Lineage tracing analysis defines erythropoietin-producing cells as a distinct subpopulation of resident fibroblasts with unique behaviors

Abstract

Erythropoietin (Epo) is produced by a subpopulation of resident fibroblasts in the healthy kidney. We have previously demonstrated that, during kidney fibrosis, kidney fibroblasts including Epo-producing cells transdifferentiate into myofibroblasts and lose their Epo-producing ability. However, it remains unclear whether Epo-producing cells survive and transform into myofibroblasts during fibrosis because previous studies did not specifically label Epo-producing cells in pathophysiological conditions. Here, we generated EpoCreERT2/+ mice, a novel mouse strain that enables labeling of Epo-producing cells at desired time points and examined the behaviors of Epo-producing cells under pathophysiological conditions. Lineage-labeled cells that were producing Epo when labeled were found to be a small subpopulation of fibroblasts located in the interstitium of the kidney, and their number increased during phlebotomy-induced anemia. Around half of lineage-labeled cells expressed Epo mRNA, and this percentage was maintained even 16 weeks after recombination, supporting the idea that a distinct subpopulation of cells with Epo-producing ability makes Epo repeatedly. During fibrosis caused by ureteral obstruction, EpoCreERT2/+-labeled cells were found to transdifferentiate into myofibroblasts with concomitant loss of Epo-producing ability, and their numbers and the proportion among resident fibroblasts increased during fibrosis, indicating their high proliferative capacity. Finally, we confirmed that EpoCreERT2/+-labeled cells that lost their Epo-producing ability during fibrosis regained their ability after kidney repair due to relief of the ureteral obstruction. Thus, our analyses have revealed previously unappreciated characteristic behaviors of Epo-producing cells, which had not been clearly distinguished from those of resident fibroblasts. Erythropoietin (Epo) is produced by a subpopulation of resident fibroblasts in the healthy kidney. We have previously demonstrated that, during kidney fibrosis, kidney fibroblasts including Epo-producing cells transdifferentiate into myofibroblasts and lose their Epo-producing ability. However, it remains unclear whether Epo-producing cells survive and transform into myofibroblasts during fibrosis because previous studies did not specifically label Epo-producing cells in pathophysiological conditions. Here, we generated EpoCreERT2/+ mice, a novel mouse strain that enables labeling of Epo-producing cells at desired time points and examined the behaviors of Epo-producing cells under pathophysiological conditions. Lineage-labeled cells that were producing Epo when labeled were found to be a small subpopulation of fibroblasts located in the interstitium of the kidney, and their number increased during phlebotomy-induced anemia. Around half of lineage-labeled cells expressed Epo mRNA, and this percentage was maintained even 16 weeks after recombination, supporting the idea that a distinct subpopulation of cells with Epo-producing ability makes Epo repeatedly. During fibrosis caused by ureteral obstruction, EpoCreERT2/+-labeled cells were found to transdifferentiate into myofibroblasts with concomitant loss of Epo-producing ability, and their numbers and the proportion among resident fibroblasts increased during fibrosis, indicating their high proliferative capacity. Finally, we confirmed that EpoCreERT2/+-labeled cells that lost their Epo-producing ability during fibrosis regained their ability after kidney repair due to relief of the ureteral obstruction. Thus, our analyses have revealed previously unappreciated characteristic behaviors of Epo-producing cells, which had not been clearly distinguished from those of resident fibroblasts. Translational StatementAlthough we and others showed previously that kidney fibroblasts, including erythropoietin (Epo)-producing cells, transdifferentiate into myofibroblasts in kidney diseases, the behavior of Epo-producing cells remains unclear, because previous studies do not specifically label Epo-producing cells. Here, we generated EpoCreERT2/+ mice to label Epo-producing cells at desired time points and thereby revealed the unique behaviors of Epo-producing cells, such as their sustained Epo-producing ability in healthy kidneys, their loss of Epo-producing ability and rapid proliferation during fibrosis, and their reacquisition of Epo-producing ability after kidney repair. Further analysis of this subpopulation will provide insights that may yield new therapeutic approaches to renal anemia. The hormone erythropoietin (Epo) is essential for erythropoiesis.1Wojchowski D.M. Sathyanarayana P. Dev A. Erythropoietin receptor response circuits.Curr Opin Hematol. 2010; 17: 169-176PubMed Google Scholar In adults, Epo is produced mainly by resident fibroblasts in the kidney and is regulated at transcriptional levels through a hypoxia-inducible factor–dependent mechanism under physiological conditions.2Haase V.H. Hypoxic regulation of erythropoiesis and iron metabolism.Am J Physiol Renal Physiol. 2010; 299: F1-F13Crossref PubMed Scopus (222) Google Scholar, 3Koury M.J. Haase V.H. Anaemia in kidney disease: harnessing hypoxia responses for therapy.Nat Rev Nephrol. 2015; 11: 394-410Crossref PubMed Scopus (179) Google Scholar, 4Obara N. Suzuki N. Kim K. et al.Repression via the GATA box is essential for tissue-specific erythropoietin gene expression.Blood. 2008; 111: 5223-5232Crossref PubMed Scopus (166) Google Scholar, 5Sato Y. Yanagita M. Renal anemia: from incurable to curable.Am J Physiol Renal Physiol. 2013; 305: F1239-F1248Crossref PubMed Scopus (35) Google Scholar Epo-producing cells are distributed mainly in the deep cortex and outer medulla,4Obara N. Suzuki N. Kim K. et al.Repression via the GATA box is essential for tissue-specific erythropoietin gene expression.Blood. 2008; 111: 5223-5232Crossref PubMed Scopus (166) Google Scholar,6Suzuki N. Obara N. Yamamoto M. Use of gene-manipulated mice in the study of erythropoietin gene expression.Methods Enzymol. 2007; 435: 157-177Crossref PubMed Scopus (28) Google Scholar the regions that are physiologically hypoxic and sensitive to subtle changes in oxygen delivery.7Brezis M. Heyman S.N. Dinour D. et al.Role of nitric oxide in renal medullary oxygenation. Studies in isolated and intact rat kidneys.J Clin Invest. 1991; 88: 390-395Crossref PubMed Scopus (234) Google Scholar Under physiological conditions, Epo-producing cells are a small subpopulation of resident fibroblasts detectable around the corticomedullary junctions, whereas under hypoxic conditions, they become detectable in a broader area of the cortex.8Kurtz A. Endocrine functions of the renal interstitium.Pflugers Arch. 2017; 469: 869-876Crossref PubMed Scopus (20) Google Scholar,9Suzuki N. Yamamoto M. Roles of renal erythropoietin-producing (REP) cells in the maintenance of systemic oxygen homeostasis.Pflugers Arch. 2016; 468: 3-12Crossref PubMed Scopus (41) Google Scholar Although the transcriptional regulation of Epo has been analyzed intensively, a remaining unanswered question is whether Epo-producing cells constitute a distinct and specialized subpopulation of resident fibroblasts, or in contrast, all resident fibroblasts possess the capacity to produce Epo. Yamazaki et al. have demonstrated that most resident fibroblasts in the kidneys of inherited super anemic mice (ISAM), which lack Epo-producing ability in the kidneys and are severely anemic, are lineage-labeled with Epo-Cre.10Yamazaki S. Souma T. Hirano I. et al.A mouse model of adult-onset anaemia due to erythropoietin deficiency.Nat Commun. 2013; 4: 1950Crossref PubMed Scopus (53) Google Scholar Although this finding suggests that all kidney fibroblasts have the potential to produce Epo, the lineage-labeled cells in the study are the cells with a history of Epo expression from the developmental period. Indeed, lineage-tracing studies analyzing the cells currently producing Epo in the adult kidneys are lacking, and the behaviors of Epo-producing cells under pathologic conditions also remain unknown. In our previous study, we demonstrated that resident fibroblasts including Epo-producing cells are lineage-labeled with myelin protein zero Cre (P0-Cre), and that, during kidney injury, they transdifferentiate into myofibroblasts and lose their potential to produce Epo, resulting in kidney fibrosis and renal anemia.11Asada N. Takase M. Nakamura J. et al.Dysfunction of fibroblasts of extrarenal origin underlies renal fibrosis and renal anemia in mice.J Clin Invest. 2011; 121: 3981-3990Crossref PubMed Scopus (263) Google Scholar,12Mack M. Yanagita M. Origin of myofibroblasts and cellular events triggering fibrosis.Kidney Int. 2015; 87: 297-307Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar We also confirmed that Epo-producing ability can be regained in myofibroblasts by the induction of severe anemia. However, what is not fully clear from our previous study is whether the cells that had been capable of producing Epo in the healthy kidney die, or rather, survive and transdifferentiate into myofibroblasts during kidney fibrosis. This lack of clarity is due to the fact that all kidney fibroblasts, including Epo-producing cells, are labeled in P0-Cre mice in the same way, so we could not trace Epo-producing cells specifically. Previous studies have identified Epo-producing cells by means of in situ hybridization or by using transgenic mice in which green fluorescent protein is knocked-in at the Epo locus.4Obara N. Suzuki N. Kim K. et al.Repression via the GATA box is essential for tissue-specific erythropoietin gene expression.Blood. 2008; 111: 5223-5232Crossref PubMed Scopus (166) Google Scholar,13Koury S.T. Bondurant M.C. Koury M.J. Localization of erythropoietin synthesizing cells in murine kidneys by in situ hybridization.Blood. 1988; 71: 524-527Crossref PubMed Google Scholar,14Lacombe C. Da Silva J.L. Bruneval P. et al.Peritubular cells are the site of erythropoietin synthesis in the murine hypoxic kidney.J Clin Invest. 1988; 81: 620-623Crossref PubMed Scopus (358) Google Scholar Therefore, Epo-producing cells could not be observed in the kidneys with impaired Epo-producing ability, and the behavior of Epo-producing cells could not be monitored while Epo production was paused. In the present study, to address these problems, we established EpoCreERT2/+ mice, a novel mouse line that allows us to label Epo-producing cells at desired time points and to trace Epo-producing cells even while Epo production is paused. Utilizing this novel mouse line, we traced the fate of Epo-producing cells under physiological and pathologic conditions and identified Epo-producing cells as being a distinct subpopulation of resident fibroblasts with unique phenotypes. All animal studies were approved by the Animal Research Committee, Kyoto University Graduate School of Medicine, and the Institutional Animal Care and Use Committee of the RIKEN Center for Biosystems Dynamic Research, Kobe branch, and performed in accordance with the guidelines of Kyoto University and the RIKEN Kobe branch, as well as US National Institutes of Health guidelines. To construct a targeting vector, genomic fragments containing the mouse Epo gene were isolated from a bacterial artificial chromosome (BAC) clone (RP23-129L22, BACPAC Resources). We inserted a CreERT2 cassette (Artemis Pharmaceuticals), polyA tail, and a FRT-flanked PGK-Neo cassette into the Epo ATG start site of exon 1 (Figure 1a). TT2 embryonic stem (ES) cells were electroporated with targeting vector.15Yagi T. Tokunaga T. Furuta Y. et al.A novel ES cell line, TT2, with high germline-differentiating potency.Anal Biochem. 1993; 214: 70-76Crossref PubMed Scopus (419) Google Scholar G418-resistant ES colonies were selected, and correctly targeted were identified by (Figure of ES cells and were into to mouse which were with mice for targeting was also confirmed by genomic of the (Figure were as EpoCreERT2/+ mice were to mice at The EpoCreERT2/+ mouse strain can be by to with the and conditions of is in the Full of for the anemia induction and the of kidney in situ and To Epo-producing Cre mice, we generated EpoCreERT2/+ with the CreERT2 cassette into the Epo at the of the (Figure 1a). ES were for by (Figure and correctly targeted ES cells were into to mouse were with mice to was also confirmed by of the (Figure The the thereby the expression of Epo. Although mouse was from the EpoCreERT2/+ mice, in with the previous that Epo mice are et of and not erythropoietin or the erythropoietin Full Text PDF PubMed Scopus Google Scholar EpoCreERT2/+ and were in the and and Epo expression in the kidney and (Figure were EpoCreERT2/+ mice and the expression levels in these were to those in anemic kidneys (Figure in situ hybridization using Epo cells in the kidney and did not Epo cells in the of in conditions we whether was a in Epo to anemia and EpoCreERT2/+ mice, and found that the Epo to anemia was in EpoCreERT2/+ mice, due to of the Epo gene (Figure However, Epo expression and increased with the of and percentage of in of in a new To the and of Cre recombination, we EpoCreERT2/+ mice with mice, in which is expressed after of the and to mice to this (Figure To Epo we anemia by during Although cells in the kidney anemia induction (Figure the number of cells increased in with the of anemia and Epo expression in the kidney (Figure and cells in severely anemic mice were located mainly in the corticomedullary they (Figure Epo cells were also observed by in situ hybridization in the corticomedullary they to those of cells To the of recombination, mice were with anemia and of the kidney mouse were In the kidneys, cells were present at whereas cells in mice with the same of were present at of in indicating that the of EpoCreERT2/+ was of cells showed that these cells were located in the interstitium and that they expressed receptor and indicating that cells were resident fibroblasts (Figure cells did not cell or a (Figure was also observed in a small number of cells in the and To that Epo-producing cells were labeled in EpoCreERT2/+ mice, we performed in situ hybridization for Epo and Cre mRNA, and found that of Cre cells expressed Epo (Figure The proportion of Cre cells in Epo-producing cells was and the number of Cre cells and cells was with the number of Epo cells and indicating the of EpoCreERT2/+ these clearly the characteristic of lineage-labeled cells in EpoCreERT2/+ EpoCreERT2/+-labeled cells were fibroblasts in the corticomedullary as previously N. Suzuki N. Kim K. et al.Repression via the GATA box is essential for tissue-specific erythropoietin gene expression.Blood. 2008; 111: 5223-5232Crossref PubMed Scopus (166) Google S. M. of erythropoietin and in cells of rat renal cortex that fibroblasts produce 1993; PubMed Scopus Google anemia induction increased the number of EpoCreERT2/+-labeled and most Cre cells in EpoCreERT2/+ mice expressed Epo and proportion of Epo-producing cells and Cre cells after the of and cells in Cre cells, cells in Epo-producing cells, are which are as are as in a new and proportion of Epo-producing cells and cells and 16 weeks after the of with cells, cells in cells, cells in Epo-producing cells, are which are as are as in a new Epo, are which are as are as Epo, are which are as are as we examined whether the same subpopulation of fibroblasts Epo a of their or whether of fibroblasts produce Epo at time points (Figure We to mice, and at time points after anemia induction or 16 weeks after the The levels at time were and anemia was for analysis at to due to of and because the mice were small at weeks of We performed in situ hybridization for Epo and mRNA, and found that of cells expressed Epo (Figure and whereas of Epo cells were for after the of cells maintained the ability to produce Epo for as as 16 the of Epo-producing cells among the cells were and at and 16 (Figure and was among these time points by analysis of that a subpopulation among resident fibroblasts that Epo in response to anemic conditions (Figure We also examined the proportion of Epo-producing cells among cells anemia induction at weeks and weeks after the (Figure and the were and (Figure We the behaviors of Epo-producing cells during kidney the ureteral To the of and we performed weeks after the of and (Figure cells were a injury, and their number was and the proportion of cells to fibroblasts in the cortex was (Figure after the number of cells increased (Figure The number of cells was after (Figure and the proportion of cells to fibroblasts had increased injury, and after after the proportion of cells among cells was that among fibroblasts expression that cells had a high capacity to during fibrosis. Although cells in healthy kidneys expressed not a of of cells to after indicating their into myofibroblasts (Figure and The of myofibroblasts in cells and in fibroblasts expression were (Figure the of cells in are the same in indicating that cells into myofibroblasts at the same as and proportion of cells, cells, cells, and cells during cells in fibroblasts, myofibroblasts in cells, myofibroblasts in fibroblasts, cells in cells in cells in fibroblasts, cells in fibroblasts, not not receptor ureteral are which are as are as in a new not not receptor ureteral obstruction. are which are as are as in the number of cells was also observed in of kidney injury, injury, and (Figure most of the cells in these expressed indicating their into myofibroblasts (Figure we examined whether cells are to produce Epo after into myofibroblasts after under anemic conditions, when of cells in the kidney expressed Epo mRNA, of cells in the kidney expressed Epo mRNA, and the proportion of Epo-producing cells among cells was (Figure and that most cells lost their Epo-producing ability after into and proportion of Epo-producing cells and cells after with anemia cells in cells, cells in Epo-producing cells, ureteral are which are as are as in a new Epo, ureteral obstruction. are which are as are as Finally, we examined whether cells have the capacity to Epo-producing ability when kidney is We a in which a was for of and be by after the anemia induction of in due to we the in this mice a by of the same of (Figure We performed analysis at time with anemia by a after ureteral by the and after The numbers of Epo-producing cells and the of Epo-producing cells among cells from to and to after obstruction, after to and (Figure and The levels were in the in the and in the (Figure and was the and The number of cells was increased in the due to also to the levels in the (Figure that cells lose Epo-producing ability during kidney Epo-producing ability after kidney and proportion of Epo-producing cells and cells in cells in cells, cells in Epo-producing cells, not ureteral are which are as are as in a new Epo, not ureteral obstruction. are which are as are as In the present study, we in labeling and Epo-producing cells at time points and the behavior of Epo-producing cells. In our previous study, we demonstrated that Epo is produced by fibroblasts in the kidney, and that Epo-producing ability is lost in the of during N. Takase M. Nakamura J. et al.Dysfunction of fibroblasts of extrarenal origin underlies renal fibrosis and renal anemia in mice.J Clin Invest. 2011; 121: 3981-3990Crossref PubMed Scopus (263) Google Scholar Although these previous the behavior of fibroblasts, the behavior of Epo-producing cells In the present study, for the we have that Epo-producing cells under physiological conditions survive and transform into myofibroblasts during fibrosis and lose their Epo-producing ability. In we showed that the same Epo-producing cells their Epo-producing ability after kidney repair. the question of whether Epo-producing cells constitute a specialized among fibroblasts, or whether all fibroblasts possess Epo-producing ability and a small number of fibroblasts produce Epo, has In the study, we found that EpoCreERT2/+-labeled fibroblasts of fibroblasts (Figure and that this subpopulation of fibroblasts produced Epo in response to hypoxic a of 16 weeks (Figure EpoCreERT2/+-labeled cells lose their Epo-producing ability during fibrosis the ability after kidney repair. the that Epo-producing cells are a unique subpopulation among the fibroblasts in the kidney. using this unique mouse line, we were to the behavior of Epo-producing cells, which has not been analyzed clearly in previous Souma et al. previously that cells with a history of Epo production expressed such as in healthy kidneys, and that those cells into myofibroblasts and lost their Epo production ability during T. Yamazaki S. T. et of renal erythropoietin-producing cells Nephrol. 2013; PubMed Scopus Google Scholar However, as their mice with mouse line severe S. Souma T. Hirano I. et al.A mouse model of adult-onset anaemia due to erythropoietin deficiency.Nat Commun. 2013; 4: 1950Crossref PubMed Scopus (53) Google Scholar all cells with a history of Epo production in were resulting in the labeling of all fibroblasts, which may not the behavior of Epo-producing cells in adult a using mice to as the of that study was to Epo-producing cell line, the analysis was in et of renal Epo-producing cell by gene rapid Epo cell and a Int. Full Text Full Text PDF PubMed Scopus Google Scholar EpoCreERT2/+ mice us for the time to trace the behavior of Epo-producing cells at time and to from fibroblasts. novel mouse line has Epo to anemia was in EpoCreERT2/+ mice, with that in To in Epo-producing cells, the CreERT2 cassette was knocked-in into the Epo at the of the which makes it for Epo to be is that the have the regulation of Epo as well as the study that Epo expression in the kidney and in the increased in response to anemia in this mouse line (Figure and that the numbers of labeled cells increased in response to anemia (Figure and the of our using this mouse line are In our novel mouse of EpoCreERT2/+-labeled cells expressed Epo after that of cells Epo is Cre However, we demonstrated the EpoCreERT2/+-labeled cells were fibroblasts in the corticomedullary as previously (Figure N. Suzuki N. Kim K. et al.Repression via the GATA box is essential for tissue-specific erythropoietin gene expression.Blood. 2008; 111: 5223-5232Crossref PubMed Scopus (166) Google S. M. of erythropoietin and in cells of rat renal cortex that fibroblasts produce 1993; PubMed Scopus Google anemia induction increased the number of EpoCreERT2/+-labeled cells (Figure and most Cre cells in EpoCreERT2/+ mice expressed Epo (Figure We also showed that in the of that is is the time the and the Epo-producing cells are to be regulated with as by the fact that the number of Epo-producing cells in the kidney with N. Suzuki N. Kim K. et al.Repression via the GATA box is essential for tissue-specific erythropoietin gene expression.Blood. 