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 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 or of the inhibitory Smad6 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 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 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- 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 between 18-23 studies because they are able to mix with host blastocysts, cycles. Ten microliters of product was analyzed on a 5% efficiently form chimeric mice and contribute to all adult polyacrylamide gel, and quantitation of the dried gel was performed tissues (Nagy et al., 1993). In addition, ES cells can be induced using a Phosphorimager and analyzed with ImageQuant software to differentiate and form embryoid bodies (EBs) that (Molecular Dynamics). Under these conditions, at least a 30-fold recapitulate hematopoiesis (along with other lineages) in a linear range was attained (data not shown). PCR primers for EKLF, HPRT, bh1, βmaj, Bra, BMP4, GATA2 and sequential pattern that mimics that seen during normal murine GATA1 have been previously described (Weiss et al., 1994; Johansson development (Keller, 1995). The morphological characteristics and Wiles, 1995; Southwood et al., 1996; Schuh et al., 1999). and expression patterns of cells in colonies formed during EB Other primer pairs were as follows: PECAM, 5′ TGCGATGGT- formation have been elegantly analyzed, and have led to the GTATAACGTCA and 5′ GCTTGGCAGCGAAACACTAA (382 bp); discovery of novel colony forming cells (transitional- and FLK1, 5′ CCATACCGCCTCTGTGACTT and 5′ ACACGATGCCA- blast-CFCs) (Faloon et al., 2000; Robertson et al., 2000), as TGCTGGTCA (503 bp); SCL, 5′ TATGAGATGGAGATTTCTGATG well as the most direct evidence to date in favor of the existence and 5′ GCTCCTCTGTGTAACTGTCC (395 bp); Smad1, 5′ TTAC- of the hemangioblast (Choi et al., 1998). Dissociated EBs can CTGCCTCCTGAAGACC and 5′ TGAAACCATCCACCAGCACG (220 bp); Smad5, 5′ TATCCCAACTCCCCAGCAAG and 5′ CCCA- be replated, and specific cellular progeny (erythroid, myeloid, ′ lymphoid) can be assayed and their numbers can be quantified GGCAGAATCTACTTTTG (331 bp); Smad8, 5 TATGCACCCCA- GCACCCC and 5′ CATGGAGACTGCGGAAACAC (606 bp). (Wiles and Keller, 1991; Keller et al., 1993). Annealing temperature in all cases was set at 2°C below the calculated Although ES cells have been extensively used for cellular denaturation temperature. studies and as an in vitro culture model for early development, they have not been used as tools to identify the inducers and Establishment of stable lines to investigate the mechanism by which inducers promote Wild-type and dominant negative (K231R) murine BMPR1B clones BMP4 induction of EKLF and GATA1 541 were obtained from Drs L. Niswander and P. ten Dijke (Zou and Niswander, 1996). Murine Smad6 was obtained from Dr Xu Cao (Bai et al., 2000). The coding sequences were subcloned downstream of the ~3 kb BamHI/StuI EKLF promoter in a vector that also contains a puromycin selection marker (L. Ouyang and J. J. B., unpublished). This promoter has been shown to drive high-level erythroid specific expression in tissue culture cells (Chen et al., 1998) and in transgenic mice (J. J. B., unpublished). ES cells were electroporated under standard conditions (BioRad Gene Pulser, 400 V/125 µF), and selection in 2 µg/ml puromycin began after a 48 hour recovery. Individual colonies were selected and expanded. As transcription from the clones are predicted to yield a 5′-untranslated region that is EKLF-derived, expression of electroporated BMPR1B was monitored by RT/PCR analysis of differentiated EBs with the following primers (the first primer of the pair is unique to expression of this clone): 5′-GGTAGGATTC- ACCATGGTC and 5′-CTCAGTCTCTCGGAACCAG.

