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Keywords:

  • signaling;
  • skeletogenesis;
  • endochondral bone;
  • embryonic

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The cranial base, located between the cranial vault and the facial bones, plays an important role in integrated craniofacial development and growth. Transgenic Shh and Sox9-deficient mice show similar defects in cranial base patterning. Therefore, in order to examine potential interactions of Shh, Ihh, another member of the Hh family, and Sox9 during cranial base development and growth, we investigated their cellular mRNA expression domains in the embryonic (E) and early postnatal (PN) cranial base from E10 to PN5 using sectional radioactive 35-S in situ hybridization. Of the Hhs, Shh was observed in the foregut epithelium and the notochord, while Sox9 showed broad expression in the loose mesenchyme of the cranial base area during E10–E11. Subsequently, from E12 onward, all genes were observed in the developing cranial base, and after birth the genes were prominently colocalized in the prehypertrophic chondrocytes of the synchondroses. Collectively, these data suggest that Hh-Sox9 auto- and paracrine signaling interactions may provide a critical mechanism for regulating the patterning of the cranial base as well as for its development and growth. © 2005 Wiley-Liss, Inc.

The cranial base is a bony structure located between the cranial vault and the facial bones. It is mainly a midline structure, composed of basioccipital, sphenoid, ethmoid, and frontal bones (Vilmann, 1969, 1971). Cranial base is an important growth site of the head and plays a central role in integrated craniofacial development and growth. The anterior cranial base rostral to the sella turcica develops from the neural crest-derived ectomesenchymal cells, whereas the posterior cranial base derives from the paraxial mesoderm (Couly et al., 1993). Unlike the other craniofacial bones, which are predominantly formed by intramembranous ossification, the cranial base is formed by endochondral ossification, in which the mesenchymal cells condense and subsequently form the cartilage model of the cranial base. The cartilage is replaced by bone except in the synchondroses, which are well-organized cartilage structures similar to long bone growth plates connecting the individual bones (Baume, 1968). The enlarging brain influences the growth of the cranial base during early stages. However, synchondroses continue to grow after cessation of brain growth (Kantomaa et al., 1991; Hilloowala et al., 1998). Thus, the formation of the cranial base is controlled by genetic and epigenetic factors. In many human syndromes, such as Down syndrome, Turner syndrome, cleidocranial dysplasia, craniosynostosis syndromes, Seckel syndrome, and Williams syndrome, the cranial base is affected (Jensen, 1985; Kreiborg et al., 1993, 1999; Kjaer et al., 2001; Quintanilla et al., 2002; Lomholt et al., 2003; Axelsson et al., 2005).

While the molecular mechanisms orchestrating axial and appendicular bone formation and growth have begun to be elucidated in great detail, less is known about the molecules regulating that of the cranial base. Genetic and experimental evidence indicate that some critical signaling pathways are shared among endochondral skeleton (Opperman, 2000; Karsenty and Wagner, 2002). Bone morphogenetic proteins (Bmp), fibroblast growth factors (Fgf), hedgehog (Hh), and Drosophila Wingless (Wnt) families, which are involved in general embryonic development, also regulate skeletal development (Opperman, 2000; Karsenty and Wagner, 2002; Ornitz and Marie, 2002; Ornitz, 2005). Inactivation or overactivation of Fgf signaling leads to cranial base anomalies (Eswarakumar et al., 2002, 2004; Rice et al., 2003). Bmp signaling has been shown to be able to regulate synchondrosis growth in an in vitro system (Shum et al., 2003).

The hedgehog signaling family consists of three members, Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Shh and Ihh play an important role in endochondral bone formation. Transgenic mice lacking Shh function suffer from severe defects in bone formation, such as absence of spinal column and defects in limb bones, and cyclopia (Chiang et al., 1996). In humans, mutations in SHH give rise to holoprosencephaly, a failure of cleavage of the prosencephalon with a deficit in midline facial development (Belloni et al., 1996; Roessler et al., 1996). Ihh null mutant mice, on the other hand, display markedly reduced chondrocyte proliferation, maturation of chondrocytes at inappropriate positions, and a failure of osteoblast development in endochondral bones (St-Jacques et al., 1999).

