BLOOD The American Society of Hematology · have been greatly facilitated by advances in mouse...

BLOOD VOL 87, NO 10

The Journal of The American Society of Hematology

MAY 15, 1996

REVIEW ARTICLE

The Transcriptional Control of Hematopoiesis

By Ramesh A. Shivdasani and Stuart H. Orkin

NDERSTANDING how pluripotential stem cells un- U dergo progressive restriction of lineage potential and acquire the characteristics of mature, terminally differentiated cells is central to developmental biology. Hematopoietic stem cells and differentiated progenitors are among the best studied and have contributed an important model system for cell differentiation. In this context, hematopoiesis is the process by which blood cells acquire defining phenotypes as a result of coordinated, cell-specific gene expression. The pattern of gene expression within a cell is established by cell-specific transcription factors that mediate the net effect of the variety of proliferation and differentiation signals which impinge on it. Hence, an understanding of transcription factor function is essential to the study of differentiation.

One convenient way to coordinate the expression of many individual genes in a terminally differentiated cell is through the aegis of one or a few cell-specific transcription factors. In this simplified scenario, pivotal steps in differentiation are linked to the activation of key transcriptional regulators. The processes actually governing cell differentiation are un- doubtedly more complex and are probably mediated through combinations between cell-specific and widely expressed proteins. Nevertheless, lineage-restricted transcription factors provide a valuable starting point in probing the molecular mechanisms of cell differentiation. Studies in this area have been greatly facilitated by advances in mouse genetic techniques, and the results and hypotheses generated by selected experiments involving hematopoietic transcription factors will be reviewed here. Our purpose is to describe representative murine studies that illustrate emerging con- cepts and to outline outstanding questions.

KNOCKOUT MICE: KEYS TO GENETIC ANALYSIS

Naturally occumng mutations have been a rich source of insight into normal gene function, particularly in the case of hematopoietic genes. Similarly, the study of fundamental developmental processes has relied heavily on induced mutations in invertebrates and lower vertebrates, particularly Caenorhabditis elegans and Drosophila. However, naturally occumng mutations highlight only a fraction of genes and, until recently, investigators were limited in their ability to study the effects of induced mutations in vivo in the context of an otherwise intact mammal. The development of trans- genesis in the mouse in the early 1980s provided an opportu- nity to manipulate the genetic make-up of a higher verte- brate. Subsequent to this, the introduction of technology

whereby mutations can be engineered into any gene in the mouse (Fig 1)'-3 has revolutionized the genetic analysis of cellular processes, particularly in the area of differentiation and embryonic development.

Gene knockout experiments are based on the use of murine embryonal stem (ES) cells, which are derived from the inner cell mass of the 32-cell stage preimplantation blastocyst and can be maintained in a totipotential state in culture. Upon reintroduction into a host blastocyst, they contribute to all tissues of a chimeric mouse, including germ cells. Accord- ingly, a mutation engineered in ES cells in tissue culture is introduced into the gennline of the mouse. Breeding of chimeras results in transmission of the mutation, initially into heterozygote mice, which can be mated to homozygosity for an autosomal locus (Fig 2). Another invaluable property of ES cells is their capacity to differentiate in vitro under appropriate culture conditions, particularly into hematopoietic precursors4-'; this permits the consequences of mutation of specific genes in ES cells to be examined in vitro.

DEVELOPMENTAL HEMATOPOIESIS: DEFINITION OF TERMS

Hematopoiesis in vertebrates occurs in distinct phases and anatomic sites during development.*-" Primitive, or embryonic, hematopoiesis represents a transient wave that begins when blood develops from the mesoderm of the yolk sac, around embryonic day 7.5 (E7.5) in mice or day 15 to 18 in humans. Erythrocytes produced at this time synthesize embryonic globin chains (C,,tIPHl) and remain nucleated. Definitive hematopoiesis is initiated in the fetal liver around El 1 in the mouse (day 35 to 42 in humans) and is characterized by the production of non-nucleated erythrocytes that express fetal globins (cr,y) in humans but adult globin genes

From the Deparhents of Medicine and Pediatrics, Dana-Farber Cancer Institute, Children's Hospital, and Harvard Medical School, Boston, MA: and the Howard Hughes Medical Institute, Boston, MA.

Submitted November 2, 1995; accepted January 9, 1996. R.A.S. and S.H.O. are recipients of awards from the National

Institutes of Health. S.H.O. is an Investigator of the Howard Hughes Medical Institute.

Address reprint requests to Ramesh A. Shivdasani, MD, PhD, 44 Binney St, Boston, MA 02115. 0 1996 by The American Society of Hematology. 0006-4971/96/8710-001 I$3.00/0

Blood, Vol 87, No 10 (May 15). 1996: pp 4025-4039 4025

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4026 SHIVDASANI AND ORKlN

5 I 2 3 1 5 3

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Fig 1. Homologous recombination. A gene locus is targeted for homologous recombination using a DNA construct that includes flanking homologous sequences W a n d a gene encoding a selectable marker. The recombination event (represented here by dashed X s l results in replacement of sequences flanked by the homologous areas (in this example, exons 1 through 5 and intervening introns) with the coding sequence of the selectable marker (in this case, neo- mycin phosphotransferase). Wild-type (top) and recombinant (bot- tom) alleles may then be distinguished using either Southern analysis or the polymerase chain reaction (not shown).

(a, Omajor) in mice. Although yolk sac-derived blood cells can reconstitute lethally irradiated mice?,”.” the origin of stable hematopoietic stem cells is postulated to be the region adjacent to the dorsal aorta, gonads, and mesonephros (AGM region).””s After birth. definitive hematopoiesis occurs in the bone marrow (also in the spleen in mice), where the red blood cells (RBCs) express adult globin genes. Primitive and definitive hematopoiesis probably have different require- ments for critical growth factors, including erythropoietin and the c-kit ligand.”

Although erythrocytes constitute the major circulating cellular blood component during embryonic development, myeloid cells and megakaryocytes are present at later stages and can be cultured from the fetal liver or bone marrow in the presence of appropriate growth factors. This feature is crucial for the analysis of several developmental mutations, as illustrated below.

The information provided by knockout mice depends in large part on thoughtful identification of candidate key regulators of cell growth and differentiation. In the choice of transcription factors for study, several features may be considered together, including patterns of expression in developing and mature cells or leukemias, known or putative lineage-specific target genes, prior in vitro or in vivo evidence for important functions. and relationships with other proteins. In the examples that follow, we discuss many of these aspects.

BLOODLESS MICE: THE CASE OF tal-1/SCL

Although key differentiation events are mediated by a panoply of transcriptional activators, one family of proteins plays a pivotal role in several cell types. Cell fate determination in myogenic” and lineages and sex determination in Drosophila’” are critically dependent on transcription factors bearing the basic helix-loop-helix (bHLH) motif.” This motif is shared among a variety of cell-specific (eg, MyoD and Mash- 1 ) as well as widely expressed (eg. c- myc and E2A gene products) transcription factors and mediates gene regulation by virtue of binding cis elements with the sequence CANNTG (E boxes).” bHLH factors expressed

in hematopoietic cells have been identified through cloning of chromosomal translocation breakpoints associated with human acute T-cell leukemia and include tal-I (SCL). tal-?.

Among the hematopoietic bHLH factors. tal- I/SCL has been of particular interest for several reasons. First. one of the leukemic clones in which it was originally identified showed the potential to differentiate along both myeloid and lymphoid lineages, ostensibly a “stem cell“ attribute.”.24 Second. expression of the gene is evident among early hematopoietic progenitor^,".^^ although it is also detected in more mature erythroid cells, mast cells, megakaryocytes, and endothelial cells. In Xenopu.y embryos. tal-l/SCL expression precedes that of other known hematopoietic genes in the earliest sites of blood formation (P. Hahn et al. unpublished data). A role for tal-I/SCL in late erythroid differentiation has also been suggested.’”

Mice homozygous for deletion of the tal-I/SCL gene die in utero as a result of the absence of blood formation‘”.‘’: no blood cells are detected within the yolk sac or embryo proper. which are consequently pale and growth-retarded (Fig 3A). However. there is no obvious effect on organogenesis or on mesodermal differentiation along nonhematopoietic paths. RBCs represent the only hematopoietic requirement for survival of an embryo. so the status of other blood lineages has been determined using in vitro colony assays. These studies show additional failure of myelopoiesis and thus implicate tal-l/SCL in a critical function very early during hematopoietic differentiation. Possibili- ties include determination of blood cell fate and development (or maintenance) of immature multipotential hematopoietic pro- geni tors.

and lyl- 1 .?’.?‘

PANACEA OR CAN OF WORMS? CAVEATS INHERENT TO GENE KNOCKOUT EXPERIMENTS

Targeted disruption of the tal-I/SCL gene shows the power of the knockout technique in ascribing an in vivo requirement for its product. These results also raise important caveats regarding knockout technology. First, a knockout phenotype shows only the earliest stage at which a gene product is required in development. The tal-I/SCL mutant mouse itself provides no direct information regarding the status of definitive hematopoiesis. AGM-derived stem cells

(--? gene targeting

Totipotenl~al ES cells

Mu!at on in one a’lele of

W

I targeted gene

Germline chimera Mulan! nmozygote (- -1

Heterozygo:es I+ - I

Fig 2. Application of ES cells in the study of development. Micro- injection of genetically manipulated totipotential ES cells into blastocysts derived from normal donors results in generation of chimeric mice; subsequent breedings generate heterozygous and homozygous mutants.




TRANSCRIPTION FACTORS IN HEMATOPOIESIS 4027

or effects on late erythroid maturation and lymphopoiesis. One approach to this deficiency is through analysis of differentiation of homozygous mutant (null) ES cells in vitro. Accordingly, we have recently shown that tal-1/SCL-null ES cells fail to produce all hematopoietic lineages as a specific consequence of the knockout?' In this and other cases, complementation of the lymphoid defect in RAG 2-deficient mice has proved to be a powerful method for assessing the role of a gene product specifically in lymphoid cell development and function.33 Alternative approaches involve engi- neering the targeted mutation in such a fashion that it is manifested only after the completion of embryonic development or exclusively in selected t i s s ~ e s . ~ ~ - ~ ~ Some of the latter techniques use the site-specific Cre recombinase and are particularly likely to find broad application in the study of hematopoiesis.

Second, the apparently complete absence of hematopoietic cells in tal-1/SCL-null embryos at the time of death pre- cludes extensive study of the nature of the developmental block. We have hypothesized, based on the above data and on the analogous role of related bHLH factors in myogenesis and neurogenesis, that tal-1/SCL functions in the initial differentiation of blood from mesoderm. However, the molecular basis of tal-1/SCL function, and its position relative to other nuclear proteins (such as GATA factors, the E2A gene products, and Rbtn2) in a hierarchy of transcriptional regulation, can only be addressed in combination with other experimental approaches.

