Histological development and dynamic expression of Bmp2 У6 mRNAs in the embryonic and postnatal mousecranial base.код для вставкиСкачать
THE ANATOMICAL RECORD PART A 288A:1250–1258 (2006) Histological Development and Dynamic Expression of Bmp2–6 mRNAs in the Embryonic and Postnatal Mouse Cranial Base PÄIVI KETTUNEN, XUGUANG NIE, INGER HALS KVINNSLAND, AND KEIJO LUUKKO* Department of Biomedicine, University of Bergen, Bergen, Norway ABSTRACT The cranial base is formed by endochondral ossiﬁcation and is characterized by the presence of the synchondrosis growth centers. The aim of this study was to describe the histological development of the mouse midsagittal cranial base area from embryonic day 10 (E10) to the postnatal age of 2 months. The Bmp family of signaling molecules serves important functions in embryo and bone development and may therefore play a signiﬁcant role in the early formation of the cranial base. To investigate this, we analyzed the mRNA pattern of expression of Bmp2–6 in the mouse cranial base from E10 to 5 days postnatally using radioactive in situ hybridization. We found that the formation of the mouse cranial base corresponds to that of rat and proceeds in a caudorostral sequence. Moreover, all Bmps studied showed distinct and overlapping developmentally regulated expression domains. Bmp2, Bmp5, and Bmp6 were expressed in the early mesenchymal condensations. Later, Bmp2, Bmp3, Bmp4, and Bmp5 were detected in the perichondrium and in the adjacent mesenchyme. Subsequently, Bmp2 and Bmp6 expressions were conﬁned to hypertrophic chondrocytes, while Bmp3, Bmp4, and Bmp5 were expressed in the osteoblasts of the trabecular bone and bone collar. Interestingly, Bmp3 was uniquely expressed postnatally in the resting zone of the synchondrosis growth center, suggesting a role in the regulation of cranial base growth. These results suggest that Bmp signaling may serve speciﬁc and synergistic functions at different key stages of cranial base development and growth. Anat Rec Part A, 288A:1250–1258, 2006. Ó 2006 Wiley-Liss, Inc. Key words: bone morphogenetic proteins; skeletogenesis; endochondral bone; hard tissue; growth; synchondrosis The cranial base, which is located between the brain and the facial bones, forms the ﬂoor of the neurocranium and is established by the coordinated development and growth of several skeletal elements. The human midline cranial base is composed of the basioccipital, sphenoid, ethmoid, and frontal bones (Scott, 1958). The cranial base is an important growth center of the head and is believed to play a signiﬁcant role in the integration of craniofacial development and growth. In contrast to the bones of the cranial vault and the face, which are formed by intramembranous ossiﬁcation, the midline cranial base bones, like the long bones, the vertebrae, and the ribcage, are formed by endochondral ossiﬁcation. However, Ó 2006 WILEY-LISS, INC. Grant sponsor: The Norwegian Cancer Society; Grant sponsor: the L. Meltzer Foundation; Grant sponsor: the University of Bergen; Grant sponsor: the Research Council of Norway. The ﬁrst two authors contributed equally to this work. *Correspondence to: Keijo Luukko, Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway. Fax: 47-55-58-63-60. E-mail: firstname.lastname@example.org Received 2 September 2006; Accepted 11 September 2006 DOI 10.1002/ar.a.20402 Published online 25 October 2006 in Wiley InterScience (www. interscience.wiley.com). Bmps IN DEVELOPING CRANIAL BASE the frontal bone is formed by intramembranous ossiﬁcation. The anterior cranial base, rostral to the sella turcica, is formed from neural crest-derived ectomesenchymal cells, whereas the cells forming the posterior cranial base derive from paraxial mesoderm (Couly et al., 1993). The mesenchymal cells condense and differentiate into chondrocytes, which proliferate and subsequently differentiate into hypertrophic chondrocytes. The cartilage model is gradually replaced by bone