Dlx3 is efficiently deleted from neural crest-derived craniofacial bones in Wnt1-cre:Dlx3F/LacZ mice
Previous studies have shown that Dlx3 is expressed in post migratory NC cells in the branchial arches as early as E9.5 (Robinson and Mahon, 1994
). Wnt1-cre mice were mated with Dlx3LacZ/WT
mice to generate Wnt1-cre:Dlx3LacZ/WT
mice that were then mated with homozygous Dlx3F/F
mice. We recently showed that this strategy led to efficient NC-specific deletion of Dlx3 in Wnt1-cre:Dlx3F/LacZ
mice (cKO) that were compared to their control Dlx3F/WT
littermates (WT) (Duverger et al., 2012
). These conditional knockout animals are viable and live a normal lifespan when fed soft food.
By analyzing LacZ staining in Dlx3LacZ/WT
mice, we determined that Dlx3 was expressed in most developing bones in the craniofacial region at E16.5 (). Strong Dlx3 expression was found in the mandible (), mostly derived from the NC (Chai et al., 2000
), as corroborated by LacZ activity detected in Wnt1cre:R26RLacZ
mice (lineage of cre-expressing cells) at E16.5 (). The level of Dlx3 mRNA in the whole mandible of cKO mice was significantly lower (7-fold) than in WT mice at P0 ().
Dlx3 expression during craniofacial development and targeted deletion using Wnt1-cre mice
mice, Dlx3 expression was also detected in frontal and parietal bones in the developing craniofacial region at E16.5 (). At this stage, a large area surrounding the sagittal sutures was devoid of LacZ staining, whereas the coronal sutures separating frontal and parietal bones exhibited a narrow line of LacZ-negative tissue (, white arrowhead). At P3, the LacZ staining in Dlx3LacZ/WT
mice progressed centrally towards the sagittal sutures and covered the whole surface of all calvaria bones (). Previous studies have shown that while frontal bones are NC-derived, parietal bones are derived from the mesoderm (Jiang et al., 2002
; Yoshida et al., 2008
). This implies that cre is not expressed in the parietal bones of Wnt1-cre mice. Analyzing Wnt1-cre:R26RLacZ
mice at E16.5 and P3, we concurred that frontal bones exhibited a very strong LacZ staining when compared to parietal bones (). The light blue staining observed in the parietal bone area has been previously shown to come from the underlying meninges and not from the bone (Jiang et al., 2002
; Yoshida et al., 2008
). The level of Dlx3 mRNA was reduced 100-fold in the frontal bones of Wnt1-cre:Dlx3F/LacZ
mice at P0, confirming that most of the cells expressing Dlx3 in the frontal bones are NC-derived (). Parietal bones, in which cre is not expressed, exhibited a milder decrease in Dlx3 mRNA levels (). This reduction is likely due to the fact that parietal bones in Wnt1-cre:Dlx3F/LacZ
mice only have one active copy of Dlx3 (Dlx3F
is active; Dlx3LacZ
is inactive), while two copies are active in the parietal bones of Dlx3F/WT
These data indicate that Dlx3 is expressed in most craniofacial bones and can be efficiently deleted from NC-derived craniofacial bones using Wnt1-cre mice. The milder decrease in the mandibles compared to the frontal bones (7- vs 100-fold) is due to the fact that Dlx3 is also expressed in the epithelial compartment of the teeth where cre is not expressed (Zhao et al., 2000
; Duverger et al., 2012
). The robust Dlx3 expression in the frontal bone and mandible suggests that Dlx3 is a significant factor driving the intramembranous process of ossification from NC progenitor cells.
