The Osteogenic Potential of the Neural Crest Lineage May Contribute to Craniosynostosis

Abstract

The craniofacial skeleton is formed from the neural crest and mesodermal lineages, both of which contribute mesenchymal precursors during formation of the skull bones. The large majority of cranial sutures also includes a proportion of neural crest-derived mesenchyme. While some studies have addressed the relative healing abilities of neural crest and mesodermal bone, relatively little attention has been paid to differences in intrinsic osteogenic potential. Here, we use mouse models to compare neural crest osteoblasts (from frontal bones or dura mater) to mesodermal osteoblasts (from parietal bones). Using in vitro culture approaches, we find that neural crest-derived osteoblasts readily generate bony nodules, while mesodermal osteoblasts do so less efficiently. Furthermore, we find that co-culture of neural crest-derived osteoblasts with mesodermal osteoblasts is sufficient to nucleate ossification centres. Altogether, this suggests that the intrinsic osteogenic abilities of neural crest-derived mesenchyme may be a primary driver behind craniosynostosis.

Keywords: Craniosynostosis; Dura mater; Neural crest; Osteogenesis; Sutures.

Fig. 1

Wnt1-cre recombination labels neural crest-derived tissues in the cranial vault. A-D Whole mount views of newborn (PN0) Wnt1-cre; Rosa26R^mTmG tissues. A Side view of the whole animal reveals predominant neural crest origin (green) of the craniofacial structure with exception of the parietal region (arrow), which is mesodermally derived. All non-neural crest tissues appear in red. B Top view of the head shows neural crest-mesoderm boundary (dashed line) between frontal and parietal bones. Neural crest contribution (green) is also seen at the interparietal bone and on the midline between paired parietals. C, D Dissected cranial vaults with adjacent membranes intact (C) or after periosteum and dura mater removal (D) reveal the neural crest origin (green) of frontal bone and the whole sagittal suture and mesodermal contribution (red) of parietal bones and part of the coronal suture. E Sagittal section at the coronal suture shows the neural crest-mesoderm boundary in detail (Inset). The neural crest frontal bone overlaps the mesodermally derived parietal (dashed lines) at the coronal suture, whereas dura mater is exclusively neural crest. F, G Primary cells isolated from dissected calvarial tissues and cultured for 24 hours. Dura mater (F) and frontal bone cells (G) are neural crest derived (green) and parietal cells (H) are mesodermal in origin (red). c, coronal suture; f, frontal; ip, interparietal; p, parietal; s, sagittal suture.

Fig. 2

Dura mater cells are more osteogenic in vitro than osteoprogenitors from the cranial vault bones. A-C Alkaline phosphatase (ALP) (red) and Von Kossa (black) staining of PN0 cultured cells after 14 days in osteogenic media. A Dura mater cells were capable of making ALP positive, mineralized, bony nodules (arrow), whereas frontal (B) and parietal (C) cells are still sparse and ungrouped at this stage. D-F mGFP and mT fluorescent cells in higher magnification (Hoechst - nuclei). D Dura mater green cells are grouped in nodule-like structure (arrow). E Frontal cells, although sparsely distributed, are more confluent than parietal cells (F) at this stage. G ALP activity from calvarial cells as measured by pixel density quantification of the red staining in A-C. Dura cells accordingly show significantly higher ALP activity when compared to frontal and parietal cells. DM, dura mater; FB, frontal bone; PB, parietal bone. * p < 0.05.

Fig. 3

Neural crest frontal bone cells are more osteogenic in vitro than mesodermally derived parietal cells. A, B Alkaline phosphatase (ALP; brown) and Von Kossa (black) staining of PN0 frontal and parietal cells after 21 days in osteogenic media. While frontal cells were largely capable of making mineralized nodules (A), cells isolated from the parietal bone show little mineralization and nodule formation (B). C, D mGFP and mT fluorescent cells in higher magnification (Hoechst - nuclei). C Even with the presence of non-neural crest contaminants (red cells) in the frontal cells culture, the mineralized nodules (red outline) are mostly made of neural crest-derived cells (green). D The rarely found nodules (yellow outline) in parietal cells culture seem to be made from neural crest contaminants. E Mineralized area of frontal and parietal cell cultures, as measured by pixel density quantification of black staining in A and B. Frontal bone cells yield significantly more mineralized cultures than parietal cells. FB, frontal bone; PB, parietal bone. *** p < 0.001.

Fig. 4

Parietal bone cells can contribute to nodule formation as long as they are co-cultured with neural crest-derived cells. A ALP/Von Kossa staining of equivalently mixed dura mater and parietal cells reveal presence of mineralized nodules (arrow) after 14 days of culture in osteogenic media. B ALP/Von Kossa staining of frontal and parietal mixed cultures; after 21 days in osteogenic media they are largely mineralized (black staining, arrow). C mGFP/mT fluorescence reveals mixed neural crest (green) and mesoderm (red) contribution for nodule (outline) formation (Hoechst - nuclei). D mTmG fluorescence reveals neural crest (green) and mesoderm (red) mixed contribution for nodule formation (yellow outline).

Fig. 5

Dura mater cells are the most osteogenic amongst neural crest-derived cranial populations. A-C ALP/Von Kossa staining of primary cells from dura mater, interfrontal suture and frontal bone, respectively. Arrows indicate mineralized nodules. D-F Contrast phase high magnification nodules reveal 3D-mineralized structure surrounded by a monolayer of cells. G-I Tomato fluorescence reveals neural crest origin of the Dura, interfrontal suture (IFS), and frontal bone (FB) populations. The nodules outlined are increased in size in dura cell cultures. J Pixel density quantification of ALP staining shows significantly increased activity of dura cells when compared to IFS and FB cells, the latter is also significantly higher than in IFS cells. K Schematics show sites of harvesting where the cells originated from. DM, dura mater; IF, interfrontal; FB, frontal bone. * p < 0.05; ** p < 0.005.