The skeletal site-specific role of connective tissue growth factor in prenatal osteogenesis

Abstract

Background: Connective tissue growth factor (CTGF/CCN2) is a matricellular protein that is highly expressed during bone development. Mice with global CTGF ablation (knockout, KO) have multiple skeletal dysmorphisms and perinatal lethality. A quantitative analysis of the bone phenotype has not been conducted.

Results: We demonstrated skeletal site-specific changes in growth plate organization, bone microarchitecture, and shape and gene expression levels in CTGF KO compared with wild-type mice. Growth plate malformations included reduced proliferation zone and increased hypertrophic zone lengths. Appendicular skeletal sites demonstrated decreased metaphyseal trabecular bone, while having increased mid-diaphyseal bone and osteogenic expression markers. Axial skeletal analysis showed decreased bone in caudal vertebral bodies, mandibles, and parietal bones in CTGF KO mice, with decreased expression of osteogenic markers. Analysis of skull phenotypes demonstrated global and regional differences in CTGF KO skull shape resulting from allometric (size-based) and nonallometric shape changes. Localized differences in skull morphology included increased skull width and decreased skull length. Dysregulation of the transforming growth factor-β-CTGF axis coupled with unique morphologic traits provides a potential mechanistic explanation for the skull phenotype.

Conclusions: We present novel data on a skeletal phenotype in CTGF KO mice, in which ablation of CTGF causes site-specific aberrations in bone formation.

Fig. 1

Growth plate malformations in connective tissue growth factor knockout (CTGF KO) mice. A: Sections through postnatal day (P) 0 wild-type (WT) and CTGF KO proximal tibiae with brackets outlining the proliferating zone (PZ), prehypertrophic zone (PHZ), and hypertrophic zone (HZ). B: Quantification of growth plate zone thicknesses are shown in the graph. **P < 0.01 and ****P < 0.0001. Scale bar = 100 μm.

Fig. 2

Skeletal site-specific effects on formed bone in connective tissue growth factor knockout (CTGF KO) mice. A–L: Micro-CT reconstructions of postnatal day (P) 0 wild-type (WT) and CTGF KO femora (A,B) and tibiae (G,H) are shown, with isolation of mid-diaphyseal femoral bone (C,D), distal femoral trabecular bone (E,F), proximal tibial trabecular bone (I,J), and mid-diaphyseal tibial bone (K,L). Analysis of bone microarchitecture in these isolated regions is displayed in Table 1.

Fig. 3

Landmarks collected from isosurfaces of micro-CT reconstructions of postnatal day (P) 0 murine skulls. A–C: All landmarks used in the analysis of the global wild-type (WT) and connective tissue growth factor knockout (CTGF KO) skulls are shown from inferior (A), superoinferior (B), and lateral (C) views; depicted is a WT skull. Landmarks used in the analysis of the three landmarks subsets are shown on the facial skeleton (blue), cranial base (yellow), and cranial vault (green). Landmarks that define the subsets of regions depicting the facial skeleton, cranial base, and cranial vault are listed in Supp. Table S1 and defined on the laboratory website: http://getahead.psu.edu.

Fig. 4

Differences in skull shape in connective tissue growth factor knockout (CTGF KO) mice. A–D: Results of Principal Components Analysis (PCA) analyses based on Procrustes coordinates from landmarks of the global skull (A), cranial vault (B), cranial base (C), and facial skeleton (D). In each figure, the shape of the cranial region of each mouse is represented as a single dot in the scatterplots of principal component 1 and principal component 2 scores before adjusting for the effects of allometry. The percentage of variation explained by each component is entered in parentheses after the axis label.

Fig. 5

Localized differences in skull morphology in connective tissue growth factor knockout (CTGF KO) mice. Distances between landmarks found to be significantly different when comparing wild-type (WT) and CTGF KO groups by EDMA are shown on a micro-CT reconstructed isosurface of a postnatal day (P) 0 WT mouse. The skull is seen from above with the rostral end to the left and caudal end to the right. Yellow lines indicate linear distances that are significantly larger in CTGF KO mice; magenta lines represent linear distances that are significantly smaller in CTGF KO mice.

Fig. 6

Morphologic traits seen in connective tissue growth factor knockout (CTGF KO) skulls. A–D: Micro-CT reconstructions of postnatal day (P) 0 wild-type (WT) and CTGF KO global skulls from lateral (A,B) and inferior views (C,D, left) are shown, with isolation of the palate (C,D right) and sphenoid bones (E,F). Increased curvature of the nasal bones is seen in CTGF KO mice (B, yellow arrow) as well as an S-shaped bend in the mandibular body (D, red arrow). A close-up of the palate shows a characteristic kink in the CTGF KO vomer (D, blue arrow), as well as failed midline convergence of the maxillary (purple arrow) and palatine processes (green arrow). Isolation of the sphenoid bones demonstrates a difference in pterygoid plate morphology (white arrow) in CTGF KO mice. Sphenoid bones (E,F) are shown from a posterior view (top row) and inferior view (bottom row) with anterior directed upward.

Fig. 7

Gene expression patterns in connective tissue growth factor knockout (CTGF KO) skeletal sites. A,B: In vivo mRNA gene expression from CTGF KO tibia (A) and parietal bone (B) is graphed relative to WT samples. Specific genes studied include those critical in osteoblast differentiation and function and CCN family members. C: In vivo expression of TGF-β ligand/receptor components from the craniofacial skeleton of CTGF KO mice is graphed relative to WT samples. Abbreviations include: runt-related transcription factor 2 (Runx-2), alkaline phosphatase (ALP), osteocalcin (Oc), TGF-β₁ ligand and TGF-β receptors 1 and 2 (TGF-β RI and RII). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.