BMP4 Induction of EKLF and GATA1 541 Were Obtained from Drs L
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Development 129, 539-549 (2002) 539 Printed in Great Britain © The Company of Biologists Limited 2002 DEV4580 The BMP/BMPR/Smad pathway directs expression of the erythroid-specific EKLF and GATA1 transcription factors during embryoid body differentiation in serum-free media Carrie A. Adelman, Subrata Chattopadhyay and James J. Bieker* Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, NY 10029, USA *Author for correspondence (e-mail: [email protected]) Accepted 26 October 2001 SUMMARY Erythroid cell-specific gene regulation during terminal could be further stimulated by the inclusion of VEGF, SCF, differentiation is controlled by transcriptional regulators, erythropoietin and thyroid hormone. EBs were competent such as EKLF and GATA1, that themselves exhibit tissue- to respond to BMP4 only until day 4 of differentiation, restricted expression patterns. Their early expression, which coincides with the normal onset of EKLF expression. already in evidence within multipotential hematopoietic The direct involvement of the BMP/Smad pathway in this cell lines, has made it difficult to determine what induction process was further verified by showing that extracellular effectors and transduction mechanisms might erythroid expression of a dominant negative BMP1B be directing the onset of their own transcription during receptor or of the inhibitory Smad6 protein prevented embryogenesis. To circumvent this problem, we have taken induction of EKLF or GATA1 even in the presence of the novel approach of investigating whether the ability of serum. Although Smad1, Smad5 and Smad8 are all embryonic stem (ES) cells to mimic early developmental expressed in the EBs, BMP4 induction of EKLF and patterns of cellular expression during embryoid body (EB) GATA1 transcription is not immediate. These data differentiation can address this issue. We first established implicate the BMP/Smad induction system as being a conditions whereby EBs could form efficiently in the crucial pathway to direct the onset of EKLF and GATA1 absence of serum. Surprisingly, in addition to mesoderm, expression during hematopoietic differentiation and these cells expressed hemangioblast and hematopoietic demonstrate that EB differentiation can be manipulated to markers. However, they did not express the committed study induction of specific genes that are expressed early erythroid markers EKLF and GATA1, nor the terminally within a lineage. differentiated β-like globin markers. Using this system, we determined that EB differentiation in BMP4 was necessary Key words: Embryoid body, Erythropoiesis, EKLF, GATA1, Serum- and sufficient to recover EKLF and GATA1 expression and free media INTRODUCTION erythroid cell production and gene expression is sequentially established at multiple sites at specific times of embryogenesis Erythroid cells are one of eight distinct blood cell lineages (Dzierzak and Medvinsky, 1995; Orkin and Zon, 1997; derived from a small population of pluripotent hematopoietic Stamatoyannopoulos and Grosveld, 2001). As erythroid tissue stem cells that are first formed during early embryogenesis is mesodermal in origin, the role of adjacent germ layers in (Metcalf, 1988). Understanding how erythroid cell-specific providing an instructive or permissive signal upon a naive cell, gene expression is accomplished has relied partly on isolation and the nature of that signal, are questions that are being of the transcription factors that play a role in inducing addressed using genetic, cellular and biochemical means in a expression of the β-like globin genes (Orkin, 1995; Baron, wide range of organisms (Choi, 1998; Beddington and 1997). This has led to identification of founders of transcription Robertson, 1999; Ray and Wharton, 2001; Zon, 2001). factor families (e.g. GATA1, EKLF (KLF1)) whose related EKLF and GATA1 are two such transcription factors that members also play important roles in other lineages (Simon, play critical roles in erythroid cell differentiation. EKLF is a 1995; Turner and Crossley, 1999; Dang et al., 2000; Tsang et C2H2 zinc-finger protein, the presence of which is crucial for al., 2000; Bieker, 2001). Because expression of many of these consolidating the switch from fetal γ-globin to adult β-globin genes is restricted to particular hematopoietic cell lineages, the expression during development (Perkins, 1999; Bieker, 2000). question quickly arises as to how these intracellular regulatory Interaction with its high-affinity site (CACCC element) at the molecules are themselves induced and regulated. Addressing proximal β-globin promoter (Miller and Bieker, 1993) helps this is necessarily an issue of developmental control, as establish the correct local chromatin structure that leads to high 540 C. A. Adelman, S. Chattopadhyay and J. J. Bieker level β-globin transcription (Armstrong et al., 1998; Zhang et expression of specific target genes. Of interest for the present al., 2001). EKLF-null mice die from a profound β-thalassemia studies, neither EKLF or GATA1 are expressed in ES cells, but at the time of the switch to adult β-globin (Nuez et al., 1995; their expression arises during EB formation before globin Perkins et al., 1995). EKLF expression is tightly erythroid expression (Simon et al., 1992; Southwood et al., 1996). This specific during development, with its onset at E7.5 (neural plate property fulfills a requirement that has been missing in stage) being strictly localized to the extra-embryonic blood attempts to investigate the onset of EKLF and GATA1 islands of the yolk sac followed by expression in the hepatic expression in hematopoietic cell lines, thus making ES cells primordia by E9.5 (Southwood et al., 1996). Paradoxically, it very appealing for such studies. Although the addition of is also transcribed early during hematopoietic differentiation cytokines can stimulate production of red cells in developing long before globin is expressed (Ziegler et al., 1999). Analysis EBs, differentiation will occur in serum alone. As a result, we of its own promoter has defined a conserved distal enhancer first established conditions where EB formation could element (~–700) that, in conjunction with the EKLF proximal efficiently proceed in the absence of serum. We then used this promoter (~–100), leads to high level, erythroid-specific system to identify extracellular inducers of EKLF and GATA1 expression in transient transfection assays as well as in erythroid genes. transgenic mice (Crossley et al., 1994; Anderson et al., 1998; Chen et al., 1998). GATA1 is a C4 zinc-finger protein whose presence is crucial MATERIALS AND METHODS for expression of numerous erythroid genes, for both primitive and definitive erythroid cell as well as megakaryocyte cell Cell lines and differentiation maturation, and for red cell viability (Weiss and Orkin, 1995; R1 ES cells were maintained on mitotically inactivated primary Tsang et al., 2000). GATA1-null cells are stalled at the fibroblast feeder cells in DMEM+15% FCS (Southwood et al., 1996). proerythroblast stage, after which they readily undergo Culture of ES cells after removal from feeder cells and EB apoptosis (Weiss et al., 1994). GATA1 is not only expressed differentiation in methylcellulose that contained IMDM+15% FCS within the hematopoietic (erythroid, mast, megakaryocytic and precisely followed established protocols (Keller et al., 1993; Weiss et eosinophilic) lineage, but also in the testes via the use of an al., 1994; Kennedy et al., 1997). Alternatively, serum was replaced by alternative promoter element (Ito et al., 1993). Erythroid- inclusion of KnockOut SR (Life Technologies) or BIT 9500 (Stem Cell Technologies) at 15%. Typically, 2000-5000 ES cells were plated restricted expression of GATA1 in both primitive and definitive in 1.5 ml in a 35 mm dish. Cytokines were included as needed, usually cells requires sequences within its first intron together with an at day 0 of differentiation: BMP4 (5 ng/ml from R&D Systems or 37.5 element located approx. –2.5 to –4.0 kb (Bieker, 1998). GATA1 ng/ml Genetics Institute), 100 ng/ml SCF (R&D), 5 ng/ml VEGF is expressed in the E7.5 extra-embryonic blood islands during (R&D), 2 U/ml erythropoietin (Amgen) and 1 µM T3 (Sigma). development (Whitelaw et al., 1990; Silver and Palis, 1997) and early in hematopoietic differentiation (Ziegler et al., 1999). RNA analysis The early cellular expression patterns of EKLF and GATA1 EBs formed in individual dishes were harvested at day 8 and total have made it difficult to directly address the mechanism by RNA was isolated after homogenization in TRI Reagent (Sigma). which they are initially induced in development and/or during Typically, one tenth of this material was used for cDNA synthesis hematopoiesis, as many multipotential cell lines already using oligo-dT (Pharmacia) and Sensiscript reverse transcriptase (Qiagen) in a volume of 20 µl. One microliter of this material was express these mRNAs (Hu et al., 1997; Reese et al., 1997). used for semi-quantitative PCR analysis with Taq polymerase However, we felt that use of the differentiating embryonic stem (Qiagen) in a volume of 50 µl that also contained [32P]dCTP as tracer. (ES) cell system might provide an alternate approach to this Cycles for each primer pair were empirically determined so as to yield problem. ES cells are derived from the inner cell mass of E3.5 product within the early exponential phase of synthesis to assure blastocysts and are the cell line of choice for genetic ablation comparative analyses in the linear range. These were