulation of RARa in hypertrophic chondrocytes, we con- ducted a second set of studies to determine whether these and/or other chondrocytes contain endog- enous retinoids serving as RAR ligands. To approach this question, we used a bioassay commonly employed to determine endogenous retinoid levels in embryonic tissues; the bioassay is very sensitive, requires small amounts of tissue, and is thus ideal for analyses of scarce specimens such as embry- onic tissues (19). It consists of an F9 cell line stably transfected with a retinoid sensitive RARE/`-galac- tosidase construct; the cell line is exposed to tissue extracts, reporter activity increases in proportion to retinoid content in the extracts, and reporter activity is finally measured biochemically or histochem- ically. Accordingly, we isolated whole cartilaginous elements from day 5.5, 8.5, and 10 embryos by Signaling Molecules and Long Bone Development 163 microsurgical procedures; for comparison, we isolated other tissues and organs from the same embryos, including the perichondrial tissues immediately adjacent to the cartilages. About 100 mg of each sample were homogenized and extracted, and extracts were added to the reporter cell line; 24 h later, cultures were stained histochemically for `-galactosidase. Standards included cultures receiving known amounts of natural retinoids, such as all-trans-retinoic acid or 9-cis-retinoic acid. We found that at each stage studied, the cartilaginous elements contained endogenous retinoids (Fig. 3A,C). These levels were higher than those in brain but much lower than those in liver. Surprisingly and unexpect- edly, extremely large amounts of retinoids were present in perichondrial tissues (Fig. 3B,D); on a tis- sue wet weight basis, these amounts were comparable to those in liver. Very similar observations were made in a recent study with a transgenic mouse carrying a RARE/`-galactosidase reporter construct that is activated by endogenous retinoids (17); the authors found that strong `-galactosidase activity (and hence high retinoid content) was present in perichondrial tissues adjacent to the prehypertrophic and hypertrophic zones of long bone growth plate as well as in hypertrophic cartilage itself. The above data, combined with the finding of a specific RARa upregulation in hypertrophic chondrocytes, set the stage for a third series of experiments in which we asked whether the endogenous retinoids and RARa are actually required for chondrocyte hypertrophy and ossification in vivo. To approach this question, we made use of powerful pharmacological agents with retinoid antagonistic activity (see Fig. 4). Beads containing such agents were placed in the vicinity of newly formed early cartilaginous elements in the chick wing, embryos were reincubated, and effects were determined over developmental time. The major advantage of this pharmacological approach is that the antagonists can be used at specific stages of development and can be placed in contact with specific skeletal elements or portions thereof. Thus, their action and developmental consequences can be studied at the local level, minimizing the possibility that the effects are global and of a systemic nature. The RAR antagonist used was RO 41- 5253 from Hoffman-LaRoche (20), which exerts antagonist effects on all RARs. Three to four beads containing the antagonist were placed around the day 4.5–5.5 humeral anlagen, and embryos were examined over time. The results were dramatic. By day 10, humerus in control embryos (implanted with beads containing vehicle) had developed normally and displayed a typical elongated morphology Fig. 2. In situ hybridization analysis of expression of indicated genes in day 10 chick embryo ulna. Arrows in C and D point to hypertrophic chondrocytes expressing RARa and type X collagen. ac, articular cap; pz, prolif- erative zone; pp, prehypertrophic zone; and hz, hypertrophic zone. Bar = 185 µm. 164 Pacifici et al. and size; in sharp contrast, the antagonist-treated humerus was about half the length. No effects were seen in radius and ulna, attesting to the fact that the effects were limited to the site of bead implantation and were not systemic. Histology and in situ hybridization provided further insights into the developmental perturbations caused by the block of retinoid signaling (Fig. 4). In control humerus, the growth plate displayed nor- mal zones of proliferating, prehypertrophic, hypertrophic, and mineralizing chondrocytes; the meta- physis was surrounded by an intramembranous bone collar (Fig. 4A, arrowheads), and the diaphysis was undergoing invasion and replacement by endochondral bone and marrow (Fig. 4A, arrow). There was strong and typical gene expression of IHH in prehypertrophic chondrocytes (Fig. 4D, arrow), RARa