Previous research has provided evidence that Shh mediates its effects on bone development by inducing Sox9 expression, a key transcription factor essential for chondrogenesis and subsequent endochondral ossification (Bi et al., 1999; Healy et al., 1999; Akiyama et al., 2002; Zeng et al., 2002; Cheung and Briscoe, 2003; Mori-Akiyama et al., 2003; Tavella et al., 2004). In humans, mutations in SOX9 result in campomelic dysplasia (CD), a skeletal dysplasia syndrome characterized by sex reversal and skeletal malformations of endochondral bones, such as bowing of the limbs and cranial defects (Foster et al., 1994; Wagner et al., 1994). In mouse, targeted inactivation of Sox9 in the neural crest cells resulted in defective presphenoid and basisphenoid bone formation, demonstrating its essential role for cranial base development (Mori-Akiyama et al., 2003).

The expression domains of Shh, Ihh, and Sox9 at successive stages of embryonic and postnatal development and growth of the cranial base are not known. Therefore, in order to investigate potential interactions between Hh signaling and Sox9 during cranial base development and growth, we studied their mRNA expression domains by radioactive in situ hybridization from mouse midsagittal tissue sections from before the visible onset of cranial base development at embryonic day 10 (E10) until the development of functional synchondrosis at 9 days postnatally (PN9).

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Preparation of Tissues

All procedures involving animals were approved by the Animal Welfare Committee of the University of Bergen. Mice (NMRI) embryos between E10 and E18, as well as newborn (NB) and PN3, PN5, and PN9 mice were dissected in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA) overnight at 4°C. The mice were killed by cervical dislocation and the embryos by decapitation. Mice embryos over E15 were decalcified with 12.5% EDTA-2.5% PFA in PBS. They were then embedded in paraffin. Sagittal sections were cut from the midline area.

Construction of Probes and In Situ Hybridization

Plasmids containing fragments of Ihh and Sox9 cDNAs were generated by RT-PCR from total RNA isolated from E18 mouse lower jaws and E13 mouse heads, respectively, and subcloned into pGEM-T Easy Vector (Promega, Madison, WI): a 405 bp Ihh fragment spanning the region between 1679 and 2083 in accession number NM 010544, and a 586 bp Sox9 fragment spanning the region between 748 and 1333 in accession number AF21878. The following primer oligonucleotides were used: Ihh F 5′-CTCTAACCACTGCCCTCCTG-3′ and R 5′-GGGAATCTAGCAGCATCGAC-3′, and Sox9 F 5′-GTTGATCTGAAGCGAGAGGG-3′ and R 5′-TCTGATGGTCAGCGTAGTCG-3′. Shh cDNA was a kind gift from Dr. Andrew McMahon (Harvard University, Cambridge, MA).

In situ hybridization on sections was performed as described previously (Luukko et al., 1996). Briefly, 35 S-UTP-labeled Shh, Ihh, and Sox9 antisense and sense cRNA probes were generated by in vitro transcription. After the hybridization procedure, the slides were dipped in NTB-2 emulsion (Eastman Kodak) for autoradiography. After 3 weeks' exposure at 4°C, the slides were developed and fixed, counterstained with hematoxylin, and mounted. Images were taken with a Spot Insight digital camera (Diagnostic Instruments, Sterling Heights, MI) mounted on a Zeiss Axioskop2 microscope (Carl Zeiss Jena, Jena, Germany). The bright-field and dark-field images of each section were digitized separately and the grains from dark fields were selected, colored red, and copied onto the bright-field image using Adobe Photoshop 7.0 software (Adobe Systems, San Jose, CA). No specific signal was detected in sections hybridized with the control sense probes (not shown).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Expression of Shh During Cranial Base Development

As the cranial base is largely a midline structure, we used serial midsagittal sections to analyze mRNA expression of Shh, Ihh, and Sox9 in the developing basioccipital and basisphenoid bones and in the spheno-occipital synchondrosis.

At E10, before the histological initiation of cranial base development, Shh was prominently expressed in the notochord (shown for E12; 1C) and in the dorsal midline area of the foregut epithelium (Fig. 1A). At E11 and E12, Shh expression continued in the foregut epithelium (Fig. 1B and C), and at E12 transcripts also appeared in the basioccipital chondroblasts (Fig. 1C). No specific expression was observed in the later developing anterior cranial base mesenchyme area at this stage. At E13, Shh transcripts were seen in the immature chondrocytes of the developing basioccipital and basisphenoid bones (Fig. 2A). One day later, Shh was observed in the differentiating chondrocytes, and by E16 expression was confined mainly to the prehypertrophic chondrocytes, but some expression was also seen in hypertrophic chondrocytes of the developing spheno-occipital and intersphenoid synchondroses (Fig. 2B–F). The Shh expression in the synchondroses continued to the last studied PN9 stage as shown for spheno-occipital synchondrosis (Fig. 2E and F). The expression of Shh in the hypertrophic cells correlates with the Shh expression in the growth plate of the chicken long bone (Wu et al., 2002).