except for the synchondroses, which are the sites where the postnatal growth and expansion of the cranial base take place. The synchondrosis is a bidirectional growth site; it is structurally analogous to the growth plate of the long bones, which allow the growth of the bone. In the midsagittal region of the human cranial base, two synchondroses, the spheno-occipital and the intersphenoidal (midsphenoidal), are present (Scott, 1958; Melsen, 1974). The spheno-occipital synchondrosis is considered to be particularly important: in addition to providing most of the growth of the cranial base, it remains active into adolescence (Ingervall and Thilander, 1972; Thilander and Ingervall, 1973; Roberts and Blackfood, 1983; Kjaer, 1990). The enlargement of the brain has also been speculated to contribute to the growth of the cranial base by affecting the proliferation and differentiation of the chondrocytes in the synchondrosis (Ingervall and Thilander, 1972; Wang and Mao, 2002a, 2002b; Tang and Mao, 2006). While there has been signiﬁcant progress in understanding the molecular regulation of long bone and cranial vault development, little is known concerning that of the cranial base (see the reviews in Opperman, 2000; Wilkie and Morriss-Kay, 2001; Karsenty and Wagner, 2002; Ornitz and Maria, 2002). Cranial base defects have been identiﬁed in many human syndromes caused by gene mutations. For instance, abnormal cranial bases in achondroplasia and Apert, Crouzon, and Muenke syndromes are caused by mutations in FGFR, indicating that FGF signaling is not only important to the formation of the cranial vault, but also for the cranial base (Horowitz, 1981; Cohen et al., 1985; Kreiborg et al., 1993, 1999; Wilkie and Morriss-Kay, 2001; Reinhart et al., 2003). Similar defects have been observed in FGFR transgenic mouse strains (Chen et al., 2001; Eswarakumar et al., 2002, 2004). Mutations in RUNX2 and TWIST give rise to cleidocranial dysplasia and Saethre-Chotzen syndromes, respectively, which are characterized by defects in the cranial base (Evans and Christiansen, 1976; Kreiborg et al., 1981; Howard et al., 1997; Lee et al., 1997). In addition, abnormalities in the cranial base have been reported in syndromes with chromosome abnormalities, such as Cri-du-Chat, Down’s, Meckel, Seckel, Turner, and Williams, as well as in human triploid fetuses (Jensen, 1985; Kjaer et al., 1997, 1999, 2001; Andersen et al., 2000; Quintanilla et al., 2002; Axelsson et al., 2005). Abnormal cranial bases have also been described in anencephalia and hydrocephalus (Kantomaa et al., 1987; Lomholt et al., 2004). Together, these results suggest that several signaling pathways are involved in the regulation of the formation of the cranial base. Interestingly, recent experimental in vitro studies have shown that the proliferation and differentiation of the chondrocytes in the synchondrosis are modulated by mechanical loading, which inﬂuences the gene expression in the synchondrosis (Wang and Mao, 2002a, 2002b; Tang and Mao, 2006). Bone morphogenetic proteins (Bmps) are a family of more than 20 secreted signaling molecules that belong to a larger transforming growth factor b (Tgfb) superfamily (for 1251 review, see Ducy and Karsenty, 2000). Bmps signal through heteromeric complexes of transmembrane type I and type II serine/threonine kinase receptors, which transfer signals from the cell surface through the Smad pathway to the nucleus (for review, see Hoffmann and Gross, 2001). Bmp signaling is modulated by interaction with antagonists such as Chordin, Dan, Follistatin, Gremlin, Noggin, and Sclerostin (for review, see Canalis et al., 2003; Avsian-Kretchmer and Hsueh, 2004; Yanagita, 2005). The ﬁrst Bmps were identiﬁed by their ability to induce ectopic bone formation in vivo (Wozney et al., 1988). Later they were found to serve broader signaling functions in embryo and organ formation (for review, see Hogan, 1996; Chen et al., 2004). In vitro experiments and the