in hypertrophic chondrocytes (Fig. 4B, arrow), and osteopontin in endochondral bone (Fig. 4C). As also shown above, osteopontin expression characterized the thin intramembranous bone surrounding the IHH-expressing prehypertrophic chondrocytes (Fig. 4C, arrowhead). In sharp contrast, the antag- onist-treated specimens were entirely cartilaginous and displayed no hypertrophic chondrocytes, no endochondral bone and marrow (Fig. 4E) and no expression of RARa (Fig. 4F). Interestingly, IHH expression was not only present but seemed broader than control (Fig. 4H, arrows), and the metaphy- seal–diaphyseal portion was surrounded by a conspicuous intramembranous bone collar (Fig. 4E, arrow- head) strongly expressing osteopontin (Fig. 4G, arrowheads). Thus, interference with retinoid signal- Fig. 3. Bioassay of endogenous retinoid content in cartilage (A and C) and perichondrial tissues (B and D) Tissue extracts were used to treat F9 cells stably transfected with a `-galactosidase/RARE reporter construct, and reporter activity was detected histochemically. A and C, day 8.5 and 10 chick embryo cartilage, respectively; B and D, day 8.5 and 10 perichondrial tissues, respectively. Signaling Molecules and Long Bone Development 165 ing has very specific consequences on long bone development and prevents completion of this process. The chondrocytes can reach the prehypertrophic IHH-expressing stage but cannot pass it; likewise, the intramembranous bone collar forms but there is no formation of endochondral bone and marrow invasion. Retinoid signaling thus appears to be required for normal progression through the terminal phases of long bone development (see our model in Fig. 6). Fig. 4. In situ hybridization analysis of expression of indicated genes in control day 10 humerus (A–D) and antagonist-treated humerus (E–H). See text for details. ep, epiphysis; me, metaphysis; and di, diaphysis. Bar = 250 µm. 166 Pacifici et al. RETINOID SIGNALING AND IHH EXPRESSION The in situ in the previous section indicate that IHH gene expression is not only maintained in antag- onist-treated skeletal anlagen but appears to be broader and more extensive than in control specimens. This led us to ask whether under normal circumstances retinoid signaling may actually represent a mech- anism to switch off IHH expression at the bottom of the prehypertrophic zone, thus favoring progres- sion to the hypertrophic phase. It is worth reiterating here that turning off IHH expression may be a very important step in chondrocyte maturation because constitutive IHH expression prevents chon- drocyte hypertrophy (6) and IHH gene ablation results in excessive and disorganized hypertrophy (10). To test our hypothesis, we conducted studies with cultured chondrocytes (these preliminary experiments have not been reported and will be described in full here). As a source of chondrocytes, we used the chick embryo sternum, which allows efficient and effective isolation of chondrocyte pop- ulations at specific stages of maturation, compared with the more cumbersome long bone growth plate (21). Accordingly, chondrocytes were isolated from the cephalic core portion of day 16 chick embryo sterna, which contains prehypertrophic-early hypertrophic chondrocytes at this stage (21). Cells were seeded in monolayer culture and allowed to grow for a few days in complete serum-con- taining medium to recover from the enzymatic isolation procedure. The cells were then treated with 30 nM all-trans-retinoic acid for 2, 4, and 6 d; RNA was isolated from each culture and processed for northern blot analysis, using a cDNA probe encoding IHH. This retinoid was chosen because it is a natural retinoid and is present in the developing limb (22); the dose used is precisely within the range seen in the developing limb as well (22). For comparison, we determined the effects of the retinoid antagonist used above, namely RO 41-5253. The results of these experiments were clear-cut. Control untreated chondrocytes displayed obvious expression of IHH (Fig. 5A, lane 1). Upon treatment with all-trans-retinoic acid, IHH RNA levels were decreased markedly (Fig. 5A, lanes 2–4); on the contrary, treatment with 50 nM retinoid antagonist boosted IHH gene expression by several fold (Fig. 5A, lanes 5–7), in good correlation with the in situ data (see Fig. 4H). Clearly, retinoid signaling appears to represent a powerful and effective switch by which IHH gene expression is inhibited in maturing chondrocytes. Retinoids should not only turn off gene expression of IHH but also should promote maturation and expression of hypertrophic chondrocyte-characteristic traits. Thus, we examined in the above cultures whether treatment with all-trans-retinoic acid or RO 41-5253 affected expression of alkaline