Third, the above findings indicate that tal-l/SCL is not required for the formation of endothelial cells or blood vessels, although it is expressed in these Several expla- nations may account for this result. One possibility is that tal- 1/SCL plays no role in these processes and that endothelial expression of this transcription factor is either irrelevant or required for aspects of endothelial cell function that are not readily apparent. Alternatively, other transcription factors with overlapping function may compensate for absence of tal-1/SCL in vascular but not in hematopoietic cells. This possibility does not preclude a primary role for a given factor in vivo; rather, it provides for assumption of critical functions by other molecules under the experimental circum- stance of targeted deficiency. However, it is probably naive to always ascribe the absence of a phenotype in knockout mice to functional redundancy. Highly conserved genes are unlikely to have survived selective pressures in the face of widespread functional overlap and absence of a phenotype probably frequently reflects the inability to detect one. Cer- tainly, there are several examples of effects observed only when knockout mice are appropriately ~ t r e s sed ,~~ .~ ' a situa- tion that is arguably more relevant when gene function is considered in an evolutionary context.

Finally, it is important to bear in mind that mutant phenotypes are likely to be influenced to various degrees by the genetic background upon which they are observed. Examples outside the hematopoietic system call for due caution in interpreting the results of gene knockouts in a single mouse ~ t ra in .4 '~~

A DIFFERENTIATION ARREST PHENOCOPY THE CASE OF rbtn2/LMO2

The Lim-domain nuclear protein rbtn2/Ttg2/LM02 has important connections with tal- l/SCL. The genes encoding

each of these proteins may be dysregulated in human T-cell acute lymphoblastic leukemia (ALL).23""5 Although rbtn21 LM02 is expressed in a wider variety of cells, message levels are highest in hematopoietic tissues, including fetal liver4; neither gene is normally expressed in T lymphocytes. Remarkably, although tal- 1/SCL forms heterodimers with the ubiquitous products of the E2A gene both in vitro and in viv0,4~,~* more than half of the immunoprecipitable rbtn2/ LM02 protein in erythroid cells exists in a complex with tal- 1/SCL?9*50 Sequence-specific DNA binding has not been shown for rbtn2LM02; thus, modulation of gene transcription through protein-protein interactions" seems plausible.

Mice lacking rbtn2LM02 function resemble the bloodless mice lacking tal-1/SCL and die in utero at approximately the same age46; erythrocytes are missing and anemia is pre- sumed to be the cause of death. In colony assays on yolk sac tissue, myeloid cell development was initially reported to be ~naf fec ted~~; however, further studies indicate that this is not the case (A. Warren and T. Rabbitts, personal commu- nication). Thus, tal-1/SCL and rbtn2LM02 are both required at a very early stage in hematopoiesis.

The similarity between the tal-1/SCL and rbtn2LM02 knockout phenotypes suggests that the physical interaction between these proteins is physiologic and that they function in a common pathway of gene regulation. Together, these factors might control or establish a primitive genetic program, perhaps one that favors cell growth in a relatively undifferentiated state. The relevant target genes during normal hematopoiesis or in leukemic cells remain unknown but may represent overlapping subsets, and their identification remains an important challenge.

A BLOCK IN ERYTHROPOIESIS: THE CASE OF GATA-1

The DNA sequence motif GATA is present in important &-regulatory elements of many erythroid-expressed genes. The zinc finger protein GATA-1,52,53 which binds this sequence, thus poses as an excellent candidate for a central regulator of erythroid-specific gene expression and differentiation. Within hematopoietic cells, expression of GATA- 1 is limited to the erythroid, megakaryocyte, eosinophil, and mast cell lineages and multipotential progenitor^.^^-^^

Targeted disruption of the mouse GATA-1 gene has provided strong evidence for its role as a key regulator of erythroid differentiation. When injected into host blastocysts, GATA-1 -negative ES cells fail to contribute to mature RBCs but are fully able to develop into other hematopoietic lineages as well as other The consequences of absence of GATA-1 have been further defined by in vitro differentiation assays,59360 which show a maturation arrest of erythroid precursors at the proerythroblast stage (Fig 4A), followed by apoptosis of these cells.61 Thus, although a role for GATA-binding factors in lineage selection is possible, as discussed below, it is survival and terminal maturation of erythroid precursors that appear to be critically dependent on GATA- 1.

GATA-1: CONCENTRATION-DEPENDENT REGULATOR OF CELL LINEAGE?

One might anticipate a putative lineage-determining factor to induce differentiation along a particular pathway when




SHIVDASANI AND ORKlN 4028 -

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expressed or activated within a naive or heterologous cell. A paradigm is provided by the myogenic bHLH factors, which can convert multiple cell types into m u s ~ l e . 6 ~ * ~ ~ Lin- eage determination of this type by hematopoietic transcription factors has been more difficult to demonstrate, in part because of the paucity of appropriate undifferentiated cell lines. Nonetheless, forced expression of GATA-1 in the multipotential cell line 416B promotes megakaryocytic differentiati~n,"~~ whereas forced expression in retrovirally transformed chicken myeloblasts promotes differentiation of thromboblasts, eosinophils, and erythroblasts at the expense of myelomonocytic lineages.% Remarkably, the latter effect may depend in part on the intracellular levels of GATA-I. Cell lines with the highest concentration exhibit megakaryocytic features, whereas those with approximately fourfold

4

Fig 4. Hematopoietic cellular abnormaliies resulting from absence of selected lineage-restricted transcription factors. (A) Matur- ing RBCs lacking GATA-1 are arrested as proerythroblast& as indi- cated here through differentiation of GATA-Mull ES cells in vitro; many arrested cells die of apoptosb (arrowhead; TdT staining not shown, photo courtesy of M. Weiss; original magnification x 425). (B) Definitive erythroid cells lacking EKLF display features associated with globin chain imbalance, including anisocytosis, microcytosis, stigmata of hemolysis, and persistence of N RBCs. The larger RBCs in the field are residual primitive (yolk sac-derived, nucleated) cells (photo courtesy of A. Perkins; original magnification x 350) IC) Mega- karyocytes that lack p45 NF-E2 exhibit an arrest in cytoplasmic maturation, characterized by a dearth of granules and failure to demarcate platelet territories (reprinted by permission of Cell Press'"; original magnification x 5,500).





Fig 3. Gross phenotypes of selected hematopoiesis-defective mice carrying targeted disruptions of transcription factor genes. Mutant mice are shown in the lower row, with normal littermates for each shown in the upper row. (A) Mice lacking the bHLH transcription factor ta l - l l SCI show a complete absence of blood formation and die around E9.5; mice lacking the LIM-domain protein rbtn2/LM02 appear essentially similar (data not shown). (BI Mice lacking the zinc finger transcription factor GATA-2 initiate hematopoiesis but succumb to severe anemia by E l l (photo courtesy of F.-Y. Tsai). (C) Mice lacking the transcriptional activator c-Myb survive a period of normal primitive erythropoiesis but exhibit a virtual failure in progression of definitive hematopoiesis and die of anemia around E15 (reprinted with permission of Cell Press).% Preliminary results point t o a similar, but probably worse, defect in the absence of the runt-related CBF subunit AML-1 (data not shown1 (D) Mice lacking the zinc-finger transcription factor EKLF show a thalassemia syndrome that results in anemic death around E16 (photo courtesy of A. Perkins). (E) Mice lacking the bZip protein p45 NF-E2 have normal fetal hematopoiesis but display almost uniformly fatal hemorrhage (arrow) at birth due to profound thrombocytopenia.

lower levels display properties of eosinophils. Such an effect, which has not been reported in the case of myogenic bHLH factors, emphasizes the complexity underlying the transcriptional basis of lineage determination. Furthermore, GATA- I . perhaps similarly to tal-I/SCL,"' may function in both lineage selection and late erythroid maturation, presumably acting through distinct target genes.

Together with the derivation of erythroid and megakaryocytic cells from a common p r e ~ u r s o r " ~ . ~ ~ and expression of GATA- 1 in these experiments suggest that it may play a role in differentiation of multiple blood lineages. However, in vitro differentiation and chimera analyses indicate that megakaryocytes and mast cells can form in the absence of GATA- 1 Although future studies may show subtle functional effects related to altered gene expression in these cells, clearly sites of transcription factor expression are not uniformly indicative of critical functions related to differentiation.

OTHER PATHS TO BLOODLESSNESS . . . ALMOST THE CASE OF GATA-2

GATA-I belongs to a small family of related zinc-finger transcription factors with different expression patterns and, presumably, distinct target geneshq Hematopoietic expression of GATA-2 overlaps with that of GATA-I, although it is also expressed more widely, including in undifferentiated

1

Lymphocytes ' +.a Osteoclast

Fig 5. Schema depicting the positions of essential function of selected transcription factors in hematopoietic development. Position- ing is based on the earliest block in differentiation resulting from absence of the designated factor. Only factors with a demonstrated role in hematopoietic differentiation (as suggested by interference with cell-specific differentiation in knockout mice) are included. As- terisks indicate examples for which the exact position in the cascade is ambiguous. See text for details.

ES cells7" and endothelial cells.7' In contrast, GATA-3 expression is widespread during embryogenesis but restricted to T lymphocytes and the nervous system in adults.72

Three aspects of GATA-2 expression and function are intriguing. First, its expression is detected in erythroid and other hematopoietic progenitors and decreases as GATA- 1 expression increases with erythroid differentiati~n.~' Second, in chicken erythroid precursors, forced expression of GATA- 2. but not of GATA-I or GATA-3, promotes proliferation and blocks differentiati~n.~' Finally, in erythroid cells cultured from GATA-I-null ES cells, GATA-2 is expressed at 50-fold greater levels than in control cells, presumably due to the absence of normal downregulation from the high levels present in progenitors.'" Together, these findings suggest that GATA-2 may serve specific functions in early hematopoietic cell development and proliferation.

Mice lacking GATA-2 exhibit a severe and early hematopoietic defect. They survive to E 10- I I , but succumb to anemia due to a marked reduction in the number of embryonic RBCs7' (Fig 3B). Results from in vitro differentiation of GATA-2-null ES cells parallel the in vivo findings, showing up to a 100-fold decrease in the frequency of primitive erythroid colonies and even greater reduction in the appearance of definitive erythroid and mast cell colonies. Furthermore, only a minimal contribution of GATA-2-null cells to the lymphoid compartment can be detected in the RAG-2-'- blastocyst complementation assay." Thus, absence of GATA-2 results in a hematopoietic defect that is broader than that observed in the absence of GATA-I; all hematopoietic lineages are profoundly affected. Loss of GATA-2 has no apparent effect on endothelial cells.

These results refine the proposed role for GATA-2 in hematopoiesis by suggesting that the factor is essential for appropriate expansion of early hematopoietic cells; in contrast, differentiation along specific hematopoietic lineages is not obviously impaired in its ab~ence.~.' One specific possibility suggested, but not proved, by the data is that GATA- 2 mediates responsiveness to growth factors, particularly the c-kit ligand. Indeed, defects in the c-kit signaling pathway, shown in part through SI and W mice, are also more promi- nent in definitive than in primitive hematop~iesis.~'.~" Al- though the data do not presently permit a clear distinction between effects manifested at the level of the stem cell or of multipotential progenitors, they indicate a requirement for GATA-2 early in the program of hematopoiesis.

