Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development
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Curated by eLife
Evaluation Summary:
This paper is of interest to scientists within the field of thymus development and function. Analysis of the data is overall rigorous and conclusions are justified. The work presented builds upon previous studies that have shown that alterations of Wnt/beta-catenin signaling in thymic epithelial cells impact the normal development and or maintenance of thymic epithelial microenvironment critical for the proper development and selection of functional self-tolerant T cell repertoire. The surprise that a thymus epithelial cell specific loss of function of beta-catenin only showed a rather minor phenotype is interesting, but it would be good to also address whether TEC recovery after a challenge, or in aging, is also affected in a minor manner or perhaps more dramatically than the steady-state situation. The author's claims are well supported by the data presented and will be of great interest to scientists and clinicians interested in understanding the signaling pathways important in thymic maintenance, as well as the development of strategies to counteract thymic involution in the aging population and cancer patients.
(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 #3 agreed to share their names with the authors.)
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Abstract
In the thymus, the thymic epithelium provides a microenvironment essential for the development of functionally competent and self-tolerant T cells. Previous findings showed that modulation of Wnt/β-catenin signaling in mouse thymic epithelial cells (TECs) disrupts embryonic thymus organogenesis. However, the role of β-catenin in TECs for postnatal T-cell development remains to be elucidated. Here, we analyzed gain-of-function (GOF) and loss-of-function (LOF) of β-catenin highly specific in mouse TECs. We found that GOF of β-catenin in TECs results in severe thymic dysplasia and T-cell deficiency beginning from the embryonic period. By contrast, LOF of β-catenin in TECs reduces the number of cortical TECs and thymocytes modestly and only postnatally. These results indicate that fine-tuning of β-catenin expression within a permissive range is required for TECs to generate an optimal microenvironment to support postnatal T-cell development.
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Author Response
Reviewer #2 (Public Review):
The authors describe how modulating the levels of beta-catenin in TECs affects thymic organization and thymopoiesis. They use a b5t-Cre to specifically stabilize or ablate beta-catenin in TECs. While stabilization of beta-catenin induces thymic dysplasia and significantly impedes development of thymocytes beyond the DN1 stage, loss of beta-catenin has a milder outcome limited to significantly reduced thymic weight starting and an overall reduction in thymocyte number but no significant effects in thymocytes subset distribution at 2weeks of age. This reduction of thymic weight is associated with a significant and selective reduction in the number of cTECs. On the basis of these findings the authors conclude that fine tuning of beta-catenin levels is essential for postnatal T cell development.
Author Response
Reviewer #2 (Public Review):
The authors describe how modulating the levels of beta-catenin in TECs affects thymic organization and thymopoiesis. They use a b5t-Cre to specifically stabilize or ablate beta-catenin in TECs. While stabilization of beta-catenin induces thymic dysplasia and significantly impedes development of thymocytes beyond the DN1 stage, loss of beta-catenin has a milder outcome limited to significantly reduced thymic weight starting and an overall reduction in thymocyte number but no significant effects in thymocytes subset distribution at 2weeks of age. This reduction of thymic weight is associated with a significant and selective reduction in the number of cTECs. On the basis of these findings the authors conclude that fine tuning of beta-catenin levels is essential for postnatal T cell development.
Overall this is an interesting but descriptive study that does not address the physiological and molecular effects of stabilizing or ablating beta-catenin in TECs. The authors suggest that stabilization of beta-catenin function in TECs results in their terminal differentiation to keratinocytes on the basis of increased expression of only two markers involucrin and loricrin, which is a limited definition and further analysis of these cells would be needed for this conclusion. On the other hand the interesting selective effect of loss of beta-catenin function on cTECs versus mTECs has not been analyzed. Are cTECs reduced due to loss of survival/proliferation/differentiation? How is their molecular profile affected by the loss of beta-catenin? Does the selective reduction of the cTEC compartment affect survival/proliferation/differentiation of the individual DN subsets? The paper would benefit from more in depth mechanistic analysis in these directions.
