Control of craniofacial development by the collagen receptor, discoidin domain receptor 2
- Department of Periodontics & Oral Medicine, University of Michigan School of Dentistry, United States
- Center for Organogenesis and Trauma, Department of Surgery, University of Texas Southwestern, United States
- Division of Cardiovascular Medicine, University of California, San Diego, United States
- Department of Diagnostic and Biomedical Sciences, University of Texas Health Science Center at Houston School of Dentistry, United States
- Department of Prosthetic Dental Sciences, College of Dentistry, King Saud University, Saudi Arabia
- Department of Biologic & Materials Science, University of Michigan School of Dentistry, United States
- Department of Biological Chemistry, School of Medicine, University of Michigan, United States
- Department of Biomedical Engineering, University of Michigan, United States
Figures
Figure 1 with 1 supplement
Ddr2 deficiency results in impaired anterior-posterior skull growth with abnormal frontal bone and suture formation.
WT and Ddr2slielsile mice were compared at 3 months (a–g) and 2 weeks (h,i). (a-c) Short snout, and reduced skull length in Ddr2slielsile mice. (a) Side (upper) and top (lower) head views of …
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Figure 1—source data 1
Skull linear measurements in Wildtype versus Ddr2 knockout mice.
- https://cdn.elifesciences.org/articles/77257/elife-77257-fig1-data1-v4.xlsx
Figure 1—figure supplement 1
Orientation of skulls in sagittal, axial, and coronal planes.
Max: Maxillary; Mand: Mandibular; M1: first molar; INC: Incisor; ISS: Intersphenoid synchondrosis; SOS: Spheno-occipital synchondrosis.
Figure 2 with 1 supplement
Cranial base hypoplasia due to chondrocyte disorganization and reduced chondrocyte proliferation in Ddr2-knockout synchondroses.
(a) H&E staining (upper) and μCT scans (lower) of WT and Ddr2slie/slie skulls showing wide cranial base synchondroses. Scale bar: 500 μm. Boxed region (red) is shown in higher magnification. Br: …
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Figure 2—source data 1
Cranial base linear measurements in Wildtype versus Ddr2 knockout mice.
- https://cdn.elifesciences.org/articles/77257/elife-77257-fig2-data1-v4.xlsx
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Figure 2—source data 2
Proliferation (EdU labeling) and apoptosis (TUNEL assay) in Wildtype versus Ddr2 knockout mice.
- https://cdn.elifesciences.org/articles/77257/elife-77257-fig2-data2-v4.xlsx
Figure 2—figure supplement 1
No change in levels of cleaved caspase 3 levels in wild-type versus Ddr2slie/slie mice.
(a) Localization of cleaved caspase 3 in ISS and SOS in 2-week-old mice using immunofluorescence. (b), quantitation of IF staining. Scale bar, 100 um.
Figure 3 with 2 supplements
ECM defects in Ddr2-deficient synchondroses.
(a-d) Immunofluorescent staining of ISS and SOS sections from 2-week-old WT and Ddr2slie/slie synchondroses. (a) Representative images of COL2 immunostaining (red) in ISS and SOS showing homogenous …
Figure 3—figure supplement 1
Ddr2 deficiency is associated with loss of collagen organization in the ISS of Ddr2slie/slie mice.
(a) Second harmonic generation imaging of fibrillar collagen in the ISS resting zone of 2-week-old WT versus Ddr2slie/slie mice. Scale bar, 100 µm (b) anisotropy was measured using ImageJ FibrilTool …
Figure 3—figure supplement 2
Quantitation of Ibsp (Bsp) and Col I immunofluorescence in WT and Ddr2slie/slie mice.
(a) Ibsp in hypertrophic chondrocyte layers. (b) Ibsp in primary spongiosa. Note: Ibsp is significantly lower only in the SOS. (c) No change in Col I in the primary spongiosa. Data are presented as …
Figure 4 with 1 supplement
Ddr2 expression in craniofacial skeleton.