Mice lacking the related factor GATA-3 also display hematopoietic abnormalities, including lethal embryonic hemorrhage. despite the presence of megakaryocytes, and defec-





tive fetal liver h e m a t o p ~ i e s i s . ~ ~ Whether these hematologic disturbances are primary or secondary, and the effect of GATA-3 loss on T-cell development, have not been reported.

PRIMITIVE BUT NOT DEFINITIVE: THE CASE OF C-Myb AND AML-1

Expression of c-myb, the cellular homolog of the v-myb nuclear oncoprotein, is abundant within immature hematopoietic cells of all lineages and decreases as these cells differentiate.7x-x" In vitro, forced expression of c-myb inhibits erythroid differentiation,R' whereas antisense c-myb oligonu- cleotides lead to growth arrest and inhibition of hematopoietic colony formation.*' The E26 virus, which contains both v-myb and v-ets, induces leukemias of erythroid and myeloid lineage~,R~.'~ and transformation of macrophages by v-myb produces immature myeloblasts.x5 Although c-myb is also expressed in cells of the nervous system and gut, its function outside hematopoietic cells has not been examined exten- sively.

Mice lacking c-myb function display a remarkable phenotype. Yolk sac (primitive) hematopoiesis progresses normally, leading to normal early development, including organogenesis, whereas fetal liver (definitive) hematopoiesis is severely impaired86; animals die in utero by E15 (Fig 3C). The number of mature circulating definitive erythrocytes and of progenitors of other hematopoietic lineages is decreased approximately IO-fold compared with normal littermates, although megakaryocyte numbers and morphology are curi- ously unaffected. Granulocytes and monocytes appear to be normal, suggesting a quantitative rather than a qualitative differentiation defect.

This phenotype has been interpreted to reflect a proliferation abnormality of definitive hematopoietic stem cells or of multipotential progenitors within the fetal liver microenvi- ronment86 and is reminiscent of the effect of the absence of GATA-2 on yolk sac hematopoie~is.~' However, the presence of megakaryocytes might argue against this simple hy- pothesis and raises the possibility that individual committed hematopoietic progenitors, with the exception of megakaryo- blasts, are separately affected by the absence of c-myb. The ability of c-myb mutant yolk sacs to generate myeloid colonies in vitro or of mutant ES cells to undergo lymphoid development has not been reported. However, transgenic mice expressing a dominant interfering myb gene in T lymphocytes show impairments in thymopoiesis and T-cell proliferation."

The gene locus encoding AML-1, the runt-related LY subunit of the heterodimeric transcription factor core-binding factor (CBF), is a frequent target of rearrangement in acute myelogenous leukemia (AML) and childhood ALL.45 88 This gene is normally expressed in myeloid and lymphoid cells, within which potential target genes include interleukin-3 (IL- 3), granulocyte-macrophage colony-stimulating factor (GM- CSF), and T-cell antigen and monocyte colony-stimulating factor (M-CSF) receptors. The preliminary analysis of AML- 1 knockout mices9 suggests a block in development of all definitive hematopoietic lineages, including megakaryocytes, leading to fetal death by E12.5; primitive hematopoiesis is thus not affected. Hemorrhage appears to contribute to this fetal wastage, raising the possibility of an additional,

perhaps vascular, defect. It will be interesting to determine whether this phenotype largely reflects absence of multiple cytokines and cytokine receptors; such evidence would provide a notable example of the relationship between a critical transcription factor and those target genes that are most relevant for normal differentiation.

The above findings support the notion that primitive and definitive hematopoiesis are controlled by distinct genetic programs and likely differ in fundamental ways other than anatomic location and response to growth factors. Unfortu- nately, these data do not allow one to conclude whether the disparate genetic programs operate on common or distinct hematopoietic stem cells, which remains a central question in developmental hematology. Nevertheless, they establish c-myb and AML-1 as pivotal regulators of definitive hematopoiesis.

REG U LATlO N 0 F MY ELO-LY M PH OPOl ESIS: THE CASE OF PU.l

The transcription factor PU. 1, the product of the Spi- I / Sfpi 1 P U . I protooncogene, is a hematopoietic-specific member of the ets fa mil^^'.^' expressed principally in monocytes/ macrophages and B lymphocytes, but also in erythroid cells and granulocytes. Potential target genes have been identified in myeloid and B cells, including the Ig h light chain and the integrin CD11b92-94; a role for the factor in P-globin gene expression and erythroid differentiation has also been p r o p ~ s e d . ~ ' , ~ ~ PU.1 is more important for myeloid than for erythroid differentiation of human CD34' progenitors in vitro."

Mice lacking PU.l function die 1 to 3 days before their expected birth.9x The principal hematopoietic defects are absence of monocytes, granulocytes, and T and B lymphocytes, as measured by flow cytometry, histochemistry, in vitro colony assays, and diagnostic Ig and T-cell receptor gene rearrangements. The basis for their highly variable anemia is unclear (erythroid precursors are present and P-globin gene expression is unaffected), and it is not an obvious cause for the uniform preterm mortality. In a separate PU.1 knockout, the mutant animals can remain viable for up to a few days after birth, and T-cell development appears not to be affected (S. McKercher and R. Maki, unpublished data). Neverthe- less, the absence of PU.1 results in apparent ablation of at least those mature hematopoietic lineages in which the gene is normally expressed (monocytes, neutrophils, and B lymphocytes).

It remains unclear whether these observations reflect defects in single or multiple distinct progenitors and whet the ontogenic relationship is between the affected lineages. One possibility is that there is a critical requirement for PU. 1 either in lineage commitment or in maturation of a common myelo-lymphoid progenitor.'x However, the existence of such a cell is controversial; a more likely possibility is an independent effect in several distinct precursor cells. As with the c-myb knockout, a distinction between these possibilities is critical for a fuller understanding of the specific require- ments for individual transcription factors in hematopoietic differentiation.

The function of adjacent cis-elements within the Ig h and K 3' enhancers requires binding by two mutually dependent





transcription factors, one of which is PU.1; the other has been designated NF-EM5.93,99 Although interaction between myb and NF-M (the chicken homologue of CIEBPP) is required for myeloid expression of the mim-1 gene," a similarly strict requirement, reflecting cooperation between proteins with distinct expression patterns, has yet to be reported for PU.1 in myeloid promoters. The recent cloning of the lymphoid-specific NF-EMSPip gene''' will permit determination of its role in myelo-lymphopoiesis.

Although the PU. 1-related transcription factor Ets-1 is required for aspects of lymphocyte h o m e o s t a ~ i s , ~ ~ ~ . ~ ~ ~ its role in other lineages has not been reported. The essential roles, if any, of other ets family transcription factors in hematopoietic differentiation also remain to be established.

SPECIFICITY VIA SELECTIVE TARGETS: THE CASE OF EKLF

The CACCC nucleotide sequence is one of the cis-regulatory motifs critical for transcription of erythroid-expressed genes, including globins.IM As with other regulatory elements, an understanding of transcriptional activation mediated through this motif has been complicated by the multi- plicity of proteins binding to it. Both erythroid-specific and ubiquitously expressed nuclear factors recognize the CACCC motif, and the contribution of the individual proteins to regulated gene expression remains an important question. Differential screening of an erythroleukemia cDNA library showed an erythroid-specific CACCC-binding protein, designated erythroid Kruppel-like factor (EKLF),Io5 which is expressed at all stages of erythropoiesis in the developing mouse. In vitro, EKLF binds preferentially to the CACCC site found in the @-globin promoter relative to similar sites present in promoters of embryonic or fetal globin genes.Io6 Moreover, the protein activates /?-globin promoter constructs through the normal CACCC site but fails to bind or activate promoters carrying mutations of the site that are seen in some patients with P-thalassemia.

Mice lacking EKLF die in utero at the fetal liver stage with a thalassemia syndrome (Fig 3D) because of inefficient p- globin Yolk sac erythropoiesis is unimpaired, and other genes whose promoters contain CACCC sites, including GATA- 1, porphobilinogen deaminase, erythropoietin receptor, and carbonic anhydrase-1, are expressed at or near normal levels. Thus, in vivo EKLF is dispensable for erythroid commitment, primitive hematopoiesis, proliferation of erythroid precursors, or expression of most erythroid genes. Rather, this factor appears to be required principally for the expression of P-globin and perhaps a limited repertoire of other, as yet unidentified, target genes in erythroid cells.

Although the effects of absence of the other transcription factors discussed in this review could in principle result from the consequent absence of a single target gene product, it is more likely that in most instances a broader program of gene expression is affected. In this regard, EKLF is fundamentally different in that it appears to be a factor required for the regulation of few genes. The phenotype of EKLF knockout mice raises additional interesting questions. Because EKLF transcripts can be detected early in hematopoiesis, before adult P-globin expression, is there a subtle role for this factor in yolk sac hematopoiesis? Because other CACCC-binding

factors are widely expressed, which of them function(s) in regulating erythroid-expressed genes, and how is erythroid specificity determined in these cases? Gene targeting is only one of several approaches toward addressing these questions.

UNEXPECTED INSIGHTS INTO THROMBOPOIESIS: THE CASE OF NF-E2

As illustrated above, analysis of transcriptional regulators that interact with cis-elements located within a- and ,&globin locus control regions (LCRs) has been a rich source of insight into erythroid differentiation. lw Besides GATA and CACCC sequences, a third motif present within these regions is related to sequences that bind the AP-1 family of regulatory proteins and has been designated NF-E2."' Studies in cultured erythroleukemia cells and in transgenic mice have consistently suggested that cell-specific enhancement of globin gene expression is mediated in large part through this DNA motif."""' The transcription factor NF-E2, which functions at this site, is a heterodimer comprised of hematopoietic-specific (p45) and widely expressed (p18) subunits, both of which belong to the basic region-leucine zipper (bZip) family of nuclear protein^."^."^ Expression of p45 NF-E2 is restricted to erythroid and mast cells, megakaryocytes, and multipotential hematopoietic progenitor^.'^."^

Mice lacking p45 NF-E2 develop normally in utero; however, the vast majority die within the first week of life (Fig 3E). Remarkably, erythroid cell development per se is only subtly affected in that rare surviving animals show a mild hypochromic anemia'I5; death results from a hemorrhagic di- athesis caused by the absence of circulating platelets."6 Mega- karyocytes are present but show profound cytoplasmic abnormalities, including a dramatic reduction in granule numbers, disorganized demarcation membranes, and a failure to delimit platelet territories (Fig 4C). Molecular and cellular studies show a differentiation arrest that occurs relatively late in megakaryocyte maturation and does not directly involve signaling by thrombopoietin. Thus, NF-E2, which is known to be present in megakaryocytes, is essential within this lineage for regulated expression of genes that are required for cytoplasmic maturation, granule formation, and platelet development. The relevant target genes are yet to be identified.