Thank you so much for stating that this is an interesting study. Thank you very much also for asking about the mechanism underlying the selective reduction in cTECs by LOF of B-catenin. According to the reviewer’s suggestion, we performed RNA sequencing analysis of cTECs and mTECs in B-cat LOF mice. We detected 92 genes that were significantly altered in B-cat LOF cTECs with the FDR corrected p-value of less than 0.05; there were 11 downregulated genes and 81 upregulated genes compared with control cTECs. We also detected 91 genes that were significantly altered in B-cat LOF mTECs; there were 53 downregulated genes and 38 upregulated genes compared with control mTECs. Thus, only less than 100 genes were significantly altered in cTECs and mTECs due to LOF of B-catenin, in agreement with the finding of no remarkable alteration in the expression of many functionally relevant molecules in cTECs and mTECs by quantitative RT-PCR analysis, as shown in the original manuscript. Nonetheless, we found that a vast majority of the genes that were significantly affected by LOF of B-catenin were different between cTECs and mTECs except Adh1 and Pglyrp1. Adh1 was upregulated in B-cat LOF cTECs and downregulated in B-cat LOF mTECs, whereas Pglyrp1 was upregulated by B-cat LOF in both cTECs and mTECs. Notably, Cdkn1a was elevated specifically in cTECs but not in mTECs in B-cat LOF mice. The expression of Cdkn1a, which encodes cyclin-dependent kinase (CDK) inhibitor p21, is linked to B-catenin activity. The upregulation of Cdkn1a in B-cat LOF cTECs compared with control cTECs was confirmed by quantitative RT-PCR analysis. We noticed no remarkable difference in other CDK family genes, including Cnnb1, Cnnb2, and Cnnd1, in cTECs and mTECs from B-cat LOF mice. These results suggest that the upregulation of Cdkn1a may contribute to the selective reduction in the number of cTECs by LOF of B-catenin. These new results are shown in new Figure 8 and explained as well as discussed in the revised manuscript.
According to your additional suggestion, we also examined the DN subsets in B-cat LOF mice and found that the frequency of DN subsets defined by CD44 and CD25 was unchanged, although the number of all four DN subsets was significantly reduced in B-cat LOF mice. These new results are shown in new Figure 9 and explained in the revised manuscript.
Thank you so much again for your comments to help improve the manuscript.
Reviewer #3 (Public Review):
The authors effectively utilized Beta5T-iCre to specifically manipulate beta-catenin expression in TECs and definitively showed that careful control of beta-catenin within TECs is needed for the proper development of TEC microenvironments critical for T cell development.
Strengths:
- The methods used allowed the authors to effectively targeted TECs while avoiding extrathymic side effects of manipulating beta-catenin in ways that impacted skin or other tissues leading to abortive development or improper separation of the thymus and parathyroid.
- The results showed that beta-catenin GOF specifically in TECs results in thymic dysplasia and loss of thymic T cell development.
- The results from the analysis of beta-catenin LOF indicate that beta-catenin in TECs is not essential for the generation of functional TECs that support T cell development but the loss of beta-catenin in TECs results in the reduction in the number of cTECs, which leads to the reduction in the number of thymocytes during the postnatal period.
- The results demonstrated that GOF of beta-catenin in TECs results in trans-differentiation of TECs into terminally differentiated keratinocytes.
Thank you so much for highlighting the strengths of this manuscript.
Weakness: The fact that beta5T expression is restricted primarily to cTECs suggests that the models used may not accurately capture the impacts of gain of function and loss of function of beta-catenin to mTECs and the maintenance of the medulla in postnatal mice. Given that beta5T-expressing cells have been shown to give rise to both cTECs and mTECs during fetal development the models may more closely demonstrate the importance of fine-tuning beta-catenin expression during fetal development while missing impacts on postnatal mTECs.
Regarding the specificity of B5t expression, it is true that B5t is abundant in cTECs but not detectable in other cells including mTECs. However, all mTECs, including mTECs in postnatal mouse and aged mouse, are derived from bipotent B5t-expressing TEC progenitors, as has been reported previously (Ohigashi et al., Cell Reports, 2015). Therefore, B5t-iCre enables efficient and conditional genetic manipulation for both cTECs and mTECs in a wide range of ontogeny, including postnatal mouse and aged mouse. A conceptually similar gene targeting strategy has been widely employed to study two major T cell lineages of CD4 T cells and CD8 T cells, as CD4-Cre is useful to delete floxed sequences in both CD4 T cells and CD8 T cells. In agreement with a recent study using the same β5t-iCre (Barthlott et al. Nature Communications, 2021), our study demonstrated the highly efficient deletion of Ctnnb1 floxed sequence in both cTECs and mTECs in postnatal mouse. The revised manuscript includes this explanation in the Discussion. Thank you very much for helping us improve the manuscript.
