We each have unique facial features that are key to our identities. These features are inherited, but the mechanisms are poorly understood. People with the genetic disease spondylo-meta-epiphyseal dysplasia, or SMED, have characteristic facial and skull abnormalities including a flattened face and shortened skull. SMED is associated with mutations that inactivate the gene encoding a protein called discoidin domain receptor 2 (DDR2), which is a receptor for collagen.

Collagen is the major structural protein in the human body, supporting the structure of cells and tissues. It also controls cell behaviors including growth, migration and differentiation, and it helps form tissues such as cartilage or bone. At least some of the effects of collagen on cells depend on its interaction with DDR2.

Since the facial and skull abnormalities in mice with mutations that stop DDR2 from working correctly resemble those of SMED patients, these mice can be used to understand the cellular basis for this disease, as well as the role of DDR2 in the embryonic development of the face and skull. Therefore, Mohamed et al. set out to understand how loss of DDR2 causes the characteristic facial and skull defects associated with SMED.

Mohamed et al. used mice that had been genetically modified so that DDR2 could be inactivated in skeletal progenitor cells, cartilage cells and bone cells (osteoblasts). Examining these mice, they found that the shortened skulls and flat face characteristic of mice lacking DDR2 are due to bones at the skull base failing to elongate correctly due to defects in the growth centers that depend on cartilage. Mohamed et al. also discovered that the cells that normally produce DDR2 are the progenitors of cartilage and bone-forming cells, which partly explains why lacking this protein leads to issues in growth of these tissues.

In addition to shedding light on the causes of SMED, Mohamed et al.’s results also provide general insights into the mechanisms controlling the formation of facial and skull bones that depend on interactions between cells and collagen. This information may help explain how other abnormalities in the face and skull emerge, and provide a basis for how the shape of the skull has changed during human evolution. In the future, it may be possible to manipulate the activity of DDR2 to correct skull defects.

Craniofacial abnormalities affecting bone formation in the skull and face are common birth defects in humans (Fish, 2016; Trainor and Richtsmeier, 2015). The craniofacial skeleton forms through regulated bone growth in the calvarium synchronized with growth of the developing brain and cranial base. Cranial sutures, fibrous tissues adjoining calvarial bones, are the principal sites for growth of the cranial vault. Sutures contain stem cells associated with several genetic markers including glioma-associated oncogene 1 (Gli1), an intermediate in hedgehog signaling, Axin-related protein 2 (Axin2), a component of the Wnt pathway, and paired related homeobox 1 (Prrx1) (Lana-Elola et al., 2007; Maruyama et al., 2016; Wilk et al., 2017; Zhao et al., 2015). The current paradigm suggests that sutural stem cells allow continued calvarial growth through osteogenic differentiation and bone formation at sutural edges of the calvarial bones. This process is coordinated with growth of the cranial base mediated by endochondral ossification centers called synchondroses. Histologically, the synchondrosis is a mirror-image growth plate with resting chondrocytes in the central zone and proliferative and hypertrophic chondrocytes on both sides. During endochondral ossification, the resting chondrocytes in the growth plate synchondrosis undergo sequential proliferation, hypertrophic differentiation, and apoptosis before being replaced by bone. The cranial base has been implicated in many human syndromes such as craniosynostosis (premature closure of cranial sutures), Down syndrome, Turner syndrome, cleidocranial dysplasia, cleft palate, and osteogenesis imperfecta (Cheung et al., 2011; McGrath et al., 2012; Nie, 2005; Paliga et al., 2014; Tahiri et al., 2014).

