Author response: Adult mouse fibroblasts retain organ-specific transcriptomic identity
Year: 2022
Type: peer-review
Abstract: Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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. Editor's evaluation The authors aim to show that fibroblasts have a heterogenous transcriptome that is retained throughout their lifetime due to their source of embryonic origin. 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. 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 advance. https://doi.org/10.7554/eLife.71008.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Fibroproliferative disorders are the main cause of mortality and morbidity in developed countries, accounting for about 45% of deaths in the United States (Bitterman and Henke, 1991). Despite the impactful prevalence of chronic organ fibrosis, current antifibrotic drugs are both inefficient and nonspecific to this condition (Friedman et al., 2013; Henderson et al., 2020). Fibroblasts, main players in fibrosis, have gained increased attention for their capacity to perform functions far beyond their canonical secretion of extracellular biological scaffolding and formation of scar tissue after injury. Recent literature poses the organ fibroblast as a major regulatory hub that senses local microenvironment imbalances and controls tissue remodeling (Gerber et al., 2018) upon activation and phenotypic differentiation into the profibrotic myofibroblast (Pakshir et al., 2020). They are also involved in immunomodulation (Van Linthout et al., 2014), by producing and responding to cytokines that activate immune cells of the innate and adaptive immune systems (Forte et al., 2018; Boyd et al., 2020), through organ-specific regulatory networks (Krausgruber et al., 2020). Organ fibroblasts have been historically difficult to identify and study in vivo, due to their vague functional definition and lack of adequate markers that label organ fibroblast pools completely and specifically (Swonger et al., 2016). Recent advances in lineage tracing and multiomics single-cell analyses have revealed a significant heterogeneity of fibroblasts within and among tissues, and we are just beginning to understand how fibroblast heterogeneity correlates with distinct functions (Henderson et al., 2020; Shaw and Rognoni, 2020; Lynch and Watt, 2018; LeBleu and Neilson, 2020; Griffin et al., 2020). Despite being morphologically similar, spindle-shaped mesenchymal cells located in stromal tissues, fibroblasts acquire specialized functions related to their anatomical position (Krausgruber et al., 2020; Slany et al., 2014; Foote et al., 2019). They appear to retain a positional memory of the embryonic developmental axis: anterior–posterior, proximal–distal, and dermal–nondermal, possibly reflecting their role in conveying positional identity in embryogenesis (Rinn et al., 2006; Rinn et al., 2008; Ackema and Charité, 2008; Chang et al., 2002). The retention of positional molecular information through to adulthood suggests organ fibroblasts respond to molecular cues that drive body compartmentalization. Fibroblast heterogeneity within an organ tends to arise from the distinct embryological origin and/or anatomical localization (Lynch and Watt, 2018; Griffin et al., 2020; Muhl et al., 2020; Forte et al., 2020), while interorgan differences have been mostly ascribed to the matrisome, as shown by the transcriptomic comparison among fibroblasts from muscular tissues (Muhl et al., 2020). Having previously reported that fibroblasts isolated from the adult mouse heart retain a cardiogenic transcriptional program (Furtado et al., 2014a), we show here that fibroblasts isolated from different adult organs similarly retain the expression of transcription factors and other gene sets involved in the determination of organ formation and patterning during embryonic development. This signature is captured in nascent embryonic organ fibroblasts and retained in adult fibroblasts under culture in isolation or in coculture with parenchymal cells from different organs. Further ectopic transplantation of fibroblasts into a different organ in vivo demonstrates the strength of the organ molecular signature despite new microenvironmental challenges. The robustness of the fibroblast organ transcriptome signature shown here supports its importance for organ interaction, connectivity, and function. In addition, knockdown of selected organ development transcription factors in cardiac fibroblasts deregulated the expression of genes involved in inflammation, fibrosis, and extracellular matrix (ECM) deposition, further supporting the relevance of these genes in fibroblasts function. In summary, our study uncovers stable expression of organ-specific, development-related signature genes in adult fibroblasts, thus offering new prospects