It is unclear whether absence of a more dramatic erythroid phenotype in the absence of NF-E2II5 reflects redundancy at the level of transcription factors, LCR cis-elements, or neither. A number of p45 NF-E2-related factors are known to be widely expressed, as are p18 NF-E2 and its homo-

not thrombocytopenic."'" Clearly, the potential for combinatorial diversity is vast, and determination of the roles of individual proteins will require a combination of experimental approaches.

loguesl 14.1 17-122. , interestingly, mice lacking p18 NF-E2 are

SPECIFICITY WITHIN UBIQUITY: THE CASE OF c-fos

Differentiation is a complex process in which the fate of a given cell depends as much on the signals that it receives as on its unique response to those signals. Although lineage- restricted transcription factors are particularly appealing can- didates as key regulators of differentiation, they do not appear to exercise a monopoly in this function. Rather, widely





expressed or even ubiquitous transcription factors may oc- cupy a special niche in a limited number of cell types in which they subserve a unique function, presumably through interactions with lineage-restricted proteins. Such is the case with the bHLH protein products of the E2A gene (see below) and with the bZip protein Fos, one component of the AP-1 transcription factor complex.

Mice carrying targeted disruptions of the c-fos gene display osteopetrosis as a primary pathology (similar to mice lacking c - s ~ c ’ ~ ~ ) and altered hematopoiesis as a secondary effect of this. 124~125 In addition, they exhibit delayed gameto- genesis and certain behavioral abnormalities that may be unrelated to the primary pathology. Thus, the absence of this widely expressed transcription factor, which has been suggested to mediate many immediate-early responses, results in a narrow spectrum of cellular abnormalities, indicat- ing a restricted range of essential functions. Interestingly, overexpression of c-fos in transgenic mice leads to dysregulated bone growth and development of sarcomas, effects that can be traced to the osteoblast and chondroblast lineages.

Osteopetrosis in c-fos-null mice results from a failure of the appropriate hematopoietic progenitor to differentiate into functional osteoclasts, although the tartrate-resistant acid phosphatase (TRAP)-positive precursor cell is present. ’’’ The defect is cell-autonomous, ie, intrinsic to the hematopoietic cell precursor, and can be reversed by restoration of c-fos expression within the mutant cells. Macrophage differentiation and function are not impaired; in fact, these cells are present in increased numbers in the bone marrow. Thus, with respect to hematopoiesis, c-fos is required for the differentiation of a specific subset of precursors, presumably by virtue of directing a tissue-specific program of gene expression. The parallels with the related, albeit lineage-restricted, bZip transcription factor p45 NF-E2 are striking. Each plays an essential role in terminal differentiation, but not in lineage commitment, of a specific subset of hematopoietic progenitors.

The ontogenic relationship among monocyte derivatives is poorly understood and it is likely that distinct groups of transcription factors function to specify individual sublin- eages. A priori, it is impossible to predict whether differentiation of each of these is under the control of lineage-restricted or widely expressed transcription factors or if a subgroup of bZip factors regulates differentiation of all monocyte derivatives. The zinc-finger transcription factor egr- l/Krox24/NGIF-A/Zif268/Tis8 has been shown to re- strict differentiation along the macrophage lineage in vitro,12’ but mice lacking this product appear to have normal numbers of monocytes. I” Similarly, mice lacking NF-IM/NF-M/C- EBPP, a candidate regulator of macrophage differentiation, do produce monocytes, although these cells show severe functional defects related in part to impaired cytokine production.13’

HOMEOBOXES AND HEMATOPOIESIS: ORGANOGENESIS THROUGH H o x l l

As discussed above, hematopoiesis occurs in defined sites at different stages of development; the disparate differentiation programs are likely influenced profoundly by the micro- environment within which they operate. Therefore, an appreciation of hematopoiesis is incomplete without consid-

ering anatomic aspects, including the control of organogenesis. Little is known about the genetic basis of yolk sac or bone marrow formation; however, the finding that formation of the spleen is regulated by a homeobox genet3’ has provided an important link between the cellular and morpho- genic aspects of hematopoiesis.

Like tal-1/SCL and rbtn2LM02, Hoxl UTCL-3 was originally identified through a chromosomal translocation found in human T-cell ALL.’” In mice, it is expressed in discrete segments of developing branchial arches and the hindbrain and in the splanchnic mesoderm.”’ Mice lacking Hoxl 1 are asplenic, which results in leukocytosis, but the body plan, other aspects of organogenesis, and extrasplenic hematopoiesis are not obviously affected. Rather, the absence of Hoxl 1 leads to a specific defect in mesodermal cells normally des- tined to form the spleen. Recent evidence indicates that these cells do initiate spleen formation but then rapidly undergo apoptosis, implicating Hox 11 in cell survival rather than lineage specification.”’ It is possible that other transcription factors similarly direct formation of the yolk sac, fetal liver, and bone marrow.

Hox 1 l/TCL-3 is not the only homeobox gene to be implicated in an aspect of hematopoiesis. Expression of several clustered HOX genes has been noted in human hematopoietic cell lines,’34 with some variation in the pattern among myeloid versus erythroid lines and a proposed role in control of cell proliferati~n.”~~”~ Expression of the clustered genes HOXB3 and HOXB4 is greatest in CD34’ progenitors and declines with overexpression of HOXB4 in murine bone marrow cells results in increased proliferation of hematopoietic (stem) cells capable of myelo- lymphoid reconstitution,’” whereas inhibition of expression in vitro appears to compromise the proliferative capacity.’” It remains to be determined whether the products of these or other clustered homeobox genes play an essential role in the differentiation of hematopoietic progenitors in vivo. Thus far, disruption of many clustered homeobox genes has not resulted in obvious hematopoietic defects.”’ However, mild anemia and thrombocytopenia without morphologic abnormalities have been reported in mice heterozygous for the mixed-lineage leukemia (MLL/ALL- 1) gene; homozygosity is lethal for other reason^.'^' MLUALL-1, a positive regulator of Hox gene expression, is also a target of dysregulation in human leukemias.

VARIATIONS ON A THEME: TRANSCRIPTION FACTORS AND LYMPHOID DEVELOPMENT

As in other hematopoietic lineages, transcription factors belonging to diverse protein families play pivotal roles in development of the lymphoid system. The principles of differentiation emerging from these studies are fundamentally similar to those reviewed above; four illustrative examples are discussed here.

The zinc-finger transcription factors encoded by the Ikaros gene were originally identified on the basis of their ability to active the enhancer of the T-cell marker gene CD3S. Ikaros is expressed exclusively within lymphocytes and early hematopoietic progenitor^,'^' wherein it is believed to regulate the expression of a number of lineage-specific genes. Mice lacking Ikaros function display a complete ab-

Ikaros.





sence of mature T and B lymphocytes and natural killer (NK) cells and of most lymphoid progenitors and die of infe~ti0n.l~' In addition, these mice exhibit splenomegaly and alterations in the ratio of myeloid to erythroid cells, which are difficult to explain but could potentially reflect a role for the factor within multipotential hematopoietic progenitors. The occasional detection of putative lymphoid pre- c u r s o r ~ ~ ~ ~ suggests an essential role for Ikaros in the progression of differentiation rather than in lymphoid cell commitment. Remarkably, mice with a mutation in a single copy of the Ikaros gene (heterozygotes) develop a lethal lymphoproliferative disease.'43 These results implicate Ikaros as a pivotal mediator of cell differentiation operating at the earliest stages of maturation of all lymphoid lineages, including NK cells; recognition of a number of putative target genes that are expressed at all stages further suggests a continued role in later or antigen-dependent maturation. Within the lymphoid system, therefore, Ikaros function is perhaps most analogous to that of tal-1/SCL or GATA-1 in other aspects of hematopoiesis.

The E12 and E47 protein products of the E2A gene, identified through cDNA cloning of factors binding to an Ig K enhancer element," are bHLH transcription factors expressed in all mammalian tissues. These proteins form homodimers and heterodimers with a wide variety of related proteins in various tissues, including tal- l/SCL in hematopoietic cells and myogenic bHLH factors in muscle tissue; sequence specificity for DNA binding is determined by the particular omb bin at ion.''.^^ In vitro studies had thus suggested that the E2A factors regulate cell growth and differentiation in many cell types. Although mice lacking the E2A gene are retarded in growth and die perinatally for unknown reasons, careful analysis has thus far shown an essential role for its products only in B cells.'"-'46 Mature B cells are absent, whereas there are no significant abnormalities of other hematopoietic and nonhematopoietic lineages, including T lymphocytes. As with many examples cited above, it is extremely difficult to pinpoint the developmental stage at which B lymphopoiesis is arrested; the presence of rare B220 (CD45)' cells might suggest that the defect lies beyond the step of lineage commitment, yet among the earliest stages of B-cell maturation. Inasmuch as this phenotype shows a highly restricted essential function for a widely expressed factor, it is reminiscent of the differentiation arrest in mice lacking c-fos.

A founding member of the POU subfamily of homeobox transcription factors, Oct-2 is expressed principally in B lymphocytes, in which it binds to the octamer motif present in all Ig promoters and most enhancer^.'^' Mice lacking Oct-2 develop normally but die shortly after birth of unknown causes.148 Early B-cell development, including rearrangement and transcription of Ig genes and the number of pre-B cells, is normal. Although the number of mature B cells in neonates is only slightly reduced, there is a dramatic reduction in the frequency of cells capable of responding to mitogens in vitro and secreting antibodies and in the amount of Ig secreted per cell. The analysis of the Oct-2-null phenotype was performed in newborn mice, in which B-cell subsets may be substantially different from adults. Interpretation of the findings is further complicated by the presence of

E2A.

Oct-2.

potentially separate effects on gene (Ig) expression and differentiation and the unexplained death of the mutant animals. Nevertheless, it is clear that Oct-2 is dispensable for early B- cell development but required for optimal antigedmitogen- dependent maturation in vitro into Ig-secreting cells. Thus far, the only target gene whose expression in B lymphocytes is critically dependent on Oct-2 encodes the cell surface glycoprotein CD36,'49 whose specific role in late B-cell maturation remains unclear.

The octamer-binding proteins Oct-1 and Oct-2 provide an instructive example of how lineage specificity of gene expression may be achieved. Their identical DNA-binding specificity and in vitro activity raise the possibility that the ubiquitously expressed factor Oct-1 can function in place of Oct-2 when the latter is absent. Oct-1 activates B-cell-specific gene expression by forming a ternary complex on DNA with the B-cell-specific coactivator OBF-1, which does not exhibit independent transcription factor function^.^^^-'^^ High-level expression of Ig genes in B cells requires the recruitment of OBF-1 to the promoter by DNA-bound Oct- 1,Oct-2, or both. Such coactivators, or adaptors, are believed to function by facilitating interactions between transcriptional activators and the basal transcription ma~hinery"~ and may underlie cell-specific gene expression in many instances.