The extracellular matrix (ECM) is critical for maintenance of stem cell niches, differentiation, organ development, and remodeling (Lu et al., 2011). During craniofacial development, skeletal stem cells interact with the collagen-rich ECM through cell-surface receptors. These interactions regulate cell proliferation, migration, differentiation, and remodeling to control and maintain shapes of individual cranial bones and the relative proportions of skeletal elements (Erlebacher et al., 1995). Disruption of these interactions can cause craniofacial abnormalities (Lu et al., 2011; Erlebacher et al., 1995; Velleman, 2000; Kuivaniemi et al., 1997). Discoidin domain receptors (DDR1 and DDR2) are a unique class of receptor tyrosine kinases (RTKs) that, together with β1 integrins, mediate interactions between cells and the ECM by specifically binding triple-helical collagens (Vogel et al., 1997; Shrivastava et al., 1997; Leitinger and Hohenester, 2007). DDRs contain a unique discoidin (DS) domain on their extracellular surface that is homologous to discoidin I-like domain of the slime mold Dictyostelium discoideum (Leitinger and Hohenester, 2007). DS domains are required for collagen recognition and specificity of DDRs such that DDR2 preferentially binds to fibrillar collagens I-III, but not nonfibrillar basement membrane type IV collagen that is exclusively recognized by DDR1 (Vogel et al., 1997; Shrivastava et al., 1997; Leitinger and Kwan, 2006).

DDR2 plays an important role during human craniofacial morphogenesis, possibly being involved in skull changes occurring during the evolution of modern humans from archaic hominins (Kuhlwilm and Boeckx, 2019). Moreover, DDR2 loss of function mutations cause the autosomal recessive growth disorder, spondylo-meta-epiphyseal dysplasia, with short limbs and abnormal calcifications (SMED, SL-AC). This disorder is associated with a number of craniofacial abnormalities including a prominent forehead, open fontanelles, hypertelorism, a short nose with a depressed nasal bridge, long philtrum and micrognathia (Alkindi et al., 2014; Ali et al., 2010; Bargal et al., 2009; Mansouri et al., 2016; Ürel-Demir et al., 2018; Smithson et al., 2009; Rozovsky et al., 2011). Ddr2-deficient mice recapitulate craniofacial phenotypes seen in SMED SL-AC patients including short snout, protruding eyes, abnormal tooth development and impaired calvarial ossification (Ge et al., 2016; Kano et al., 2008; Labrador et al., 2001; Mohamed et al., 2020). However, the basis for these abnormalities is not understood.

We recently described cell autonomous functions of Ddr2 in growth of the appendicular skeleton (Mohamed et al., 2022). Preferential localization of DDR2 in GLI1-positive skeletal progenitor populations associated with resting/growth zone chondrocytes of the growth plate and bone marrow was demonstrated while conditional inactivation of Ddr2 in Gli1-expressing cells and chondrocytes phenocopied the dwarf bone phenotype of globally Ddr2-deficient mice. In the present study, we focus on DDR2 functions in craniofacial development. We show that DDR2 is associated with putative suture stem cells that can serve as precursors for osteoblasts/osteocytes in the cranial vault and with synchondrosis-associated resting/proliferating chondrocytes that form hypertrophic chondrocytes, osteoblasts, and osteocytes in the cranial base. Furthermore, as shown by global and conditional inactivation approaches, DDR2 controls suture formation, frontal bone thickness and anterior/posterior growth of the skull. Notably, these DDR2 functions involve activity in different cell populations and are correlated with changes in collagen organization and chondrocyte polarity.

The ECM is a critical regulator of skeletal development and has been implicated in a wide spectrum of human skeletal disorders, many of which involve craniofacial structures (Lu et al., 2011; Erlebacher et al., 1995; Velleman, 2000; Kuivaniemi et al., 1997). In this study, we highlight the importance of collagen-cell interactions with emphasis on cell-autonomous functions of the cell surface collagen receptor, DDR2, during growth of the craniofacial skeleton. Characterization of craniofacial phenotypes of globally Ddr2-deficient mice revealed two types of defects; (1) abnormalities in development of the flat bones of the skull including defective frontal suture formation/mineralization and thinning of frontal bones, (2) reduced anterior-posterior skull growth related to delayed endochondral ossification at cranial base synchondroses. To understand the basis for these defects, we conducted a systematic characterization of the distribution and lineage of Ddr2-expressing cells and used a conditional deletion approach to resolve the cellular sites of action of Ddr2. These studies complement our previous analysis of DDR2 function in development of the appendicular skeleton (Mohamed et al., 2022).

As a prerequisite for understanding function, we determined the spatial distribution of Ddr2 expression during craniofacial development. Analysis of LacZ knock-in reporter mice revealed expression in developing midface as early as E11.5 with staining initially seen in cartilage primordia of the cranial base, nasal septum, and Meckel’s cartilage in developing mandible. Later during development, Ddr2 expression was localized in neonatal calvariae in developing periosteum, dura mater, and cranial sutures where craniofacial stem cells reside. In the adult, it was also localized to cranial sutures, periosteum, dura mater and in bone marrow lining cells. Ddr2 expression was either low or undetected in osteocytes. Ddr2 expression was also localized to the resting and proliferative zones of neonatal cranial base synchondroses but was not detected in hypertrophic chondrocytes. These findings led us to hypothesize that Ddr2 regulates development of the craniofacial skeleton through its function in skeletal progenitor cells and chondrocytes.

This concept is further supported by lineage tracing studies conducted in Ddr2^Mer-icre-Mer;Rosa26^LSL-tdTomato mice where an initial tamoxifen pulse labeled cells in cranial suture mesenchyme, periosteum and dura as well as resting and proliferating cells of synchondroses. Over a two-month chase period, the TdTomato label increased in sutures and expanded into lining cells of bone marrow and calvarial osteocytes while, in the cranial base, labeled cells persisted in the central resting zone and expanded to proliferative and hypertrophic zones of synchondroses and into adjacent bone. While these studies do not prove Ddr2 is expressed in stem/progenitor populations, they show DDR2-positive cells can serve as precursors to cells of the bone lineage. Significantly, in related studies reported in preprint form, a DDR2 +cranial suture cell population was described (Greenblatt et al., 2021) that is distinct from previously characterized CTSK +suture stem cells (Debnath et al., 2018). These DDR2 + cells have several stem cell properties including capacity for self-renewal, tri-lineage differentiation potential to osteoblasts, adipocytes and chondrocytes, expression of several SC markers including GLI1 and capacity to generate all DDR2 + cells present in the native suture. However, further studies will be required to determine whether the DDR2 + cells we identified in synchondroses also share these stem cell properties.

Consistent with DDR2 functioning in skeletal progenitors, we observed substantial colocalization of DDR2 with a subset of GLI1 + cells in calvarial sutures, periosteum and synchondroses. GLI1-positive sutural stem cells (SuSCs) are of great interest because of their contribution to the development and regeneration of craniofacial bones and possible role in craniofacial abnormalities. Ablation of GLI1 +SuSCs leads to craniosynostosis, a premature closure of cranial sutures resulting in arrested skull growth (Zhao et al., 2015; Durham et al., 2019), while impaired cell proliferation and osteogenic differentiation of SuSCs is associated with wide-open fontanels due to reduced or delayed bone growth (Goto et al., 2004; Qin et al., 2019). To determine if DDR2 functions in GLI1 + cells, we conditionally inactivated Ddr2 using Gli1^CreERT; Ddr2^fl/fl mice. Loss of Ddr2 in Gli1-expressing cells faithfully recapitulated the cranial phenotype of Ddr2^slie/slie mice including the presence of open frontal sutures (analogous to the metopic suture in humans) and thinning of frontal bones. This phenotype also mimics the open fontanels seen in SMED, SL-AC patients. These results are consistent with a function for Ddr2 in Gli1 +sutural cells to regulate osteoblast differentiation during intramembranous bone formation.

Intriguingly, our lineage tracing studies identified Gli1^CreERT-induced tdTomato labeling beyond previously reported sites in cranial sutures, long bone growth plates and metaphyses (Zhao et al., 2015; Mohamed et al., 2022; Shi et al., 2017). Specifically, this Cre also marked chondrocyte lineages and co-localized with Ddr2 in cranial base synchondroses. Consistent with this distribution, tamoxifen treated Gli1^CreERT; Ddr2^fl/fl mice had cranial base defects that were similar in type and magnitude to those seen in Ddr2^slie/slie mice. Since it has been proposed that changes in the cranial base can indirectly affect suture formation (Kreiborg et al., 1993), we conducted a second conditional knockout of Ddr2 in chondrocytes using Col2a1^Cre that would be expected to be preferentially active during endochondral bone formation in synchondroses, but not during intramembranous bone formation in sutures (Figure 3). The synchondroses of Col2a1^Cre;Ddr2^f/f and Gli1^CreERT;Ddr2^fl/fl mice were similar. However, Col2a1^Cre;Ddr2^f/f mice had normal frontal sutures. Therefore, the contribution of Ddr2 to endochondral bone formation in the synchondrosis is independent from its function in cranial sutures.

An unexpected finding was the observed thinning of frontal bones in Col2a1^Cre;Ddr2^f/f mice. This thinning, which was first detected in Ddr2^slie/slie and Gli1^CreERT;Ddr2^fl/fl mice, would not normally be expected at a site of intramembranous bone formation that is not a major site of type 2 collagen synthesis. However, interpretation of this result is confounded by earlier studies showing endogenous Col2a1 expression and Col2a1^Cre activity can be detected in calvarial osteoblasts and suture mesenchymal cells (Szabova et al., 2009; Sakagami et al., 2017). It is therefore possible that this Cre could inactivate Ddr2 in a select population of Col2a1-expressing cells in intramembranous bone, thereby explaining the reduced frontal bone thickness in Col2a1^Cre;Ddr2^fl/fl mice. However, the absence of a posterior frontal suture defect in Col2a1^Cre;Ddr2^fl/fl mice argues against Ddr2 having a function in Col2a1-expressing cells necessary for normal suture formation.

Significant similarities and differences are apparent when results of the present study are compared with our previous work on DDR2 functions in the appendicular skeleton (Mohamed et al., 2022). At both sites, DDR2 was localized to resting and proliferative zone cartilage of synchondroses or long bone growth plates, respectively, where co-localization with the Hh intermediate, GLI1, was observed. Lineage tracing at both sites suggested that DDR2 positive cells could serve as progenitors for hypertrophic chondrocytes, osteoblasts, and osteocytes of endochondral bone. Consistent with this cartilage localization, inactivation of Ddr2 in Gli1-positive progenitors (Gli1^CreERT) or in chondrocytes (Col2a1^Cre) inhibited endochondral bone formation in synchondroses and tibial growth plates and this inhibition was related to inhibition of chondrocyte proliferation rather than an increase in apoptosis. Type II collagen matrix organization and chondrocyte alignment was also disrupted at both sites although this phenotype was more severe in the synchondrosis where chondrocytes formed an ectopic hypertrophic zone. DDR2 functions in endochondral bone formation likely explain the dwarf phenotype, reduced A-P skull length and shortened snout of Ddr2 deficient mice and as well as the dwarfism and the depressed facial features of SMED, SL-AC patients. In mice, Ddr2 was also expressed adjacent to intramembranous bone in the periosteum of calvaria and tibial cortical bone as well as cranial sutures. However, important differences in intramembranous bone formation were seen when comparing these two sites. In the cranial vault, Ddr2 deficiency inhibited frontal suture formation and reduced frontal bone thickness. In contrast, Ddr2 deficiency had little effect on thickness of tibial cortical bone, which also forms through an intramembranous process (Mohamed et al., 2022). The basis for these differences is not clear. On one hand, differences in rates of formation of calvarial versus cortical bone may mask DDR2-related activity in cortical bone. Alternatively, they may reflect inherent differences between these two tissue sites. Functions of DDR2 in cortical bone may be more apparent in regeneration models which involve more rapid bone formation than is seen during development. As we recently showed, Ddr2 deficiency severely disrupts intramembranous bone healing of subcritical-sized calvarial defects (Binrayes et al., 2021). It is not known if healing of related cortical bone defects would also be disrupted.

Although the basis for DDR2 actions on cranial and skeletal development are not well-understood, we hypothesize that it may function by controlling properties of the ECM such as collagen distribution, fibril orientation and stiffness, which in turn alters cell behavior via regulation of integrin signaling. In the present study, we evaluated type II collagen properties in synchondroses using immunofluorescence and SHG microscopy. In wild type mice, uniform Col II staining in the extraterritorial and territorial chondrocyte matrix and a high degree of collagen orientation (increased anisotropy) was observed throughout the synchondrosis resting zone. However, Ddr2-deficient mice showed a non-homogenous Col II staining pattern, predominantly in pericellular space adjacent to chondrocytes as well as decreased anisotropy. These ECM changes were accompanied by decreased chondrocyte polarization as measured by GM130 Golgi staining, reduced proliferation, and formation of an ectopic hypertrophic zone. GM130 is a peripheral membrane protein tightly bound to the cytoplasmic face of the Golgi apparatus, the central organelle for secretory protein trafficking and cargo modification (Arita et al., 2015). GM130 is required for maintaining Golgi structure, spindle assembly, cell cycle progression, and cytoskeletal dynamics (Wei et al., 2015; Sanders and Kaverina, 2015; Colanzi and Corda, 2007; Farquhar and Palade, 1998; Liu et al., 2017). Moreover, disruption of GM130 in mitosis impairs spindle assembly and cell division (Wei et al., 2015). This may explain the underlying deficient chondrocyte proliferation seen in Ddr2-deficient synchondroses, although these mechanisms require more clarification. In our previous study on long bone development, comparable changes in Col II distribution and chondrocyte proliferation were observed in the tibial growth plate (Mohamed et al., 2022).

In other experimental systems, similar DDR2-dependent changes in collagen fibril orientation and distribution have been observed and shown to modify cellular behavior by altering integrin signaling. For example, DDR2 in cancer-associated fibroblasts (CAFs) increases tumor stiffness by reorganizing type I collagen fibrils at the tumor-stromal boundary, thereby promoting lung metastasis of breast tumors. These ECM changes are associated with increased integrin activity and mechanotransduction by CAFs (Bayer et al., 2019). Also, in a recent collaborative study on trauma-induced heterotopic ossification (HO) in mice, we showed that loss of DDR2 was associated with decreased collagen fibril orientation as measured by SHG as well as dramatic inhibition of HO (Pagani et al., 2022). The loss of collagen alignment was accompanied by a reduction in integrin signaling as measured by reduced FAK phosphorylation, decreased nuclear translocation of TAZ and reduced YAP/TAZ signaling, consistent with DDR2 being required for force transduction, subsequent osteoblast differentiation and HO.

Further evidence that changes in Col II orientation/distribution might explain the cartilage phenotype of Ddr2-deficient mice comes from studies with Col2a1 mutant mice (Brown et al., 1981; Pace et al., 1997; Seegmiller et al., 1971; Spranger et al., 1994; Vikkula et al., 1994). Both Col2a1expDmm/Dmm mice harboring a mutation in the C-propeptide globular domain of type II collagen (Col2a1) (Pace et al., 1997) as well as mice containing an R992C mutation associated with spondyloepiphyseal dysplasia Arita et al., 2015 have defects in the assembly and folding of type II collagen, that alter collagen distribution/orientation (Arita et al., 2015; Brown et al., 1981; Pace et al., 1997). In both cases, mutant mice have a similar phenotype to Ddr2-nulls; they are dwarf, have a shortened vertebral column and small rib cage as well as craniofacial defects characterized by skull dysmorphogenesis, reduced skull length, reduced cranial base length, cleft palate, and short mandible. Interestingly, mice harboring the R992C mutation also exhibit disrupted chondrocyte polarity (Arita et al., 2015). Most importantly, the altered distribution of cartilage matrix was also reported in a SMED, SL-AC patient (Borochowitz et al., 1993). Histological analysis of the resting zone of costochondral cartilage from a SMED, SL-AC patient showed abnormally distributed chondrocytes with noticeable dark staining in surrounding matrix filling lacunar space (Borochowitz et al., 1993). The altered distribution of cartilage matrix highlights the similarity between histopathological features of the patient samples and our results in Ddr2-deficient mice, and provides important insights into potential mechanisms underlying Ddr2 regulation of bone development mediated by changes in ECM organization.

In summary, this study establishes a critical function of Ddr2 in skeletal progenitor cells, including GLI1 + cells, and chondrocytes to control bone formation and cartilage growth during postnatal growth of the craniofacial skeleton, and advances our understanding of the roles of cell-matrix interactions in craniofacial morphogenesis. Our findings provide further insight into the cellular basis of cranial defects in SMED SL-AC patients as well as evolutionary changes in craniofacial structure related to altered DDR2 activity.

All data generated or analysed during this study are included in the manuscript and source data files.

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Author details

1. Fatma F Mohamed

   Department of Periodontics & Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

2. Chunxi Ge

   Department of Periodontics & Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, United States

   Contribution

   Investigation, Supervision, Conceptualization, Formal analysis, Writing – original draft

   Contributed equally with

   Shawn A Hallett

   Competing interests

   No competing interests declared

3. Shawn A Hallett

   Department of Periodontics & Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, United States

   Contribution

   Conceptualization, Writing – original draft

   Contributed equally with

   Chunxi Ge

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1472-7502

4. Alec C Bancroft

   Center for Organogenesis and Trauma, Department of Surgery, University of Texas Southwestern, Dallas, United States

   Contribution

   Conceptualization

   Competing interests

   No competing interests declared

5. Randy T Cowling

   Division of Cardiovascular Medicine, University of California, San Diego, San Diego, United States

   Contribution

   Resources, Writing – review and editing

   Competing interests

   No competing interests declared

6. Noriaki Ono

   Department of Diagnostic and Biomedical Sciences, University of Texas Health Science Center at Houston School of Dentistry, Houston, United States

   Contribution

   Resources, Formal analysis, Writing – original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3771-8230

7. Abdul-Aziz Binrayes

   Department of Prosthetic Dental Sciences, College of Dentistry, King Saud University, Riyadh, Saudi Arabia

   Contribution

   Conceptualization, Writing – original draft

   Competing interests

   No competing interests declared

8. Barry Greenberg

   Division of Cardiovascular Medicine, University of California, San Diego, San Diego, United States

   Contribution

   Resources, Writing – original draft

   Competing interests

   No competing interests declared

9. Benjamin Levi

   Center for Organogenesis and Trauma, Department of Surgery, University of Texas Southwestern, Dallas, United States

   Contribution

   Conceptualization, Writing – original draft

   Competing interests

   No competing interests declared

10. Vesa M Kaartinen

    Department of Biologic & Materials Science, University of Michigan School of Dentistry, Ann Arbor, United States

    Contribution

    Supervision, Writing – original draft

    Competing interests

    No competing interests declared

11. Renny T Franceschi

    1. Department of Periodontics & Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, United States
    2. Department of Biological Chemistry, School of Medicine, University of Michigan, Ann Arbor, United States
    3. Department of Biomedical Engineering, University of Michigan, Ann Arbor, United States

    Contribution

    Conceptualization, Formal analysis, Funding acquisition, Project administration, Supervision, Validation, Writing – original draft, Writing – review and editing

    For correspondence

    rennyf@umich.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-1405-2541

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