Our morphometric results demonstrated a significant decrease in the length of the proliferating zone in CTGF KO mice. This zone is normally maintained by both adequate PTH-rP production in the resting zone as well as Ihh production in the prehypertrophic and hypertrophic zones (Chung et al., 2001
). Our findings are consistent with the previous report of decreased PTH-rP and Ihh gene expression in the CTGF KO growth plate (Kawaki et al., 2008a
). Furthermore, we found that while the hypertrophic zone length was increased, the prehypertrophic zone length was unaffected. This expansion is likely due to the previously reported decrease in Ihh production by these cells (Kawaki et al., 2008a
). It has been shown that abrogation of PTH-rP signaling can still result in proper progression from proliferating through prehypertrophic to hypertrophic chondrocytes (Provot and Schipani, 2005
). This could explain why the length of the prehypertrophic zone is unaffected in CTGF KO mice while hypertrophic zone expansion occurs.
Although bone forms through both endochondral (replacing a preexisting cartilage anlage) and intramembranous (de novo
) ossification processes, individual bones may form from a combination of the two processes, as seen in the occipital bone of the skull (Rice, 2008
). Furthermore, both chondrocytes and osteoblasts originate from precursor mesenchymal cells, the location of their condensations during prenatal skeletal development determines the embryonic cell lineage of the future bone—craniofacial bones from neural crest and paraxial mesoderm, the remaining axial skeleton from somatic mesoderm of the sclerotomes, and the appendicular skeleton from lateral and intermediate mesoderm (Karsenty, 1998
). Taking this into account, we examined various skeletal sites to determine if CTGF ablation produces global or site-specific changes in osteogenesis.
Our micro-CT and gene expression analyses demonstrated site-specific differences in bone formation within the appendicular skeleton. Using micro-CT, we found significant decreases in metaphyseal trabecular bone in both the phenotypically abnormal (kinked) tibiae and phenotypically normal (straight) femora in CTGF KO mice compared with WT littermates. These differences indicate defective endochondral ossification, as the trabeculae in this region form through this process. Nonetheless, we cannot discount that altered chondrogenesis could be contributing to these changes in bone mass as we and others have demonstrated dysregulation of the growth plate zones as well as production of crucial cartilage ECM components, such as aggrecan and collagen type X (Ivkovic et al., 2003
; Kawaki et al., 2008a
). However, it is less likely that aberrant osteoclast function is solely responsible for the bone phenotype presented herein. As it was previously demonstrated that CTGF KO mice have fewer osteoclasts in the metaphyseal region (Ivkovic et al., 2003
), this decrease in resorption would be expected to cause a resultant increase in trabecular bone; this is not the case in the CTGF KO mice.
Our analyses of the midshaft of these same bones demonstrated different trends in bone formation, such that it was normal in the femur and increased in the tibia. In the phenotypically normal femur, the percent of bone at the midshaft was not significantly different in CTGF KO mice compared with WT littermates. On the contrary, in the phenotypically abnormal tibia, the midshaft showed a dramatic increase in total bone volume in CTGF KO mice compared with WT littermates. These results demonstrate specific differences in the bone volume at these various sites. To confirm if this was due to an increase in osteoblast function, we analyzed the gene expression of Runx-2
(marker of early osteoblast commitment), ALP
(marker of osteoblast maturation), and OC
(marker for terminal osteoblast differentiation). Our qPCR analyses demonstrated increased expression of all three osteoblast markers, consistent with increased bone formation at the cellular level. We also found a significant increase in Nov/CCN3 expression, which has previously been shown in the growth plate of CTGF KO mice (Kawaki et al., 2008a
). These results demonstrate that not only is bone formation variably affected depending on the skeletal site, and in this case the site within an individual bone, but also that CTGF ablation does not result in a global decrease in bone formation.