Activation of gene expression involves the formation of a nucleic acid-protein complex that permits or facilitates RNA transcription. Among the DNA- binding proteins that participate in these complexes are the architectural transcription factors, which bend DNA and serve to bring DNA-bound proteins in app~sition."~ The high-mobility group (HMG) family of nuclear proteins includes the closely related architectural transcription factors LEF-1 and TCF-1, whose expression is widespread during embryogenesis but largely restricted to T and B lymphocytes p~stnatally. '~~ Mice lacking TCF- 1 function display thymic hypoplasia with cortical hypocellularity, 10- to 100-fold reduction in the total number of thymocytes, and threefold to 10-fold reduction in the numbers of splenic and lymph node T cells.156 Numbers of B and NK cells, rearrangement of the T-cell receptor p chain gene, the T-cell response to mitogens, and overall health and viability are unaffected. These findings, which reflect a unique differentiation block occumng at the transition between immature CD8+ precursors and the more mature CD4+CD8+ T lymphocyte subpopulation, provide the only example to date in which absence of a transcription factor exclusively affects the T-cell lineage. In contrast, mice lacking LEF-1 die as a result of a number of abnormalities in organogenesis but appear to have an intact lymphoid compartment,"' possibly compensated by TCF- 1.

Gene disruption experiments have established a crucial role for transcription factors in many aspects of lymphopoiesis, ranging from putative lineage specification to the regulation of antigen-driven maturation, as illustrated above and summarized in Table 1. Thus, lineage-restricted transcription factors appear to function in principally similar ways in many aspects of hematopoiesis. Furthermore, parallels between the transcriptional control of cell differentiation in other tissues and in hematopoie~is '~~ . '~~ underscore the essential conservation of molecular mechanisms underlying development of disparate organs and cell types.

TCF-1 and LEF-1.





Table 1. Transcription Factors Required for Aspects of Normal Lymphoid Development

Transcription Factor Protein Family Lineages Affected Comments on the Knockout Phenotype

lkaros

PU.1

Ets-1

Zinc-finger

Ets

Ets

E2A (E12/€47) bHLH Pax5/BSAP Paired EBF (early B-cell factor) Zinc-finger

Oct-2

NFKB

~ 5 0 RelA

RelB

POU domain

Re1

CIEBPP (NF-M/NF-ILG) bZip

TCF-1 HMG-box

T and B; precursors

B (and T?);

T and B (myelomonocytes)

B only B only B only

B only

~ _ _ _ _ ~

No mature lymphocytes or NK cells; early differentiation defect

No detectable B lymphocytes; T cells require further characterization

Abnormal pools of resting lymphocytes characterized in Rag-2.'- background; T-cell proliferation defect

Early defect in B-cell maturation Early defect in B-cell maturation Block in B-cell development after lineage

commitment but before lg gene rearrangement

maturation Late defect in antigen-dependent

B only Abnormal mitogenic activation ?

? Multiorgan inflammation; normal

Embryonic lethality due to apoptosis in the fetal liver

lymphoid development; impaired delayed-type hypersensitivity; myeloid hyperplasia

directly to impaired IL-6 synthesis

CD4'CD8- T lymphocytes

B only

T only

Defective B-cell production related

Block in transition from CD8' to (macrophages)

References

141

98

102, 103

144, 145 171 172

148

173 174

170

175

156

CONCLUSIONS AND FUTURE DIRECTIONS

As shown in Fig 5, key transcription factors regulate multiple aspects of hematopoietic differentiation-from lineage commitment through terminal maturation. As outlined, this scheme reflects the premise that differentiation is fundamentally a process of ordered gene regulation that culminates in the expression of a unique complement of specific and widely expressed genes in each cell type. Even in the absence of reliable experimental methods for identification of the relevant target genes of a given transcription factor, study of critical lineage-specific factors has provided an invaluable window into the molecular basis of cell differentiation.

Although based on the results reviewed above, the scheme depicted in Fig 5 may be deceptively simple. Because knockout phenotypes show only the earliest block in a pathway, the true hierarchy among regulatory factors is in fact specula- tive, and it is likely that multiple interactions among the listed proteins and among other lineage-restricted or ubiquitous factors occur throughout the developmental cascade. Indeed, the genetic program of a cell must be determined by combinations of transcription factors rather than by single proteins.

The phenotypic analysis of knockout mice is usually a simple study at the level of organs and cells. As such, fundamental questions related to the mechanisms by which loss of critical target genes interferes with cell differentiation remain largely unanswered. The most important outcome of the knockout experiments reviewed above has been the establishment of candidate proteins as essential regulators of differentiation. Gene knockouts have thus set the stage for future identification of crucial target genes and for the

study of how transcriptional regulation of individual genes impacts on cell differentiation.

The factors discussed here were originally identified either through recurring chromosomal translocation in human leukemia or on the basis of their binding to critical cis-regulatory elements of lineage-specific genes. These are likely to remain important sources for characterization of novel transcription factors that regulate development.

Genes encoding proteins other than transcription factors are also required for various aspects of cell differentiation. Within hematopoietic cells, an essential role has been shown for several signal transduction molecules, including the growth factors G-CSF;' GM-CSF,I5' thrombopoietin,'6n and erythropoietin," the receptor tyrosine kinase Flk-1 , I h ' and the Janus kinase Jak3.'62,'63 Hematopoietic defects have been noted in diverse knockout models, including the Retinoblas- toma,'".''' C-myc,'" platelet-derived growth factor,'" and RelB "' genes. Indeed, transcription factor function is modulated by numerous influences, including signals delivered by cytokines and growth factors, cell-cell interactions, and the stage of the cell cycle. Although the impact of some of these influences on transcriptional regulators is known, their effect on lineage-specific factors has largely been unexplored and promises to be a fruitful area of study in the future.

How the expression of key transcription factors is initiated in progenitor cells is unclear. Possibilities include a "sto- chastic" (unregulated) process or a finely tuned response to external signals. Study of the regulation of some key transcription factors will allow investigators to begin addressing this question. Finally, selection between alternate lineages likely involves both activation and silencing of dia-





tinct subsets of genes; the competing activities of various transcriptional regulators may lie at the heart of this process. An important goal for future research is to define the mechanisms by which transcription factors operate within regulatory networks to orchestrate lineage selection and hematopoietic development.

ACKNOWLEDGMENT

We are grateful to Peter Dini, Mitchell Weiss, and Andrew Perkins for helpful comments; to Alan Warren, Dan Tenen, Richard Maki, and Leonard Zon for sharing unpublished findings; and to Mitchell Weiss, Fong-Ying Tsai, Andrew Perkins, Michael Mucenski, and Dorothea Zucker-Franklin for providing materials for figures. Knockout strains generated by the authors have been deposited with the Induced Mutant Resource at the Jackson Laboratory (Bar Harbor, ME).

REFERENCES

1. Thomas KR, Capecchi MR: Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51:503, 1987

2. Mansour S, Thomas K, Capecchi M: Disruption of the proto- oncogene int-2 in mouse embryo-derived stem cells: A general strat- egy for targeting mutations to non-selectable genes. Nature 336:348, 1988

3. Capecchi MR: Altering the genome by homologous recombination. Science 244:1288. 1989

4. Doetschman T, Eistetter H, Katz M, Schmidt W, Kemler R: The in vitro development of blastocyst-derived embryonic stem cell lines: Formation of visceral yolk sac, blood islands, and myocardium. J Embryo1 Exp Morph 87:27, 1985

5. Schmitt RM, Bruyns E, Snodgrass HR: Hematopoietic development of embryonic stem cells in vitro: Cytokine and receptor gene expression. Genes Dev 5:728, 1991

6. Keller G, Kennedy M, Papayannopoulou T, Wiles MV: Hema- topoietic differentiation during embryonic stem cell differentiation in culture. Mol Cell Biol 13:472, 1993

7. Weiss M, Orkin SH: In vitro differentiation of embryonic stem cells: New approaches to old problems. J Clin Invest 97:591, 1996

8. Moore MSA, Metcalf D: Ontogeny of the haemopoietic system: Yolk sac origin of in vivo and in vitro colony forming cells in the developing mouse embryo. Br J Haematol 18:279, 1970

9. Tavassoli M: Embryonic and fetal hemopoiesis: An overview. Blood Cells 1:269, 1991

10. Zon LI: Developmental biology of hematopoiesis. Blood 86:2876, 1995

1 I . Toles JF, Chui DHK, Belbeck LW, Starr E, Barker JE: Hema- topoietic stem cells in murine embryonic yolk sac and peripheral blood. Proc Natl Acad Sci USA 86:7456, 1989

12. Huang H, Auerbach R: Identification and characterization of hematopoietic stem cells from the yolk sac of the early mouse embryo. Proc Natl Acad Sci USA 90:10110, 1993

13. Medvinsky AL, Samoylina NL, Muller AM, Dzierzak EA: An early pre-liver intraembryonic source of CFU-S in the developing mouse. Nature 364:64, 1993

14. Godin IE, Garcia-Porrero JA, Coutinho A, Dieterlen-Lievre F, Marcos MAR: Para-aortic splanchnopleura from early mouse embryos contains B 1 a cell progenitors. Nature 364:67, 1993

15. Muller AM, Medvinsky A, Strouboulis J, Grosveld F, Dzier- zak E: Development of hematopoietic stem cell activity in the mouse embryo. Immunity 1:291, 1994

16. Wu H, Liu X, Jaenisch R, Lodish H: Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor. Cell 8359, 1995

17. Rudnicki MA, Schnegelsberg PNJ, Stead RH, Braun T, Ar-

nold H-H, Jaenisch R: MyoD or Myf-5 is required for the formation of skeletal muscle. Cell 75:1351, 1993

18. Villares R, Cabrera CV: The achaete-scute gene complex of D. melanogaster: Conserved domains in a subset of genes required for neurogenesis and their homology to c-myc. Cell 50:415, 1987

19. Lee JE, Hollenberg SM, Snider L, Turner DL, Lipnick N, Weintraub H: Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix-loop-helix protein. Science 268:836, 1995

20. Caudy M, Vassin H, Brand M, Tuma R, Jan LY, Jan YN: daughterless, a Drosophila gene essential for both neurogenesis and sex determination, has sequence similarity to myc and the achae- tescute complex. Cell 55:1061, 1988

21. Murre C, McCaw PS, Baltimore D: A new DNA binding and dimerization motif in immunoglobin enhancer binding, daughterless, MyoD, and myc proteins. Cell 56:777, 1989

22. Murre C, McCaw PS, Vaessin H, Caudy M, Jan LY, Jan YN, Cabrera CV, Buskin JN, Hauschka SD, Lassar AB, Weintraub H, Baltimore D: Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell 58537, 1989

23. Begley CG, Aplan PD, Davey MP, Nakahara K, Tchorz K, Kurtzberg J, Hershfield MS, Haynes BF, Cohen DI, Waldmann TA, Kirsch IR: Chromosomal translocation in a human leukemic stem- cell line disrupts the T-cell antigen receptor d-chain diversity region and results in a previously unreported fusion transcript. Proc Natl Acad Sci USA 86:2031, 1989