for possible antifibrotic therapies. Results Cell homeostasis and ECM components comprise a generic fibroblast gene signature To compare the gene signature of fibroblasts from different organs and eliminate potential RNA contaminants from other organ cell types, dissociated adult murine tissues were cultured for 5 days, followed by sorting for CD45−CD31−CD90+ fibroblasts (Furtado et al., 2014a; Figure 1—figure supplement 1a). High-throughput gene expression profiling identified 1281 highly expressed genes common to all fibroblast types, comprising the generic fibroblast signature (Figure 1—source data 1). Through Ingenuity Pathway Analysis (IPA, Qiagen), we classified the commonly expressed genes based on cellular function (Figure 1—figure supplement 1b) and cellular localization (Figure 1—figure supplement 1c). Top functions included mechanisms of cell maintenance, such as proliferation, cytoskeletal arrangement, and cell movement, as well as general metabolic processes, including carbohydrate, nucleic acid protein, and small molecule biochemistry. Common fibroblast identifier genes were encountered within various IPA process classification groups. As an example, the cell surface receptor CD90 (Thy1 gene) belongs with cellular assembly and organization, growth and proliferation and protein synthesis, while the myofibroblast marker smooth muscle actin (Acta2 gene) was found in functions of cellular movement. The presence of CD90 and absence of CD31 (Pecam1 gene)/CD45 (Ptprc gene) in all organ groups validated our positive/negative selection strategy for cell isolation, indicating a generally consistent population of cells in all organs. ECM elements, including collagens, were included in several functional annotations, such as to cell morphology, assembly and organization, cellular compromise, function, and maintenance or cell signaling. Organ fibroblasts retain Hox codes The Hox code defines body segmental identity and is highly conserved from flies to mammals. Hox genes show colinear expression and undergo chronological activation in the embryo, where upstream genes successively activate downstream genes in an anteroposterior fashion, such that upstream genes are activated first in more anterior segments of the body. In mammals, the Hox cluster has undergone a series of duplications and deletions that led to the formation of four paralogous clusters a, b, c, and d (Figure 1a). Figure 1 with 2 supplements see all Download asset Open asset Positional code of organ fibroblasts. (a) Murine Hox cluster code, showing proximally expressed Hox genes in pink and distal ones in purple. (b) Bar plots showing the average raw signal for all expressed Hox genes in organ-specific fibroblast samples. Data are mean ± standard error of the mean (SEM) of three biological replicates for each fibroblast type from the microarray analysis. Refer to Figure 1—figure supplements 1–2, Figure 1—source data 1, and Figure 1—source data 2. Figure 1—source data 1 Microarray data: highly expressed genes common to all organ fibroblast populations, classified based on cellular process or cellular localization. https://cdn.elifesciences.org/articles/71008/elife-71008-fig1-data1-v2.xlsx Download elife-71008-fig1-data1-v2.xlsx Figure 1—source data 2 Microarray data: average raw expression and standard errors of Hox code genes across all fibroblast samples from the microarray analysis (n = 3). https://cdn.elifesciences.org/articles/71008/elife-71008-fig1-data2-v2.xlsx Download elife-71008-fig1-data2-v2.xlsx Site-specific HOX expression has been previously reported in human skin fibroblasts (Rinn et al., 2006; Rinn et al., 2008; Chang et al., 2002) and mouse mesenchymal cells isolated from different organs (Ackema and Charité, 2008), and it has been shown to be cell autonomous and epigenetically maintained, suggesting a source of positional memory to differentially pattern tissue-specific homeostasis and regeneration. To determine if fibroblasts isolated from other adult mouse organs retain a distinct Hox signature, we plotted the average raw expression of all Hox genes per each fibroblast type (Figure 1b, Figure 1—figure supplement 2, Figure 1—source data 2). Among profiled organs, five patterns of Hox expression were identified: lung and liver showed expression of anterior Hox genes, in particular genes from the clusters 1–7, although liver had lower Hox4–7 expression when compared with lung. A second group including skin and gonad displayed high Hox6 expression, with skin from thoracic and abdominal ventral skin areas also expressing high Hox9 gene levels. The third classification group was represented by the heart, characterized by low Hox gene expression. This may reflect embryonic developmental processes, as Hox genes are known to exert minimal influence on heart formation, and are generally not