An adhesion G protein-coupled receptor is required in cartilaginous and dense connective tissues to maintain spine alignment
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Evaluation Summary:
This is an interesting and thorough study of the developmental roles of the G-coupled protein receptor Adgr6 in spine development that contributes both to the understanding of spine morphogenesis and the etiology of common types of scoliosis that are of unknown origin. Using conditional mouse knockouts, the authors dissect the contributions of Adgr6 in each spine-associated tissue. In addition to the use of state-of-the-art genetic tools, the authors show beautiful histological and tomography data illustrating developmental processes and phenotypes with great detail. Their results also implicate cAMP signaling and CREB activity in the regulation of mechanical properties of dense spine tissues.
(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 #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
Adolescent idiopathic scoliosis (AIS) is the most common spine disorder affecting children worldwide, yet little is known about the pathogenesis of this disorder. Here, we demonstrate that genetic regulation of structural components of the axial skeleton, the intervertebral discs, and dense connective tissues (i.e., ligaments and tendons) is essential for the maintenance of spinal alignment. We show that the adhesion G protein-coupled receptor ADGRG6, previously implicated in human AIS association studies, is required in these tissues to maintain typical spine alignment in mice. Furthermore, we show that ADGRG6 regulates biomechanical properties of tendon and stimulates CREB signaling governing gene expression in cartilaginous tissues of the spine. Treatment with a cAMP agonist could mirror aspects of receptor function in culture, thus defining core pathways for regulating these axial cartilaginous and connective tissues. As ADGRG6 is a key gene involved in human AIS, these findings open up novel therapeutic opportunities for human scoliosis.
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Reviewer 3 (Public Review):
In this work, the authors study the role of Adgrg6 in spine alignment. Using a battery of tissue-specific Cre deleter lines, they show that Adgrg6 activity in intervertebral disc (IVD), ligament and tendon cells is necessary to prevent spine misalignment. The finding that the phenotype appears around postnatal day 20 associates it with the human disorder adolescent idiopathic scoliosis. The authors show reduction in the phosphorylation of CREB in cartilage cells from Adgrg6 KO spine, suggesting a molecular entry point into this regulatory mechanism. Additionally, loss-of-function of Adgrg6 leads to a postnatal phenotype, indicating that its activity is important already in the embryo. Finally, they show involvement of Adgrg6 in regulating mechanical properties of tendons.
It would have been interesting to see …
Reviewer 3 (Public Review):
In this work, the authors study the role of Adgrg6 in spine alignment. Using a battery of tissue-specific Cre deleter lines, they show that Adgrg6 activity in intervertebral disc (IVD), ligament and tendon cells is necessary to prevent spine misalignment. The finding that the phenotype appears around postnatal day 20 associates it with the human disorder adolescent idiopathic scoliosis. The authors show reduction in the phosphorylation of CREB in cartilage cells from Adgrg6 KO spine, suggesting a molecular entry point into this regulatory mechanism. Additionally, loss-of-function of Adgrg6 leads to a postnatal phenotype, indicating that its activity is important already in the embryo. Finally, they show involvement of Adgrg6 in regulating mechanical properties of tendons.
It would have been interesting to see if there is early indication for abnormality before the onset of scoliosis by showing histology and gene expression in cartilage, tendon and IVD of P10 mice.
Overall, it is an interesting and important paper, but it would have benefited from better organisation of the Results section.
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Reviewer 2 (Public Review):
This work is significant in that it carefully dissects the tissue dependency of the function of Adgrg6 through use of conditional loss of function in different components of the skeleton. The precision of the work, both in characterization of the anatomy through histological, tomography, and genetic analysis of expression is quite exceptional and allowed fine grained dissection of the regionality of Adgrg6 action as it pertains to formation and maintenance of the spine, as well as the temporal manifestation of its phenotypes.
The authors find that although Adgrg6 has important functions in differentiation of chondrogenic cells, its role affecting the susceptibility to AIS stems from its function in dense connective tissues such as ligaments. Notably, the authors do not find an effect when altered in …
Reviewer 2 (Public Review):
This work is significant in that it carefully dissects the tissue dependency of the function of Adgrg6 through use of conditional loss of function in different components of the skeleton. The precision of the work, both in characterization of the anatomy through histological, tomography, and genetic analysis of expression is quite exceptional and allowed fine grained dissection of the regionality of Adgrg6 action as it pertains to formation and maintenance of the spine, as well as the temporal manifestation of its phenotypes.