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Figure 1. Comparison of Shh and Sox9 mRNA expression in the developing cranial base at E10–E12 analyzed by radioactive in situ hybridization on midsagittal sections. Red coloring, which indicates hybridization signal, is superimposed onto the hematoxylin-stained histology image. Shh transcripts are seen in the foregut epithelium in the dorsal midline area between E10 and E12 (AC). At E12, Shh is seen in the chondroblasts of the posterior part of the future basioccipital bone and in the notochord (C). Sox9 shows broad expression in the loose mesenchyme at E10 (D), which subsequently, during E11 and E12, becomes restricted to the mesenchymal condensates of the future cranial base bones (E and F). b, brain; ba1, first branchial arch; bo, basioccipital cartilage; m, mesenchymal cell condensate; n, notochord; pi, pituitary gland; r, Rathke's pouch; to, tongue. Scale bars = 400 μm in A (applies to E10 stages) and B (applies to E11 and E12).

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Figure 2. Comparison of Shh and Sox9 mRNA expression in E13 (A and G), E14 (B and H), and E16 (C and I) developing cranial base, as well as in NB (D and J), PN5 (E and K), and PN9 (F and L) spheno-occipital synchondrosis analyzed by radioactive in situ hybridization on midsagittal sections. Red coloring, which indicates hybridization signal, is superimposed onto the hematoxylin-stained histology image. A: Shh is observed in the immature chondrocytes of the developing basioccipital and basisphenoid bones at E13. B: At E14, Shh expression is seen in the differentiating chondrocytes. C–F: At E16 and postnatal stages, Shh expression is evident in the prehypertrophic chondrocytes. Some expression is also seen in the hypertrophic chondrocytes of the developing spheno-occipital and intersphenoid synchondroses. G: Sox9 is seen in the cartilage model of the cranial base at E13. H–L: During subsequent development and cranial base ossification between E14 and PN9, Sox9 expression gradually becomes restricted to the resting, proliferating, and prehypertrophic chondrocytes of the synchondroses. b, brain; bo, basioccipital cartilage; bs, basisphenoid cartilage; e, erosive zone; h, hypertrophic chondrocytes; is, intersphenoid syndchondrosis; pi, pituitary gland; pr, proliferating chondrocytes; pre, prehypertrophic chondrocytes; rc, resting chondrocytes; so, spheno-occipital synchondrosis; t, trabecular bone; to, tongue. Scale bars = 400 μm in A (applies to E13–E16) and D (applies to postnatal stages).

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Expression of Sox9 in Developing Cranial Base

In contrast to Shh, Sox9 showed broad expression in the loose head mesenchyme at E10 (Fig. 1D). At E11, Sox9 expression had become restricted to the mesenchymal condensates of the developing basioccipital and basisphenoid bones. At the anterior part of the developing cranial base, the expression largely continued throughout the loose mesenchyme (Fig. 1E). At E12, Sox9 transcripts were restricted to the cartilage templates of the cranial base bones (Fig. 1F). From E14 onward, Sox9 expression was gradually downregulated from the chondrocytes as they became hypertrophic (Fig. 2H–L). The expression became restricted to the synchondroses with high expression in the prehypertrophic chondrocytes. In addition, some early hypertrophic chondrocytes showed Sox9.

Expression of Ihh in Cranial Base

Ihh, which is expressed in the developing endochondral bones, is an essential regulator of their formation (St-Jacques et al., 1999; Karp et al., 2000; Long et al., 2001, 2004; Minina et al., 2001, 2002; Jeong et al., 2004), but characterization of its cellular expression in the cranial base has been limited to E15 stage (Iwasaki et al., 1997). We therefore analyzed cellular expression domains of Ihh mRNAs during E10–PN5. In agreement with a previous RT-PCR result (Kronmiller and Nguyen, 1996), we did not observe Ihh expression in the developing cranial base area or adjacent tissues during E10–E11 (Fig. 3A and B). A prominent Ihh expression in the cartilage model of the developing basioccipital cartilage was detected at E12 (Fig. 3C). Subsequently, at E13 and E14, the expression was also observed in basisphenoid cartilage (Fig. 3D and data not shown). By E16, transcripts were confined to the differentiating chondrocytes of the future spheno-occipital, intersphenoid, and sphenoethmoidal synchondroses (Fig. 3E). After birth, Ihh expression in the synchondroses was evident in the prehypertrophic chondrocytes and was also observed in some early hypertrophic chondrocytes. This expression pattern continued in the later postnatal stages (Fig. 3F and G). It is interesting to note that Ihh was also observed in the developing intramembranous mandibular and maxillary bones at E13 and E14, as shown for E13 (Fig. 3H).