phosphatase (APase), a typical hypertrophic cell trait. Northern hybridization showed that treatment with all-trans-retinoic acid led to a powerful increase in APase gene expression (Fig. 5B, lanes 2–4) Fig. 5. Northern blot analysis of IHH and APase gene expression in cultured chondrocytes. Cells were left untreated (lane 1) or were treated with all-trans-retinoic acid (lanes 2–4) or antagonist (lanes 5–7) for 2, 4, and 6 d. Signaling Molecules and Long Bone Development 167 compared with control values (Fig. 5B, lane 1), whereas antagonist treatment decreased it (Fig. 5B, lanes 5–7). Thus, retinoid signaling downregulates traits characteristic of prehypertrophic chondrocytes (i.e., IHH) and induces expression of hypertrophic traits (i.e., APase). PERICHONDRIAL TISSUES AS POSITIVE REGULATORS OF CHONDROCYTE MATURATION Perichondrial tissues adjacent to the prehypertrophic to hypertrophic zones of growth plate con- tain large amounts of endogenous retinoids (see Fig. 3), which in turn could exert a positive effect on neighboring chondrocytes and favor their maturation. To gain support for our hypothesis, we con- ducted the following studies. We reasoned that if perichondrial tissues were to provide positive signals for chondrocyte matura- tion, hypertrophic chondrocytes should first emerge along the chondroperichondrial border in an early developing long bone anlage because chondrocytes in that location would be closer to the source of positive perichondrially derived signals. To test this possibility, we systematically examined the development of long bone anlagen between day 7.5 and day 9.0 of chick embryogenesis. We knew from previous observations that a day 7.5 anlage contains chondrocytes up to the prehypertrophic stage but does not contain hypertrophic cells yet; conversely, a day 9 anlage displays a clear hyper- trophic zone in the diaphysis. Thus, we prepared longitudinal sections of limbs from day 7.5 through day 9 chick embryos and processed them for histology and in situ hybridization by using type X collagen as a molecular marker of chondrocyte hypertrophy. We found that the first hypertrophic type X collagen-expressing hypertrophic chondrocytes emerged on day 8.5 of development and were indeed located along the chondroperichondrial border; no such cells were present in the center where the distance from the border is greater (not shown; see Fig. 8 in ref. 8). By day 9, hypertrophic chon- drocytes had formed a “zone,” that is, they were uniformly present from border to border. To corroborate this finding, we performed another experiment. We implanted a single bead con- taining the retinoid antagonist RO 41-5253 next to the incipient diaphysis of a day 5.5 humerus anlage and reincubated the embryos until day 8.5. Because the antagonist emanates from a single bead, it creates a concentration gradient in its surroundings (23), including one from the near side (closest to the bead) to the far site of anlage’s diaphysis. If so, the antagonist should block the emergence of type X collagen-expressing chondrocytes in the near side but may not do so in the far side. In situ hybrid- ization on longitudinal sections of day 8.5 control and antagonist-treated humerus showed that this prediction was correct. Type X collagen-containing chondrocytes were present only on the far side and were absent in the near side (not shown; manuscript in preparation). Together, the data clearly indicate that the chondroperichondrial border serves as the initial site for emergence of hypertrophic chondrocytes. This site may thus have special promaturation properties, including presence of pro- maturation retinoids. CONCLUSIONS AND A MODEL The lines of evidence presented here and in previous reports provide strong evidence that the hedgehog and retinoid signaling pathways participate in, and regulate, long bone development. These pathways act within zones of the growth plate to regulate behavior and function of resident cells as well as amongst different growth plate zones. The latter is exemplified by the ability of IHH produced in the prehypertrophic zone to influence mitotic activity in the preceding proliferative zone. In addi- tion, these pathways appear to be able to mediate interactions between chondrocytes and surrounding perichondrial tissues. This is suggested by the involvement of chondrocyte-derived IHH in bone collar formation and of perichondrium-derived retinoids in chondrocyte function. Thus, these pathways represent critical signaling mechanisms that are interrelated and interdependent, counteract and counter- balance each other’s actions, and ultimately orchestrate long bone development. Their respective roles are depicted in the model shown in Fig. 6, which can be summarized as follows: (1) In the growth 