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  1. eLife

    Author Response:

    We thank the reviewers for their thorough evaluation of our work. We apologize for the lengthy time needed for submission of this revision, since it required considerable new experimental work, testing reagents for eventual application, breeding and analysis of additional mutant mouse embryos to address the reviewer comments as well as delving further into underlying mechanisms. As a result, the revised manuscript contains 11 new figures (2 additional data figures, Figures 9, 10 and a summary graphic, Figure 11) to address the reviewers concerns and provide new findings to extend understanding of how ADAMTS6, through cleavage of fibrillin-2, serves in skeletal development. Below, we provide an itemized response to each of the reviewers’ comments.

    Reviewer #1:

    In this study, Mead and colleagues report that global loss of ADAMTS6 causes a severe chondrodysplasia that is significantly worsened by concomitant loss of ADAMTS10 and, conversely, almost fully prevented by haploinsufficiency for fibrillin 2, a substrate of ADAMTS6. Of note, haploinsufficiency for fibrillin 1 does not affect the chondrodysplasia of ADAMTS6 null mice. The authors use a variety of in vivo and in vitro assays for the testing of their hypothesis.

    The paper is informative as it expands and deepens our current undertstanding of proteases and their substrates in endochondral bone development.

    Though the phenotype is interesting and the rescue experiment is compelling, it remains completely elusive how the loss of ADAMTS6 and the increased accumulation of fibrillin 2 cause severe chondrodysplasia, which negatively impinges on the novelty of the paper.

    The new data (Figure 9 and Figure 10, summarized in Figure 11) shows that fibrillin-2 accumulation is associated with mesenchymal/perichondrial matrix sequestration of GDF5 with impaired BMP (but not TGFb) signaling observed in the developing long bones. We conclude that ADAMTS6 (a protease) functions in skeletal development via cleavage of fibrillin-2 as a primary mechanism. In its absence, the increased fibrillin-2 sequesters growth factors such as GDF5 and we propose, other BMP/TGFb superfamily members previously shown to bind fibrillin-2 (cited references). The role of fibrillin-2 in release of GDF5 is proposed as a secondary mechanism, based upon imaging of sequestered GDF5 on fibrillin-2 microfibrils. Finally, we propose that through these primary and downstream effects, ADAMTS6 promotes chondrocyte differentiation in the developing long bones by indirectly modulating Sox9 expression and production of cartilage proteoglycan (a tertiary mechanism).

    Reviewer #2:

    This work investigated the role of Adamts6, a protease closely related to Adamts10, in fibrillin proteolysis and demonstrates that Fbn2 digestion by Adamts10 and Adamts6 plays a critical role in skeletal development. The study shows the overlapping roles of these proteinases in fibrillin digestion and elimination and in skeletal development. Using mouse genetic approaches, the authors show that doubly mutants for Adamts10 and Adamts6 suffer more severer skeletal developmental defects than single mutants. The authors also show that Adamts6 directly binds to Fbn2 and cleaves Fbn2 via a biochemical approach. Lastly, analysis of compound mutants of Adamts6 and Fbn2 or Fbn1 demonstrates that prevention of Fbn2 accumulation, but not Fbn1, rescues that Adamts6 KO skeletal phenotype and the reversed the aberrant BMP signaling.

    This study elegantly shows the physiologic importance of Fbn2 proteolysis by Adamts6 in skeletal development. On the other hand, the role of Adamts10 in Fbn2 proteolysis was previously demonstrated in vitro, and the mouse phenotype of a mutant Adamts10, including accumulation of Fbn2 and dampened BMP signaling with normal TGF signaling, was previously reported. In this regard, the major findings in this paper are somewhat expected.

    The strength of this paper is that it demonstrates the critical role of Adamts6 in Fbn2 proteolysis in skeletal development by combining mouse genetic, cell biological, and biochemical approaches. The experiments were conducted rigorously, and the conclusions are solid. The major weakness is that because the results are somewhat expected based on the knowledge from previous studies, this work gives an impression of being incremental. Also, as it is narrowly focusing on the role of Adamts6 on Fbn2 proteolysis, the significance of the findings does not seem very clear to a broader audience.

    The experiments were performed very well and the presented evidence supports the authors' conclusions.