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Figure 3. Expression of Ihh mRNA in E10–E13 and E16 cranial base as well as in newborn and 5-day postnatal spheno-occipital synchondrosis analyzed by radioactive in situ hybridization on midsagittal sections. Red coloring, which indicates hybridization signal, is superimposed onto the hematoxylin-stained histology image. A and B: No Ihh expression is seen in the future site of the cranial base or adjacent tissues at E10 and E11. C: At E12, Ihh transcripts are seen in the chondroblasts of the posterior part of the future basioccipital bone. D: At E13, Ihh expression are observed in the chondrocytes of the developing basioccipital and basisphenoid bones. E: At E16, transcripts are seen in the prehypertrophic and in some early hypertrophic chondrocytes. F and G: In newborn and 5-day postnatal spheno-occipital synchondrosis, Ihh is expressed in the prehypertrophic and in some hypertrophic chondrocytes. H:Ihh transcripts are present in the developing mandibular bone (arrow), which forms through intramembranous ossification. b, brain; ba1, first branchial arch; bo, basioccipital cartilage; bs, basisphenoid cartilage; is, intersphenoid synchondrosis; m, mandibular bone; mc, Meckel's cartilage; pre, prehypertrophic chondrocytes; h, hypertrophic chondrocytes; rc, resting chondrocytes; pi, pituitary gland; so, spheno-occipital synchondrosis; t, trabecular bone; to, tongue. Scale bars = 400 μm. Scale bar in D applies to E13 and E16 stages. Scale bar in F applies to postnatal stages.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

In mouse, the histological initiation of cranial base formation is visible when the mesenchyme condensation of the posterior part of the future basioccipital cartilage appears at E11. The synchondrosis growth centers are functional and express all the cell types typical for them during postnatal ages in murine (Baume, 1968, and this study). Therefore, our study, spanning from E10 to PN9 covers all the critical stages of cranial base development such as patterning, chondrogenesis and osteogenesis, and growth by synchondrosis. Hh signaling and Sox9 have been shown to be essential for proper cranial base formation (Bi et al., 1999; Hu and Helms, 1999; Akiyama et al., 2002; Jeong et al., 2004), but the cellular localization of Shh, as well as Ihh and therefore the exact roles of Hh signaling during cranial base development, has remained unknown. We found that Shh, Ihh, and Sox9 showed developmentally regulated expression patterns in sites that suggest potential regulatory interactions for them at different stages of the cranial base formation.

Possible Interactions Between Shh and Sox9 During Patterning of Cranial Base

The condensation process of mesenchymal cells started at the sites of future basioccipital bone at about E11 and proceeded rostrally. In line with earlier reports (Chiang et al., 1996; Zeng et al., 2002), we observed Shh in the notochord at E10–E12. However, we also found that Shh was specifically expressed in the dorsal midline of the foregut epithelium. In contrast, Sox9 showed prominent expression in the adjacent loose mesenchyme throughout the presumptive cranial base area. Later at E12, Shh was also expressed in the cartilage model of basioccipial bone while Sox9 continued to show broader expression.

Sox9 regulates the site and shape of the skeletal condensation (Bi et al., 1999). Earlier studies have shown that paracrine Shh signaling from the notochord and floor plate induces Sox9 in sclerotome, which is subsequently maintained by Bmp signaling (Zeng et al., 2002). Targeted inactivation of the common hedgehog receptor Smoothened in mouse has demonstrated that Hh signaling is not necessary for cranial neural crest cell generation and migration but is essential for later developmental steps of cranial patterning, including proper patterning and formation of the cranial base (Jeong et al., 2004). The observed defects in the cranial base of the Smoothened mutant mice correlate remarkably with the defects in Sox9−/− mice, both of which show defective/absence of presphenoid and basisphenoid bones (Mori-Akiyama et al., 2003; Jeong et al., 2004). Based on these experimental results and our gene localization data, we suggest a model where local paracrine Shh signaling from the foregut epithelium and the notochord controls Sox9 in forming mesenchyme condensations. Later autocrine Shh signaling regulates Sox9 in the cartilage model. Therefore, we propose that Shh-Sox9 signaling interaction is a critical mechanism in regulating the patterning and formation of the cranial base. Our detailed analysis also showed that Ihh mRNAs were absent from the developing cranial base during the early stages of cranial base patterning and formation. Thus, the functions of Shh at these earliest stages appear not to be redundant with Ihh, as also supported by the fact that Ihh null mutant mice show normal early patterning of the head skeleton (St-Jacques et al., 1999). Furthermore, because Dhh is not reported in the developing skeleton or head and no defects have been observed in the head in the mutant mice, the function of Hh signaling in the earliest stages of cranial base development may be solely mediated by Shh (Bitgood and McMahon, 1995; Bitgood et al., 1996).