analysis of transgenic mouse strains have shown that Bmps regulate skeletal patterning and bone development, but that their functions are largely redundant. Bmp7 null mutation leads to multiple developmental defects in the skeleton, including polydactyly, defective basisphenoid bone and xyphoid cartilage, fused ribs and vertebrae, and retarded ossiﬁcation of bones (Luo et al., 1995; Jena et al., 1997). Deletion of Bmp3, Bmp5, and Bmp6 in mice resulted in mild abnormalities in bones (Kingsley et al., 1992; King et al., 1994; Solloway et al., 1998; Daluiski et al., 2001). Bmp2/7 and Bmp5/7 double heterozygotes have normal skeletons, while Bmp4/7 double heterozygotes showed minor defects in the rib cage and digits (Katagiri et al., 1998). Disruption of Bmp2, Bmp4, and Bmpr1A (Alk3) did not produce much information about their roles in skeletogenesis due to early embryonic lethality (Mishina et al., 1995; Winnier et al., 1995; Zhang and Bradley, 1996). Defects in bone development have been reported in Bmp receptor and antagonist mutant mice such as ActR1 (Alk2), Bmpr1B (Alk6), and Noggin (Brunet et al., 1998; Yi et al., 2000; Dudas et al., 2004). The histological development and growth of the cranial base have been described earlier for rat (Baume, 1968; Dorenbos, 1973; Roberts and Blackwood, 1983) and humans (Baume, 1968). As an increasing number of transgenic mice are now produced for use as in vivo models for human syndromes and developmental disorders, this prompted us to investigate whether mouse cranial base development and ossiﬁcation are similar to that in humans and rat. In addition, knowledge of the gene expression patterns is essential for unraveling the molecular control of cranial base formation. In the present study, therefore, we systematically investigated the histological development of the mouse cranial base in midsagittal tissue sections between embryonic day 10 (E10) and the postnatal age of 2 months. Bmp signaling is important for skeletal development and therefore most likely also plays a signiﬁcant role in cranial base formation. Although expression domains of Bmp receptor mRNAs have been previously reported in the developing cranial base (Dewulf et al., 1995; Verschueren et al., 1995), the expression patterns of Bmps have not been analyzed systematically. We therefore compared the expression of Bmp2–6 mRNAs during cranial base development in mice from before the visible onset of its development from E10 until the presence of functional synchondroses 5 days postnatally (5PN). MATERIALS AND METHODS Preparation of Tissues and Histology The study was approved by the Animal Welfare Committee of the Biomedical Institute, University of Bergen. 1252 KETTUNEN ET AL. NMRI mice were mated overnight and the appearance of a vaginal plug was taken as day 0 of embryogenesis (E0). Delivery of NMRI mice takes place at E19, which corresponds to the newborn (NB) stage. Embryonic mouse heads between E10 and E18, NB heads, and 5-day-old (PN5), 9-day-old, and 2-month-old cranial bases were used in this study. The adult mice were killed by cervical dislocation. Embryos and postnatal mice were killed by decapitation. The age of the embryos was determined by the day of appearance of the vaginal plug and further conﬁrmed by morphological criteria according to Theiler (1989). Heads and cranial bases were dissected in Dulbecco’s phosphatebuffered saline (PBS) and ﬁxed in 4% paraformaldehyde (PFA) overnight at 48C. Heads and cranial bases older than E14 were decalciﬁed with 12.5% EDTA/2.5% PFA in PBS. They were then rinsed in PBS, dehydrated in ethanol, cleared in xylene, and embedded in parafﬁn. For histological analysis, 7 mm midsagittal sections of E10–E14, E16, NB, PN5, PN9, and 2-month-old tissues were cut with a microtome (Leica Microsystems, Wetzlar, Germany). The sections were deparafﬁnized in xylene, rehydrated in graded ethanol, and stained with hematoxylin and eosin (Shandon instant hematoxylin and eosin; Thermo