(a) Whole-mount X-gal staining (green) of Ddr2Lacz/+ skulls showing of Ddr2 expression in midfacial region, cranial vault, and cranial sutures. Scale bar: 50 μm. (b) X-gal staining of cryostat …
Figure 4—figure supplement 1
Ddr2-LacZ localization in craniofacial skeleton.
(a) In Ddr2+/LacZ embryos harvested at E13.5, intense X-gal staining can be seen in the maxilla (Mx), mandible (Mn), cartilage primordia of cranial base (Cb, black arrowhead), nasal septum and tooth …
Figure 5 with 2 supplements
Ddr2mer-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 …
Figure 5—figure supplement 1
Ddr2mer-iCre-mer induced recombination in cranial sutures and cranial base synchondrosis.
Neonatal Ddr2Mer-icre-Mer;Rosa26LSL-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: …
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+.
Figure 6
Loss of Ddr2 in Gli1-expressing cells resulted in a craniofacial phenotype similar to Ddr2slie/slie mice.
(a) Protocol used for induction of Cre-recombination upon tamoxifen injection. (b) Genotyping PCR showing WT (lower band) and Ddr2 floxed alleles (upper band) near 1650 bp and recombined knockout …
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Figure 6—source data 1
Skull linear measurements in Ddr2fl/fl versus Gli1CreERT ;Ddr2 fl/fl mice.
- https://cdn.elifesciences.org/articles/77257/elife-77257-fig6-data1-v4.xlsx
Figure 7 with 2 supplements
Ddr2 conditional knockout in Col2a1-expressing chondrocytes causes cranial base hypoplasia and alters synchondroses without affecting cranial sutures.
(a-d) μCT scans of Ddr2fl/fl and Col2a1Cre;Ddr2fl/fl skulls (3-month-old) showing reduced anterior-posterior skull length, length of individual bones and increased anterior and, to a lesser extent, …
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Figure 7—source data 1
Skull linear measurements in Ddr2fl/fl versus Col2a1Cre;Ddr2fl/fl mice.
- https://cdn.elifesciences.org/articles/77257/elife-77257-fig7-data1-v4.xlsx
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Figure 7—source data 2
Measurement of spacing between chondrocytes in Ddr2fl/fl versus Col2a1Cre;Ddr2fl/fl mice.
- https://cdn.elifesciences.org/articles/77257/elife-77257-fig7-data2-v4.xlsx
Figure 7—figure supplement 1
Ddr2 loss in mature osteoblasts using BglapCre did not result in craniofacial abnormalities.
(a-d) Quantification using μCT scans shows no difference between Ddr2fl/fl and BglapCre; Ddr2fl/fl skull length, width and height at age of 3 months. Scale bar: 1 mm in a. (e–g) quantification …
Figure 7—figure supplement 2
Synchondroses in Col2a1Cre:Ddr2ff mutants exhibited deficient chondrocyte proliferation and abnormal type II collagen distribution.