The authors achieved their aims and the results strongly support their conclusions. The work clearly demonstrates the importance of proper regulation of Wnt/beta-catenin signaling in the development and maintenance of TEC microenvironments and should lead to more interest in defining the specific Wnts and Frizzleds that are important in the development and postnatal maintenance of specific TEC subsets. This work will be important in identifying clinical strategies to counteract thymic involution and the subsequent loss of T cell function.
Thank you so much for the supportive comments.
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Reviewer #3 (Public Review):
The authors effectively utilized Beta5T-iCre to specifically manipulate beta-catenin expression in TECs and definitively showed that careful control of beta-catenin within TECs is needed for the proper development of TEC microenvironments critical for T cell development.
Strengths:
The methods used allowed the authors to effectively targeted TECs while avoiding extrathymic side effects of manipulating beta-catenin in ways that impacted skin or other tissues leading to abortive development or improper separation of the thymus and parathyroid.
The results showed that beta-catenin GOF specifically in TECs results in thymic dysplasia and loss of thymic T cell development.
The results from the analysis of beta-catenin LOF indicate that beta-catenin in TECs is not essential for the generation of functional TECs that …
Reviewer #3 (Public Review):
The authors effectively utilized Beta5T-iCre to specifically manipulate beta-catenin expression in TECs and definitively showed that careful control of beta-catenin within TECs is needed for the proper development of TEC microenvironments critical for T cell development.
Strengths:
The methods used allowed the authors to effectively targeted TECs while avoiding extrathymic side effects of manipulating beta-catenin in ways that impacted skin or other tissues leading to abortive development or improper separation of the thymus and parathyroid.
The results showed that beta-catenin GOF specifically in TECs results in thymic dysplasia and loss of thymic T cell development.
The results from the analysis of beta-catenin LOF indicate that beta-catenin in TECs is not essential for the generation of functional TECs that support T cell development but the loss of beta-catenin in TECs results in the reduction in the number of cTECs, which leads to the reduction in the number of thymocytes during the postnatal period.
The results demonstrated that GOF of beta-catenin in TECs results in trans-differentiation of TECs into terminally differentiated keratinocytes.
Weakness:
The fact that beta5T expression is restricted primarily to cTECs suggests that the models used may not accurately capture the impacts of gain of function and loss of function of beta-catenin to mTECs and the maintenance of the medulla in postnatal mice. Given that beta5T-expressing cells have been shown to give rise to both cTECs and mTECs during fetal development the models may more closely demonstrate the importance of fine-tuning beta-catenin expression during fetal development while missing impacts on postnatal mTECs.
The authors achieved their aims and the results strongly support their conclusions.
The work clearly demonstrates the importance of proper regulation of Wnt/beta-catenin signaling in the development and maintenance of TEC microenvironments and should lead to more interest in defining the specific Wnts and Frizzleds that are important in the development and postnatal maintenance of specific TEC subsets. This work will be important in identifying clinical strategies to counteract thymic involution and the subsequent loss of T cell function.
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Reviewer #2 (Public Review):
The authors describe how modulating the levels of beta-catenin in TECs affects thymic organization and thymopoiesis. They use a b5t-Cre to specifically stabilize or ablate beta-catenin in TECs. While stabilization of beta-catenin induces thymic dysplasia and significantly impedes development of thymocytes beyond the DN1 stage, loss of beta-catenin has a milder outcome limited to significantly reduced thymic weight starting and an overall reduction in thymocyte number but no significant effects in thymocytes subset distribution at 2weeks of age. This reduction of thymic weight is associated with a significant and selective reduction in the number of cTECs . On the basis of these findings the authors conclude that fine tuning of beta-catenin levels is essential for postnatal T cell development.
Overall this is …
Reviewer #2 (Public Review):
The authors describe how modulating the levels of beta-catenin in TECs affects thymic organization and thymopoiesis. They use a b5t-Cre to specifically stabilize or ablate beta-catenin in TECs. While stabilization of beta-catenin induces thymic dysplasia and significantly impedes development of thymocytes beyond the DN1 stage, loss of beta-catenin has a milder outcome limited to significantly reduced thymic weight starting and an overall reduction in thymocyte number but no significant effects in thymocytes subset distribution at 2weeks of age. This reduction of thymic weight is associated with a significant and selective reduction in the number of cTECs . On the basis of these findings the authors conclude that fine tuning of beta-catenin levels is essential for postnatal T cell development.
