Figure 5: Fusion of vertebra progenitors (centra) in zebrafish mutants completely lacking the retinoic acid-metabolizing enzyme Cyp26b1 (amorphic mutation), and spatial correlation between bone matrix-synthesizing osteogenic cells (in green) and ossified centra (in red).
Figure 6: Ectopic ossification of coronal suture (cs; indicated by arrows) in the skulls of zebrafish mutant (column 2) and human patient (column 3) with reduced activity of the retinoic acid-metabolizing enzyme Cyp26b1 (hypomorphic mutations), leading to a fusion of the anterior and posterior calvarial plates. In contrast, the sagittal suture between the left and right hemisphere of the skull is widely open in the mutants. In addition, both the fish mutant and the human patient display midfacial dysplasia. AR, alizarin red staining of mineralized bone.
Figure 7: Fragmentation of calvarial plate of skull in zebrafish treated with retinoic acid (middle panel), and in human patient (right panel) that completely lacks the retinoic acid-metabolizing enzyme Cyp26b1 (amorphic mutation). Holes are indicated by arrows. Mineralized bone of the zebrafish was stained with alizarin red before the treatment (left panel), and with calcein (green) at 7 days after the treatment (middle panel), revealing that the holes had initially contained bone (red staining in left panel), and that the roles have started to be refilled by new bone (green margins in middle panel). ss, sagittal suture.
BMPs were initially discovered based on their effect to induce ectopic ossification when applied to skeletal muscle. In zebrafish larvae, transgenic overexpression of BMPs causes enhanced maturation of bone-forming osteoblasts, hyperossification and fusions of skeletal elements, including fusions of the vertebra precursors (centra) of the developing spine. Very similar defects occur in zebrafish mutants in the retinoic acid (RA)-metabolizing enzyme Cyp26b1 (Laue et al., Development 2008).
In collaboration with human geneticists, CYP26b1 mutations could also be identified in human patients with severe synostoses (fusion of bones), including fusions of the calvarial plates of the skull (coronal craniosynostosis), as well as calvarial fragmentation at other sites of the skull. Functional studies in zebrafish indicate that both calvarial fusion and calvarial fragmentation, two seemingly opposite effects, are due to an RA-induced precocious differentiation of osteoblasts to pre-osteocytes. Osteoblasts generate the proteins of the bone matrix, while pre-osteocytes stimulate the biomineralization of this matrix. Fusion of calvaria is thus due to an ectopic ossification of the sutures between the clavarial plates, which normally act as joints and growth zones of these flat bones (Laue et al., Am. J. Human Genet. 2011). Calvarial fragmentation, on the other side, involves bone-resorbing osteoclasts, which according to our data are much more strongly activated by pre-osteocytes, rather than osteoblasts (Jeradi and Hammerschmidt, Development 2016). Ongoing studies aim to understand why RA-induced calvarial fragmentation only occurs in regions containing cartilage directly underneath the bony skull. In addition, we aim to elucidate the mechanisms underlying the metameric organization of vertebra progenitors along the anterior-posterior axis of the developing spine. First results suggest that bone-forming osteoblastic cells are not organized in a metameric (segmented) manner, but uniformly present in the axis, while there seem to be metameric sources of RA and, possibly, BMPs and/or Fibroblast Growth factors (FGFs). Finally, we are working on zebrafish mutants isolated during our forward genetic screens, which display defects in the adult spine, including scoliosis, a severe and rather common, but little understood pathology in human. Whole genome sequencing is ongoing to identify the causative DNA lesions in the different zebrafish mutants, combined by phenotypic analyses at the molecular and cellular level.
