Abstract
BackgroundLimb bones develop and grow by endochondral ossification, which is regulated by specific cell and molecular pathways. Changes in one or more of these pathways can have severe effects on normal skeletal development, leading to skeletal dysplasias. Many skeletal dysplasias are known to result from mis-expression of major genes involved in skeletal development, but the etiology of many skeletal dysplasias remains unknown. We investigated the morphology and development of a mouse line with an uncharacterized mutation exhibiting a skeletal dysplasia-like phenotype (Nabo).MethodsWe used µCT scanning and histology to comprehensively characterize the phenotype and its development, and to determine the developmental stage when this phenotype first appears.ResultsNabo mice have shorter limb elements compared to wildtype mice, while clavicles and dermal bones of the skull are not affected. Nabo embryos at embryonic stage E14 show shorter limb cartilage condensations. The tibial growth plate in Nabo mice is wider than in wildtype, particularly in the proliferative zone, however proliferative chondrocytes show less activity than wildtype mice. Cell proliferation assays and immunohistochemistry against the chondrogenic marker Sox9 suggest relatively lower, spatially-restricted, chondrocyte proliferation activity in Nabo. Bone volume and trabecular thickness in Nabo tibiae are also decreased compared to wildtype.DiscussionOur data suggest that the Nabo mutation affects endochondral ossification only, with the strongest effects manifesting in more proximal limb structures. The phenotype appears before embryonic stage E14, suggesting that outgrowth and patterning processes may be affected. Nabo mice present a combination of skeletal dysplasia-like characteristics not present in any known skeletal dysplasia. Further genomic and molecular analysis will help to identify the genetic basis and precise developmental pathways involved in this unique skeletal dysplasia.
Highlights
The mammalian skeleton develops primarily through two mechanisms: endochondral ossification and intramembranous ossification
We chose skeletal elements that develop by endochondral ossification, intramembranous ossification or a combination of both and compared them to CD1 wildtype mice
The scapula at P125 shows a 25.5% reduction in element size compared to age-matched wildtype mice, and the length is significantly different for all post-natal stages except for P28 where the p-value is p = 0.085 (ANCOVA, see Table S1 for details) (Fig. 3)
Summary
The mammalian skeleton develops primarily through two mechanisms: endochondral ossification and intramembranous ossification. This cartilage anlage is replaced with bone, which grows longitudinally under the control of the growth plate (Kronenberg, 2003). Direct differentiation of osteoblasts from mesenchymal cells results in direct apposition of bone without a cartilaginous model (Hall, 2005) Disruption of these mechanisms can produce severe phenotypes with defects in both endochondral and intramembranous ossification (Eames, De la Fuente & Helms, 2003; Krakow & Rimoin, 2010). These phenotypes are collectively known as skeletal dysplasias (Rimoin et al, 2007). Further genomic and molecular analysis will help to identify the genetic basis and precise developmental pathways involved in this unique skeletal dysplasia
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