Specific deletion of Axin1 leads to activation of β-catenin/BMP signaling resulting in fibular hemimelia phenotype in mice
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Curated by eLife
Evaluation Summary:
This manuscript describes the mesenchymal cells expressed Axin1 as a key regulator for Wnt and BMP signaling pathway which is essential for lower limb development. Fibular hemimelia (FH) is a rare genetic disorder with unknown mechanisms. Their data clearly demonstrated that inhibition of β-catenin and BMP signaling genetically and pharmacologically could largely reverse fibular hemimelia phenotype in mice. In general, the manuscript is clear, well written, and concise, the study is well-structured and various techniques have been used to validate the data. It presents as a thorough study highlighting the importance of Axin1/ β-catenin/BMP signaling in FH development, and, furthermore, the interpretation of the results and the following conclusions are convincing.
(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. The reviewers remained anonymous to the authors.)
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Abstract
Axin1 is a key regulator of canonical Wnt signaling pathway. Roles of Axin1 in skeletal development and in disease occurrence have not been fully defined. Here, we report that Axin1 is essential for lower limb development. Specific deletion of Axin1 in limb mesenchymal cells leads to fibular hemimelia (FH)-like phenotype, associated with tarsal coalition. Further studies demonstrate that FH disease is associated with additional defects in Axin1 knockout (KO) mice, including decreased osteoclast formation and defects in angiogenesis. We then provide in vivo evidence showing that Axin1 controls limb development through both canonical β-catenin and BMP signaling pathways. We demonstrate that inhibition of β-catenin or BMP signaling could significantly reverse the FH phenotype in mice. Together, our findings reveal that integration of β-catenin and BMP signaling by Axin1 is required for lower limb development. Defect in Axin1 signaling could lead to the development of FH disease.
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Author Response
Reviewer #2 (Public Review):
Fibular hemimelia (FH) is a rare genetic disorder with unknown mechanisms. In this study, the authors generated Axin1 conditional knockout (cKO) mice by depleting Axin1 gene specifically in Prx-1 expressing mesenchymal cells and demonstrated that Axin1 cKO mice developed FH phenotype with various severities. FH phenotype in Axin1 cKO mice can be rescued by either β-catenin or BMP inhibition if the inhibition was applied to the pregnant mother at E9.5 to E12.5. For mechanistic study, the authors showed elevated expression of BMP signaling molecules in limb tissue of Axin1 cKO mice and Axin1 regulated the degradation of pSmad5 in mesenchymal cells.
The study has many strengths. 1) The study was performed with high rigor. Utilization of various cre lines to conditional KO Anix1 in different …
Author Response
Reviewer #2 (Public Review):
Fibular hemimelia (FH) is a rare genetic disorder with unknown mechanisms. In this study, the authors generated Axin1 conditional knockout (cKO) mice by depleting Axin1 gene specifically in Prx-1 expressing mesenchymal cells and demonstrated that Axin1 cKO mice developed FH phenotype with various severities. FH phenotype in Axin1 cKO mice can be rescued by either β-catenin or BMP inhibition if the inhibition was applied to the pregnant mother at E9.5 to E12.5. For mechanistic study, the authors showed elevated expression of BMP signaling molecules in limb tissue of Axin1 cKO mice and Axin1 regulated the degradation of pSmad5 in mesenchymal cells.
The study has many strengths. 1) The study was performed with high rigor. Utilization of various cre lines to conditional KO Anix1 in different cell types to formally demonstrate the expression of Anix1 in Prx-1-expressing mesenchymal cells, but not in Sox9-, Col2-, and Osx-expressing cells is required for normal fibular development. 2) Treatment of Axin1 cKO mice with β-catenin and BMP inhibitor at different time points to demonstrate that inhibitors should be given during the early embryonic development, a very important point for considering the translational potential of the study. 3) Detailed in vitro experiments were performed to investigate the molecular mechanisms of Axin 1 on Smad 5 stability. 4) Both β-canenin and BMP signaling pathways are important, including skeletal development, this study used Axin1 cKO mice to integrate these two pathways together, which is a important and new contribution.
Weaknesses of the study have been described below:
- Authors need to report/describe findings/pheotypes in bones other than fibula in Axin1 cKO mice (4-8-week-old) first, and then focus on fibular development. From the X-ray data shown in Figure 1D-E, it appears that Axin1 cKO mice have high bone mass or osteopetrosis. Thus, histology of bones (femur, tibia, knee joint) other than fibula should be provided.
We have performed histology in femur, tibia and knee joint in Axin1 KO mice as the reviewer suggested.
- Fig. 2 described Axin1/2 dKO mice. I suggest to remove Figure 2 or move it to supplemental data. Including Axin1/2 dKO mice in the main text makes the story complicated and difficult to explain because the most of figures in this manuscript were on Axin 1, such as rescue experiments and molecular mechanistic study. Further, various severity of FH in Axin1 cKO mice are closer to human FH cases (various severity) than Axin1/2 dKO mice that have a completed loss of fibula. The title is also on Axin1. If Axin1/2 dKO mouse data are included in the main text, authors need to provide molecular explanation why Axin1/2 dKO mice have more severe phenotypes.
To make the entire story more straightfoward, we have removed the Axin1/2 double KO data (Fig. 2) as the reviewer suggested.
- Please include a paragraph in the discussion regarding the limitation of the study. Is there any human report that FH patients have mutation in Axin 1 and its related downstream signal proteins such as β-catenin and BMP? Can FH being directed before birth and to treat pregnant mother? Do authors plan to use unbiased approaches such as RNAseq or proteomics to discover new gene/proteins that are regulated by Axin1 in mesenchymal cells?