    We thank the reviewer for the overall positive review of our work. However, we respectfully disagree that the regulation of fibrillin-2 by ADAMTS6 is somewhat expected. The present work, focusing as it does primarily on ADAMTS6 (and not ADAMTS10), identifies three new ADAMTS6 substrates and shows genetic evidence that fibrillin-2, but not fibrillin-1 cleavage by ADAMTS6 is consequential in skeletal development. The new work on ADAMTS6 is fully novel and could not have been predicted from any prior work. The manuscript also demonstrates how the uncleaved fibrillin-2 acts, functioning via GDF5 sequestration (and potentially other related growth factors) to limit chondrogenic function in cartilage. These findings are of broad general interest because they provide a specific, genetically validated illustration of growth factor regulation by ECM. The present work only features ADAMTS10 as a secondary player, to illustrate the transcriptional adaptation and its cooperative impact with ADAMTS6 in skeletal development. Moreover, none of the work presented on Adamts10 overlaps with prior publications.

    We would also like to emphasize the use of BIAcore to show intermolecular affinities between ADAMTS6 and fibrillins, and application of a sophisticated proteomics method to determine the precise cleavage sites of ADAMTS6 in fibrillin-2, fibrillin-1 and fibronectin. These applications are novel and in-depth and the findings are non-obvious. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD027096 and 10.6019/PXD027096.

    Reviewer #3:

    This paper explores the shared and unique functions of the structurally related proteases ADAMTS6 and ADAMTS10 in the developing cartilage growth plate, using genetic and biochemical approaches to show they are both required, but use distinct mechanisms to promote the switch from embryonic prevalence of fibrillin-2 microfibrils to postnatal prevalence of fibrillin-1. This conclusion is supported by a phenotypic analysis documenting an essential function for ADAMTS6 in cartilage and a significant genetic interaction between Adamts6 and Adamts10 in this tissue that is clear from the greater severity of defects in Adamts6/10 double mutants. The phenotypic analysis of the growth plate defects is not comprehensive, but clearly documents elevated fibrillin-2 levels in the cartilage matrix, consistent with an essential role for both proteases in clearance of fibrillin-2. These findings are coupled with mechanistic in vitro and biochemical studies showing that ADAMTS6 directly interacts with and cleaves Fibrillin-1 and -2. Most significantly, the work demonstrates that Fibrillin-2 is an essential substrate of ADAMTS6 because the skeletal defects in Adamts6-/- mice are significantly rescued in Adamnts6-/-;Fbn2+/- mutants. Thus, the Adamts6 mutant phenotype can be largely attributed to inappropriate accumulation of Fibrillin-2. The paper also investigates whether the greater severity of skeletal manifestations in Adamts6-/- mice compared to Adamts10-/- mice may be due in part to compensatory transcriptional upregulation of Adamts6 in Adamts10-/- mice. However, although the authors argue that transcriptional upregulation of Adamts6 contributes to the milder skeletal phenotype of Adamts10 mutants, whether the observed upregulation in mRNA levels translates to elevated ADAMTS6 protein levels is unknown, and whether the less severe Adamts10 phenotype might reflect the presence of different ADAMTS protein that has similar function to ADAMTS10, is unclear. Nonetheless, the data represent an important contribution to our understanding of the regulation of fibrillin microfibril deposition and clearance.

    We thank the reviewer for the comprehensive and overall positive review of our work.

  2. eLife

    Evaluation Summary:

    The study by Mead and colleagues proves that the in vivo genetic interactions between Adamts 6 and fibrillin 2 is critical for normal endochondral bone development. In particular, the authors show that global loss of ADAMTS6 causes a severe chondrodysplasia that is significantly worsened by concomitant loss of ADAMTS10 and, conversely, almost fully prevented by haploinsufficiency for fibrillin 2, a substrate of ADAMTS6. The paper expands and deepens our current undertstanding of proteases and their substrates in endochondral bone development.

    (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 #3 agreed to share their name with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    In this study, Mead and colleagues report that global loss of ADAMTS6 causes a severe chondrodysplasia that is significantly worsened by concomitant loss of ADAMTS10 and, conversely, almost fully prevented by haploinsufficiency for fibrillin 2, a substrate of ADAMTS6. Of note, haploinsufficiency for fibrillin 1 does not affect the chondrodysplasia of ADAMTS6 null mice. The authors use a variety of in vivo and in vitro assays for the testing of their hypothesis.

