Adult mouse fibroblasts retain organ-specific transcriptomic identity
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Evaluation Summary:
This paper is of general interest to a broad audience of scientists working in regenerative medicine, tissue engineering, disease modelling, and stem cell fields. It reveals organ fibroblast heterogeneity and shows that the organ-specific identity is preserved in vitro and during ectopic transplantation in vivo. The right choice of fibroblasts might therefore be critical in the fields mentioned above.
(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
Organ fibroblasts are essential components of homeostatic and diseased tissues. They participate in sculpting the extracellular matrix, sensing the microenvironment, and communicating with other resident cells. Recent studies have revealed transcriptomic heterogeneity among fibroblasts within and between organs. To dissect the basis of interorgan heterogeneity, we compare the gene expression of murine fibroblasts from different tissues (tail, skin, lung, liver, heart, kidney, and gonads) and show that they display distinct positional and organ-specific transcriptome signatures that reflect their embryonic origins. We demonstrate that expression of genes typically attributed to the surrounding parenchyma by fibroblasts is established in embryonic development and largely maintained in culture, bioengineered tissues and ectopic transplants. Targeted knockdown of key organ-specific transcription factors affects fibroblast functions, in particular genes involved in the modulation of fibrosis and inflammation. In conclusion, our data reveal that adult fibroblasts maintain an embryonic gene expression signature inherited from their organ of origin, thereby increasing our understanding of adult fibroblast heterogeneity. The knowledge of this tissue-specific gene signature may assist in targeting fibrotic diseases in a more precise, organ-specific manner.
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Evaluation Summary:
This paper is of general interest to a broad audience of scientists working in regenerative medicine, tissue engineering, disease modelling, and stem cell fields. It reveals organ fibroblast heterogeneity and shows that the organ-specific identity is preserved in vitro and during ectopic transplantation in vivo. The right choice of fibroblasts might therefore be critical in the fields mentioned above.
(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):
A summary of what the authors were trying to achieve:
The authors aim to show that fibroblasts have a heterogenous transcriptome that is retained throughout their lifetime due to their source of embryonic origin. They have previously shown that there is transcriptomic lineage retained in cardiomyocytes and are attempting to show this across many organ types.- An account of the major strengths and weaknesses of the methods and results:
1. Major strengths,
a. Figure 7 transplant data were strong.
b. The authors have provided extensive data.
c. Functional confirmation in cardiac lineage was very convincing.2. Weaknesses
a. Although the Hox code hypothesis was mentioned, the manuscript did not closely follow the hypothesis. Many analyses were superficial, for example, IPA was used and many genes and pathways …Reviewer #1 (Public Review):
A summary of what the authors were trying to achieve:
The authors aim to show that fibroblasts have a heterogenous transcriptome that is retained throughout their lifetime due to their source of embryonic origin. They have previously shown that there is transcriptomic lineage retained in cardiomyocytes and are attempting to show this across many organ types.- An account of the major strengths and weaknesses of the methods and results:
1. Major strengths,
a. Figure 7 transplant data were strong.
b. The authors have provided extensive data.
c. Functional confirmation in cardiac lineage was very convincing.2. Weaknesses
a. Although the Hox code hypothesis was mentioned, the manuscript did not closely follow the hypothesis. Many analyses were superficial, for example, IPA was used and many genes and pathways were listed.
b. A weakness in the initial gene analysis is there are so many genes and pathways mentioned and used (Figure 2a) that it is difficult to determine why the genes in Figure 2 (b-g) were the ones chosen to be validated. The qPCR validation seems to support the hypothesis that these genes have organ specific expressions but their from the initial analysis is unclear. It would help to simplify the schematic for Figure 2a and highlight the specific genes that are being validated. This is further compounded by the figure 3 data, which shows mixed results (PAX8 does not really seem to be expressed in the kidney and FOXD1 seems to have an odd pattern of expression; FOXA2 seems to be expressed in some nuclei and not others of the lung) and non-direct comparison between multiple organs.
c. One of the flaws is the fibroblast signatures generated with CD90 sorted fibroblasts. CD90 (Thy1) is expressed by a small fraction of fibroblasts in many organs, even from the Ref30, the main source of single cell data. Therefore, the generic signature was CD90+ fibroblast gene signature, not fibroblast signature.
d. The clusters of the scRNA-seq from both freshly isolated and cultured fibroblasts seem to be due to the batch effects, as it is not very possible that not a single overlapped cell was identified. The listed organ specific genes in heatmaps were hand-picked? as they are identical.
e. Immunocytochemistry validation should also include the staining on the negative fibroblasts to confirm the "organ specific markers" in Figure 3.
f. Poor presentation. Many figure panels were not described in the Results. These could have been either removed or better organized.- An appraisal of whether the authors achieved their aims, and whether the results support their conclusions.