RESULTS

Establishment and analysis of embryoid body formation in the absence of serum Our first question was to determine whether EB differentiation could be established in methylcellulose in the absence of serum. Although hematopoietic cytokines can stimulate erythroid production within EBs as they differentiate in serum- containing media, their presence is not absolutely required (Wiles and Keller, 1991). At the same time, EB development is unlike that of Xenopus, which develops faithfully in a simple buffer (Nieuwkoop and Faber, 1967). One published report Fig. 1. Formation and expression pattern of embryoid bodies (EBs). established a chemically defined medium for EB formation (A) EBs were differentiated for 10 days in methylcellulose with (Johansson and Wiles, 1995); however, this was performed serum (FBS) or with two serum-free substitutes, knockout SR (SR1) under suspension conditions, where EBs can form from or BIT 9500 (SR2). A single representative EB is shown for each aggregates of ES cells. As our earlier studies (Southwood et case, as is the plating efficiency. (B) Total RNA from day 8 EBs differentiated in FBS, SR1 or SR2 (as indicated) was monitored by al., 1996) followed protocols that establish EB formation in semi-quantitative RT/PCR for expression of EKLF, GATA1, adult semi-solid media (Keller et al., 1993), we wished to monitor βmaj globin, embryonic βh1 globin and HPRT. serum-free EB differentiation under those conditions. In addition to providing a stringent test for any putative inducers, this protocol demands that single, physically separated cells form individual EBs, such that all cells within the developing indicate that although EBs can be formed in serum-substituted EB are clonal. In addition, reproducibility of temporal conditions, hemoglobinization is considerably reduced development occurs more synchronously than in suspension. qualitatively in the absence of serum. Finally, expansion of hematopoietic cells occurs in close To obtain a more precise idea what occurs at a molecular proximity to the original colony, minimizing dispersion and level under these conditions, we monitored expression of making scoring and secondary plating significantly easier several genes by semi-quantitative RT/PCR analysis. (Wiles, 1993). Two sources of commercial serum-free Expression of erythroid-specific markers (EKLF, GATA1, βh1 substitutes (Knockout SR from Life Technologies and BIT and βmaj) from single-dish pools of day 8 EBs indicate that 9500 from Stem Cell Technologies) were tested for this these genes are expressed in EBs that have been differentiated purpose and compared with EB formation in 15% FBS. in FBS, but not in EBs differentiated in either SR1 or SR2 (Fig. R1 ES cells were removed from feeder cell culture and 1B). These data show that, although morphologically normal allowed to differentiate in methylcellulose (Southwood et al., EBs can be formed in the absence of FBS, neither EKLF nor 1996), aiming for ~50-100 EBs/35mm dish. Inspection of the GATA1, in addition to the β-like globin genes, is expressed. resultant EBs (data not shown) indicated that: first, the As the efficiency of EB formation was more robust in SR1, we efficiency of EB formation was comparable in FBS or the generated EBs with this serum substitute for the rest of the knockout SR (SR1) mix (both ~6%), but was considerably experiments. lower in the BIT 9500 (SR2) mix (≤1%). Second, the Although these data were encouraging, the issue remained morphology of EBs were comparable, although the size of the of how lack of EKLF expression caused by non-induction in a SR2-derived EBs was variably smaller than those derived from committed erythroid cell could be distinguished from lack of FBS or SR1. Third, EBs grown in FBS attained a robust EKLF expression caused by a simple absence of blood cell redness after ten days differentiation (Fig. 1A); those grown in formation. In the extreme, a lack of EKLF could arise as a SR1 or SR2 were very pale at the equivalent time. These data trivial consequence of deficient mesoderm formation, such that 542 C. A. Adelman, S. Chattopadhyay and J. J. Bieker the induction system would essentially become an assay of We next split this cocktail into two subgroups. Inclusion of mesoderm inducers. VEGF, EPO and T3 were not sufficient for EKLF or GATA1 To help formulate a way to address this issue, Fig. 2A lays induction (Fig. 4A). However, inclusion of BMP4, SCF and out a scheme of erythroid commitment (Orkin and Zon, 1997) VEGF yielded a weak but detectable signal for EKLF and and molecular expression markers that can be used to follow GATA1 (Fig. 4B). Of particular interest was that BMP4 