Mesenchyme-dependent BMP signaling directs the timing of mandibular osteogenesis

Abstract

To identify molecular and cellular mechanisms that determine when bone forms, and to elucidate the role played by osteogenic mesenchyme, we employed an avian chimeric system that draws upon the divergent embryonic maturation rates of quail and duck. Pre-migratory neural crest mesenchyme destined to form bone in the mandible was transplanted from quail to duck. In resulting chimeras, quail donor mesenchyme established significantly faster molecular and histological programs for osteogenesis within the relatively slower-progressing duck host environment. To understand this phenotype, we assayed for changes in the timing of epithelial-mesenchymal interactions required for bone formation and found that such interactions were accelerated in chimeras. In situ hybridization analyses uncovered donor-dependent changes in the spatiotemporal expression of genes, including the osteoinductive growth factor Bmp4. Mesenchymal expression of Bmp4 correlated with an ability of quail donor cells to form bone precociously without duck host epithelium, and also relied upon epithelial interactions until mesenchyme could form bone independently. Treating control mandibles with exogenous BMP4 recapitulated the capacity of chimeras to express molecular mediators of osteogenesis prematurely and led to the early differentiation of bone. Inhibiting BMP signaling delayed bone formation in a stage-dependent manner that was accelerated in chimeras. Thus, mandibular mesenchyme dictates when bone forms by temporally regulating its interactions with epithelium and its own expression of Bmp4. Our findings offer a developmental mechanism to explain how neural crest-derived mesenchyme and BMP signaling underlie the evolution of speciesspecific skeletal morphology.

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

“…In particular, neural crest mesenchyme directs species-specific morphology of the face 7,13,51,59 , feather pattern 52 , muscle pattern 56 , and cartilage 53,57 through the regulation of host gene expression. For example, neural crest mesenchyme dictates when bone forms in the mandible by temporally regulating Bmp4 expression 55 .…”

A simple PCR-based strategy for estimating species-specific contributions in chimeras and xenografts

“…Mandibles were isolated as described (Merrill et al, 2008) and digested for 30 minutes at 4°C in 0.25% trypsin with EDTA in Saline A (UCSF Cell Culture Facility). Digestion was stopped in RNase-free 1ϫ PBS.…”

“…4A) (Merrill et al, 2008). However, when we collect multiple samples of chimeras, we often see a range of Runx2 expression phenotypes, probably as a consequence of varying amounts of donor cells.…”

“…We did find some variation in Runx2 expression levels within each group, probably due to the (A) Quail-duck mandible shown schematically and after performing whole-mount in situ hybridization for Runx2. Expression is elevated on the donor side although the number of quail cells remains unknown (Merrill et al, 2008). Runx2 fold change is measured relative to calculated percentages of quail donor cells in duck hosts.…”

“…We employ quail-chick and quail-duck chimeras to understand patterning of neural crest mesenchyme (NCM), ectoderm, and mesoderm (Eames and Schneider, 2005;Eames and Schneider, 2008;Merrill et al, 2008;Schneider, 1999;Schneider, 2005;Solem et al, 2011;Tokita and Schneider, 2009). A goal is to define molecular programs that these embryonic precursors use to communicate with one another (Fig.…”