E with DAPI-stained nuclei (blue) was performed on coronal mouse E12.5 head sections. (A ) Box outlines indicate area in inset and white hatched line in insets demarcates cranial ectoderm from mesenchyme. (I ) Lateral view of whole-mount skeletal preps or gray-scaled vibrant field images of embryonic mouse heads. Ey, eye. Scale bars for sections represent 100 mm. Scale bar (K) for complete mount images (I ) represents 5 mm. Diagram inset in (D) depicts lateral view of embryonic head with box outlining area of interest. doi:10.1371/journal.pgen.1004152.gexpression of the differentiation marker, Keratin 14 (K14) was unaffected (Figure S4E,F). Subsequent, we examined formation of dermal fibroblast progenitors in Crect; RR; Wls fl/fl mutant embryos. Cranial dermal fibroblast progenitors expressed the markers, Twist2 [3,37] and Insulin Development aspect 2 (IGF2) by E12.five in supraorbital mesenchyme (Figure 4O, P), but mutant embryoslacked Twist2 and IGF2 expression (Figure 4S, T). Twist2 expression became far more progressively restricted to upper dermal fibroblasts during differentiation in controls, but was absolutely absent from cranial supraorbital mesenchyme of mutants (Figure 4 R, V). The altered cell fate marker expression at E12.5 (Figure four, S4 I, J) promptly following deletion of ectoderm Wls (Figure S4K)PLOS Genetics | plosgenetics.orgWnt Sources in Cranial Dermis and Bone FormationFigure 3. Distinct needs for Wntless in the cranial ectoderm and mesenchyme. (A, B, C, D, C9, D9) Von Kossa staining, or (E ) alcian blue staining was performed on coronal mouse embryonic head sections and counterstained with eosin. Br, brain, fb, frontal bone, vhf, supraorbital vibrissae hair follicle, mn, meningeal progenitors. Black arrowheads indicate guard hair follicles (hf), red arrowheads indicate dorsal extent of ossified frontal bone, and open black arrows indicate ectopic cartilage. (C9, D9 C0, D0) Black dotted line demarcates the reduce limit in the dermal layer and the black bracket shows dermal thickness. Diagrams inset (B) figure depicts lateral view of E15.five embryonic head with plane of section and area of interest. Red regions in diagram represent bone primordia. Scale bars (A,E) represent one hundred mm.5-Bromo-3,3-dimethyl-1-indanone web doi:10.Formula of 13252-13-6 1371/journal.PMID:26644518 pgen.1004152.gwas indicative of primary defects in mesenchymal cell fate selection. With each other, our information suggest ectoderm Wnts type a non-cell autonomous inductive signal for the underlying mesenchyme for specification of osteoblast and dermal fibroblast progenitors, and for repression of chondrogenesis. Next, we determined if mesenchyme Wls deletion resulted within a later defect in differentiation of cranial bone and dermal fibroblast progenitors. In En1Cre; RR; Wls fl/fl mutants, Runx2 expression in osteoblast progenitors was intact devoid of ectopic Sox9 expression, but showed diminished expression of the skeletal differentiation marker, Osx and ossification (Figure S3). Wnt responsiveness by Axin2 expression was comparable in control and mutant cranial mesenchyme at E14.five (Figure S3). In Dermo1Cre; RR; Wls fl/fl mutants, Runx2 expression was also unaffected throughout fate choice stages (Figure 5A, G, B, H). Nevertheless, throughout later osteoblast progenitor differentiation (E15.5), Osx was diminished in mutants at E15.5 (Figure 5C, I). In dermal progenitors undergoing specification, Twist2 expression was unaffected (Figure 5D,J), and surface ectoderm differentiation marker, K14, was appropriately expressed (Figure S6C, D). In addition at la.