The authors find that although Adgrg6 has important functions in differentiation of chondrogenic cells, its role affecting the susceptibility to AIS stems from its function in dense connective tissues such as ligaments. Notably, the authors do not find an effect when altered in osteoblasts, underscoring a mechanical deficiency model of AIS in which non-osteogenic tissues may drive the presentation and expression of scoliosis. Lastly, although preliminary analysis in KO and WT cells in vitro, the authors show ability to restore Agrgr6 regulated genes by treatment with small molecule mediators of CREB, which functions downstream. Such targeted modulation and restoration of components of Agrgr6 function within skeletogenic cells may prove to be an effective means of prevention in treatment of this disorder - possibly even in cases of diverse genetic, or environmental causes. This however is not directly tested in the animal model presented.
The data is quite clear and directly addresses their attempts to understand the etiology of progressive, and late deterioration of the spine. Weaknesses of the manuscript lie in the integration of their approach and data within a logical framework in which to apply their findings. It is unclear why this gene was a target of analysis, such as its prevalence in AIS, or ability to serve as a unique model for the disease - unique as in why this gene rather than other models of AIS in mouse or zebrafish. The impact of the findings here could be greatly strengthened by discussion of why experiments were initiated and how the data addressed the overall question of cause of AIS by Agrgr6 and by integration within and between sections of the results.
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Reviewer 1 (Public Review):
In the manuscript by Liu et al., the authors investigate the role of Adgr6 in spine development in mice and dissect tissue specific contributions leading to late onset scoliosis in knockouts. Furthermore, they implicate Adgr6 in regulating gene expression and the mechanical properties of dense connective tissues via cAMP signaling that are linked to de development of scoliosis.
Overall, this is an interesting and thorough study of the developmental roles of Adgr6 in spine development that contributes both to the understanding of spine morphogenesis and the etiology of common types of scoliosis that are of unknown origin (i.e., idiopathic). Through the use of various tissue specific drivers, the authors generate conditional mouse knockouts that allowed them to dissect the respective contribution of Adgr6's …
Reviewer 1 (Public Review):
In the manuscript by Liu et al., the authors investigate the role of Adgr6 in spine development in mice and dissect tissue specific contributions leading to late onset scoliosis in knockouts. Furthermore, they implicate Adgr6 in regulating gene expression and the mechanical properties of dense connective tissues via cAMP signaling that are linked to de development of scoliosis.
Overall, this is an interesting and thorough study of the developmental roles of Adgr6 in spine development that contributes both to the understanding of spine morphogenesis and the etiology of common types of scoliosis that are of unknown origin (i.e., idiopathic). Through the use of various tissue specific drivers, the authors generate conditional mouse knockouts that allowed them to dissect the respective contribution of Adgr6's function in each spine associated tissue. In addition to the use of state-of-the-art genetic tools, the authors show beautiful histological and micro-CT data illustrating developmental processes and phenotypes with great detail. Their results also implicate cAMP signaling and CREB activity in the regulation of mechanical properties of dense spine tissues.
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Evaluation Summary:
This is an interesting and thorough study of the developmental roles of the G-coupled protein receptor Adgr6 in spine development that contributes both to the understanding of spine morphogenesis and the etiology of common types of scoliosis that are of unknown origin. Using conditional mouse knockouts, the authors dissect the contributions of Adgr6 in each spine-associated tissue. In addition to the use of state-of-the-art genetic tools, the authors show beautiful histological and tomography data illustrating developmental processes and phenotypes with great detail. Their results also implicate cAMP signaling and CREB activity in the regulation of mechanical properties of dense spine tissues.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive …
Evaluation Summary:
This is an interesting and thorough study of the developmental roles of the G-coupled protein receptor Adgr6 in spine development that contributes both to the understanding of spine morphogenesis and the etiology of common types of scoliosis that are of unknown origin. Using conditional mouse knockouts, the authors dissect the contributions of Adgr6 in each spine-associated tissue. In addition to the use of state-of-the-art genetic tools, the authors show beautiful histological and tomography data illustrating developmental processes and phenotypes with great detail. Their results also implicate cAMP signaling and CREB activity in the regulation of mechanical properties of dense spine tissues.
(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 #1 and Reviewer #2 agreed to share their names with the authors.)
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