Growth factor signaling alters the morphology of the zebrafish ethmoid plate.

Craniofacial development relies on coordinated tissue interactions that allow for patterning and growth of the face. We know a priori that the Wingless, fibroblast growth factor, Hedgehog and transforming growth factor-beta growth factor signaling pathways are required for the development of the face, but how they contribute to the shape of the face is largely untested. Here, we test how each signaling pathway contributes to the overall morphology of the zebrafish anterior neurocranium. We tested the contribution of each signaling pathway to the development of the ethmoid plate during three distinct time periods: the time of neural crest migration [10 hour post fertilization (hpf)]; once the neural crest is resident in the face (20 hpf); and finally at the time at which the cartilaginous condensations are being initiated (48 hpf). Using geometric morphometric analysis, we conclude that each signaling pathway contributes to the shape, size and morphology of the ethmoid plate in a dose-, and time-dependent fashion.

Prickle1 regulates differentiation of frontal bone osteoblasts.

Enlarged fontanelles and smaller frontal bones result in a mechanically compromised skull. Both phenotypes could develop from defective migration and differentiation of osteoblasts in the skull bone primordia. The Wnt/Planar cell polarity (Wnt/PCP) signaling pathway regulates cell migration and movement in other tissues and led us to test the role of Prickle1, a core component of the Wnt/PCP pathway, in the skull. For these studies, we used the missense allele of Prickle1 named Prickle1Beetlejuice (Prickle1Bj). The Prickle1Bj/Bj mutants are microcephalic and develop enlarged fontanelles between insufficient frontal bones, while the parietal bones are normal. Prickle1Bj/Bj mutants have several other craniofacial defects including a midline cleft lip, incompletely penetrant cleft palate, and decreased proximal-distal growth of the head. We observed decreased Wnt/ß-catenin and Hedgehog signaling in the frontal bone condensations of the Prickle1Bj/Bj mutants. Surprisingly, the smaller frontal bones do not result from defects in cell proliferation or death, but rather significantly delayed differentiation and decreased expression of migratory markers in the frontal bone osteoblast precursors. Our data suggests that Prickle1 protein function contributes to both the migration and differentiation of osteoblast precursors in the frontal bone.

Accelerated enamel mineralization in Dspp mutant mice.

Dentin sialophosphoprotein (DSPP) is one of the major non-collagenous proteins present in dentin, cementum and alveolar bone; it is also transiently expressed by ameloblasts. In humans many mutations have been found in DSPP and are associated with two autosomal-dominant genetic diseases - dentinogenesis imperfecta II (DGI-II) and dentin dysplasia (DD). Both disorders result in the development of hypomineralized and mechanically compromised teeth. The erupted mature molars of Dspp(-/-) mice have a severe hypomineralized dentin phenotype. Since dentin and enamel formations are interdependent, we decided to investigate the process of enamel onset mineralization in young Dspp(-/-) animals. We focused our analysis on the constantly erupting mouse incisor, to capture all of the stages of odontogenesis in one tooth, and the unerupted first molars. Using high-resolution microCT, we revealed that the onset of enamel matrix deposition occurs closer to the cervical loop and both secretion and maturation of enamel are accelerated in Dspp(-/-) incisors compared to the Dspp(+/-) control. Importantly, these differences did not translate into major phenotypic differences in mature enamel in terms of the structural organization, mineral density or hardness. The only observable difference was the reduction in thickness of the outer enamel layer, while the total enamel thickness remained unchanged. We also observed a compromised dentin-enamel junction, leading to delamination between the dentin and enamel layers. The odontoblast processes were widened and lacked branching near the DEJ. Finally, for the first time we demonstrate expression of Dspp mRNA in secretory ameloblasts. In summary, our data show that DSPP is important for normal mineralization of both dentin and enamel.

Cholesterol lipids of Borrelia burgdorferi form lipid rafts and are required for the bactericidal activity of a complement-independent antibody.

Borrelia burgdorferi, the agent of Lyme disease, is unusual as it contains free cholesterol and cholesterol glycolipids. It is also susceptible to complement-independent bactericidal antibodies, such as CB2, a monoclonal IgG1 against outer surface protein B (OspB). We find that the bactericidal action of CB2 requires the presence of cholesterol glycolipids and cholesterol. Ultrastructural, biochemical, and biophysical analysis revealed that the bacterial cholesterol glycolipids exist as lipid raft-like microdomains in the outer membrane of cultured and mouse-derived B. burgdorferi and in model membranes from B. burgdorferi lipids. The order and size of the microdomains are temperature sensitive and correlate with the bactericidal activity of CB2. This study demonstrates the existence of cholesterol-containing lipid raft-like microdomains in a prokaryote, and we suggest that the temperature dependence of B. burgdorferi lipid raft organization may have significant implications in the transmission cycle of the spirochetes which are exposed to a range of temperatures.