The skin, tail, and heart fibroblasts seem to have distinct patterns of gene expression that correspond to the reported Hox gene expression identified in development. As a paper that mainly addresses cardiac lineage, it is effective. Their profiling of other organs is less convincing, and their emphasis on kidney transcription seems suspect. However, their attempt to begin to establish an organ specific lineage for fibroblasts is an important step in fibroblast development and cell biology.- Any additional context you think would help readers interpret or understand the significance of the work.
The text on many of the figures is difficult to read. It becomes difficult to follow the cardiac lineage story with the congested figures (5 & 7). Figure 2 could use clarification or the text should explain more explicitly where the genes of interest came from. -
Reviewer #2 (Public Review):
In this paper, the authors performed a thorough analysis of fibroblasts isolated from different mouse tissues. They demonstrate that fibroblasts display tissue-specific gene expression signatures and functions. They further show that the source of fibroblasts affects the functionality of three-dimensional (3D) cardiac microtissues. Interestingly, upon ectopic transplantation under the kidney capsule, fibroblasts retain their tissue-specific signature. However, the kidney microenvironment did drive specific adaptations, such as a change in the HOX genes expression and changes in the expression of genes associated with the adaptation to the new microenvironment.
Strengths:
1. This study compares fibroblasts isolated from several different tissues and identifies common and differentially expressed genes. The …
Reviewer #2 (Public Review):
In this paper, the authors performed a thorough analysis of fibroblasts isolated from different mouse tissues. They demonstrate that fibroblasts display tissue-specific gene expression signatures and functions. They further show that the source of fibroblasts affects the functionality of three-dimensional (3D) cardiac microtissues. Interestingly, upon ectopic transplantation under the kidney capsule, fibroblasts retain their tissue-specific signature. However, the kidney microenvironment did drive specific adaptations, such as a change in the HOX genes expression and changes in the expression of genes associated with the adaptation to the new microenvironment.
Strengths:
1. This study compares fibroblasts isolated from several different tissues and identifies common and differentially expressed genes. The authors demonstrate that fibroblasts isolated from different organs show differential expression of HOX and organ parenchyma genes. Importantly the expression of these genes is preserved after isolation and culture, as the analysis is performed on fibroblasts culture for 5 days.
2. In addition to the tissue-specific signature, the authors also demonstrate that fibroblasts isolated from different tissue might differ in their functionality after comparing 3D cardiac microtissues that are formed wither with heart or kidney fibroblasts. This experiment found that kidney fibroblasts failed to homogeneously distribute in cardiac tissues and interconnect with cardiomyocytes.
3. Ectopic transplantation under the kidney capsule showed that fibroblasts isolated from the heart, tail and kidney retained their tissue-specific identity. However, the authors also observed adaptation of the fibroblasts gene expression signature to a new microenvironment and changes in the expression of HOX genes.
Weaknesses:
1. The present study utilized cultured fibroblasts, and it would have been more informative to look at freshly isolated fibroblasts from the same study. The authors mention that cultured fibroblasts present an activated/myofibroblast-like phenotype. However, it is hard to dissect those without a direct side-by-side comparison.
2. The authors compare their dataset to the previously published single-cell RNA-seq mouse dataset. However, cross-comparison with human datasets is lacking, and it would have been interesting to have more insights into humans.
3. Both in vitro and in vivo experiments are done only after three days, mainly comparing heart vs kidney fibroblasts (3D cardiac microtissues) or tail, heart vs kidney (ectopic transplantation). It would have been helpful to have these comparisons done across the entire range of fibroblasts and also looked at the effect of a longer co-culture/transplantation.
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Reviewer #3 (Public Review):
Forte et al. show a clear organ-specific functionalization of fibroblasts, based on transcriptomics. Of great interest is that compelling evidence is provided that these transcriptional signatures have direct translational consequences. This is shown through coculture experiments, where coculture of cardiomyocytes with non-cardiac fibroblasts impairs integration and contractility, while cardiac fibroblasts integrate with cardiomyocyte cultures to create functional beating tissue.
This memory is shown to be malleable: three days post implantation in the renal capsule, explanted fibroblasts largely maintained their original transcriptomic signature, while also showing the onset of adaptation to a new microenvironment. Longer implantation times will be necessary to determine to which extent the core …
Reviewer #3 (Public Review):
Forte et al. show a clear organ-specific functionalization of fibroblasts, based on transcriptomics. Of great interest is that compelling evidence is provided that these transcriptional signatures have direct translational consequences. This is shown through coculture experiments, where coculture of cardiomyocytes with non-cardiac fibroblasts impairs integration and contractility, while cardiac fibroblasts integrate with cardiomyocyte cultures to create functional beating tissue.
This memory is shown to be malleable: three days post implantation in the renal capsule, explanted fibroblasts largely maintained their original transcriptomic signature, while also showing the onset of adaptation to a new microenvironment. Longer implantation times will be necessary to determine to which extent the core organ-specific signature is preserved after prolonged exposure to ectopic environments.
In addition, markers are identified which allow the separation of fibroblasts based on their anatomical origin. Considering the lack of tissue-specific markers for fibroblasts, this is a significant advancement.
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