Deletion of Dlx3 in the neural crest alters the shape of the calvaria
To analyze the effects of Dlx3 deletion on the development and patterning of the craniofacial skeleton, we performed a skeletal staining (Alizarin red and Alcian blue) of WT and cKO embryos at E18.5. The development of the skull appeared normal in cKO mice and no major patterning defects were observed (). However, the calvaria appeared more rounded in the cKO than in the WT, with a visible shortening of the frontal bones along the sagittal axis (). At that stage the shape and structure of the mandibles also appeared normal in cKO animals (). All the bones of the basal part of the skull were present and no significant change in their shape was observed (data not shown). Additionally, the histology of the developing bone was not visibly altered in the frontal bone and the mandible of newborn animals ().
Structure of the developing skull of Wnt1-cre:Dlx3F/LacZ mice
These observations demonstrate that the absence of Dlx3 in the NC does not lead to major patterning defects (missing or transformed bone) in skull development but does alter the shape of the developing calvaria, possibly due to a disproportion between frontal and parietal bone development. This suggests that Dlx3 regulates the rate of frontal bone formation to maintain the normal pace of coordinate formation of parietal and frontal bones.
Changes in gene expression in frontal bones in Wnt1-cre:Dlx3F/LacZ mice predict an increase in bone formation and mineralization
To address mechanisms related to Dlx3 regulation of the frontal bone, genes affected by the absence of Dlx3 function in NC-derived craniofacial bones were identified by cDNA microarray analysis. We performed the study on frontal bones from cKO and WT animals at the P0 stage, in order to focus on early molecular effects of Dlx3 deletion on bone development. Using a threshold of >1.5 for the fold change, and <0.05 for the p value, 250 genes were found to be significantly affected (107 down-regulated; 143 up-regulated) in the frontal bones from cKO animals ( and Supplemental Table S1
). Ingenuity Pathway Analysis performed on this list of affected genes revealed significant alteration of functions related to bone development, with a predominant prediction for increased bone formation and mineralization. Table I presents the list of affected genes previously shown to have an effect on bone formation and/or mineralization in vivo
. Based on the change in the level of expression of these genes in cKO mice and their function in bone development in vivo
, we were able to predict the potential effects of these variations on bone formation and/or mineralization in our conditional knockout model. Consistent with the global prediction from Ingenuity Pathway Analysis, the majority of the genes listed in Table I are affected in a way that predicts an increase in bone formation and mineralization in cKO mice.
Bone markers affected in frontal bones and/or mandibles in Wnt1-cre:Dlx3F/LacZ mice
Among the eighteen genes listed in , one encodes a golgi protein (Galnt3), three encode nuclear proteins (Trps1, Mef2c and FosB), three encode membrane proteins or receptors (Dlk1, Pthr1 and Cd200), while the eleven remaining genes all encode secreted proteins. The three most strongly downregulated genes are Lect1, Calca and Sost. Lect1 (Leucocyte cell derived chemotaxin 1), also known as ChM-I (Chondromodulin-I), was revealed as a major bone mineralization factor in chondromodulin null mice that exhibit increased bone mineral density (Nakamichi et al., 2003
). Tissue-specific alternative splicing of the Calca
gene gives rise to two proteins, calcitonin and α-calcitonin gene-related peptide, that are distinctively involved in bone resorption and vascular regulation, respectively (Huebner et al., 2008
). Sost (Sclerostin) is secreted by osteocytes and have been shown to have an inhibitory effect on bone formation and mineralization in vivo
(Li et al., 2008
). Among the most strongly upregulated genes encoding secreted proteins were Agt, Ibsp and Ihh. The Agt gene encodes angiotensinogen that accelerates osteoporosis when injected in mice (Shimizu et al., 2008
). Ibsp, more commonly known as Bsp (Bone sialoprotein), is a member of the SIBLING (small, integrin-binding ligand N-linked glycoprotein) family and is a promoter of bone formation and mineralization (Malaval et al., 2008
). Ihh (Indian Hedgehog) signaling has also been shown to promote osteoblast maturation and bone formation in vivo
(St-Jacques et al., 1999
; Lenton et al., 2011
Genes affected in the frontal bones of Wnt1-cre:Dlx3F/LacZ mice with known oriented effects on bone formation and/or mineralization in vivo
Altogether, these data suggest that the absence of Dlx3 in frontal bone osteoblasts leads to molecular changes that would predict an increase in bone formation and mineralization in Wnt1-cre:Dlx3F/LacZ mice.
