Do β-globin, GATA-1,or EpoR regulatory domains specifically mark erythroid progenitors in transgenic reporter mice?

We read with interest the article by Heinrich et al¹  on apparent erythroid-specific expression of green fluorescent protein (GFP) and Cre-recombinase as a result of a “knock-in” mutation into erythropoietin receptor (EpoR) gene. Currently, evaluation of erythroid progenitors relies on the colony-forming assay that is often hampered by difficulty in interpretation, as it depends on both the proliferation and differentiation of the progenitors. In addition, there is a shortage of surface antigens suitable for detecting erythroid progenitors.

To overcome these problems, several lines of mice, in addition to that described by Heinrich et al,¹  have been established that express fluorescent proteins in the erythroid lineage. The fluorescent protein transgenes are under different regulatory influences, but all are active in the erythroid lineage. First, the 24-kilobase (kb) β-globin gene fragment was used to direct the expression of a cyan fluorescent protein transgene in the erythroid lineage.²  Second, we exploited the 8-kb GATA-1 gene hematopoietic regulatory domain (G1-HRD) to express GFP and found that erythroid progenitors are efficiently labeled with this method.³  In these studies, through the analysis of GFP-positive cells in the transgenic mice in combination with several known surface markers, we delineated 2 classic erythroid progenitors, CFU-E and BFU-E (erythroid colony-forming unit and burst-forming unit, respectively), as 2 discrete subfractions (late erythroid progenitor [LEP] and early erythroid progenitor [EEP]) separable by the flow cytometer.³  Third, as noted above, Heinrich et al established a knock-in mouse line that expresses GFP-Cre fusion protein under the EpoR gene regulatory influence.¹ 

How distinctly do these reporter transgenes mark the erythroid lineage? We believe that detailed assessments of their expression profiles are crucial for the use of these transgene products as internal erythroid markers. For instance, GATA-1 is expressed in the erythroid, megakaryocytic, eosinophilic, and mast cell lineages as well as Sertoli cells.⁴  Our analyses also unveiled that G1-HRD is active in the erythroid and megakaryocytic lineage cells, but not in Sertoli cells.⁴  In contrast, Heinrich et al reported that GFP-Cre transgene from the EpoR locus is expressed specifically in the erythroid lineage,¹  even though the endogenous EpoR gene is expressed in a variety of tissues.⁵  In addition, whereas EpoR is expressed in both embryonic and hematopoietic stem cells,⁶  the cells harboring Cre-mediated gene recombination were detected only in the erythroid lineage and embryonic vasculature, suggesting that valid Cre expression may start from the erythroid progenitor stage.¹  The precise reason for the apparent acquirement of erythroid specificity is unknown as expression of EpoR has been noted in other tissues.^1,7  While we recognize that the GFP-Cre mouse line could be very useful for the study of erythropoiesis, the precise expression profiles of GFP-Cre, especially those in megakaryocytes and embryonic cells, need to be clarified in more detail before claiming strict erythroid specificity. With progress in these areas, we may be able to approach novel concepts on the mechanisms of erythropoiesis in the near future.