An interesting point worth noting is the specificity and reproducibility of the abnormal bones in the CTGF KO skeleton. The CTGF KO phenotype involves distinct kinks in appendicular long bones, specifically bones of the embryonic zeugopod
region, which comprises the radius and ulna in the forelimb (FL) and the tibia and fibula of the hindlimb (HL). However, the bones of the stylopod
(humerus [FL] and femur [HL]), and autopod
(carpus [FL], tarsus [HL], and digits [FL&HL]) remain unchanged. Why CTGF ablation only affects the zeugopod
region could be due to changes in skeletal patterning, mechanobiological alterations, or a combination of the two. One of the key classes of genes involved in limb development includes the Hox genes, and it has been shown that specific mutations in Hoxa11, Hoxc11, and Hoxd11 deleteriously affect normal development of the zeugopod
(Wellik and Capecchi, 2003
; Zakany and Duboule, 2007
; Koyama et al., 2010
). Mechanobiological cues are also important in proper limb development, where muscle-induced mechanical load is necessary for proper bone formation in utero
(Nowlan et al., 2007
; Sharir et al., 2011
The craniofacial skeleton is unique in terms of skeletal development in that it derives from multiple cell types and involves both forms of ossification (Noden and Trainor, 2005
; Sperber et al., 2010
). Crania are historically analyzed by subdividing them into three regions—the cranial vault or calvaria, the cranial base, and the facial skeleton. During development, a dual-layered capsular membrane known as the ectomeninx
surrounds the developing brain. This ectomeninx
, of both paraxial mesoderm and neural crest origin, forms the dura mater, which has an outer layer with chondro-/osteogenic properties. In the region of the future calvarium, this membrane will undergo intramembranous ossification, while in the area of the future cranial base this membrane will undergo endochondral ossification. The facial skeleton is of solely neural crest origin and forms through only intramembranous ossification (Sperber et al., 2010
). Therefore, generally speaking, global aberrations in endochondral ossification are seen only in the cranial base, while effects on intramembranous ossification present as defects in cranial vault or facial bones, the differences between which can result from their different ossification processes.
Micro-CT, gene expression, and landmark analyses of the axial skeleton of CTGF KO mice demonstrated multiple aberrations in the bone formation these mice. It has been previously shown that the ribcage of CTGF KO mice presents with characteristic kinks in the bone (Ivkovic et al., 2003
). When assessing bony elements in the axial skeleton of CTGF KO and WT mice, we analyzed the amount of bone at two sites within the craniofacial skeleton and two locations within the vertebral column. We saw a dramatic difference within the vertebral column, such that the more caudal vertebral bodies (L1) had decreased bone while the more rostral (T8) were not significantly affected. Ossification of the vertebral column at birth is most prominent in the thoracic region of normal newborn mice while other more rostral and caudal sites fully ossify perinatally (Theiler, 1989
). Therefore, the decreased ossification found at L1 and not T8 could have resulted from a delay in ossification in CTGF KO mice.
We also analyzed the parietal bones, which form solely from intramembranous ossification. These bones demonstrated decreased ossification in CTGF KO compared with WT mice. This coincides with our findings of decreased expression osteogenic markers in CTGF KO parietal bones (). These results are in agreement with a previous study that showed a reduction in the expression of some of these markers during osteogenic differentiation of primary osteoblast cultures derived from CTGF KO mice compared with WT littermates (Kawaki et al., 2008b
). Furthermore, we demonstrated through histomorphometric analyses that the number of osteoblasts in CTGF KO parietal bones is similar to that in WT parietal bones. Therefore, the decreased expression in osteoblast markers was not due to a decrease in osteoblast numbers but rather a decrease in gene expression on a per cell basis. We also demonstrated a decrease in expression of closely related CCN family member, Cyr61/CCN1 in CTGF KO parietal bones. Since it has been demonstrated that CCN1 can stimulate osteogenesis in vitro
(Su et al., 2010
), the absence of CTGF coupled with decreased CCN1 expression is consistent with the aforementioned reduction in the expression of osteogenic markers.
We determined phenotypic differences between CTGF KO and WT skulls using 3D coordinates of cranial landmarks for the entire skull, and then analyzed landmarks representing regions of the skull separately. Our PCA analysis of the global skull landmark set demonstrated a clear separation between CTGF KO and WT littermates with both allometric and nonallometric skull shape differences contributing to the changes seen in the skulls of CTGF KO mice. PCA analyses of the landmark subsets representing the three cranial regions also revealed separation between groups.