alone the presence/absence of particular cell types within the appeared to be sufficient to induce this level of expression. pathway. Expression of each gene by RT/PCR analysis was Quantitation of this data (after normalization to HPRT) used as a means to determine how far along the path the revealed that the level of EKLF expression in EBs formed with differentiating EBs have proceeded in the absence of serum BMP4 alone was ~2% that seen in EBs formed in FBS. It is of (Fig. 2B). Not surprisingly, is expressed, indicating note that expression of β-globin was observed only in the that mesoderm is formed in the serum-free EBs. More combined presence of BMP4, VEGF and SCF. surprising was the extent to which erythroid commitment To optimize the level of BMP4 needed to generate proceeded in the absence of serum, as hemangioblast (FLK1, reasonable levels of EKLF and GATA1 expression, and to PECAM) and hematopoietic progenitor (SCL, GATA2) assess the contribution of SCF and EPO to the results of Fig. markers were also expressed. In combination with the 4, we titrated BMP4 that was obtained from two suppliers. We observation that GATA1, EKLF and the β-like globins are not found that the level of EKLF and GATA1 expression is present, we conclude that EBs, differentiated in the absence of proportional to the amount of BMP4 added, and that this FBS, provide a suitable assay system to screen for inducers of reaches a plateau whose optimum concentration varies EKLF and GATA1 expression in hematopoietic cells. depending on the BMP4 supplier (data not shown). We then formed EBs using this optimal concentration of BMP4, and Resolution of cytokines involved in induction of also tested whether EB formation with only SCF and EPO EKLF and GATA1 expression could direct EKLF and/or GATA1 expression. The quantitated Using the serum-free EB system, we tested whether selected and averaged results of three sets of experiments shown in Fig. cytokines could reconstitute EKLF and GATA1 expression. 4C demonstrate that BMP4 alone can yield EKLF and GATA1 Serum-free culture conditions for primary hematopoietic stem levels that are up to 15-20% the level seen with serum. This cells contain a mix of cytokines that typically include IL3, GM- level can be boosted to ~50% by the inclusion of the other four CSF, IL6, SCF and erythropoietin (EPO). However, our choice cytokines. The data of Fig. 4 thus demonstrate that BMP4 is was directed by three considerations. First, as hematopoietic necessary and sufficient to induce EKLF and GATA1, and that progenitors were already formed, we excluded IL3, GM-CSF any combination of SCF, VEGF, EPO and T3 are not able to and IL6 from consideration. Second, the compelling data that substitute for this requirement. indicate the importance of VEGF (Kennedy et al., 1997) and BMP4 expression increases endogenously in EBs that are the BMP family (Hogan, 1996) for erythroid differentiation formed in the presence of serum (Faloon et al., 2000). Our data directed us to include these in our tests. Finally, thyroid imply that this level must be significantly lower when EBs are hormone was also included (Bauer et al., 1998). As a result, formed in serum-free conditions. This was directly tested by we formed EBs in the presence or absence of a cytokine monitoring BMP4 expression in EBs that were differentiated ‘cocktail’ that included BMP4, SCF, VEGF, EPO and T3, and in the various combinations of cytokines shown in Fig. 5. found that these were sufficient to enable EBs to express EKLF Quantitation of these data demonstrate that levels of BMP4 are and GATA1 (Fig. 3). approximately 10-fold less in EBs differentiated in the

Fig. 2. Temporal pathway of erythroid commitment during development and expression of the markers used for analysis of EBs (based on that described by Orkin and Zon (Orkin and Zon, 1997). (A) ‘Cellular status’ denotes stages in lineage determination from uncommitted mesoderm to the terminally differentiated erythroid cell that are useful as a working model for analysis. Below each stage are their corresponding expression markers. The sets in brackets serve as endothelial, rather than hematopoietic, markers for hemangioblast cells. (B) Total RNA from day 8 EBs differentiated with 15% serum (+) or serum-substitute SR1 (–) was monitored for expression of the indicated hematopoietic markers by semi-quantitative RT/PCR. BMP4 induction of EKLF and GATA1 543