expressed in the heart, except for the residual expression carried over by neural crest cells that invade the arterial pole of the heart and promote aorticopulmonary septation (Kirby et al., 1983). The great vessels were excluded from our sample collection, and therefore cells of neural crest origin were likely not captured in the analyses. The fourth classification group was represented by the kidney, which expressed intermediate to high levels of most anterior Hox genes up to Hox11, consistent with previous observations for the developing kidney (Patterson and Potter, 2004). The fifth category, represented by the mouse tail, had a posterior Hox code signature, represented by Hox13, which correlates with previous findings for human distal segment fibroblasts, represented by feet skin fibroblasts (Rinn et al., 2006; Rinn et al., 2008). Taken together with previous observations, these analyses confirm that adult organ fibroblasts retain positional Hox gene expression signatures, generally reflecting the embryological segmental identity of organ fibroblasts. Organ fibroblasts show unique molecular signatures To highlight the unique transcriptomic signatures of these positionally distinct fibroblast pools, we performed a differential expression analysis and considered genes that were enriched by tenfold change or more in single organ fibroblasts relative to tail fibroblasts (Figure 2, Figure 2—source data 1). Gene ontology (GO) annotation revealed organ development programs; processes such as epithelial development, hepatoblast differentiation, lung lobe development, kidney development, reproductive process, and heart development were found enriched in their respective organ fibroblast pools (Figure 2a). Strikingly, signature embryonic transcription factors, that is, genes with established involvement in organ development, were enriched in organ-specific subsets, including Tbx20, a crucial transcription factor for heart development previously described in cardiac fibroblasts (Furtado et al., 2014a). Likewise, genes essential for lung morphogenesis (Foxf1) (Costa et al., 2001), liver development (Hhex) (Keng et al., 2000), early kidney formation (Pax8) (Bouchard et al., 2002), and gonad development (Lhx9) (Birk et al., 2000) were all specifically enriched in fibroblasts from their respective organs. Expression of signature genes was validated by quantitative polymerase chain reaction (qPCR) (Figure 2b, Figure 2—figure supplement 1) and immunocytochemistry (Figure 3, Figure 3—figure supplement 1). In general, signature gene expression patterns in embryonic fibroblasts were retained in fibroblasts from adult tissues (Krt4, Krt6a, Serpinb5, and Hp for skin; Tbx20 and Col2a1 for heart; Foxf1 for lung; Hhex and Foxa2 for liver, Bmp7 and Pax8 for kidney, Cyp11a1 and Lbx9 for gonad). Significant expression of Hhex and Bmp7 were also found in several organs during embryonic development but were restricted to a single organ in adulthood. As an exception to single organ enrichment, Foxa2 was also substantially upregulated in lung fibroblasts (~20-fold), in addition to liver fibroblasts (~30-fold). Figure 2 with 7 supplements see all Download asset Open asset Embryological molecular signature of organ fibroblasts. (a) Cytoscape representation of network of genes (dots) singularly expressed in organ fibroblasts (only one gray edge between the gene and the organ) or shared among organs (multiple gray edges linking the gene to several organs). Genes involved in organ development are highlighted. (b, c) Validation of the expression of selected organ-enriched, developmental related genes using qPCR on cultured organ-derived fibroblasts isolated from adult mice (top row) or E16.5 embryos (bottom row). Data are mean ± standard error of the mean (SEM) of the e−DDCt values, on three technical replicates of merged biological samples. The housekeeping gene is Hprt and the reference sample is tail fibroblasts. Refer to Figure 2—figure supplements 1–7, Figure 2—source data 1. Figure 2—source data 1 Microarray data: expression of genes that were enriched by tenfold change or more in single organ fibroblasts compared to tail fibroblasts (n = 3). https://cdn.elifesciences.org/articles/71008/elife-71008-fig2-data1-v2.xlsx Download elife-71008-fig2-data1-v2.xlsx Figure 3 with 1 supplement see all Download asset Open asset Adult fibroblasts express organ-specific transcription factors. Immunocytochemistry for development-related organ-enriched markers (KRT14, TBX20, FOXA2, HHEX, FOXD1, PAX8, and RSPO) on adult fibroblasts obtained from different organs of Col1a1-GFP mice and cultured for 5 days: (a) Skin fibroblasts stained with a mouse monoclonal anti-KRT14 antibody; (b) heart fibroblasts stained with a mouse monoclonal anti-TBX20 antibody; (c) lung fibroblasts stained with a rabbit monoclonal anti-FOXA2 