2008; 111: 5223-5232Crossref PubMed Scopus (166) Google Scholar,13Koury S.T. Bondurant M.C. Koury M.J. Localization of erythropoietin synthesizing cells in murine kidneys by in situ hybridization.Blood. 1988; 71: 524-527Crossref PubMed Google Scholar et al. cell line of Epo-producing cells and showed that Epo production by hypoxia in even in et of renal Epo-producing cell by gene rapid Epo cell and a Int. Full Text Full Text PDF PubMed Scopus Google Scholar Therefore, cells that had been producing Epo at the time of have Epo production by the time of which was after the the expression of CreERT2 in EpoCreERT2/+ mice, which is by Epo not be for in all cells, and be in the cells of Epo. the that a Epo, with a that is or the hypoxic conditions in their Indeed, do the of Epo-producing cells. et al. have that induction of anemia in mice increased the number of Epo-producing cells, indicating that a of Epo-producing cells may be et of cells renal Clin Invest. 2016; PubMed Scopus Google Scholar showed that of kidney fibroblasts produced Epo in response to such as of hypoxia-inducible and J. et of kidney cells produce erythropoietin and supporting in response to hypoxia in Int. Full Text Full Text PDF PubMed Scopus Google Scholar is the of labeling of this mouse of Epo-producing cells were labeled in the EpoCreERT2/+ mice, indicating that the in this mouse line was not This labeling may be of EpoCreERT2/+ mice, because in a previous EpoCreERT2/+ mice, the number of labeled cells was around after hypoxic et of renal Epo-producing cell by gene rapid Epo cell and a Int. Full Text Full Text PDF PubMed Scopus Google Scholar whereas the number of labeled cells in our study was around that the hypoxic are we a we can that the in EpoCreERT2/+ mice is The of CreERT2 is well to the of CreERT2 M.J. et for and fibroblasts in the 2016; PubMed Scopus Google Scholar and expression by the Epo may not be for we had knocked-in in the Epo we have been to the of recombination, the regulation of Cre expression have been In of the of this study, the EpoCreERT2/+ mice revealed the unique behaviors of Epo-producing cells, and these mice will as a for the analysis of Epo-producing cells. We previously that such as and Epo production in renal N. Takase M. Nakamura J. et al.Dysfunction of fibroblasts of extrarenal origin underlies renal fibrosis and renal anemia in mice.J Clin Invest. 2011; 121: 3981-3990Crossref PubMed Scopus (263) Google Scholar and demonstrated that hormone and erythropoietin from rat M. Y. hormone and erythropoietin from the kidneys of adult PubMed Scopus Google Scholar study suggests that Epo production in J. et of in resident cells gene expression and erythropoietin production during kidney fibrosis in Int. Full Text Full Text PDF PubMed Scopus Google Scholar that hypoxia could Epo production or the of Epo-producing cells. are to be a new therapeutic for renal and of these have demonstrated their in the of renal P. et of erythropoietin production in Nephrol. 2010; PubMed Scopus Google Scholar, A. I. et of anemia in Nephrol. 2016; PubMed Scopus Google Scholar, N. C. et for anemia in with kidney not J PubMed Scopus Google Scholar, N. C. et for anemia in J PubMed Scopus Google Scholar, M. K. Y. et study with for anemia in with Nephrol. Scopus Google Scholar However, a is that the systemic of hypoxia-inducible could such as or V.H. targeting of the from 2017; PubMed Scopus Google Scholar, M. T. M. as a novel therapeutic anemia in kidney Int. 2017; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. T. M. in the of anemia in kidney Opin PubMed Scopus Google Scholar The analysis of EpoCreERT2/+ mice will provide insights into the behaviors and of Epo-producing cells, and may to the of novel for renal anemia. is by the which is a Kyoto University and from and All the This was by the for Research and under numbers and for Research for Research and and from the of and of and from the the and the This was by the Research Center and the and the and generated the EpoCreERT2/+ and the and analyzed the the All to the and approved the with In this of in with the for in of with is et al. examined and to the of in may identified with and with extrarenal had been to of the and of with extrarenal PDF The fate of erythropoietin-producing of the this et al. the of a mouse line that allows for the labeling of cells under of the erythropoietin (Epo) gene The that Epo-producing cells become cells after kidney and lose their ability to produce Epo. they that the cells can their Epo synthesis to a period. 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