Overall this is an interesting but descriptive study that does not address the physiological and molecular effects of stabilizing or ablating beta-catenin in TECs. The authors suggest that stabilization of beta-catenin function in TECs results in their terminal differentiation to keratinocytes on the basis of increased expression of only two markers involucrin and loricrin, which is a limited definition and further analysis of these cells would be needed for this conclusion. On the other hand the interesting selective effect of loss of beta-catenin function on cTECs versus mTECs has not been analyzed. Are cTECs reduced due to loss of survival/proliferation/differentiation? How is their molecular profile affected by the loss of beta-catenin? Does the selective reduction of the cTEC compartment affect survival/proliferation/differentiation of the individual DN subsets? The paper would benefit from more in depth mechanistic analysis in these directions.
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Reviewer #1 (Public Review):
The work by Fujimori et al. addresses the role of downstream WNT signaling in thymus epithelial cell (TEC) differentiation and function. A TEC-specific beta5t-cre driver allowed for the generation of gain of function (GoF) or loss of function (LoF) mouse models. The specificity of the beta5t-cre driver system was key in allowing the authors to focus on TEC effects. Of note, the LoF of beta-catenin showed a smaller thymus with fewer cortical TEC, but generally no changes in the thymus morphology or in the ability to support normal percentages of thymocyte subsets. These results clearly establish that WNT signaling plays a minor role in TEC differentiation and function. Nevertheless, the GoF approach led to thymus dysplasia with a loss of TEC identity, due to a loss of FOXN1 expression, as well as a failure to …
Reviewer #1 (Public Review):
The work by Fujimori et al. addresses the role of downstream WNT signaling in thymus epithelial cell (TEC) differentiation and function. A TEC-specific beta5t-cre driver allowed for the generation of gain of function (GoF) or loss of function (LoF) mouse models. The specificity of the beta5t-cre driver system was key in allowing the authors to focus on TEC effects. Of note, the LoF of beta-catenin showed a smaller thymus with fewer cortical TEC, but generally no changes in the thymus morphology or in the ability to support normal percentages of thymocyte subsets. These results clearly establish that WNT signaling plays a minor role in TEC differentiation and function. Nevertheless, the GoF approach led to thymus dysplasia with a loss of TEC identity, due to a loss of FOXN1 expression, as well as a failure to support T cell development. These results point to a role for WNT signaling in inducing the TEC differentiation into other non-T-cell-development-supporting epithelial subsets.
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Evaluation Summary:
This paper is of interest to scientists within the field of thymus development and function. Analysis of the data is overall rigorous and conclusions are justified. The work presented builds upon previous studies that have shown that alterations of Wnt/beta-catenin signaling in thymic epithelial cells impact the normal development and or maintenance of thymic epithelial microenvironment critical for the proper development and selection of functional self-tolerant T cell repertoire. The surprise that a thymus epithelial cell specific loss of function of beta-catenin only showed a rather minor phenotype is interesting, but it would be good to also address whether TEC recovery after a challenge, or in aging, is also affected in a minor manner or perhaps more dramatically than the steady-state situation. The author's …
Evaluation Summary:
This paper is of interest to scientists within the field of thymus development and function. Analysis of the data is overall rigorous and conclusions are justified. The work presented builds upon previous studies that have shown that alterations of Wnt/beta-catenin signaling in thymic epithelial cells impact the normal development and or maintenance of thymic epithelial microenvironment critical for the proper development and selection of functional self-tolerant T cell repertoire. The surprise that a thymus epithelial cell specific loss of function of beta-catenin only showed a rather minor phenotype is interesting, but it would be good to also address whether TEC recovery after a challenge, or in aging, is also affected in a minor manner or perhaps more dramatically than the steady-state situation. The author's claims are well supported by the data presented and will be of great interest to scientists and clinicians interested in understanding the signaling pathways important in thymic maintenance, as well as the development of strategies to counteract thymic involution in the aging population and cancer patients.
(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 #3 agreed to share their names with the authors.)
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