Distinct requirements for cranial ectoderm and mesenchyme-derived wnts in specification and differentiation of osteoblast and dermal progenitors - PubMed (original) (raw)

Distinct requirements for cranial ectoderm and mesenchyme-derived wnts in specification and differentiation of osteoblast and dermal progenitors

L Henry Goodnough et al. PLoS Genet. 2014.

Abstract

The cranial bones and dermis differentiate from mesenchyme beneath the surface ectoderm. Fate selection in cranial mesenchyme requires the canonical Wnt effector molecule β-catenin, but the relative contribution of Wnt ligand sources in this process remains unknown. Here we show Wnt ligands are expressed in cranial surface ectoderm and underlying supraorbital mesenchyme during dermal and osteoblast fate selection. Using conditional genetics, we eliminate secretion of all Wnt ligands from cranial surface ectoderm or undifferentiated mesenchyme, to uncover distinct roles for ectoderm- and mesenchyme-derived Wnts. Ectoderm Wnt ligands induce osteoblast and dermal fibroblast progenitor specification while initiating expression of a subset of mesenchymal Wnts. Mesenchyme Wnt ligands are subsequently essential during differentiation of dermal and osteoblast progenitors. Finally, ectoderm-derived Wnt ligands provide an inductive cue to the cranial mesenchyme for the fate selection of dermal fibroblast and osteoblast lineages. Thus two sources of Wnt ligands perform distinct functions during osteoblast and dermal fibroblast formation.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Expression of Wnt ligands, Wntless, and Wnt signaling response in cranial ectoderm and mesenchyme.

(A, B) RT-PCR for individual Wnt ligands was performed on cDNA from purified mouse embryonic cranial mesenchyme and surface ectoderm. (C, D G, H) Indirect immunofluorescence with DAPI counterstained nuclei (blue), (E) in situ hybridization, or immunohistochemistry (F, I) was performed on coronal mouse embryonic head sections. (G, H, I) Boxes indicate region in insets at higher magnification. White arrowheads indicate co-expression of (G) Wls/Runx2 or (D,H) Lef1/Runx2, (I) red arrowheads indicate osteoblast progenitors, and blue arrowheads indicate dermal progenitors. (F–I) White hatched lines demarcate ectoderm from mesenchyme. (J) Summary scheme of E12.5 supraorbital cranial mesenchyme. (J) Embryonic axes, figure depicts lateral view of embryonic head, region of interest in sections used in figures are shown. Scale bars represent 100 µm.

Figure 2

Figure 2. Deletion of Wntless in the cranial ectoderm and mesenchyme.

(A, C, E, G) β-galactosidase staining with eosin counterstain or (B, D, F, H) indirect immunofluorescence with DAPI-stained nuclei (blue) was performed on coronal mouse E12.5 head sections. (A–H) Box outlines indicate region in inset and white hatched line in insets demarcates cranial ectoderm from mesenchyme. (I–L) Lateral view of whole-mount skeletal preps or gray-scaled bright field images of embryonic mouse heads. Ey, eye. Scale bars for sections represent 100 µm. Scale bar (K) for whole mount pictures (I–L) represents 5 mm. Diagram inset in (D) depicts lateral view of embryonic head with box outlining region of interest.

Figure 3

Figure 3. Distinct requirements for Wntless in the cranial ectoderm and mesenchyme.

(A, B, C, D, C′, D′) Von Kossa staining, or (E–H) 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. (C′, D′ C″, D″) Black dotted line demarcates the lower limit of the dermal layer and the black bracket shows dermal thickness. Diagrams inset (B) figure depicts lateral view of E15.5 embryonic head with plane of section and region of interest. Red regions in diagram represent bone primordia. Scale bars (A,E) represent 100 µm.

Figure 4

Figure 4. Ectoderm deletion of Wntless leads to loss of cranial bone and dermal lineage markers in the mesenchyme.

Indirect immunofluorescence with DAPI-stained (blue) nuclei was performed on coronal mouse embryonic head sections at E12.5 or as indicated (A,B, F, G, H, I, M, N, P, R, T, V). Alkaline Phosphatase staining (C, J), in situ hybridization (D, E, K, L, O, S), or β-galactosidase staining with eosin counterstain (Q, U) was performed on coronal tissue sections. Diagram in (A) demonstrates plane of section and region of interest for E12.5-E13.5 (A–T). Box and dashed lines in (Q, U) demonstrate the region of high magnification, and β-galactosidase stained sections were included for perspective for (R, V). Diagram inset in high magnification photograph from (Q) shows plane of section and region of interest for E15.5. Red arrows indicate changes in marker expression and black arrows in (U) high magnification indicate ectopic cartilage. Scale bars represent 100 µm.

Figure 5

Figure 5. Mesenchyme deletion of Wntless leads to diminished differentiation and Wnt responsiveness in the bone lineage.

Indirect immunofluorescence with DAPI-stained (blue) nuclei (A, B, D, F, G, H, J, L, P, T) and immunohistochemistry (M,Q) was performed on coronal mouse embryonic head sections In situ hybridization (C, I, N, O, R, S) or eosin counterstain (E, K), was performed on coronal tissue sections of embryonic murine heads at the indicated stages. Diagram in (A) demonstrates plane of section and region of interest for E11.5-E12.5. Box in (D, J) demonstrate the region of high magnification. (I, S, T) Red arrows highlight changes in marker expression in osteoprogenitor domain. (E,K) vhf: subraorbital vibrissae hair follicle and black bracket indicates the dermal layer. (A,G) Scale bars represent 100 µm.

Figure 6

Figure 6. Generation of Wnt responsiveness in the cranial mesenchyme.

In situ hybridization (A–E, H, Q, R), immunohistochemistry (S, T) or indirect immunofluorescence with DAPI-stained nuclei (blue) was performed on coronal mouse embryonic head sections (F, G, I–P). (S, T) White dotted line demarcates ectoderm from mesenchyme. Embryonic head diagram depicts region of interest and plane of section. Embryonic axes for the sections are presented. Scale bars represent 100 µm.

Figure 7

Figure 7. Mesenchyme Wnt ligand expression is dependent on ectoderm Wls and mesenchymal β-catenin.

(A–S) In situ hybridization was performed on coronal mouse embryonic head sections. Diagram of embryonic head in (A) inset depicts region of interest and plane of section. Insets in (K, L) show β-galactosidase staining and eosin counterstaining on serial sections. (T) A working model for role of tissue sources of Wnt ligands during cranial mesenchymal lineage fate selection. Scale bars represent 100 µm.

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