harvesting all samples at day 8. We found that the ability of BMP4 to induce EKLF expression is lost if it is added after day 3, even though it is present for four days in these samples (Fig. 6A). As GATA1 (Robertson et al., 2000) and EKLF (Fig. 6B) normally comes on by day 4 in this system, whether grown in serum or BMP4 alone, these data indicate that the competence of cells present in the EB to respond to BMP4 and express EKLF and GATA1 is transient, and that its timing coincides within the same temporal frame as when transitional and blast colony forming cells arise in developing EBs (Choi, 1998; Faloon et al., 2000; Robertson et al., 2000). Fig. 3. EKLF and GATA1 gene expression in the presence of selected cytokines. Gene expression was monitored (by semi- Involvement of the BMP receptor/Smad pathway in quantitative RT/PCR) in EBs differentiated for 8 days in SR1 in the EKLF induction absence (–) or in the presence (+) of a cytokine cocktail that included Our data implicate BMP molecules, particularly BMP4, as BMP4, SCF, VEGF, T3 and erythropoietin. Pooled EBs from a single being important inducers of EKLF. BMP4 interacts with the dish are shown in each lane. BMP receptor, enabling the interaction between its two subunits (BMPR-IA or IB and II) which then leads to phosphorylation of Smad1, Smad5 and/or Smad8 prior to their knockout SR mix versus that seen in the presence of FBS. association with Smad4 and translocation to the nucleus (Dijke Importantly, inclusion of exogenous BMP4 alone is sufficient et al., 2000; Massague and Chen, 2000). We therefore checked to recover to ~100% the level of endogenous BMP4 expression three predictions based on this scheme. seen in FBS. First, we monitored whether the appropriate Smads are We next addressed whether the timing of BMP4 addition present and/or induced in our serum-free EBs that were grown was crucial for EKLF and GATA1 induction by adding BMP4 in BMP4. Fig. 7A shows that all are present, and that inclusion at varying times after initiating EB formation, and then of BMP4 results in a two-fold increase in the Smad1 level.

Fig. 4. Cytokine requirements for EKLF and GATA1 expression. EBs were differentiated with SR1 and various combinations of cytokines, focusing on VEGF, T3 and erythropoietin (A), or BMP4, SCF and VEGF (B). Total RNA was analyzed by semi-quantitative RT/PCR at day 8 of differentiation. For comparison, samples from EBs differentiated in serum (FBS) or in SR1 and all five cytokines were also analyzed. ‘Water’ indicates a no-RNA negative control. (C) EKLF and GATA1 gene expression in embryoid bodies differentiated for 8 days with in SR1 and the indicated cytokines (top), and quantitation/average of three experiments (bottom) after normalization to HPRT levels from the same samples. A previously determined, optimal concentration of BMP4 was used for these analyses. Signal with FBS was given an arbitrary level of ‘1’ (lane 8). ‘Water’ indicates a no-RNA negative control (lane 9). 544 C. A. Adelman, S. Chattopadhyay and J. J. Bieker

Second, we tested the involvement of the BMP receptor in constitutively in the developing EB. As a result, we used the EKLF activation by disrupting its activity. Expression of a EKLF promoter, which contains its erythroid specificity BMPR-IB point mutant that has lost its ability to bind ATP elements within the 950 base pairs proximal to its start site of yields a powerful dominant negative protein that effectively transcription (Anderson et al., 1998; Chen et al., 1998), to drive disrupts BMP receptor function (Zou and Niswander, 1996). expression of the BMP receptor K231R mutant (BMPR-DN) However, we did not wish to express such a derivative only in the erythroid cell (see Discussion). This promoter drives expression of a linked lacZ reporter specifically to yolk sac erythroblasts and to the developing fetal liver in transgenic mice (J. J. B., unpublished). Stable ES lines were established that express either the wild-type (648-3) or dominant negative (649-6) BMP receptor constructs, and these were differentiated in the presence of serum. The results (Fig. 7B) show that EKLF is not expressed and GATA1 is barely detectable in the 649-6 line. We next revisited our previous concern and addressed how far along the hematopoietic pathway these EBs had proceeded. The results (Fig. 7B) show that mesodermal (Bra), hemangioblast (PECAM) and hematopoietic precursor (SCL, GATA2) markers are all expressed. Intriguingly, FLK1 is not detectable. Third, we tested whether interference with the downstream Fig. 5. Expression of endogenous BMP4 in differentiating EBs. EBs signalers of the BMP pathway would also alter EKLF and were differentiated for eight days in SR1 and the indicated cytokines GATA1 expression. By a similar design to that discussed and total RNA was monitored for expression of BMP4. Expression above, we established stable ES lines that expressed the after differentiation in