A small subset of genes are similarly affected in the mandibles and frontal bones of Wnt1-cre:Dlx3F/LacZ mice
To determine if a similar gene signature was also found in other NC-derived bones, microarray analysis was performed on mandibles. We found 185 genes to be significantly affected (140 down-regulated; 45 up-regulated) in the mandibles of cKO animals at P0. By overlapping the data from frontal bones and mandibles, 20 genes were found to be affected in both tissues (). Importantly, all these genes were affected in a similar way (up- or down-regulated) in both tissues, as shown in . shows the list of these genes with their fold change in mandibles and frontal bones. Among them, four genes were also listed in (Sost, Mepe, Ihh and Agt), three of which were affected in a way that predicts increased bone formation and/or mineralization. Like Sost, Mepe (Matrix Extracellular Phosphoglycoprotein, also known as OF45) is secreted by osteoblasts and play an inhibitory role on bone formation and mineralization in mouse (Gowen et al., 2003
). Bglap was significantly downregulated in both frontal bones and mandibles, even though it did not pass the threshold of 1.5-fold change for the frontal bones (not listed in ). Bglap encodes osteocalcin, the most abundant non-collagenous protein in bone extracellular matrix. Osteocalcin plays a crucial role in the regulation of bone formation, with Bglap−/−
mice exhibiting increased bone formation (Ducy et al., 1996
). Interestingly, Lect1 and Calca, the two bone markers that were most strongly downregulated in frontal bones were not affected in mandibles.
Genes affected by Dlx3 deletion in both mandibles and frontal bones
These results show that, although a subset of genes were similarly affected by the absence of Dlx3 in the mandible and the calvaria, these two tissues exhibit a specific molecular signature in Wnt1-cre:Dlx3F/LacZ mice.
The osteoblast markers affected in the frontal bones and/or mandibles of Wnt1-cre:Dlx3F/LacZ mice are not affected in parietal bones
To validate the microarray data and demonstrate that the molecular changes observed in cKO mice are specific to NC-derived bones, we performed qPCR analysis for a subset of relevant genes and used parietal bones as a control tissue (not NC-derived). qPCR analysis confirmed that Sost and Mepe were downregulated in frontal bones and mandibles, while these two genes were not affected in parietal bones (). Similarly, Bglap expression was significantly reduced in frontal bones and mandibles, while no significant change was measured in parietal bones ().
qPCR analysis confirmed a significant downregulation of Lect1 and Calca expression in frontal bones while these two markers were unaffected in mandibles and parietal bones (). Interestingly, the basal expression level of Lect1 was significantly higher in parietal bones than in frontal bones (). The specific increase in Alpl expression in frontal bones was also confirmed by qPCR analysis (). Microarray analysis revealed a significant increase in Ibsp expression in frontal bones () but not in mandibles. qPCR analysis confirmed Ibsp up-regulation in frontal bones, but also showed a slight but significant increase in mandibles, while parietal bones showed no change in the expression of this marker (). We confirmed the specificity of these molecular effects by analyzing the expression of two common osteogenic-related genes that, according to the microarray analyses, showed no variation in expression: Collagen 1a1 (Col1a1) and Dlx5. The expression of these markers was unaffected in all the tissues analyzed (data not shown).
These data demonstrate that the molecular effects of the absence of Dlx3 in the NC are specific to NC-derived bones.