24. Finger LR, Kagan J, Christopher G, Kurtzberg J, Hershfield MS, Nowell PC, Croce CM: Involvement of the TCL5 gene on human chromosome 1 in T-cell leukemia and melanoma. Proc Natl Acad Sci USA 865039, 1989

25. Xia Y, Brown L, Yang CY-C, Tsna JT, Siciliano MJ, Es- pinosa R 111, LeBeau MM, Baer RJ: Ta12, a helix-loop-helix gene activated by the (7;9)(q34;q32) translocation in human T-cell leukemia. Proc Natl Acad Sci USA 88:11416, 1991

26. Mellentin JD, Smith SD. Cleary ML: lyl-I, a novel gene altered by chromosomal translocation in T cell leukemia, codes for a protein with a helix-loop-helix DNA binding motif. Cell 58:77, 1989

27. Begley CG, Aplan PD, Denning SM, Haynes BF, Waldmann TA, Kirsch IR: The gene SCL is expressed during early hematopoiesis and encodes a differentiation-related DNA-binding motif. Proc Natl Acad Sci USA 86:10128, 1989

28. Mouthon M-A, Bernard 0, Mitjavila M-T, Romeo P-H, Vain- chenker W, Mathieu-Mahul D: Expression of tal-I and GATA-binding proteins during human hematopoiesis. Blood 81547, 1993

29. Aplan PD, Nakahara K, Orkin SH, Kirsch IR: The SCL gene product: A positive regulator of erythroid differentiation. EMBO J 11:4073, 1992

30. Shivdasani RA, Mayer EL, Orkin SH: Absence of blood formation in mice lacking the T-cell leukemia oncoprotein tal- 1 /SCL. Nature 373:432, 1995

31. Robb L, Lyons 1, Li R, Hartley L, Kontgen F, Harvey RP, Metcalf D, Begley CG: Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene. Proc Natl Acad Sci USA 92:7075, 1995

32. Porcher C, Swat W, Rockwell K, Fujiwara Y, Shivdasani RA, Alt FW, Orkin SH: Complete block to hematopoiesis in the absence of the T-cell leukemia oncoprotein SCLITAL- I . Blood 86:253a, 1995 (abstr, suppl 1)

33. Chen J, Lansford R, Stewart V, Young F, Alt FW: RAG-2- deficient blastocyst complementation: An assay of gene function in lymphocyte development. Proc Natl Acad Sci USA 90:4528, 1993

34. Gu H, Marth JD, Orban PC, Mossmann H, Rajewsky K: Deletion of a DNA polymerase p gene segment in T cells using cell type-specific gene targeting. Science 265: 103, 1994

35. Metzger D, Clifford J, Chiba H, Chambon P: Conditional site-





specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase. Proc Natl Acad Sci USA 92:6991, 1995

36. Kuhn R, Schwenk F, Aguet M, Rajewsky K: Inducible gene targeting in mice. Science 269:1427, 1995

37. Kallianpur AR, Jordan JE, Brandt SJ: The SCLITAL-1 gene is expressed in progenitors of both the hematopoietic and vascular systems during embryogenesis. Blood 83: 1200, 1994

38. Hwang L-Y, Siegelman M, Davis L, Oppenheimer-Marks N, Baer R: Expression of the TALI proto-oncogene in cultured endothelial cells and blood vessels of the spleen. Oncogene 8:3043, 1993

39. Lowell CA, Soriano P, Varmus HE: Functional overlap in the src gene family: Inactivation of hck and fgr impairs natural immunity. Genes Dev 8:387, 1994

40. Lieschke GJ, Grail D, Hodgson G, Metcalf D, Stanley E, Cheers C, Fowler KJ, Basu S, Zhan YF, Dunn AR: Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization. Blood 84: 1737, 1994

41. Baribault H, Penner J, Iozzo RV, Wilson-Heiner M: Colo- rectal hyperplasia and inflammation in keratin 8-deficient FVB/N mice. Genes Dev 8:2964, 1994

42. Threadgill DW, Dlugosz AA, Hansen LA, Tennenbaum T, Lichti U, Yee D, LaMantia C, Mourton T, Hermp K, Harris RC, Bamard JA, Yuspa SH, Coffey RJ, Magnuson T: Targeted disruption of mouse EGF receptor: Effect of genetic background on mutant phenotype. Science 269:230, 1995

43. Sibilia M, Wagner EF: Strain-dependent epithelial defects in mice lacking the EGF receptor. Science 269:234, 1995

44. Boehm T, Foroni L, Kaneko Y, Perutz MP, Rabbits TH: The rhombotin family of cysteine-rich LIM-domain oncogenes: Distinct members are involved in T-cell translocations to human chromo- somes llp15 and llp13. Proc Natl Acad Sci USA 88:4367, 1991

45. Rabbits TH: Chromosomal translocations in human cancer. Nature 372: 143, 1994

46. Warren AJ, Colledge WH, Carlton MBL, Evans MJ, Smith AJH, Rabbits TH: The oncogenic cysteine-rich LIM domain protein Rbtn2 is essential for erythroid development. Cell 78:45, 1994

47. Hsu H-L, Cheng J-T, Chen Q, Baer R: Enhancer-binding activity of the tal-1 oncoprotein in association with the E47E12 helix-loop-helix proteins. Mol Cell Biol 11:3037, 1991

48. Voronova AF, Lee F: The E2A and tal-1 helix-loop-helix proteins associate in vivo and are modulated by Id proteins during interleukin 6-induced myeloid differentiation. Proc Natl Acad Sci USA 915952, 1994

49. Valge-Archer VE, Osada H, Warren AJ, Forster A, Li J, Baer R, Rabbits TH: The LIM protein RBTN2 and the basic helix-loop- helix protein TALI are present in a complex in erythroid cells. Proc Natl Acad Sci USA 91:8617, 1994

SO. Wadman I, Li J, Bash RO, Forster A, Osada H, Rabbits TH, Baer R: Specific in vivo association between the bHLH and LIM proteins implicated in human T cell leukemia. EMBO J 13:4831, 1994

51. Sadler I, Crawford AW, Michelsen JW, Beckerle MC: Zyxin and cCRP: Two interactive LIM domain proteins associated with the cytoskeleton. J Cell Biol 119:1573, 1992

52. Tsai SF, Martin DI, Zon LI, D’Andrea AD, Wong GG, Orkin SH: Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells. Nature 339:446, 1989

53. Evans T, Felsenfeld G: The erythroid-specific transcription factor eryfl: A new finger protein. Cell 58:877, 1989

54. Martin DIK, Zon LI, Mutter G, Orkin SH: Expression of an erythroid transcription factor in megakaryocytic and mast cell lineages. Nature 344:444, 1990

55. Romeo P-H, Prandini M-H, Joulin V, Mignotte V, Prenant

M, Vainchenker W, Marguerie G, Uzan G: Megakaryocytic and erythrocytic lineages share specific transcription factors. Nature 344:447, 1990

56. Zon LI, Yamaguchi Y, Yee K, Albee EA, Kimura A, Bennett JC, Orkin SH, Ackerman SJ: Expression of mRNA for the GATA- binding proteins in human eosinophils and basophils: Potential role in gene transcription. Blood 81:3234, 1993

57. Pevny L, Simon MC, Robertson E, Klein WH, Tsai S-F, D’Agati V, Orkin SH, Costantini F: Erythroid differentiation in chimeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature 349:257, 1991

58. Simon MC, Pevny L, Wiles MV, Keller G, Costantini F, Orkin SH: Rescue of erythroid development in gene targeted GATA- 1-mouse embryonic stem cells. Nat Genet 1:92, 1992

59. Weiss MJ, Keller G, Orkin SH: Novel insights into erythroid development revealed through in vitro differentiation of GATA- 1 - embryonic stem cells. Genes Dev 8: 1184, 1994

60. Pevny L, Lin C-S, D’Agati V, Simon MC, Orkin SH, Costan- tini F: Development of hematopoietic cells lacking transcription factor GATA-1. Development 121:163, 1995

61. Weiss MJ, Orkin SH: Transcription factor GATA-1 permits survival and maturation of erythroid precursors by preventing apoptosis. Proc Natl Acad Sci USA 92:9623, 1995

62. Lassar AB, Patterson BM, Weintraub H: Transfection of a DNA locus that mediates the conversion of 10T112 fibroblasts to myoblasts. Cell 47:649, 1986

63. Davis RL, Weintraub H, Lassar AB: Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 5 1:987, 1987 64. Visvader JE, Elefanty AG, Strasser A, Adam JM: GATA-1

but not SCL induces megakaryocytic differentiation in an early myeloid line. EMBO J 11:4557, 1992

65. Visvader J, Adams JM: Megakaryocytic differentiation induced in 416B myeloid cells by GATA-2 and GATA-3 transgenes or 5-azacytidine is tightly coupled to GATA-1 expression. Blood 82:1493, 1993

66. Kulessa H, Frampton 3, Graf T: GATA-1 reprograms avian myelomonocytic cell lines into eosinophils, thromboblasts, and erythroblasts. Genes Dev 9: 1250, 1995

67. Suda T, Suda J, Ogawa M: Single-cell origin of mouse hemopoietic colonies expressing multiple lineages in variable combinations. Proc Natl Acad Sci USA 80:6689, 1983

68. Ogawa M: Effects of hemopoietic growth factors on stem cells in vitro. Hematol Clin North Am 3:453, 1989

69. Orkin SH: GATA-binding transcription factors in hematopoietic cells. Blood 80575, 1992

70. Yamamoto M, KO LJ, Leonard MW, Beug H, Orkin SH, Engel JD: Activity and tissue-specific expression of the transcription factor NF-E1 multigene family. Genes Dev 4:1650, 1990

71. Dorfman DM, Wilson DB, Bruns GA, Orkin SH: Human transcription factor GATA-2. Evidence for regulation of preproendo- thelin-1 gene expression in endothelial cells. J Biol Chem 267:1279, I992

72. Oostenvegel M, Timmerman J, Leiden J, Clevers H: Expres- sion of GATA-3 during lymphocyte differentiation and mouse embryogenesis. Dev Immunol 3:1, 1992

73. Briegel K, Lim K-C, Plank C, Beug H, Engel J, Zenke M:Ec- topic expression of a conditional GATA-2/estrogen receptor chimera arrests erythroid differentiation in a hormone-dependent manner. Genes Dev 7:1097, 1993

74. Tsai F-Y, Keller G, Kuo FC, Weiss MJ, Chen J-Z, Rosenblatt M, Alt F, Orkin SH: An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature 37 1 :22 I , 1994

75. Chui DHK, Russell ES: Fetal erythropoiesis in steel mutant mice. I. A morphological study of erythroid cell development in fetal liver. Dev Biol 40:256, 1974





76. Ogawa M, Nishikawa S, Yoshinaga K, Hayashi S-I, Kunisada T, Nakao J, Kina T, Sudo T, Kodama H, Nishikawa S-I: Expression and function of c-Kit in fetal hemopoietic progenitor cells: Transition from the early c-Kit-independent to the late c-Kit-dependent wave of hemopoiesis in the murine embryo. Development 117:1089, 1993