We have added a paragraph to discuss the limitation in the discussion section as the reviewer suggested. We have collaborated with Dr. Qinglin Kang and collected 9 samples from patients with FH disease and identified a mutation of β-catenin gene, encoding a potential phosphorylation site, which may lead to upregulation of β-catenin protein levels. In the future, we will investigate if the mutation of β-catenin affects its function in mesenchymal cells. We are currently planning to perform the RNA-Seq and proteomics experiments to identify novel downstream target gene(s) of Axin1.
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Evaluation Summary:
This manuscript describes the mesenchymal cells expressed Axin1 as a key regulator for Wnt and BMP signaling pathway which is essential for lower limb development. Fibular hemimelia (FH) is a rare genetic disorder with unknown mechanisms. Their data clearly demonstrated that inhibition of β-catenin and BMP signaling genetically and pharmacologically could largely reverse fibular hemimelia phenotype in mice. In general, the manuscript is clear, well written, and concise, the study is well-structured and various techniques have been used to validate the data. It presents as a thorough study highlighting the importance of Axin1/ β-catenin/BMP signaling in FH development, and, furthermore, the interpretation of the results and the following conclusions are convincing.
(This preprint has been reviewed by eLife. We include …
Evaluation Summary:
This manuscript describes the mesenchymal cells expressed Axin1 as a key regulator for Wnt and BMP signaling pathway which is essential for lower limb development. Fibular hemimelia (FH) is a rare genetic disorder with unknown mechanisms. Their data clearly demonstrated that inhibition of β-catenin and BMP signaling genetically and pharmacologically could largely reverse fibular hemimelia phenotype in mice. In general, the manuscript is clear, well written, and concise, the study is well-structured and various techniques have been used to validate the data. It presents as a thorough study highlighting the importance of Axin1/ β-catenin/BMP signaling in FH development, and, furthermore, the interpretation of the results and the following conclusions are convincing.
(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. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The findings reported in this paper demonstrate that integration of Wnt and BMP signaling by Axin1 is crucial for lower limb development, and defects in Axin1 and Axin2 signaling could lead to the development of FH disease.
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Reviewer #2 (Public Review):
Fibular hemimelia (FH) is a rare genetic disorder with unknown mechanisms. In this study, the authors generated Axin1 conditional knockout (cKO) mice by depleting Axin1 gene specifically in Prx-1 expressing mesenchymal cells and demonstrated that Axin1 cKO mice developed FH phenotype with various severities. FH phenotype in Axin1 cKO mice can be rescued by either β-catenin or BMP inhibition if the inhibition was applied to the pregnant mother at E9.5 to E12.5. For mechanistic study, the authors showed elevated expression of BMP signaling molecules in limb tissue of Axin1 cKO mice and Axin1 regulated the degradation of pSmad5 in mesenchymal cells.
The study has many strengths. 1) The study was performed with high rigor. Utilization of various cre lines to conditional KO Anix1 in different cell types to …
Reviewer #2 (Public Review):
Fibular hemimelia (FH) is a rare genetic disorder with unknown mechanisms. In this study, the authors generated Axin1 conditional knockout (cKO) mice by depleting Axin1 gene specifically in Prx-1 expressing mesenchymal cells and demonstrated that Axin1 cKO mice developed FH phenotype with various severities. FH phenotype in Axin1 cKO mice can be rescued by either β-catenin or BMP inhibition if the inhibition was applied to the pregnant mother at E9.5 to E12.5. For mechanistic study, the authors showed elevated expression of BMP signaling molecules in limb tissue of Axin1 cKO mice and Axin1 regulated the degradation of pSmad5 in mesenchymal cells.
The study has many strengths. 1) The study was performed with high rigor. Utilization of various cre lines to conditional KO Anix1 in different cell types to formally demonstrate the expression of Anix1 in Prx-1-expressing mesenchymal cells, but not in Sox9-, Col2-, and Osx-expressing cells is required for normal fibular development. 2) Treatment of Axin1 cKO mice with β-catenin and BMP inhibitor at different time points to demonstrate that inhibitors should be given during the early embryonic development, a very important point for considering the translational potential of the study. 3) Detailed in vitro experiments were performed to investigate the molecular mechanisms of Axin 1 on Smad 5 stability. 4) Both β-canenin and BMP signaling pathways are important, including skeletal development, this study used Axin1 cKO mice to integrate these two pathways together, which is a important and new contribution.
Weaknesses of the study have been described below:
Authors need to report/describe findings/pheotypes in bones other than fibula in Axin1 cKO mice (4-8-week-old) first, and then focus on fibular development. From the X-ray data shown in Figure 1D-E, it appears that Axin1 cKO mice have high bone mass or osteopetrosis. Thus, histology of bones (femur, tibia, knee joint) other than fibula should be provided.
Fig 2 described Axin1/2 dKO mice. I suggest to remove Figure 2 or move it to supplemental data. Including Axin1/2 dKO mice in the main text makes the story complicated and difficult to explain because the most of figures in this manuscript were on Axin 1, such as rescue experiments and molecular mechanistic study. Further, various severity of FH in Axin1 cKO mice are closer to human FH cases (various severity) than Axin1/2 dKO mice that have a completed loss of fibula. The title is also on Axin1. If Axin1/2 dKO mouse data are included in the main text, authors need to provide molecular explanation why Axin1/2 dKO mice have more severe phenotypes.
Please include a paragraph in the discussion regarding the limitation of the study. Is there any human report that FH patients have mutation in Axin 1 and its related downstream signal proteins such as beta-catenin and BMP? Can FH being directed before birth and to treat pregnant mother? Do authors plan to use unbiased approaches such as RNAseq or proteomics to discover new gene/proteins that are regulated by Axin1 in mesenchymal cells?
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