Calvaria osteoblasts isolated from frontal bones from Wnt1-cre:Dlx3F/LacZ mice exhibit increased differentiation and mineralization capacity in culture
The gene signature obtained for Wnt1-cre:Dlx3F/LacZ mandibles and calvaria predicted an increase in bone formation in these mice. To check the validity of these predictions, we performed ex vivo cultures of primary osteoblasts isolated from dissected frontal bones from WT and cKO neonates. The cells were grown to subconfluence in proliferating medium before being induced to differentiate and mineralize using osteogenic medium. Cultures were analyzed 0, 7, 14, 21 and 28 days after induction. While no morphological differences could be observed at days 0 and 7 (), the mineralization nodules that were visible from day 14 onwards showed a different appearance in the cultures from WT and cKO mice. Indeed, we observed higher density and opacity of the nodules in cultures from cKO mice under a bright field microscope (). From day 14 onwards, Alkaline phosphatase (ALP) staining was stronger in cKO cultures when compared to WT (, upper panel), which is consistent with the increase in Alpl expression detected by microarray and qPCR analysis of frontal bones at P0 ( and ). In order to measure the extent of mineralization in these cultures, we performed Von Kossa staining to detect calcium phosphate deposits. We showed that cKO cultures formed more minerals than WT cultures, with a significantly broader staining at days 21 and 28 ().
Ex vivo differentiation of osteoblasts isolated from neonatal frontal bones
Taken together, these observations demonstrate that, in the absence of Dlx3, calvaria osteoblasts exhibit a higher cell autonomous mineralization capacity ex vivo. These results are consistent with the gene signature of Wnt1-cre:Dlx3F/LacZ frontal bones at P0.
Adult Wnt1-cre:Dlx3F/LacZ mice exhibit morphological defects and decreased bone mineral density in mandibles and calvaria
In light of the molecular and ex vivo data presented above, we analyzed the structure of adult craniofacial bones in Wnt1-cre:Dlx3F/LacZ mice using high resolution X-ray and micro-CT analysis.
Mandibles at 8 weeks exhibited morphological and structural defects. High-resolution X-ray analysis showed a shortening of the distance between the incisor apical loop and the gingival margin (). Micro-CT cross sections taken at different positions along the proximal-distal axis of the mandible (, insets 1–4) showed high porosity of the mandibular bone in cKO mice compared to controls. This was further supported by a significant increase in the ratio between bone surface and bone volume in mandibles from cKO mice (), and by 3D reconstruction images of the alveolar region (). Of interest, the average bone mineral density of the mandible was lower in cKO mice than in WT mice ().
Structural defects in mandibles in adult Wnt1-cre:Dlx3F/LacZ mice
Bone mineral density measurements on Dlx3F/WT and Wnt1cre:Dlx3F/LacZ mice at 8 weeks
High-resolution X-ray and micro-CT analysis of the calvaria at 8 weeks confirmed a more rounded shape for the calvaria in cKO mice (). Micro-CT sections showed a significant reduction in the thickness of both frontal (76.4±2.6 μm for cKO versus 94.9±3.1 μm for WT; NcKO=4; NWT=6; p=0.008) and parietal bones (81.4±4.5 μm for cKO versus 97.8±3.7 μm for WT; NcKO=4; NWT=6; p=0.03) in cKO mice, with a stronger decrease for frontal bones (, insets, and 6B). Bone mineral density measurements showed a significant decrease in mutant animals for both frontal bones and parietal bones (), with a stronger decrease in frontal bones.
Structural defects in calvaria in adult Wnt1-cre:Dlx3F/LacZ mice
These data indicate that, in contrast to our molecular and ex vivo observations, adult Wnt1-cre:Dlx3F/LacZ mice exhibit decreased bone mineralization. Also, despite their mesodermal origin, parietal bones exhibited decreased bone thickness and mineral density. This could potentially be due to a moderate reduction in Dlx3 expression levels in this bone (). However, the molecular data showing that the markers significantly affected in frontal bones and/or mandible are unaffected in parietal bones, suggest that Dlx3 haploinsufficiency has no significant effect on the expression of these genes. Therefore, the effects observed on parietal bones are likely due to a late systemic effect.