Figure 5. Ddr2^mer-iCre-mer marks progenitors of the skeletal lineage during postnatal craniofacial development.

(a) Protocol used for induction of Cre-recombination and expression of tdTomato fluorescent protein (red) upon tamoxifen injection. (b) Fluorescent tdTomato on cryostat sections of calvaria (upper panel) at the postnatal P5, P14, and P60 showing labeling (white arrowheads) in suture mesenchyme, periosteum (Ps) and dura mater (Du), subsequently contributing to osteocytes (Ocy) (inset box) and bone marrow (Bm) of calvarial bones over time. Scale bar: 100 μm. Lower panel, cranial base synchondrosis (ISS) showing labeling at P5 in resting (RZ) and proliferative (PZ) chondrocyte zones, but not in the hypertrophic zone (HZ) (cyan dotted lines), consistent with X-gal staining. Lineage trace at P14 shows an increase in tdTomato + cells in all synchondrosis regions and associated bone marrow (Bm). At P60, tdTomato labeling is persistent in the middle zone and shows a clone of tdTomato labeling in proliferative and hypertrophic chondrocytes (yellow asterisk) and appears in the lining of bone marrow (Bm) and osteocytes (Ocy) of cortical bone (Ct). Scale bar: 100 μm (P14 and P60) and 50 μm (P5). Gray: cell nuclei. (c) Protocol used for induction of Cre-recombination and expression of tdTomato fluorescent protein (red) in Gli1^CreERT; Rosa26^LSL-tdTomato mice. (d) Fluorescent tdTomato (red) on cryostat sections of calvaria (right) and cranial base synchondroses (ISS and SOS, left) from 2-week-old Gli1^CreERT; Rosa26^LSL-tdTomato mice showing labeling in a similar cell population to that seen with Ddr2^mer-iCre-mer. Scale bar: 200 μm (Suture; left) and 100 μm (ISS and SOS; right). Boxed region is shown in higher magnification (bottom). RZ: resting zone; PZ: proliferative zone; HZ: hypertrophic zone. (e) Representative immunofluorescence images showing Gli1 (green) and Ddr2 (red) immunostaining of coronal sutures, SOS and ISS from 2-week-old mice. Scale bar: 100 μm. Boxed region is shown in higher magnification (bottom). White arrowheads indicate co-localization. Cell nuclei were stained with DAPI (blue). Bm: Bone marrow; Tb; trabecular bone.

Figure 5—figure supplement 1. Ddr2^mer-iCre-mer induced recombination in cranial sutures and cranial base synchondrosis.

Neonatal Ddr2^Mer-icre-Mer;Rosa26^LSL-tdTomato mice were treated with tamoxifen as described in Figure 5. (a) Whole mounts (left) at P14 and P60 show Ddr2 tdTomato labeling in all cranial sutures: frontal, sagittal, coronal, and lambdoid sutures (left). Cryosections (right) show distribution of tdTomato-labeled cells at P60 in the suture mesenchyme, bone marrow lining cells and osteocytes. Scale bar: 50 μm. (b) Cryostat sections of the cranial base spheno-occipital synchondrosis (SOS) shows tdTomato labeling initially in resting and proliferative chondrocyte zones (P5). At later times (P14, P60), progeny of Ddr2-positive cells form single or two column clones along the axis of cranial base growth extending into the hypertrophic zone and osteocytes. Scale bar: 50 μm.

Figure 5—figure supplement 2. Co-localization of DDR2 and GLI1 in cranial suture and synchondroses in 2-week-old mice.

(a) Coronal suture. (b) SOS. (c) ISS. Colocalization is expressed as the percentage of GLI1 + cells that are also DDR2+.