Electron, Waltham, MA) for 2 and 5 min, respectively, according to the manufacturer’s instructions. The sections were then dehydrated, cleared in xylene, and mounted with DePeX (BDH, Poole, U.K.). In Situ Hybridization We used 7 mm midsagittal sections of E10–E14, E16, NB, and PN5 tissues for in situ hybridization analysis of Bmp2– 6 mRNA expression. The mouse Bmp2–6 cDNAs, which were a kind gift of Dr. Wozney of the Genetics Institute (Cambridge, MA), have been described earlier in Vainio et al. (1993) and Aberg et al. (1997). In vitro transcription and in situ hybridization were performed as described in Luukko et al. (1996). In brief, the cDNA fragments containing plasmids were linearized, and antisense and sense RNA probes were generated using 35S-labeled UTP (Amersham, Buckinghamshire, U.K.) and appropriate RNA polymerases. Alkaline hydrolysis was omitted as the length of the probes was considered to be appropriate for hybridization. The probes were passed through Sephadex G-50 NICK columns (Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s instructions, ethanol-precipitated, airdried, and dissolved in 1 M dithiothreitol (DTT; Sigma Chemical, Dorset, U.K.) and Wilkinson’s hybridization buffer at 1:9. The probes were diluted to the ﬁnal concentration of 5 3 104 cpm/ml, and 50–100 ml of the hybridization buffer with the labeled cRNA was added to each slide. Hybridization was carried out for 15–20 hr at 528C and the glasses were washed with 60% formamide (Merck, Darmstadt, Germany) and 30 mM of DTT at 658C. For autoradiography, the dehydrated slides were coated with NTB-2 emulsion (Eastman Kodak, New Haven, CT), dried, and exposed for 3 weeks at 48C. The sections were developed in Kodak D-19, ﬁxed with Uniﬁx (Eastman Kodak), counterstained with hematoxylin (Thermo Electron, Waltham, MA), and mounted with DePeX (BDH). Photomicrography was performed using a Zeiss Axioskop 2 microscope (Carl Zeiss, Jena, Germany) and a SPOT Insight digital camera (Diagnostic Instruments, Sterling Heights, MI). Brightand dark-ﬁeld images were taken from hybridized sections. Digital images were processed with Adobe Photoshop 7.0 software (Adobe Systems, San Jose, CA). Silver grains were selected from the dark-ﬁeld images, colored red, and superimposed onto the bright-ﬁeld images. RESULTS Histological Development of Mouse Cranial Base We used midsagittal sections to investigate the histological development of the mouse cranial base during embryonic and early postnatal development (E10 to 5PN). We further examined the histology of the spheno-occipital synchondrosis of 9-day-old and 2-month-old mice. In our analysis, we focused particularly on the basioccipital and sphenoid bones and the spheno-occipital synchondrosis. At E10, the notochord, which ﬁrst deﬁnes the midline axis, was observed in the area between the developing hindbrain and foregut. In addition, Rathke’s pouch was observed at the roof of the foregut, but no sign of cell condensation was detected in the loose mesenchyme in the presumptive cranial base area between the foregut epithelium and the developing brain (Fig. 1A). Mesenchymal cell condensation of the developing cranial base began to be visible at E11 in the caudal end of the future basioccipital bone (Fig. 1B). During subsequent development, the mesenchymal condensations of the anterior part of the presumptive basioccipital, basisphenoid, and presphenoid bones appeared (Fig. 1C). At E13, the chondrocranium was largely formed and chondrocyte differentiation had started in the middle of each cartilage template. Perichondral mesenchyme condensation surrounding the cartilage models was clearly visible on the brain side, whereas on the oral side the perichondrium was not fully developed along the whole chondrocranium (Fig. 1D). At E14, the sites of the future synchondroses began to be recognizable. Terminally differentiated hypertrophic chondrocytes were present in the caudal and middle part of basioccipital