168 Pacifici et al. plate of developing long bone anlagen, IHH expression is turned on in prehypertrophic chondrocytes (step 1); (2) IHH diffuses, reaches chondrocytes in the preceding proliferative zone, and regulates their mitotic activity and maturation rates (step 2); (3) IHH also reaches perichondrial cells and induces intramembranous ossification. This would require a transition from perichondrium to periosteum, angio- genesis and/or vessel recruitment, and osteogenesis (step 3); (4) The intramembranous process causes or is accompanied by a marked upregulation of retinoid synthesis or delivery of retinoids from perichon- drium/periosteum-associated blood vessels (step 4); and (5) The retinoids diffuse into the adjacent cartilage, switch off IHH expression and turn on RARa expression, and promote terminal maturation of chondrocytes and endochondral ossification (step 5). The model correlates well with recent data on the roles of IHH in chondrocyte proliferation and osteogenesis in developing long bones (24–27). For example, we and others have shown that IHH is a direct stimulator of chondrocyte proliferation (25–27) and that IHH is located in both prehyper- trophic and proliferative zones of the growth plate (28,29). In addition, the importance of retinoid signaling in cartilage maturation, matrix mineralization and osteogenesis has been reiterated by ele- gant recent studies (30–32). One particularly interesting insight is that retinoid signaling regulates expression of Cbfa1/Runx2 (31–33), a master regulator of chondrocyte hypertrophy and osteoblast differentiation (34). The model prescribes also that angiogenesis is an important aspect of long bone development and may actually have previously unsuspected roles (8). Blood vessels have long been known to be required for osteogenesis and marrow formation. In step 4 of the above model, however, we speculate that blood vessels may also be required at an earlier step during long bone development that is at the level of prehypertrophic/hypertrophic chondrocytes where the vessels would deliver retinoids or stimulate local production of them. The resulting increase in retinoid signaling would pro- mote further cartilage maturation, hypertrophy and endochondral ossification. Indeed, we and others have shown recently that pharmacological or genetic interference with angiogenesis has severe reper- cussions on not only osteogenesis but also chondrocyte maturation in developing long bones (28,35). When blood vessels did not form normally, chondrocyte maturation was delayed and the cells failed to display traits of their terminally mature phenotype. Fig. 6. Model depicting the distinct but interrelated roles of IHH and retinoid signaling in long bone develop- ment. See text for details. Signaling Molecules and Long Bone Development 169 It is important to point out here work by others indicating that perichondrium is a negative regulator of cartilage maturation (36,37) rather than a positive regulator as we propose. Before addressing this important issue, it should be remembered that perichondrium is not a homogeneous and static struc- ture. It is composed of cell layers with different organization, phenotype and function (38,39), and its phenotype and gene expression patterns change dramatically depending on its location along the epi- physeal–diaphyseal axis (40,41). Thus, it is actually possible that perichondrium has both negative and positive roles in long bone development and such differing functions depend on its topographical location/phenotype. In the epiphyses and proximal metaphyses where immature chondrocytes reside, perichondrium could have a negative role on maturation, would help the cells to remain proliferative and immature, and would clearly demarcate the cartilage boundary. In distal metaphyseal and dia- physeal regions instead, perichondrium would undergo a phenotypic change and favor/permit matu- ration as well as invasion of hypertrophic cartilage by progenitor bone and marrow cells and vessels. Although speculative at the moment, this possibility offers an explanation for the fact that cartilage does become hypertrophic and thus, mechanisms must exist to allow it to do so. Should perichondrium be such a powerful negative regulator of maturation as suggested by others, cartilage would never mature. These considerations and speculations underline the fact that much remains to be learned about long bone development and that exciting insights are to be expected by current unabating interest in limb skeletogenesis. ACKNOWLEDGMENTS We thank Dr. W. Abrams for help with preparation of figures and our colleagues Drs. S. L. Adams, T. Kirsch, and M. Iwamoto, who participated in the original studies upon which this chapter is based. 