77. Pandolfi PP, Roth ME, Karis A, Leonard MW, Dzierzak E, Grosveld FG, Engel JD, Lindenbaum MH: Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis. Nat Genet 11:40, 1995

78. Gonda TJ, Sheiness DK, Bishop JM: Transcripts from the cellular homologs of retroviral oncogenes: Distribution among chicken tissues. Mol Cell Biol 2:617, 1982

79. Westin EH, Gallo RC, Arya SK, Eva A, Souza LM, Baluda MA, Aaronson SA, Wong-Staal F Differential expression of the amv gene in human hematopoietic cells. Roc Natl Acad Sci USA 79:2194, 1982

80. Kastan MB, Stone KD, Civin CI: Nuclear oncoprotein expression as a function of lineage, differentiation stage, and proliferation status of normal human hematopoietic cells. Blood 74:1517, 1989

81. Clarke MF, Kukowska-Latallo JF, Westin E, Smith M, Pro- chownik EV: Constitutive expression of a c-myb cDNA blocks Friend murine erythroleukemia cell differentiation. Mol Cell Biol 8:884, 1988

82. Gewirtz AM, Calabretta B: A c-myb antisense oligodeoxy- nucleotide inhibits normal human hematopoiesis in vitro. Science 242:1303, 1988

83. Radke K, Beug H, Komfeld S, Graf T: Transformation of both erythroid and myeloid cells by E26, an avian leukemia virus that contains the myb gene. Cell 31:643, 1982

84. Graf T, McNagny K, Brady G, Frampton J: Chicken “erythroid” cells transformed by the gag-myb-ets-encoding E26 leukemia virus are multipotent. Cell 70:201, 1992

85. Beug H, Blundell P, Graf T: Reversibility of differentiation and proliferative capacity in avian myelomonocytic cells transformed by tsE26 leukemia virus. Genes Dev 1:277, 1987

86. Mucenski ML, McLain K, Kier AB, Swerdlow SH, Schreiner CM, Miller TA, Pietryga DW, Scott JW Jr, Potter SS: A functional c-myb gene is required for normal fetal hepatic hematopoiesis. Cell 65:677, 1991

87. Badiani P, Corbella P, Kioussis D, Marvel J, Weston K Dominant interfering alleles define a role for c-Myb in T-cell development. Genes Dev 8:770, 1994

88. Stegmaier K, Pendse S, Barker GF, Bray-Ward P, Ward DC, Montgomery KT, Krauter KS, Reynolds C, Sklar J, Donnelly M, Bohlander S, Rowley JD, Sallan SE, Gilliland DG, Golub TR: Fre- quent loss of heterozygosity at the TEL gene locus in acute lymphoblastic leukemia of childhood. Blood 86:38, 1995

89. Okuda T, van Deursen J, Hiebert SW, Grosveld G, Downing JR: A m - 1 , the target of multiple chromosomal translocations in human leukemia, is essential for normal murine fetal liver hematopoiesis. Blood 86:596a, 1995 (abstr, suppl 1)

90. Moreau-Gachelin F, Ray D, Tambourin P, Tavitian A, Klemsz MJ, McKercher SR, Celada A, Van Beveren C, Maki RA: The Pu.1 transcription factor is the product of the putative oncogene Spi-1. Cell 61:1166, 1990

91. Klemsz MJ, McKercher SR, Celada A, Van Beveren C, Maki RA: The macrophage and B cell-specific transcription factor PU.1 is related to the ets oncogene. Cell 61:113, 1990

92. Pahl HL, Scheibe RJ, Zhang D-E, Chen H-M, Galson DL, Maki RA, Tenen DG: The proto-oncogene PU. 1 regulates expression of the myeloid-specific CDllb promoter. J Biol Chem 268:5014, 1993

93. Pongubala JMR, Van Beveren C, Nagulapalli S, JKM, McKercher SR, Maki RA, Atchison ML: Effect of PU.1 phosphory- lation on interaction with NF-EM5 and transcriptional activation. Science 259:1622, 1993

94. Shin MK, Koshland ME: Ets-related protein PU.l regulates expression of the immunoglobulin J-chain gene through a novel Ets- binding element. Genes Dev 7:2006, 1993

95. Schuetze S, Paul R, Gliniak BC, Kabat D: Role of the PU.1 transcription factor in controlling differentiation of Friend erythroleukemia cells. Mol Cell Biol 12:2967, 1992

96. Galson DL, Hensold JO, Bishop TR, Schalling M, D’Andrea A, Jones C, Auron PE, Housman DE: Mouse b-globin DNA-binding protein BI is identical to a proto-oncogene, the transcription factor Spi-1PU.1, and is restricted in expression to hematopoietic cells and the testis. Mol Cell Bioi 13:2929, 1993

97. Voso MT, Bum TC, Wulf G, Lim B, Leone G, Tenen DG: Inhibition of hematopoiesis by competitive binding of transcription factor PU.l. Proc Natl Acad Sci USA 91:7932, 1994

98. Scott EW, Simon MC, Anastasi J, Singh H: Requirement of transcription factor PU. 1 in the development of multiple hematopoietic lineages. Science 265:1573, 1994

99. Eisenbeis CF, Singh H, Storb U: PU.1 is a component of a multiprotein complex which binds an essential site in the murine immunoglobulin lambda 2-4 enhancer. Mol Cell Biol 136452, 1993

100. Ness SA, Kowens-Leutz E, Casini T, Graf T, Leutz A: Myb and NF-M: Combinatorial activators of myeloid genes in heterologous cell types. Genes Dev 7:749, 1993

101. Eisenbeis CF, Singh H, Storb U: Pip, a novel IRF family member, is a lymphoid-specific, PU. I-dependent transcriptional activator. Genes Dev 9:1377, 1995

102. Bones J-C, Willerford DM, Grevin D, Davidson L, Camus A, Martin P, Stehelin D, Alt FW: Increased T-cell apoptosis and terminal B-cell differentiation induced by inactivation of the Ets- 1 proto-oncogene. Nature 377:635, 1995

103. Muthusamy N, Barton K, Leiden JM: Defective activation and survival of T cells lacking the Ets-1 transcription factor. Nature 377:639, 1995

104. Orkin SH: Globin gene regulation and switching: Circa 1990. Cell 63:665, 1990

105. Miller IJ, Bieker JJ: A novel, erythroid cell-specific murine transcription factor that binds to the CACCC element and is related to the Kruppel family of nuclear proteins. Mol Cell Biol 13:2776, 1993

106. Feng WC, Southwood CM, Bieker JJ: Analyses of @-thalassemia mutant DNA interactions with erythroid Kruppel-like factor (EKLF), an erythroid cell-specific transcription factor. J Biol Chem 269:1493, 1994

107. Nuez B, Michalovich D, Bygrave A, Ploemacher R, Gros- veld F Defective haematopoiesis in fetal liver resulting from inactivation of the EKLF gene. Nature 375:316, 1995

108. Perkins AC, S h q e AH, Orkin SH: Lethal @-thalassemia in mice lacking the erythroid CACCC-transcription factor EKLF. Na- ture 375:318, 1995

109. Orkin SH: Transcription factors and hematopoietic development. J Biol Chem 270:4955, 1995

110. Mignotte V, Wall L, deBoer E, Grosveld F, Romeo P-H: Two tissue-specific factors bind the erythroid promoter of the human porphobilinogen deaminase gene. Nucl Acids Res 17:37, 1989

111. Ney PA, Sorrentino BP, Lowrey CH, Nienhuis AW: Induc- ibility of the HS I1 enhancer depends on binding of an erythroid specific nuclear protein. Nucl Acids Res 18:6011, 1990

112. Talbot D, Grosveld F: The 5’ HS 2 of the globin locus control region enhances transcription through the interaction of a multimeric complex binding at two functionally distinct NF-E2 binding sites. EMBO J 10:1391, 1991

113. Andrews NC, Erjument-Bromage H, Davidson MB, Tempst P, Orkin SH: Erythroid transcription factor NF-E2 is a haematopoietic-specific basic-leucine zipper protein. Nature 362:722, 1993

114. Andrews NC, Kotkow KJ, Ney PA, Erdjument-Bromage H, Tempst P, Orkin SH: The ubiquitous subunit of erythroid transcrip-




4038 SHlVDASANl AND ORKlN

tion factor NF-E2 is a small basic-leucine zipper protein related to the v-mufoncogene. Proc Natl Acad Sci USA 90:11488, 1993

1 15. Shivdasani RA, Orkin SH: Erythropoiesis and globin gene expression in mice lacking the transcription factor NF-E2. Proc Natl Acad Sci USA 92:8690, 1995

1 16. Shivdasani RA, Rosenblatt MF, Zucker-Franklin D, Jackson CW, Hunt P, Saris C, Orkin SH: Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development. Cell 8 1 :695, 1995

117. Chan JY, Han X-L, Kan YW: Cloning of Nrfl, an NF-E2- related transcription factor, by genetic selection in yeast. Proc Natl Acad Sci USA 90:11371, 1993

118. Caterina JJ, Donze D, Sun C-W, Ciavatta DJ, Townes TM: Cloning and functional characterization of LCR-FI: A bZIP transcription factor that activates erythroid-specific, human globin gene expression. Nucleic Acids Res 22:2383, 1994

119. Moi P, Chan K, Asunis I, Cao A, Kan YW: Isolation of NF-E2-related factor 2 (Nfl) , a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/API repeat of the P-globin locus control region. Proc Natl Acad Sci USA 91:9926, 1994

120. Chui DHK, Tang W, Orkin SH: cDNA cloning of murine Nrf2 gene, coding for a p45 NF-E2 related transcription factor. Biochem Biophys Res Commun 209:40, 1995

121. Igarashi K, Kataoka K, Itoh K, Hayashi N, Nishizawa M, Yamamoto M: Regulation of transcription by dimerization of erythroid factor NF-E2 p45 with small Maf proteins. Nature 367568, 1994

122. Kataoka K, Igarashi K, Itoh K, Fujiwara KT, Noda M, Ya- mamoto M, Nishizawa M: Small Maf proteins heterodimerize with Fos and may act as competitive repressors of the NF-E2 transcription factor. Mol Cell Biol 15:2180, 1995

122a. Kotkow, KJ, Orkin SH: Complexity of the erythroid transcription factor NF-E2 as revealed by gene targetting of the mouse p18 NF-E2 locus. Proc Natl Acad Sci USA (in press)

123. Soriano P, Montgomery C, Geske R, Bradley A: Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell 64:693, 1991

124. Johnson RS, Spiegelman BM, Papaioannou V: Pleiotropic effects of a null mutation in the c-fos proto-oncogene. Cell 71577, 1992

125. Wang 2-Q, Ovitt C, Grigoriadis AE, Mohle-Steinlein U, Ruther U, Wagner EF: Bone and haematopoietic defects in mice lacking c-fos. Nature 360:741, 1992