antibody; (d) liver fibroblasts stained with a rabbit monoclonal anti-HHEX antibody; (e) kidney fibroblasts stained with a rabbit polyclonal anti-FOXD1 antibody; (f) kidney fibroblasts stained with a mouse monoclonal anti-PAX8 antibody; (g) testis fibroblasts stained with a goat polyclonal anti-RSPO1 antibody. As secondary antibodies, goat anti-mouse Alexa Fluor 568 was used for (a, b), (f); goat anti-rabbit Alexa Fluor 555 for (c, d) and donkey anti-goat Alexa Fluor 568 for (f). Representative images from three independent experiments on three biological replicates. Scale bar = 100 µm. Refer to Figure 3—figure supplement 1. IPA analysis delineated top canonical pathways, diseases, functions, and networks associated with selectively enriched genes in each fibroblast population and supported the argument that fibroblasts retain molecular identity of their developmental organ of origin (Figure 2—figure supplements 2–7). Among organ-related processes enriched in fibroblast subsets were dermatological diseases and conditions and morphogenesis of the epithelial tissue for skin fibroblasts (Figure 2—figure supplement 2), respiratory system development for lung fibroblasts (Figure 2—figure supplement 3), liver development for liver fibroblasts (Figure 2—figure supplement 4), acute renal failure, metanephros development and kidney formation, and abnormal kidney development, disease and function for kidney fibroblasts (Figure 2—figure supplement 5), reproductive system development, function and disease, morphology of genital organs, and primary sex determination networks and reproductive system dysfunction for gonad fibroblasts (Figure 2—figure supplement 6), cardiovascular disease development and function, cardiac enlargement and disease, and cardiac developmental processes for heart fibroblasts (Figure 2—figure supplement 7). To establish the translational relevance of our findings, the presence of 42 genes uniquely enriched in cardiac fibroblasts (log2, tenfold, False Discovery Rate (FDR) < 0.01) was determined in left ventricular heart biopsies from healthy (N = 5) and chronic ischemic heart failure patients (N = 5) (Figure 2—figure supplement 7—source data 1). Ischemic heart failure was chosen for analysis due to the likelihood of replacement fibrosis as a pathological signature. Out of the 42 murine cardiac fibroblast genes, 28 were present in both control and heart failure samples, including Tbx20, which was unchanged between control and heart failure. FNDC1, FRZB, MFAP4, and OLFML1 were significantly upregulated in ischemic heart failure; while MFSD2A, PNP, and SERPINA3N were downregulated. Four genes had no human homolog and ten were not found in the human heart transcriptome dataset. These findings confirm general commonalities across species and further identify potential candidates of fibrotic cardiovascular disease interest for future investigation. Organ-enriched gene expression is retained at the single-cell level in freshly isolated and cultured fibroblasts Single-cell RNAseq (scRNAseq) is a powerful tool to determine granularity of gene expression at the population level. To assess how organ signatures are reflected in freshly isolated fibroblasts, we reanalyzed the stromal cell dataset from a publicly available multiorgan scRNAseq study (the Mouse Cell Atlas) (Han et al., 2018). Focusing on lung, testis, kidney, liver, and neonatal heart cells, we unbiasedly identified eight populations, including three lung and two kidney subclusters (Figure 4a). Pairwise differential expression analysis supported a previously reported classification of lung fibroblasts populations (Xie et al., 2018), with two types of matrix fibroblasts – 'Lung A' (Col14a1, Pi16, Dcn enriched), 'LungB' (Col13a1, Cxcl14, Tcf21 enriched) – and a group of myofibroblasts – 'LungC' (Acta2, Myl9, Tagln positive) (Figure 4—figure supplement 1 a, b). For kidney clusters, 'Kidney B' showed higher levels of canonical fibroblast markers Dcn, Gsn, and Col1a2, while the larger 'Kidney A' expressed relatively higher levels of genes involved in response to injury, or in renal carcinoma metastasis and progression (Spp1 and Krt8), suggesting that this cluster is composed of tubular cells acquiring a mesenchymal phenotype in vivo (Rudman-Melnick et al., 2020; Figure 4—figure supplement 1c, d), as kidney epithelial cells are known to undergo dedifferentiation in vivo and in vitro to repair tubular injuries (Kusaba et al., 2014; Van der Hauwaert et al., 2013). Figure 4 with 2 supplements see all Download asset Open asset Analysis of organ-specific signatures at the single-cell level. (a–c) Reanalysis of the Mouse Cell Atlas stromal cell dataset. (a) Uniform Manifold Approximation and Projection (UMAP) visualization of selected stromal cell populations (682 cells). (b) the expression of genes, identified from the