cartilage, but were not detected in the basisphenoid and presphenoid cartilages at this stage (Fig. 1E). At E16, the areas of hypertrophic chondrocytes had expanded in the presumptive basioccipital and basisphenoid bones. However, differentiation of hypertrophic chondrocytes was not yet observed in the presphenoid cartilage. At E16, bone formation was detected in the perichondrium of the future basioccipital and basisphenoid bones. In addition to the bone collar, trabecular bone formation was visible in the developing basioccipital bone. At this stage, the angle between the anterior and posterior cranial base was greater compared to earlier stages, and the straight shape of the skull base was observed (Fig. 1F). During postnatal development, the future cranial base became increasingly ossiﬁed except in the areas of the synchondroses (Fig. 1G–J). In the middle part of the synchondrosis, there was a narrow band of resting cells, surrounded by the proliferation, prehypertrophic, and hypertrophic zones on each side. At PN5, as at the earlier stages, the intersphenoidal synchondrosis was still broader than the spheno-occipital synchondrosis. In the young adult mouse (2 months), the basisphenoidal and intersphenoidal synchondroses had become a thin band of chondrocytes embedded in cartilage matrix (Fig. 1K). Expression of Bmp2–6 mRNAs in Developing Cranial Base We systematically compared the expression of Bmp2–6 at consecutive developmental stages between E10 and Bmps IN DEVELOPING CRANIAL BASE Fig. 1. Histological development of the embryonic (E10–E14 and E16) cranial base (A–F) as well as newborn (G), 5-day and 9-day postnatal and 2-month-old spheno-occipital synchondrosis (I–K). Midsagittal sections stained with hematoxylin and eosin. Higher magniﬁcations of the developing E13, E14, E16, and NB spheno-occipital synchondrosis (marked with a black asterisk) are provided in smaller photomicrographs (D–F, H). The intersphenoidal synchondrosis is marked with a turquoise asterisk. b, brain; ba1, ﬁrst branchial arch; bo, basioccipital 1253 cartilage; bob, basioccipital bone; bs, basisphenoid cartilage; bsb, basisphenoid bone; cm, calciﬁed cartilaginous matrix; e, erosive zone; h, hypertrophic chondrocytes; m, mesenchymal cell condensate; n, notochord; p, perichondrium; pe, periosteum; pi, pituitary gland; pm, perichondral mesenchyme condensation; pr, proliferating chondrocytes; pre, prehypertrophic chondrocytes; ps, presphenoid cartilage; rc, resting chondrocytes; r, Rathke’s pouch; t, trabecular bone; to, tongue. Scale bars ¼ 200 mm. 1254 KETTUNEN ET AL. Fig. 2. Expression of mRNAs for Bmp2, Bmp3, and Bmp4 in E10–14 and E16 (A–F, I–N, Q–V) developing cranial base as well as in newborn (G, O, W) and 5 days postnatal (H, P, X) developing spheno-occipital synchondrosis. Midsagittal sections. The expression of Bmp2–4 is also seen in the developing dura mater (arrowheads). Scale bars ¼ 200 mm in A (applies to A, I, Q and G, H, O, P, W, X) and 200 mm in B (applies to B–F, J–N, R–V). PN5. At E10, before any sign of cranial base development, Bmp2 mRNAs were detected in the loose mesenchyme next to the foregut epithelium in the prospective cranial base area (Fig. 2A). At E11, Bmp2 expression was seen in the ﬁrst mesenchymal condensations marking the sites of the future basioccipital and basisphenoidal bones (Fig. 2B). At E12, its expression extended to the anterior cranial base. Interestingly, in the caudal end of the basioccipital cartilage, Bmp2 mRNAs were downregulated in the differentiating chondrocytes, while the expression persisted in the surrounding mesenchymal condensate (Fig. 2C), and by E13 transcripts were mostly conﬁned to Bmps IN DEVELOPING CRANIAL BASE the perichondrium (Fig. 2D). At E14, Bmp2 expression was clustered in hypertrophic chondrocytes in the future basioccipital bone (Fig. 2E). Between E16 and PN5, transcripts were mostly seen in the hypertrophic chondrocytes in the spheno-occipital synchondrosis and in the perichondrium next to the developing synchondrosis (Fig. 2F–H). Bmp3 showed restricted expression in the epithelium of the foregut adjacent to the future anterior cranial base at E10 (Fig. 2I). From E11 onward, transcripts became conﬁned to the perichondrium (Fig. 2J–M). At E16, Bmp3 mRNAs also appeared in the developing trabecular bone in the middle of the basioccipital cartilage (Fig. 2N). At birth, Bmp3 was expressed in the osteoblasts of trabecular bone and periosteum (Fig. 2O). Interestingly, a weak expression was also observed in the future site of the resting zone of the synchondrosis, which became more prominent by PN5 (Fig. 2P). Bmp4 expression was detected in the foregut epithelium and had a diffuse presence in the adjacent mesenchyme in the cranial base area at E10 (Fig. 2Q). From E11 onward, expression was found in the mesenchyme surrounding the cartilage templates (Fig. 2R–U). From E16 onward, Bmp4 mRNAs were observed in the trabecular bone and in the perichondrium (Fig. 2V–X). Bmp5 was seen in the notochord of the E10 cranial base area (Fig. 3A). At E11, expression appeared in condensed mesenchyme and, 1 day later, also in the prospective perichondrium (Fig. 3B and C). At E13 and later stages, Bmp5 expression was conﬁned to the perichondrium and subsequently to the periosteum (Fig. 3D–G). In addition, some transcripts were observed in the trabecular bone at E16 and later stages (Fig. 3E–G). Bmp6 expression ﬁrst appeared in the developing cranial base at E11. Transcripts were seen in the mesenchymal condensations of the caudal part of the cranial base during E11 and E12, but not at the rostral end (Fig. 3H–J). At E13, transcripts were observed in the early hypertrophic chondrocytes (Fig. 3K). From E16 onward, Bmp6 expression was conﬁned to the hypertrophic chondrocytes of the developing synchondrosis (Fig. 3L–N). In addition, some transcripts were seen in the trabecular bone and in the perichondrium/periosteum at NB and PN5 (Fig. 3M and N). DISCUSSION The cranial base is an important growth center of the head, integrating the growth of the cranial vault and facial bones. However, the molecular mechanisms of its formation and growth have remained largely unknown. In this study, we have described the histological formation of the mouse cranial base in the midsagittal area between E10 and 2 months after birth. We found that the histological development of the embryonic and early postnatal mouse cranial base corresponds to that of rats (Baume, 1968; Dorenbos, 1973; Roberts and Blackwood, 1983). Mouse cranial base development was observed to start at the caudal end as a mesenchymal condensation and, as in humans and rats, proceeded in a caudorostral sequence (Scott, 1958; Baume, 1968; Dorenbos, 1973; Roberts and Blackwood, 1983; Kjaer, 1990). Moreover, analysis of the expression of Bmp2–6 mRNAs in embryonic and early postnatal stages using radioactive in situ hybridization demonstrated that all the genes studied had a distinct, developmentally regulated expression domain. This sug- 1255 Fig. 3. Expression of mRNA for Bmp5 and Bmp6 in E10–E13 (A–D, H–K) and E16 (E, L) developing cranial base as well as in NB (F, M) and PN5 (G, N) spheno-occipital synchondrosis. Midsagittal sections. In addition to the cranial base, the expression of Bmp5 and Bmp6 is seen in the developing dura mater (arrowheads). Scale bars ¼ 200 mm in A and H; 200 mm in N (applies to B–G, I–N). gests that Bmp signaling plays an important role at different stages of cranial base development and growth. Possible Functions of Bmps in Condensation of Cranial Base Mesenchyme The ﬁrst event of the skeletogenesis is the migration of undifferentiated mesenchymal cells to the areas destined to become bone. This is followed by the condensation of the cells, which determines the site, size, and shape of future bones (Ducy and Karsenty, 1998; Hall and Miyake, 2000). During the initial stages of mouse cranial base development, two types of mesenchyme condensations were observed. One was the mesenchymal condensation for cartilage templates; the other was the perichondral mesenchymal condensation that surrounds the cartilage templates. Bmp signaling has been shown to be an important patterning signal during body development (for review, see Hogan, 1256 KETTUNEN ET AL. 1996) and may be involved in prepatterning of neural crest skeletal lineage (Kanzler et al., 2000). We found that Bmp2 and Bmp4 showed restricted expression in the mesenchymal area of the cranial base before the condensations appeared. Subsequently, at E11, Bmp2 expression continued in the condensate where Bmp5 and Bmp6 were also upregulated, while Bmp3 and Bmp4 were expressed in the surrounding mesenchyme. Bmp2 and Bmp5 have been reported in the mesenchymal condensates preﬁguring the future skeleton (Ducy and Karsenty, 2000), and their signaling appears to regulate the size and shape of the condensations (King et al., 1994; Zou et al., 1997; Pizette and Niswander, 2000). Bmp signaling, including Bmp2, regulates Sox9, which is essential for formation of the mesenchymal condensation and subsequent cartilage formation (Bi et al., 1999, 2001; Healy et al., 1999). In vitro, the Bmp antagonist Noggin inhibits aggregation of mesenchymal cells into prechondrogenic condensations (Pizette and Niswander, 2000). On the other hand, Bmp3 has been shown to antagonize osteogenic Bmps (Daluiski et al., 2001). Thus, the combined signaling activity of Bmp2 and Bmp5, and probably Bmp3, Bmp4, and Bmp6, may serve patterning functions in cranial base development by regulating the site, size, and shape of the mesenchymal condensates. This may take place through activation of Sox9 in the developing cranial base anlage (Nie et al., 2005) and through coordinated action with Shh, which is a patterning signal for the head that includes the cranial base and facial bones and, like Sox9, is expressed in the developing cranial base area (Chiang et al., 1996; Zeng et al., 2002; Jeong et al., 2004, Nie et al., 2005). Mouse embryos lacking Hh signaling or Sox9 in the neural crest cells show similar patterning defects in the cranial base (Mori-Akiyama et al., 2003; Jeong et al., 2004). Interestingly, we observed Bmp6 in the mesenchymal condensation of the presumptive basioccipital bone, which derives from the paraxial mesoderm, but no expression was seen in the basisphenoid and presphenoid condensations derived from the neural crest (Couly et al., 1993). Thus, the early Bmp6 expression may reﬂect the differing origin of the cranial base bones. Possible Roles of Bmps in Chondrocyte Proliferation and Differentiation in Cranial Base After the mesenchymal condensation, the cells in the condensate differentiate into chondrocytes, which form the cartilage templates for the cranial base bones. There is a substantial body of in vitro and genetic evidence that Bmp signaling is involved in the regulation of chondrocyte development and differentiation (Zou et al., 1997; Ducy and Karsenty, 2000; Pizette and Niswander, 2000; Yi et al., 2000; Minina et al., 2002; Shum et al., 2003). We found that expression patterns of the studied Bmps in the developing cartilage templates of the cranial base were largely similar to those of other developing bones after condensation and prior to osteogenesis (Duprez et al., 1996; Solloway et al., 1998; Minina et al., 2001). The perichondrium and the surrounding mesenchyme showed Bmp2, Bmp3, Bmp4, and Bmp5 expression. Bmp2 and Bmp6 expressions were gradually conﬁned to the hypertrophic chondrocytes. Thus, these results suggest that Bmp signaling, possibly in interaction with Fgfs and Ihh/Pthlh, regulates chondrocyte differentiation, proliferation, maturation, and hypertrophy during cranial base development, as has also been proposed in relation to long bones (Minina et al., 2002; Kobayashi et al., 2005). Recently, Ihh, which controls chondrocyte proliferation and differentiation, was shown to transduce mechanical signals and upregulate Bmp2/4 in chondrocytes (Wu et al., 2001). Moreover, mechanical loading was proposed to induce cartilage formation in the mesenchymal tissue in vivo by downregulating BMP3 (Aspenberg et al., 2000). In addition, mechanical stress was shown to promote the proliferation of the cranial base cartilage in vivo (Wang and Mao, 2002b). Thus, Bmps may be involved in mediation of the possible mechanical inﬂuence of the expanding brain on cartilage formation during cranial base development and growth. Possible Roles of Bmps in Ossiﬁcation of Cranial Base The developing cranial base is formed through two types of ossiﬁcation processes. The cartilage templates of the cranial base bones undergo endochondral ossiﬁcation, whereby hypertrophic chondrocytes are replaced by invading osteoprogenitor cells that subsequently differentiate into osteoblasts and produce bone. The periosteum lining the bone proper forms through intramembranous ossiﬁcation, whereby the mesenchymal cells differentiate directly into osteoblasts (Karsenty and Wagner, 2002). Bmps were originally identiﬁed by their ability to induce ectopic bone formation (Wozney et al., 1988), but their actual in vivo roles and the molecular basis of their functions in bone formation have remained largely unknown, partly due to the fact that several Bmp mutant mouse strains have only mild skeletal patterning and joint formation anomalies or early lethality (Karsenty and Wagner, 2002). However, targeted inactivation of the antiproliferative protein gene Tob in transgenic mice resulted in greater bone mass due to an increased number of osteoblasts, providing indirect evidence that Bmp/Smad signaling controls osteoblast proliferation and differentiation in vivo (Yoshida et al., 2000). We found the studied Bmps in sites or adjacent to sites that contain osteoblast precursors and osteoblasts that largely correspond to their reported expression patterns in the long bones and ribs (Duprez et al., 1996; Solloway et al., 1998; Minina et al., 2001). Bmp2 and Bmp4 share the same receptors and apparently regulate bone formation (Yoshida et al., 2000; Minina et al., 2001; Wright et al., 2002). We found Bmp2 and Bmp4 mRNAs in the perichondrium and adjacent mesenchyme, respectively. In addition, the developing trabecular bone showed Bmp4 transcripts. Thus, the proteins might regulate osteoblast proliferation and differentiation and subsequent ossiﬁcation in the cranial base. In contrast, Bmp3, transcripts of which were found in the developing periosteum and trabecular bone, and which acts as an antagonist to osteogenic Bmps, especially to Bmp2 (Daluiski et al., 2001), might modulate the activity of osteogenic Bmps and negatively regulate the density of the cranial base bones as shown for the long bones (Daluiski et al., 2001). Possible Roles of Bmps in Postnatal Growth of Cranial Base The postnatal lengthening of the cranial base takes place predominantly in the spheno-occipital synchondrosis. Earlier Bmp signaling has been shown to control chondrocyte proliferation in cultured cranial bases (Shum et al., 2003). Interestingly, we observed Bmp3 expression in the resting zone of the spheno-occipital syn- Bmps IN DEVELOPING CRANIAL BASE chondrosis from the newborn stage onward. The resting zone of the growth plate in the long bones has been suggested to contain stem cell-like cells or precursor cells of proliferating chondrocytes (Abad et al., 2002). The in vivo function of Bmp3 in chondrocytes is unknown, but recent in vivo data suggest that it inhibits cell differentiation during bone formation (Aspenberg et al., 2000; Daluiski et al., 2001). Therefore, it is tempting to propose that Bmp3 may act to keep resting zone chondrocytes and adjacent proliferating chondrocytes undifferentiated, which would control the proliferation rate of the chondrocytes and thereby inﬂuence the growth of the cranial base. 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