    The paper is informative as it expands and deepens our current undertstanding of proteases and their substrates in endochondral bone development.

    Though the phenotype is interesting and the rescue experiment is compelling, it remains completely elusive how the loss of ADAMTS6 and the increased accumulation of fibrillin 2 cause severe chondrodysplasia, which negatively impinges on the novelty of the paper.

    In additon, numerous technical issues should be addressed to strengthen the authors' conclusions and their biological relevance.

  4. eLife

    Reviewer #2 (Public Review):

    This work investigated the role of Adamts6, a protease closely related to Adamts10, in fibrillin proteolysis and demonstrates that Fbn2 digestion by Adamts10 and Adamts6 plays a critical role in skeletal development. The study shows the overlapping roles of these proteinases in fibrillin digestion and elimination and in skeletal development. Using mouse genetic approaches, the authors show that doubly mutants for Adamts10 and Adamts6 suffer more severer skeletal developmental defects than single mutants. The authors also show that Adamts6 directly binds to Fbn2 and cleaves Fbn2 via a biochemical approach. Lastly, analysis of compound mutants of Adamts6 and Fbn2 or Fbn1 demonstrates that prevention of Fbn2 accumulation, but not Fbn1, rescues that Adamts6 KO skeletal phenotype and the reversed the aberrant BMP signaling.

    This study elegantly shows the physiologic importance of Fbn2 proteolysis by Adamts6 in skeletal development. On the other hand, the role of Adamts10 in Fbn2 proteolysis was previously demonstrated in vitro, and the mouse phenotype of a mutant Adamts10, including accumulation of Fbn2 and dampened BMP signaling with normal TGF signaling, was previously reported. In this regard, the major findings in this paper are somewhat expected.

    The strength of this paper is that it demonstrates the critical role of Adamts6 in Fbn2 proteolysis in skeletal development by combining mouse genetic, cell biological, and biochemical approaches. The experiments were conducted rigorously, and the conclusions are solid. The major weakness is that because the results are somewhat expected based on the knowledge from previous studies, this work gives an impression of being incremental. Also, as it is narrowly focusing on the role of Adamts6 on Fbn2 proteolysis, the significance of the findings does not seem very clear to a broader audience.

    The experiments were performed very well and the presented evidence supports the authors' conclusions.

  5. eLife

    Reviewer #3 (Public Review):

    This paper explores the shared and unique functions of the structurally related proteases ADAMTS6 and ADAMTS10 in the developing cartilage growth plate, using genetic and biochemical approaches to show they are both required, but use distinct mechanisms to promote the switch from embryonic prevalence of fibrillin-2 microfibrils to postnatal prevalence of fibrillin-1. This conclusion is supported by a phenotypic analysis documenting an essential function for ADAMTS6 in cartilage and a significant genetic interaction between Adamts6 and Adamts10 in this tissue that is clear from the greater severity of defects in Adamts6/10 double mutants. The phenotypic analysis of the growth plate defects is not comprehensive, but clearly documents elevated fibrillin-2 levels in the cartilage matrix, consistent with an essential role for both proteases in clearance of fibrillin-2. These findings are coupled with mechanistic in vitro and biochemical studies showing that ADAMTS6 directly interacts with and cleaves Fibrillin-1 and -2. Most significantly, the work demonstrates that Fibrillin-2 is an essential substrate of ADAMTS6 because the skeletal defects in Adamts6-/- mice are significantly rescued in Adamnts6-/-;Fbn2+/- mutants. Thus, the Adamts6 mutant phenotype can be largely attributed to inappropriate accumulation of Fibrillin-2. The paper also investigates whether the greater severity of skeletal manifestations in Adamts6-/- mice compared to Adamts10-/- mice may be due in part to compensatory transcriptional upregulation of Adamts6 in Adamts10-/- mice. However, although the authors argue that transcriptional upregulation of Adamts6 contributes to the milder skeletal phenotype of Adamts10 mutants, whether the observed upregulation in mRNA levels translates to elevated ADAMTS6 protein levels is unknown, and whether the less severe Adamts10 phenotype might reflect the presence of different ADAMTS protein that has similar function to ADAMTS10, is unclear. Nonetheless, the data represent an important contribution to our understanding of the regulation of fibrillin microfibril deposition and clearance.

  6. Version 2 published on bioRxiv
  7. Version 1 published on bioRxiv