RNAseq on cells in the single-cell RNAseq (scRNAseq) dataset. (c) showing the average expression per population of organ development-related genes as shown in Figure 3). scRNAseq analysis of cultured stromal cells of different origins. (d) visualization of the captured cells. (e) the expression of genes, identified from the RNAseq on cells. (c) showing the average expression per population of organ development-related genes as shown in Figure 3). Refer to Figure 4—figure supplements 1–2, Figure data 1, and Figure data 2. Figure data 1 Analysis of the stromal cell from the Mouse Cell markers genes per each and markers identified by comparison of the two kidney and three lung Download Figure data 2 Analysis of single-cell RNAseq (scRNAseq) data of merged cultured fibroblasts from different markers genes per each and markers identified by comparison of the two kidney and two cardiac Download the expression of organ-specific (Figure and developmental related genes (Figure previously identified in the cultured cell analysis was despite the and heterogeneity at the single-cell level (Figure when subclusters were present in lung and kidney the expression of the organ-specific genes was enriched in myofibroblasts expressed relatively higher levels of or activated fibroblasts A expressed higher levels of Pax8 and (Figure Figure 4—figure supplement 1, Figure data 1). Further of the organ-enriched program was obtained with scRNAseq of primary cultures from different liver, lung, heart, skin, testis, and (Figure 1–2, 1–2, and fibroblasts. clusters were based on organ although by the expression of genes and proliferation genes (Figure Figure data 2). expressed (Figure and development-related genes (Figure from original analysis were in these organ cultured kidney clusters expressed the epithelial response marker and were to freshly isolated 'Kidney A' (Figure 4—figure supplement possibly two of tubular cells et al., had higher expression of myofibroblast genes Myl9, and and the gene 2 expressed epithelial genes and (Figure 4—figure supplement Figure data 2). As for the cultured heart fibroblasts, 1 displayed myofibroblast genes (Acta2, and and 2 had signature of activated fibroblasts and (Figure 4—figure supplement Figure data Forte et al., 2020). scRNAseq experiments showed that cultured cells present an phenotype compared to freshly isolated cells and the retention of an organ-specific transcriptome identity on both cultured and freshly isolated cells. Organ-enriched transcriptome is involved in the fibrotic response To the functional relevance of organ-enriched fibroblast a knockdown was used to organ transcription factors, the heart as a (Figure et al., adult cardiac fibroblasts were with and for determination of and knockdown (Figure of was in of cells (Figure by and expression was by over when compared with (Figure was also by (Figure et al., Figure 5 Download asset Open asset In vitro knockdown of cardiac transcription factors. (a) of adult cardiac fibroblasts from and with two for and (b) showing the expression on in of cells of three independent (c) qPCR of by expression, in cells with or plots showing genes differentially expressed in and Tbx20 change in expression of and Tbx20 in cells with through RNA (f) showing the among genes by or Tbx20 knockdown and of genes upregulated by tenfold or more in heart fibroblasts compared to other organs. (g) showing in expression of genes by or Tbx20 Genes by both and Tbx20 in manner. analyses. Top genes in both Tbx20 and genes by genes by Tbx20 – in – upregulated pathways, and – genes in processes known to the cardiac fibrotic response for or Tbx20 data are represented as over control ± standard error of the mean from RNAseq of three biological replicates per significant genes have an < Refer to Figure data 1. Figure data 1 of the differentially expressed genes between and and analysis. Download this and Tbx20, transcription factors essential for heart formation in embryonic development et al., were in cultured cardiac fibroblasts, followed by RNAseq analysis (Figure is expressed by all organ fibroblasts while fibroblast Tbx20 expression is restricted to the Despite for both Figure a higher of genes on compared with Tbx20 Among genes upregulated by tenfold in the original organ analysis Figure were by and five by Tbx20 two of these genes were in both including Tbx20, suggesting as a possible upstream of A of genes showed between and Tbx20 (Figure the of These included cytokines and and genes involved in and cell and and Of among the genes selectively upregulated in is a key of organ fibrosis, possibly downstream of et al., Among genes upregulated in Tbx20 the downstream of is involved in and is to
Source: eLife
Language: English
Subfield: Hepatology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $2,500
OpenAlex ID: W4220780871