the presence of serum was included as a inhibitory Smad6 protein in the erythroid cell under control of positive control, and ‘water’ indicates a no-RNA negative control. the EKLF promoter and allowed these to differentiate in the presence of serum. The results (Fig. 7C) from two stable lines (Smad6-4 and Smad6-5) show that EKLF and GATA1 levels are virtually nil in these lines, but that all the other markers (Bra, PECAM, SCL, GATA2 and FLK1) are expressed. In toto, the data of Fig. 7 enable us to conclude that EKLF and GATA1 expression are dependent upon an intact BMP receptor function, and that the likely downstream molecules are not only in place to transmit this signal but play a necessary role in this process. EKLF induction with BMP4 is not immediate We next addressed whether the BMP/Smad pathway directly induces EKLF and GATA1 expression. Initially, we examined the kinetics of its induction by isolating differentiating EBs at daily intervals from d2 through d6, leaving them intact or dispersing them into single-cell suspensions (Kanatsu and Nishikawa, 1996), and incubating them with BMP4 for 24 hours. In no case did we see induction of EKLF or GATA1, even though the dispersion protocol left the cells biologically viable (data not shown). Based on the induction kinetics of Fig. 6, we therefore alternatively focused on isolating d2 or d3 EBs and incubating them for varying lengths of time with BMP4. In each case (Fig. 8) we found that at least 2 days (and optimally 3 days) was required for significant induction of EKLF and GATA1. These results demonstrate that EKLF induction by BMP4 is not an immediate-early response, but rather uses a less direct mechanism. In combination with Fig. Fig. 6. Kinetics of EKLF and GATA1 expression during EB 7, the results suggest that successful EKLF and GATA1 differentiation. (A) EBs were differentiated for varying lengths of induction probably requires the synthesis and/or activation of time (days, as indicated) in SR1 before addition of BMP4. All an additional factor in the erythroid cell. samples were harvested at day 8 and total RNA was analyzed for EKLF, GATA1 or HPRT expression. Expression in the presence of serum was included as a positive control, and ‘water’ indicates a no- RNA negative control. (B) The onset of EKLF expression was DISCUSSION monitored in EBs differentiating in FBS or in SR1 and BMP4, as indicated for varying lengths of time (days). Asterisk indicates a no- Induction of lineage-specific genes that are already expressed RNA negative control. early during differentiation has been difficult to analyze owing BMP4 induction of EKLF and GATA1 545

Fig. 7. Tests of the BMP receptor/Smad pathway in EKLF and GATA1 expression. (A) EBs were differentiated for eight days in SR1 + the indicated cytokines, and total RNA was monitored for expression of Smad1, Smad5, Smad8 or HPRT. Expression after differentiation in the presence of serum was included as a positive control, and ‘water’ indicates a no-RNA negative control. (B,C) EBs derived from stable ES cell lines expressing wild-type BMPR-1B (wild-type line 648- 3) or a dominant negative BMPR-1B (K231R line 649-6) in (B), or a mock transfected (mock) or Smad6-expressing ES lines (Smad6-4 and Smad6- 5) in (C), were differentiated in FBS for 8 days before the harvest of total RNA and expression analysis for hematopoietic markers described in Fig. 2. ‘Water’ indicates a no-RNA negative control. to limitations in finding appropriate cell lines in which to differentiation system as a means to identify potential address their onset in expression. To circumvent this problem, extracellular mediators of EKLF and GATA1 expression. We we have taken the novel approach of using the EB find that EBs can differentiate, in the absence of serum, significantly down the hematopoietic pathway, and express hemangioblast and hematopoietic markers. However, erythroid markers are not present, thus providing a suitable assay for extracellular factors needed to establish their expression. Using this system we find that the BMP/BMP receptor/Smad pathway plays a crucial role in induction of EKLF and GATA1 expression. Our approach with the EB system has enabled us to show this in two ways by showing that (1) addition of BMP4 in the absence of serum was sufficient to induce EKLF and GATA1; and (2) erythroid-specific interference of the BMP transduction pathway, even in the presence of serum, abrogated EKLF and GATA1 expression. These experiments extend the (already remarkable) range of analyses that can be obtained from differentiating EBs. BMP4 and Smad1 play a role in EKLF and GATA1 induction Although our effects are manifested by BMP4, our