(a) EdU staining (green) showed a significant reduction of chondrocyte proliferation in Col2a1Cre; Ddr2fl/fl synchondrosis. (b) Bar graph shows quantification of EdU-positive cells in ISS and SOS. (c…
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Mus musculus) | Discoidin Domain Receptor2 (Ddr2) | Genbank | Gene ID: 18214 | |
| Strain, strain background (Mus musculus, C57BL/6 J) | Ddr2slie/slie | Jackson lab | JAX:008172 | Breeding with C57BL/6 J mice at least 8 generations. |
| Strain, strain background (Mus musculus, C57BL/6 J) | Ddr2LacZ mice | Generated from ‘knockout-first’ Ddr2tm1a(EUCOMM)Wtsi -ES cells(European Mutant Mouse Repository) | EPD0607__B01 PMID:35140200 | ‘knockout-first’ mice crossed with Sox2Cre mice |
| Strain, strain background (Mus musculus, C57BL/6 J) | Ddr2fl/f | Generated from ‘knockout-first’ Ddr2tm1a(EUCOMM)Wtsi -ES cells(European Mutant Mouse Repository) | EPD0607__B01 PMID:35140200 | ‘knockout-first’ mice crossed with FlpO mice |
| Strain, strain background (Mus musculus, C57BL/6 J) | Ddr2mer-iCre-mer | Generated in UCSD Transgenic Animal Model Core and Embryonic Stem Cell shared resource | PMID:35140200 | |
| Strain, strain background (Mus musculus, C57BL/6 J) | Rosa26LSL-tdTomato | Jackson lab | JAX:007914 | |
| Strain, strain background (Mus musculus, C57BL/6 J) | Gli1CreERT | Jackson lab | JAX:007913 | |
| Strain, strain background (Mus musculus, C57BL/6 J) | Col2a1Cre | Generated at Richard R. Behringer lab | PMID:10686612 | |
| Strain, strain background (Mus musculus, C57BL/6 J) | BglapCre | Generated at Thomas L. Clemens lab | PMID:12215457 | |
| Sequence-based reagent | Ddr2fl/f _F | Renny T. Franceschi lab | PMID:35140200 | |
| Sequence-based reagent | Ddr2fl/f _R | Renny T. Franceschi lab | PMID:35140200 | |
| Sequence-based reagent | Ddr2-LacZ-F | Renny T. Franceschi lab | PMID:35140200 | |
| Sequence-based reagent | Ddr2-LacZ-R | Renny T. Franceschi lab | PMID:35140200 | |
| Sequence-based reagent | Ddr2mer-iCre-mer -F | Renny T. Franceschi lab | PMID:35140200 | |
| Sequence-based reagent | Ddr2mer-iCre-mer -R | Renny T. Franceschi lab | PMID:35140200 | |
| Antibody | Anti-human/mouse DDR2(Rabbit polyclonal) | LS Bio | LS-B15752 | IF(1:200) |
| Antibody | Anti-human/mouse Y740P-DDR2(Rabbit monoclonal) | R&D systems | MAB25382 | IF(1:200) |
| Antibody | Anti-Human/mousecleaved caspase 3(Rabbit polyclonal) | Cell Signaling | 9661 | IF(1:200) |
| Antibody | Anti-Human/mouse COL2(Rabbit polyclonal) | ABCam | Ab34712 | IF(1:100) |
| Antibody | Anti-Human/mouse GLI1(Rabbit polyclonal) | Novus Biologicals | NBP1-78259 | IF(1:100) |
| Antibody | Anti-Mouse IBSP(Rabbit polyclonal) | Renny T Franceschi lab | IF(1:200) | |
| Antibody | Anti-Human/mouse COL10(Rabbit polyclonal) | ABCam | Ab58632 | IF(1:100) |
| Antibody | Anti-Mouse COL1(Rabbit polyclonal) | Millipore | AB765P | IF(1:100) |
| Antibody | Anti-Mouse GM130(Mouse polyclonal) | BD Biosciences | 610822 | IF(1:100) |
| Commercial assay or kit | Click-iT EdU Alexa Fluor 488 Imaging Kit | Invitrogen | C10337 | |
| Commercial assay or kit | FragEL DNA Fragmentation Detection Kit | CalBiochem | QIA33-1 | |
| Chemical compound, drug | Tamoxifen | Renny T Franceschi lab | PMID:35140200 | |
| Chemical compound, drug | Aqua-Poly/Mount | Polysciences Inc | 18606 | |
| Chemical compound, drug | ProLong Gold Antifade Mountant with DAPI | Life technologies | P36931 | |
| Software, algorithm | Prism 9 | GraphPad | GraphPad Software, San Diego, CA | https://www.graphpad.com/ |
| Software, algorithm | ImageJ | ImageJ FibrilTool-Plugin | PMID:24481272 | https://imagej.nih.gov/ij/ |
| Other | Scano μCT 100 | MicroView software version 2.5.0 | PMID:35140200 | Methods-Skull morphometric analysis |