126. Ruther U, Komitowski D, Schubert FR, Wagner EF: c-fos expression induces bone tumors in transgenic mice. Oncogene 4:861, 1989

127. Grigoriadis AE, Wang Z-Q. Cecchini MG, Hofstetter W, Felix R, Fleisch HA, Wagner EF: c-Fos: A key regulator of osteoclast-macrophage lineage determination and bone remodeling. Sci- ence 266:443, 1994

128. Nguyen HQ, Hoffman-Liebermann B, Liebermann DA: The zinc finger transcription factor egr-l is essential for and restricts differentiation along the macrophage lineage. Cell 72:197, I993

129. Lee SL, Tourtellotte LC, Wesselschmidt RL, Milbrandt J: Growth and differentiation proceeds normally in cells deficient in the immediate early gene NGFI-A. J Biol Chem 270:9971, 1995

130. Tanaka T, Akira S, Yoshida K, Umemoto M, Yoneda Y, Shirafuji N, Fujiwara H, Suematsu S, Yoshida N, Kishimoto T: Targeted disruption of the NF-IL6 gene discloses its essential role in bacteria killing and tumor cytotoxicity by macrophages. Cell 80:353, 1995

131. Roberts CWM, Shutter JR, Korsmeyer SJ: Hoxl I controls the genesis of the spleen. Nature 368:747, 1994

132. Hatano M, Roberts CWM, Minden M, Crist WM, Kors-

meyer SJ: Deregulation of a homeobox gene, HOXl I , by the t(10;14) in T cell leukemia. Science 253:79, 1991

133. Dear TN, Colledge WH, Carlton MBL, Lavenir 1, Larson T, Smith AJH, Warren AJ, Evans MJ, Sofroniew MV, Rabbitts TH: The Hoxll gene is essential for cell survival during spleen development. Development 121 :2909, 1995

134. Lawrence HJ, Largman C: Homeobox genes in normal hematopoiesis and leukemias. Blood 80:2445, 1992

135. Perkins AC, Cory S: Conditional immortalization of mouse myelomonocytic, megakaryocytic and mast cell progenitors by the Hox-2.4 homeobox gene. EMBO J 12:3835, 1993

136. Sauvageau G, Lansdorp PM, Eaves CJ, Hogge DE, Dragow- ska WH, Reid DS, Largman C, Lawrence HJ, Humphries RK: Differ- ential expression of homeobox genes in functionally distinct CD34+ subpopulations of human bone marrow cells. Proc Natl Acad Sci USA 91:12223, 1994

137. Sauvageau G, Thorsteinsdottir U, Eaves CJ, Lawrence HJ, Largman C, Lansdorp PM, Humphries RK: Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo. Genes Dev 9:1753, 1995

138. Giampaolo A, Sterpetti P, Bulocrini D, Samoggia P, Pelosi P, Valtieri F, Peschle C: Key functional role and lineage-specific expression of HOXB cluster genes in purified hematopoietic progenitor differentiation. Blood 84:3637, 1994

139. Davis AP, Witte DP, Hsieh-Li HM, Potter SS, Capecchi MR: Absence of radius and ulna in mice lacking hoxa- I I and hoxd- I 1. Nature 375:791, I995

140. Yu BD, Hess JL, Homing SE, Brown GAJ, Korsmeyer SJ: Altered Hox expression and segmental identity in MII-mutant mice. Nature 378:505, 1995

141. Georgopoulos K, Morgan BA, Moore DD: Ikaros, an early lymphoid restricted transcription factor, a putative mediator for T cell commitment. Science 258:808, 1992

142. Georgopoulos K, Bigby M, Wang J-H, Molnar A, Wu P, Winandy S, Sharpe A: The Ikaros gene is required for the development of all lymphoid lineages. Cell 79:143, 1994

143. Winandy S, Wu P, Georgopoulos K: A dominant mutation in the Ikaros gene leads to rapid development of leukemia and lymphoma. Cell 83:289, 1995

144. Bain G, Maandag ECR, Izon DJ, Amsen D, Kruisbeek A, Weintraub BC, Krop I, Schlissel MS, Feeney AJ, van Roon M, van der Valk M, te Riele HPJ, Bems A, Murre C: E2A proteins are required for proper B cell development and initiation of immunoglobulin rearrangements. Cell 79:885, 1994

145. Zhuang Y, Soriano P, Weintraub H: The helix-loop-helix gene E2A is required for B cell formation. Cell 792375, 1994

146. Zhuang Y, Kim C, Bartelmez S, Cheng P-F, Groudine M, Weintraub H: Helix-loop-helix transcription factors E12 and E47 are not essential for skeletal or cardiac myogenesis, erythropoiesis, chondrogenesis, and neurogenesis. Proc Natl Acad Sci USA 89:12132, 1992

147. Staudt LM, Clerc RG, Singh H, LeBowitz JH, Sharp PA, Baltimore D: Cloning of a lymphoid-specific cDNA encoding a protein binding the regulatory octamer DNA motif. Science 241 577, 1988

148. Corcoran LM, Karvelas M, Nossal GJV, Ye 2 - S , Jacks T, Baltimore D: Oct-2, although not required for early B-cell development, is critical for later B-cell maturation and for postnatal survival. Genes Dev 7570, 1993

149. Konig H, Pfisterer P, Corcoran LM, Wirth T: Identification of CD36 as the first gene dependent on the B-cell differentiation factor Oct-2. Genes Dev 9:1598, 1995

150. Strubin M, Newel1 JW, Matthias P: OBF-1, a novel B cell- specific coactivator that stimulates immunoglobulin promoter activ-





ity through association with octamer-binding proteins. Cell 80:497, 1995

15 1. Luo Y, Roeder RG: Cloning, functional characterization, and mechanism of action of the B-cell-specific transcriptional coactivator OCA-B. Mol Cell Biol 15:4115, 1995

152. Gstaiger M, Knoepfel L, Georgiev 0, Schaffner W, Hovens CM: A B-cell coactivator of octamer-binding transcription factors. Nature 373:360, 1995

153. Tanese N, Tjian R Coactivators and TAFs: A new class of eukaryotic transcription factors that connect activators to the basal machinery. Cold Spring Harb Symp Quant Biol 58:179, 1993

154. Grosschedl R, Giese K, Page1 J: HMG domain proteins: Architectural elements in the assembly of nucleoprotein structures. Trends Genet 10:94, 1994

155. van Genderen C, Okamura RM, Farinas I, Quo R-G, Parslow TG, Bruhn L, Grosschedl R: Development of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient mice. Genes Dev 8:2691, 1994

156. Verbeek S, Izon D, Hofhuis F, Robanus-Maandag E, te Riele H, van de Wetering M, Oosterwegel M, Wilson A, MacDonald HR, Clevers H: An HMG-box-containing T-cell factor required for thymocyte differentiation. Nature 374:70, 1995

157. Zuker CS: On the evolution of eyes: Would you like it simple or compound? Science 265:742, 1994

158. Jonsson J, Carlsson L, Edlund T, Edlund H: Insulin-promoter-factor 1 is required for pancreas development in mice. Nature 371:606, 1994

159. Dranoff G, Crawford AD, Sadelain M, Ream B, Rashid A, Bronson RT, Dickersin GR, Bachurski CJ, Mark EL, Whitsett JA, Mulligan RC: Involvement of granulocyte-macrophage colony-stimulating factor in pulmonary homeostasis. Science 264:7 13, 1994

160. Gumey AL, Carver-Moore K, de Sauvage FJ, Moore MW: Thrombocytopenia in c-mpl-deficient mice. Science 265: 1445, 1994

161. Shalaby F, Rossant J, Yamaguchi TP, Gertstenstein M, Wu X-F, Breitman ML, Schuh AC: Failure of blood island formation and vasculogenesis in Flk-1-deficient mice. Nature 376:62, 1995

162. Thomis DC, Gumiak CB, Tivol E, Sharpe AH, Berg LJ: Defects in B lymphocyte maturation and T lymphocyte activation in mice lacking Jak3. Science 270:794, 1995

163. Nosaka T, van Deursen JMA, Tripp RA, Thierfelder WE, Witthuhn BA, McMickle AP, Doherty PC, Grosveld GC, Ihle JN: Defective lymphoid development in mice lacking Jak3. Science 270:800, 1995

164. Lee EY-HL, Chang C-Y, Hu N, Wang Y-CJ, Lai C-C, Hermp K, Lee W-H, Bradley A: Mice deficient for Rb are nonviable and show defects in neurogenesis and haematopoiesis. Nature 359:288, 1992

165. Jacks T, Fazeli A, Schmitt EM, Bronson RT, Goodell MA, Weinberg RA: Effects of an Rb mutation in the mouse. Nature 359:295, 1992

166. Clarke AR, Maandag ER, van Roon M, van der Lugt NMT, van der Valk M, Hoopex ML, Bems A, te Riele H: Requiremeu for a functional Rb-1 gene in murine development. Nature 359:328, 1992

167. Davis AC, Wims M, Spotts GD, Hann SR, Bradley A: A null c-myc mutation causes lethality before 10.5 days of gestation in homozygotes and reduced fertility in heterozygous female mice. Genes Dev 7:671, 1993

168. Leveen P, Pekny M, Gebre-Medhin S, Swolin B, Larsson E, Betsholtz C: Mice deficient for PDGF B show renal, cardiovascu- lar, and hematologic abnormalities. Genes Dev 8:1875, 1994

169. Soriano P: Abnormal kidney development and hematologi- cal disorders in PDGF B-receptor mutant mice. Genes Dev 8: 1888, 1994

170. Weih F, Carrasco D, Durham SK, Barton DS, Rizzo CA, Ryseck R-P, Lira SA, Bravo R: Multiorgan inflammation and hematopoietic abnormalities in mice with a targeted disruption of RelB, a member of the NF-KB/Rel family. Cell 80:331, 1995

171. Urbanek P, Wang Z-Q, Fetka I, Wagner E, Busslinger M: Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking PaxS/BSAP. Cell 79:901, 1994

172. Lin H, Grosschedl R Failure of B-cell differentiation in mice lacking the transcription factor EBF. Nature 376:263, 1995

173. Sha WC, Liou H-C, Tuomanen EI, Baltimore D: Targeted disruption of the p50 subunit of NF-KB leads to multifocal defects in immune responses. Cell 80:321, 1995

174. Beg AA, Sha WC, Bronson RT, Ghosh S, Baltimore D: Embryonic lethality and liver degeneration in mice lacking the RelA component of NF-KB. Nature 376:167, 1995

175. Screpanti I, Romani L, Musiani P, Modesti A, Fattori E, Lazzaro D, Sellitto C, Scarpa S, Bellavia D, Lattanzio G, Bistoni F, Frati L, Cortese R, Gulino A, Ciliberto G, Costantini F, Poli V: Lymphoproliferative disorder and imbalanced T-helper response in CEBPP-deficient mice. EMBO J 14:1932, 1995




1996 87: 4025-4039

RA Shivdasani and SH Orkin The transcriptional control of hematopoiesis [see comments]

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