We further computed localized differences in craniofacial shape using the nonparametric bootstrap algorithm of EDMA (Lele and Richtsmeier, 2001
). Overall differences in craniofacial shape were significant in CTGF KO cranial vault and facial skeleton regions, but not so for the cranial base. Confidence interval tests for each linear distance demonstrated relative increases along the medio-lateral axis for the facial skeleton and anterior cranial vault and relative decreases in distances along the rostro-caudal axes of the cranial vault.
In addition to our landmark analyses, we also noted several obvious morphologic differences in CTGF KO skulls; these included changes in the nasal bones, mandibles, palate and vomer, and pteryogoid plates of the sphenoid bone. These abnormal phenotypic traits were conserved in all CTGF KO mice studied. To relate the site specificity of these changes to any potential underlying mechanisms, we need to take into account (1) the embryonic cell origins of these bones, and (2) the signaling pathways known to involve CTGF in craniofacial development. The nasal bones, mandibles, palate, and vomer derive entirely from the cranial neural crest population, while the sphenoid has a dichotomous embryonic cell origin such that the sphenoid body is derived from paraxial mesoderm, while the pterygoid plates are derived from neural crest cells (Noden and Trainor, 2005
The mandibles of CTGF KO mice, while aberrant in phenotype (), displayed decreased percent bone volume compared with WT mice. The mandible has a complex process of bone formation involving both intramembranous and endochondral ossification, as it forms around Meckel's cartilage (Lee et al., 2001
; Shimo et al., 2004
). Additionally, it has been shown that CTGF is required for Meckel's cartilage development (Shimo et al., 2004
). Therefore, this decrease in bone could be due to a combination of alterations in the preexisting Meckel's cartilage, decreased ossification at this site, or both.
The TGF-β signaling family has been shown to be involved in CTGF expression and signaling in bone development (Arnott et al., 2011
). A role for CTGF in TGF-β signaling-mediated craniofacial development was shown by the Chai laboratory using a cranial neural crest-specific knockout of the TGF-β receptor II (Tgfbr2fl/fl; Wnt1-Cre
) (Ito et al., 2003
; Oka et al., 2007
; Iwata et al., 2010
). These mice demonstrated decreased CTGF expression in developing Meckel's cartilage, concomitant with a mandibular phenotype similar to that of the CTGF KO mice (Oka et al., 2007
). Analysis of the craniofacial phenotype in the Tgfbr2fl/fl;Wnt1-Cre
mice also demonstrated similarities with the CTGF KO mice in the development of their nasal bones, vomer, and palate (Ito et al., 2003
; Iwata et al., 2010
). Bones formed by endochondral ossification were targeted in mice in which the TGF-β receptor II was conditionally inactivated in chondrocytes under the type II collagen, alpha 1 (Col2a1) promoter (Col2acre+/−
). These mice demonstrated changes in the sphenoid body (Baffi et al., 2004
), which were not observed in our analysis of CTGF KO skulls. Therefore, the effects of CTGF ablation on TGF-β RII signaling in neural crest cells may provide an explanation for the craniofacial phenotype seen in our analyses.
We found aberrations in the TGF-β signaling pathway at both the receptor and ligand levels in CTGF KO parietal bones and mandibles. We demonstrated reductions in the expression of both TGF-β RI and RII; TGF-β RI is responsible for propagating the TGF-β signal through phosphorylation of Smads 2 and 3 (Hendy et al., 2005
). It is interesting to note that in Tgfbr2fl/fl;Wnt1-Cre
mice (discussed above), the expression of CTGF was decreased in developing Meckel's cartilage, and that the abnormal craniofacial phenotype was partially rescued by adding back exogenous CTGF (Oka et al., 2007
). These results demonstrate the importance of CTGF as a necessary downstream player in TGF-β-mediated craniofacial development. A comparison of phenotypic changes seen in CTGF KO skulls with those in the skulls of Tgfbr2fl/fl;Wnt1-Cre
mice highlights a potentially crucial role of the TGF-β-CTGF signaling loop in craniofacial development. We postulate that the absence of CTGF in mice results in a dysregulation of TGF-β signaling, thus providing a possible mechanistic explanation for the abnormal skull phenotype.