Possible Roles of Shh Signaling During Chondrocyte Life Cycle

During subsequent development, the mesenchymal cells in the future cranial base condensations differentiate into chondroblasts, which proliferate, differentiate, and become hypertrophic. We observed that at E12 and E13 Shh was coexpressed with Sox9 in the cartilage model of the developing basioccipital bone. Subsequently, similar expression was also observed in the rostral developing cranial bone anlages. Thereafter, Shh expression became restricted to the prehypertrophic chondrocytes, while Sox9 continued in resting, proliferating, and prehypertrophic chondrocytes. The Shh expression pattern largely correlated with that of Ihh, in particular in the prehypertrophic chondrocytes. Genetic experiments have shown that both Sox9 and Ihh are needed for chondrocyte proliferation and differentiation (St-Jacques et al., 1999; Karp et al., 2000; Long et al., 2001; Minina et al., 2001, 2002; Akiyama et al., 2002). Shh and Ihh share the same signaling receptor in the developing bones (Long et al., 2001; Jeong et al., 2004), and recently it has been shown that continuous Shh overexpression in chondrocytes resulted in prominent Sox9 expression and absence of exoccipital and supraoccipital bones in mice (Tavella et al., 2004). Thus, the observed overlapping expression domains of Ihh, Shh, and Sox9 suggest that in addition to Ihh, Shh acts to maintain Sox9 in the undifferentiated chondrocytes by autocrine signaling and thereby controls proper chondrocyte proliferation and differentiation and subsequent bone formation.

Possible Function of Hh Signaling and Sox9 in Synchondrosis

Synchondroses are bidirectional growth centers, analogous structures to the growth plates of the long bones (Abad et al., 2002). The proliferation of the cartilage cells in both sides of the resting zone, which contain stem cell-like cells, contributes to the postnatal growth and expansion of the cranial base. Subsequently, the cells become hypertrophic, undergo apoptosis, and are replaced by the trabecular bone. As cranial base development proceeded, the expression of Shh and Sox9 as well as Ihh was gradually restricted to the synchondroses. Their expression patterns are virtually similar to that of long bone growth plates, suggesting the same regulatory functions. Knowing the essential function of Ihh for cartilage and bone development (St-Jacques et al., 1999; Karp et al., 2000; Long et al., 2001, 2004; Minina et al., 2001, 2002) and that both Shh and Ihh were prominently expressed in the prehypertrophic chondrocytes, we propose that Shh may regulate chondrocyte proliferation and differentiation in the synchondrosis and thereby control its development and growth. In support of this, overexpression of Shh leads to defects in growth plates with cessation of chondrocyte differentiation at the prehypertrophic stage (Tavella et al., 2004). Sox9 was broadly expressed in developing synchondrosis, and after birth its expression continued in all chondrocytes except for the late hypertrophic ones, with the highest expression in the prehypertrophic chondrocytes. Sox9 function is essential for various stages of cartilage development. Therefore, it is possible that Sox9, regulated by auto- and paracrine Shh and Ihh, may serve important roles in maintaining the organization and function of cartilaginous synchondroses during cranial base development and growth. Together, our results provide further evidence that the signaling pathways regulating long bone and cranial base development and growth by the epiphyseal growth plates and bidirectional synchondrosis, respectively, are shared. However, it is obvious that, like the formation of bones with different size and shape in general, the formation of the growth plate and synchondrosis is a result of differential molecular regulation. The future challenge is to uncover the synchondrosis and growth plate-specific signaling pathways, which regulate their formation and function.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The authors thank Ms. Kjellfrid Haukanes and Ms. Helen Olsen for their skilful technical assistance.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED