Control of craniofacial development by the collagen receptor, discoidin domain receptor 2
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Curated by eLife
Evaluation Summary:
The authors report that discoidin domain receptor 2 (DDR2), a non-integrin collagen receptor, is required in Gli1+ cells for the development of the craniofacial skeleton. It is known that mutations in DDR2 are associated with craniofacial abnormalities, such as midface hypoplasia and open fontanels. This paper is of potential interest to craniofacial skeletal developmental researchers. While the data quality is high, the paper helps to confirm what has been recently published by the same authors.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Development of the craniofacial skeleton requires interactions between progenitor cells and the collagen-rich extracellular matrix (ECM). The mediators of these interactions are not well-defined. Mutations in the discoidin domain receptor 2 gene ( DDR2 ), which encodes a non-integrin collagen receptor, are associated with human craniofacial abnormalities, such as midface hypoplasia and open fontanels. However, the exact role of this gene in craniofacial morphogenesis is not known. As will be shown, Ddr2 -deficient mice exhibit defects in craniofacial bones including impaired calvarial growth and frontal suture formation, cranial base hypoplasia due to aberrant chondrogenesis and delayed ossification at growth plate synchondroses. These defects were associated with abnormal collagen fibril organization, chondrocyte proliferation and polarization. As established by localization and lineage-tracing studies, Ddr2 is expressed in progenitor cell-enriched craniofacial regions including sutures and synchondrosis resting zone cartilage, overlapping with GLI1 + cells, and contributing to chondrogenic and osteogenic lineages during skull growth. Tissue-specific knockouts further established the requirement for Ddr2 in GLI +skeletal progenitors and chondrocytes. These studies establish a cellular basis for regulation of craniofacial morphogenesis by this understudied collagen receptor and suggest that DDR2 is necessary for proper collagen organization, chondrocyte proliferation, and orientation.
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Author Response
Reviewer #1 (Public Review):
The authors characterized the expression of DDR2 in the developing craniofacial skeleton. The authors showed that Ddr2-deficient mice exhibited defects in craniofacial bones including impaired calvarial growth and frontal suture formation, cranial base hypoplasia due to aberrant chondrogenesis, and delayed ossification at growth plate synchondroses. The histological studies are well done. However, the studies as shown in this manuscript do not provide cellular and molecular mechanisms beyond what is already known, particularly beyond what the authors have already published in a similar study in Bone Research (Mohamed et al., 2022 Feb 9;10(1):11). With the same Cre lines and analytic approaches, the authors already showed in the Bone Research paper that Ddr2 in the Gli1+ cells is required …
Author Response
Reviewer #1 (Public Review):
The authors characterized the expression of DDR2 in the developing craniofacial skeleton. The authors showed that Ddr2-deficient mice exhibited defects in craniofacial bones including impaired calvarial growth and frontal suture formation, cranial base hypoplasia due to aberrant chondrogenesis, and delayed ossification at growth plate synchondroses. The histological studies are well done. However, the studies as shown in this manuscript do not provide cellular and molecular mechanisms beyond what is already known, particularly beyond what the authors have already published in a similar study in Bone Research (Mohamed et al., 2022 Feb 9;10(1):11). With the same Cre lines and analytic approaches, the authors already showed in the Bone Research paper that Ddr2 in the Gli1+ cells is required for chondrocyte proliferation and polarity in growth plate development and osteoblast differentiation. Cartilage development and bone formation occur in both long bones and craniofacial skeleton, the authors showed similar functions of Ddr2 in similar skeletal tissues, although the location is different. One new point in this manuscript might be: the authors indicated that loss of Ddr2 led to ectopic chondrocyte hypertrophic (Fig. 7I). But what the data actually showed was delayed chondrocyte hypertrophy and abnormal location of the delayed hypertrophic chondrocytes, which could be well caused by abnormal chondrocyte polarity. This interesting defect was superficially described with no mechanistic investigation at cellular or molecular level.
New data is now provided showing that Ddr2 deficiency is associated with abnormal collagen organization and orientation as measured by second harmonic generation (SHG) (Fig 3-figure supplement 1). Specifically, collagen orientation as reflected by SHG anisotropy measurements was disrupted in Ddr2-deficient synchondroses. This result complements data showing that the distribution of type II collagen as measured by immunofluorescence changes with Ddr2 deficiency such that no collagen is seen in the interterritorial matrix between chondrocyte bundles (Fig 3a). This loss of collagen organization provides a potential mechanism to explain the disruption of chondrocyte polarity and altered localization of hypertrophic cells in synchondroses. In further support of this concept, other recently published studies described in the Discussion have shown that Ddr2 deficiency is associated with disruption of collagen fibril orientation in other experimental systems such as in CAF cells surrounding breast tumors as well as at sites of heterotopic ossification and that these abnormalities are associated with defective integrin signaling. Additional studies beyond the scope of the present communication will be required to determine if these matrix changes can explain the observed phenotypes. However, we believe this proposed mechanism is the most likely explanation for DDR2 effects based on current data.
Reviewer #2 (Public Review):
DDR2 is a collagen-binding receptor that is required for proper skull development. Ddr2 loss-of-function in humans is associated with the developmental disease spondylo-meta-epiphyseal dysplasia (SMED). Here, the authors aim to elucidate the role of DDR2 in skull development. In this work, the role of DDR2 in skull and face development is studied in mice, which exhibit SMED-like symptoms in the absence of Ddr2. Histological studies showed that Ddr2 knockout disrupts organization and proper differentiation within progenitor-rich regions of the skull from which bone growth occurs. Histology and lineage tracing studies revealed that DDR-expressing cells in/around these zones 1) generally also express the proliferation regulator Gli1, and 2) eventually contribute to osteogenic and chondrogenic lineages. Cell-type specific knockout studies were used to show that DDR2 has a development-specific role: knockout of Ddr2 in Gli+ cells re-capitulated the developmental abnormalities observed in global Ddr2 knockout mice; knockout in chondrocytes partially recapitulated developmental abnormalities, and osteoblast-specific knockout mice were indistinguishable from their wild-type littermates. This work also catalogues the locations of Ddr2 positive cells and their lineages at various stages of development. Additionally, the anatomical effects of loss of DDR2 function on skull and face development are thoroughly described in global and cell-type specific knockouts.
This work is a vital and stimulating contribution to the scientific literature. The authors' claims and conclusions are well supported by the evidence they present.
The scientific approach is sound and the conclusions important. However, a limitation of the work's discussion is a lack of attention paid to the specific biophysical mechanism that DDR2 is playing during development. The discussion of the positioning of the golgi is nice, but a lack of golgi polarity is likely a downstream effect of processes occurring within the cell adhesion and mechanotransduction machinery. Perhaps, like integrins, DDR2 is a mechanosensor that the cell needs to properly sense local collagen orientation, polarize, and secrete properly-organized COL2. It would be beneficial to put up some guideposts that will facilitate engagement from the molecular biophysics/mechanobiology community.
Thank you for this suggestion. In response, we added new studies showing that DDR2 is necessary for ECM organization (please see reviewer 1 comments and additions to the Discussion section). In addition, the Discussion has been revised to include speculation on the relationship between DDR2-dependent ECM organization, mechanical properties of the matrix and cell differentiation. Because very little is known about DDR2 from a mechanistic perspective, much of what we propose is currently conjecture, but hopefully can guide future study.
Reviewer #3 (Public Review):
From this work, the authors investigated a number of parameters in order to profoundly understand and demonstrate the vital role of ongoing interaction between components of extracellular matrix and particular stem cells to induce normal Craniofacial development. Thus, there was a focus on the genetic manipulation (knockout) impact of molecules behind the above-mentioned interaction, and on determining how such modification would be reflected on skull bone morphogenesis.
Strengths and Weaknesses
• Using different animals' backgrounds in the same experiment might impact work outcomes.
• Better to have (ethical approval) at the beginning of the material and methods in separate paragraphs.
• It is great that the authors precisely explain all the measurements.
• Supplementary file to have details of used antibodies might be required.
• All methods have been written in academic and clear ways.
• It is nice that there is a conclusion sentence by end of the results paragraph, which made it easy for readers to fully remember and understand.
• It is possible to see a reduction in proliferative chondrocyte, with no change in apoptosis rate?
Reductions in proliferation are certainly seen in many systems. Proliferation and apoptosis are not necessarily coupled.
• Results are supposed to be compatible.
• Very nice and representative images from the immunofluorescence protocol.
• Using different techniques to confirm observations is clearly manifested in methods and results.
It is clear that the author has used different methods and techniques in order to meet his work's objectives. Importantly, there was more than one procedure to confirm observations that are related to one or more than one aim.
Although determining to what extent the outcomes of this work could be applied to community need might require a subspecialist physician's opinion, it seems that observations of the present study are likely to require a series of further investigations in order to take it to the level of human users. Notably, identification of molecules and pathways behind skull development abnormalities would open a door to early diagnosis reasons for such deformities, thus mitigating future abnormalities either by developing new prevention methods or discovering unique medications.
Thank you for these comments. Additional commentary has been added to the Discussion to provide a more mechanistic interpretation of our results, however speculative they may be at this time. Ln 555-605
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Evaluation Summary:
The authors report that discoidin domain receptor 2 (DDR2), a non-integrin collagen receptor, is required in Gli1+ cells for the development of the craniofacial skeleton. It is known that mutations in DDR2 are associated with craniofacial abnormalities, such as midface hypoplasia and open fontanels. This paper is of potential interest to craniofacial skeletal developmental researchers. While the data quality is high, the paper helps to confirm what has been recently published by the same authors.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The authors characterized the expression of DDR2 in the developing craniofacial skeleton. The authors showed that Ddr2-deficient mice exhibited defects in craniofacial bones including impaired calvarial growth and frontal suture formation, cranial base hypoplasia due to aberrant chondrogenesis, and delayed ossification at growth plate synchondroses. The histological studies are well done. However, the studies as shown in this manuscript do not provide cellular and molecular mechanisms beyond what is already known, particularly beyond what the authors have already published in a similar study in Bone Research (Mohamed et al., 2022 Feb 9;10(1):11). With the same Cre lines and analytic approaches, the authors already showed in the Bone Research paper that Ddr2 in the Gli1+ cells is required for chondrocyte …
Reviewer #1 (Public Review):
The authors characterized the expression of DDR2 in the developing craniofacial skeleton. The authors showed that Ddr2-deficient mice exhibited defects in craniofacial bones including impaired calvarial growth and frontal suture formation, cranial base hypoplasia due to aberrant chondrogenesis, and delayed ossification at growth plate synchondroses. The histological studies are well done. However, the studies as shown in this manuscript do not provide cellular and molecular mechanisms beyond what is already known, particularly beyond what the authors have already published in a similar study in Bone Research (Mohamed et al., 2022 Feb 9;10(1):11). With the same Cre lines and analytic approaches, the authors already showed in the Bone Research paper that Ddr2 in the Gli1+ cells is required for chondrocyte proliferation and polarity in growth plate development and osteoblast differentiation. Cartilage development and bone formation occur in both long bones and craniofacial skeleton, the authors showed similar functions of Ddr2 in similar skeletal tissues, although the location is different. One new point in this manuscript might be: the authors indicated that loss of Ddr2 led to ectopic chondrocyte hypertrophic (Fig. 7I). But what the data actually showed was delayed chondrocyte hypertrophy and abnormal location of the delayed hypertrophic chondrocytes, which could be well caused by abnormal chondrocyte polarity. This interesting defect was superficially described with no mechanistic investigation at cellular or molecular level.
-
Reviewer #2 (Public Review):
DDR2 is a collagen-binding receptor that is required for proper skull development. Ddr2 loss-of-function in humans is associated with the developmental disease spondylo-meta-epiphyseal dysplasia (SMED). Here, the authors aim to elucidate the role of DDR2 in skull development. In this work, the role of DDR2 in skull and face development is studied in mice, which exhibit SMED-like symptoms in the absence of Ddr2. Histological studies showed that Ddr2 knockout disrupts organization and proper differentiation within progenitor-rich regions of the skull from which bone growth occurs. Histology and lineage tracing studies revealed that DDR-expressing cells in/around these zones 1) generally also express the proliferation regulator Gli1, and 2) eventually contribute to osteogenic and chondrogenic lineages. …
Reviewer #2 (Public Review):
DDR2 is a collagen-binding receptor that is required for proper skull development. Ddr2 loss-of-function in humans is associated with the developmental disease spondylo-meta-epiphyseal dysplasia (SMED). Here, the authors aim to elucidate the role of DDR2 in skull development. In this work, the role of DDR2 in skull and face development is studied in mice, which exhibit SMED-like symptoms in the absence of Ddr2. Histological studies showed that Ddr2 knockout disrupts organization and proper differentiation within progenitor-rich regions of the skull from which bone growth occurs. Histology and lineage tracing studies revealed that DDR-expressing cells in/around these zones 1) generally also express the proliferation regulator Gli1, and 2) eventually contribute to osteogenic and chondrogenic lineages. Cell-type specific knockout studies were used to show that DDR2 has a development-specific role: knockout of Ddr2 in Gli+ cells re-capitulated the developmental abnormalities observed in global Ddr2 knockout mice; knockout in chondrocytes partially recapitulated developmental abnormalities, and osteoblast-specific knockout mice were indistinguishable from their wild-type littermates. This work also catalogues the locations of Ddr2 positive cells and their lineages at various stages of development. Additionally, the anatomical effects of loss of DDR2 function on skull and face development are thoroughly described in global and cell-type specific knockouts.
This work is a vital and stimulating contribution to the scientific literature. The authors' claims and conclusions are well supported by the evidence they present.
The scientific approach is sound and the conclusions important. However, a limitation of the work's discussion is a lack of attention paid to the specific biophysical mechanism that DDR2 is playing during development. The discussion of the positioning of the golgi is nice, but a lack of golgi polarity is likely a downstream effect of processes occurring within the cell adhesion and mechanotransduction machinery. Perhaps, like integrins, DDR2 is a mechanosensor that the cell needs to properly sense local collagen orientation, polarize, and secrete properly-organized COL2. It would be beneficial to put up some guideposts that will facilitate engagement from the molecular biophysics/mechanobiology community.
-
Reviewer #3 (Public Review):
From this work, the authors investigated a number of parameters in order to profoundly understand and demonstrate the vital role of ongoing interaction between components of extracellular matrix and particular stem cells to induce normal Craniofacial development. Thus, there was a focus on the genetic manipulation (knockout) impact of molecules behind the above-mentioned interaction, and on determining how such modification would be reflected on skull bone morphogenesis.
Strengths and Weaknesses:
• Using different animals' backgrounds in the same experiment might impact work outcomes.
• Better to have (ethical approval) at the beginning of the material and methods in separate paragraphs.
• It is great that the authors precisely explain all the measurements.
• Supplementary file to have details of used …
Reviewer #3 (Public Review):
From this work, the authors investigated a number of parameters in order to profoundly understand and demonstrate the vital role of ongoing interaction between components of extracellular matrix and particular stem cells to induce normal Craniofacial development. Thus, there was a focus on the genetic manipulation (knockout) impact of molecules behind the above-mentioned interaction, and on determining how such modification would be reflected on skull bone morphogenesis.
Strengths and Weaknesses:
• Using different animals' backgrounds in the same experiment might impact work outcomes.
• Better to have (ethical approval) at the beginning of the material and methods in separate paragraphs.
• It is great that the authors precisely explain all the measurements.
• Supplementary file to have details of used antibodies might be required.
• All methods have been written in academic and clear ways.
• It is nice that there is a conclusion sentence by end of the results paragraph, which made it easy for readers to fully remember and understand.
• It is possible to see a reduction in proliferative chondrocyte, with no change in apoptosis rate?
• Results are supposed to be compatible.
• Very nice and representative images from the immunofluorescence protocol.
• Using different techniques to confirm observations is clearly manifested in methods and results.
It is clear that the author has used different methods and techniques in order to meet his work's objectives. Importantly, there was more than one procedure to confirm observations that are related to one or more than one aim.
Although determining to what extent the outcomes of this work could be applied to community need might require a subspecialist physician's opinion, it seems that observations of the present study are likely to require a series of further investigations in order to take it to the level of human users. Notably, identification of molecules and pathways behind skull development abnormalities would open a door to early diagnosis reasons for such deformities, thus mitigating future abnormalities either by developing new prevention methods or discovering unique medications.
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