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(1997) Interleukin-6 with its soluble receptor enhances the expression of insulin-like growth factor-I in osteoblasts Endocrinology 138, 52 48 52 55 19 Ohlsson, C., Bergtsson, B.-A., Isoksson, O G P., Andreassen, T T., and Slootweg, M C (1998) Growth hormone and bone Endocr Rev 19, 55 –79 20 Rosen, C J (2002) Growth hormone and insulin-like growth factor-I treatment for metabolic bone diseases, in Principles... proteins, Smad1 through Smad8, have been identified and characterized Smads are divided into three classes according to their signaling functions: receptor-regulated Smads (R-Smad), common-Smads (Co-Smad), and inhibitory Smads (I-Smad; refs 1,4,7) R-Smads are ligand specific and include Smad1, Smad2, Smad3, Smad5, and Smad8 Among these R-Smads, Smad1, 5, and 8 are involved in the BMPs-activated signaling... blocks BMP-2-induced Runx-2 expression but not the activation of caspases or apoptosis induced by BMP-2, indicating that the BMP-2-induced apoptosis was independent of the Smad1 signaling pathway They found that the proapoptotic effect of BMP-2 was dependent on activation of protein kinase C (PKC) The inhibition of PKC by the selective PKC inhibitor calphostin C blocked the BMP-2-induced increase in the... the stromal cells in response to From: The Skeleton: Biochemical, Genetic, and Molecular Interactions in Development and Homeostasis Edited by: E J Massaro and J M Rogers © Humana Press Inc., Totowa, NJ 1 95 196 Reddy and Roodman Fig 1 Life cycle of the osteoclast 1,2 5- ( OH)2D3, is involved in osteoclast development by potentiating M-CSF-dependent proliferation of bone marrow cells and induction of osteoclast... cells Endocrinology 136, 33–38 32 Pash, J M and Canalis, E (1996) Transcriptional regulation of insulin-like growth factor-binding protein -5 by prostaglandin E2 in osteoblast cells Endocrinology 137, 23 75 2382 33 Raisz, L G., Fall, P M., Gabbitas, B Y., McCarthy, T L., Kream, B E., and Canalis, E (1993) Effects of prostaglandin E2 on bone formation in cultured fetal rat calvariae: role of insulin-like growth... Bax/Bcl-2 ratio, caspase activity, and apoptosis In contrast, the cAMP-dependent protein kinase A (PKA) inhibitor H89, the p38 MAPK inhibitor SB20 358 0, and the MEK inhibitor PD-98 059 did not affect BMP-2-induced apoptosis Therefore, Hay et al (42) proposed that BMP-2 modulated apoptosis through a Smad-independent, PKC-dependent pathway that uses Bax/Bcl-2, cytochrome c, and a caspase cascade that involves... exogenous PGE2 in a dose-dependent manner At 0 .5 nM PGE2, an approx 3- and 1 . 5- fold stimulation was observed, compared with the control and the OP-1-induced activity, respectively The enhancement of the OP-1-induced AP activity by PGE2 approached saturation at 0 .5 nM The effect of a long-term exposure of PGE2 on OP-1-treated FRC cells was also examined Figure 5 shows that the number of OP-1-induced bone... required for normal bone remodeling (see reviews in refs 19 and 20) At the cellular level, the high-affinity GH receptors are present in osteoblasts and the binding capacity is higher in differentiated cells The binding of GH stimulates osteoblastic cell proliferation (23), IGF-I synthesis (24), and IL-6 synthesis ( 25) Conversely, the IGF binding protein -5 stimulates GH synthesis in the osteosarcoma UMR cells... pathway between GH and IGF-I has been proposed (27) Additionally, GH induces BMP-2 and -4 expression and BMPR-IA in developing rat periodontium (28) Prostaglandins Ample data show that prostaglandins have important physiological roles in skeletal metabolism (21) PGE2 stimulates IGF-I synthesis, upregulates the IGF-I receptor, and increases both the synthesis and the degradation of IGFBP -5 (29–34) PGE2... and its activator, TAB1 (TGF- -activated kinase-binding protein), have been identified as components of BMP-mediated kinase-signaling pathways Additionally, X-linked inhibitor of apoptosis protein (XIAP), a broad inhibitor of the caspases, has been found to interact with the BMP type I receptor It appears that XIAP functions to connect BMP-mediated signaling to downstream signaling molecules TAB1 and . We implanted a single bead con- taining the retinoid antagonist RO 4 1 -5 253 next to the incipient diaphysis of a day 5. 5 humerus anlage and reincubated the embryos until day 8 .5. Because the antagonist. Nah, H D., and Pacifici, M. (19 95) Syndecan-3, tenascin-C, and the development of cartilaginous skeletal elements and joints in chick limbs. Dev. Dyn. 203, 152 –162. Synergy Between OP-1 and Osteotropic. between GH and IGF-I has been proposed (27). Additionally, GH induces BMP-2 and -4 expression and BMPR-IA in developing rat periodontium (28). Prostaglandins Ample data show that prostaglandins have