studies have not necessarily differentiated between different, yet functionally very similar, BMP molecules. BMP4, BMP2 and BMP7 are closely-related TGFβ-family ligands that interact Fig. 8. Kinetics of EKLF and GATA1 induction by BMP4. EBs were with a distinct pair of BMP receptor membrane (of differentiated in SR1 until day 2 or day 3 as indicted before the type I and II), which then dimerize and induce the serine/ addition of BMP4. After an additional incubation for the indicated threonine kinase activity of the type I receptor (Hogan, 1996; lengths of time in BMP4, total RNA was harvested and analyzed for Dijke et al., 2000). This enables the dimeric receptor to bind EKLF, GATA1 and HPRT expression. and phosphorylate the intracellular mediators Smad1, Smad5 546 C. A. Adelman, S. Chattopadhyay and J. J. Bieker or Smad8, which then interact with Smad4 before shuttling to to dorsal (low level) or ventral (high level) fates within the the nucleus (Kretzschmar and Massague, 1998). Our studies responding mesoderm (Dale, 2000; Zon, 2001), and ventral implicate the BMP/BMP receptor/Smad pathway in specific mesoderm differentiating into blood. Our data are concordant erythroid gene induction. But which of the many components with this observation in two ways. First, the highest level of in this pathway are likely players? Genetic and developmental EKLF and GATA1 expression is attained with the highest studies enable us to parse this list. levels of input BMP4. Second, endogenous BMP4 levels in The role of BMP4 in blood formation was initially suggested serum-free EBs must not be sufficient to induce EKLF and because of its strong ventralizing activity in the Xenopus GATA1; however, an increase of 8- to 10-fold (owing to animal cap assay (Harland, 1994) and further verified by positive autoregulation by BMP4) leads to successful dominant-negative studies (Xu et al., 1999). Its importance for induction. hematopoietic differentiation in the mouse has been established The inability to detect FLK1 expression in the BMPR-DN from effects of its genetic ablation (Winnier et al., 1995), which EBs was initially surprising, as FLK1 is expressed during disrupts mesoderm and blood cell formation in the yolk sac. serum-free EB differentiation. However, one explanation is that Similarly, disruption of one of the BMP receptor molecules the low level of endogenous BMP4 present in serum-free EBs (BMPR1A) disrupts mesoderm formation (Mishina et al., is sufficient for FLK1 transcript accumulation, while 1995). BMP2 and BMP7 have also been implicated in expression of the BMPR-DN construct depletes BMP4 below mesoderm related induction and activation of Smad1 and this threshold and thus prevents any detectable FLK1 Smad5 (Massague et al., 2000). However, disruption of BMP7 expression. As a BMPR-DN construct has been shown to leads to kidney, eye and skeletal problems, but does not affect neuralize ventral tissue in Xenopus (Graff et al., 1994), it is hematopoiesis (Dudley et al., 1995; Luo et al., 1995). Ablation also possible that cells within these EBs are not able to fully of BMP2 leads to malformation of the amnion and chorion and differentiate into endothelial cells. defects in cardiac development (Zhang and Bradley, 1996). Using the EKLF promoter to drive expression of dominant Smad4 is essential for mesoderm induction (Yang et al., 1998), negative BMPR1B and Smad6 proteins circumscribes their but Smad5 deficiency leaves hematopoietic precursors and effects in two ways. First, the impact of these molecules can blood cell formation unaffected, even though vasculogenesis only occur after induction of the EKLF gene. As a result, these and angiogenesis are disrupted (Chang et al., 1999; Yang et al., negative effectors will not be expressed until after 1999). Disruption of murine Smad1 or Smad8 have not yet hematopoiesis has begun. Limiting their presence in this way been reported; however, the role of Smad8 in mesodermal assures that a wide-range, and thus less directed, effect is patterning may be complex, as there is evidence in Xenopus avoided. Second, even when their expression is eventually that it negatively modulates signaling by BMP (Nakayama et downregulated (in the same way that EKLF is downregulated), al., 1998). it will be beyond the window of opportunity to turn on EKLF In terms of developmental profile, BMP4 is expressed before and GATA1. gastrulation in the extra-embryonic ectoderm adjacent to the epiblast, in position to influence the adjacent mesoderm that Smad target sites are present in EKLF and GATA1 emerges from the posterior primitive streak (Waldrip et al., promoters 1998). Slightly later, the posterior mesoderm itself is Implication of the BMP/BMP receptor/Smad pathway in expressing BMP4 (Winnier et al., 1995). In addition, a EKLF regulation begs the question of whether there are any hedgehog/BMP4 pathway has been implicated in the ability of appropriate Smad binding sites in the EKLF promoter. A visceral endoderm to respecify ectodermal cells to a posterior search for Smad consensus 5′CAGAC sites in both the murine mesodermal fate (Belaoussoff et al., 1998; Bhardwaj et al., and human EKLF and GATA1 promoters reveals their 2001; Dyer et al., 2001). Intriguingly, Smad1 is also induced presence; however, Smad proteins bind this site with relatively after gastrulation within the mesodermal cell region of the low affinity, and usually require interaction with another DNA primitive streak (Waldrip et al., 1998). Our data demonstrate binding co-factor to effect a high-affinity interaction with DNA that both endogenous BMP4 and Smad1 levels increase in (Dijke et al., 2000; Massague and Wotton, 2000; Wrana, 2000). developing EBs in the presence of exogenous BMP4. As a Of particular interest is the recent identification of OAZ as a result, from these genetic and developmental studies, our Smad co-factor for BMP2 signaling (Hata et al., 2000). OAZ tentative model is that the BMP4/BMP receptor/Smad1 is a large protein with 30 zinc fingers that associates with pathway is the crucial one for EKLF and GATA1 expression. activated Smad1/Smad 4 and mediates induction of the Such a scenario would also be consistent with studies in Xenopus Xvent-2 gene by binding to a DNA site lower vertebrates. For example, BMP4 (Dale et al., 1992; Jones adjacent to the Smad-binding site. However, not all BMP2 et al., 1992) and Smad1 (Wilson et al., 1997) are potent responsive genes have these sites and use OAZ, and the lack ventralizing agents in injected Xenopus embryos. In addition, of other examples precludes using an established OAZ induction of Xenopus blood cell gene expression in ectoderm consensus element to search for such a site in the EKLF and by ectopic GATA1 requires an intact BMP pathway (Huber et GATA1 promoters. Also relevant is the ability of Smads to al., 1998). Studies in the chick (Connolly et al., 2000) and in interact with members of the AML family of transcription zebrafish (Hammerschmidt et al., 1996) also support a factors (Pardali et al., 2000). AML (Runx1) is a transcription conserved role for BMP-mediated signaling in directing a factor that is commonly rearranged in acute myeloid and ventral fate as part of a dorsal/ventral patterning process. lymphocytic leukemias and plays a crucial role in Consistent with this idea, the downstream effects of BMP4 hematopoiesis (Speck and Dzierzak, 2000). Although the on patterning are known to be crucially sensitive to its Smad/AML study focused on the TGFβ/Smad3 pathway concentration, with gradients of effective BMP4 levels leading induction of IgA, the other interactions observed may be BMP4 induction of EKLF and GATA1 547 relevant to EKLF expression, particularly that of Smad1 with that EKLF is no longer induced if BMP4 is added after day 3. AML1b (Pardali et al., 2000). Nakayama et al. stated that EBs grown in BMP4 were white, However, we have not yet proven that the effect of BMP4 but when VEGF (and SCF) were added they were red. Our data on EKLF and GATA1 expression is conveyed directly by Smad provide a molecular explanation for this, as BMP4 alone protein. Although the ability of erythroid-driven dominant induced EKLF and GATA1, but β-globin expression was seen negative constructs to interfere with EKLF expression is only when SCF and VEGF were also included. consistent with this idea, it is possible that an intermediate step, such as transcriptional activation of another factor, may occur We thank Drs Liaohan Ouyang, Lee Niswander, Peter ten Dijke and first. Indeed, our kinetic data imply that such a scenario may Xu Cao for plasmids, Dr Gordon Keller for differentiation protocols, very well be operant. and Genetics Institute for BMP4. This work was supported by PHS In any case, the inability to use hematopoietic cell lines for grant DK48721 to J. J. B. analysis of EKLF and GATA1 promoters means that analyses Note added in proof designed to delimit the BMP responsive element will have to Tremblay et al. recently demonstrated that Smad1-deficient rely on stable reporter genes, which contain selected promoter mice, although embryonic lethal, are able to form blood cells deletions, that are integrated into ES cells and then tested after (Tremblay et al., 2001). As a result, the molecular details of differentiation into EBs. 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