Margaret A. Baron - US grants

DESCRIPTION: In vertebrates, blood development begins during gastrulation and results from the induction of extraembryonic (ventral) mesoderm to form hematopoietic tissue. Although the precursors that give rise to blood cells are mesodermal in origin, tissue recombination studies with chick embryos suggest that their differentiation depends on inductive factors from the visceral yolk sac. Bone morphogenetic proteins (BMP) are members of the TGF-B family that bind to type I and II serine/threonine kinase receptors. A variety of experimental evidence has shown that BMPs have important roles in embryonic development other than bone formation. For example, BMP-4 has been demonstrated to be important in the induction of extraembryonic hematopoietic mesoderm. Targeted disruption of BMP-4 causes embryonic lethality between days 6.5 and 9.5 of gestation and results in severe defects in mesoderm formation/differentiation, including visceral yolk sac hematopoietic mesoderm. Mice homozygous null for the type I Bmp-2/4 receptor are unable to form mesoderm. BMPs may function in vivo as heterodimers. BMP-7 is expressed in many of the same or adjacent tissues as BMP-4 and BMP-4/BMP-7 heterodimers have been shown to have potent ventralizing activity in Xenopus animal cap assays. This proposal will examine how BMP-4 and related proteins function in the induction of yolk sac hematopoietic mesoderm.

DESCRIPTION (Adapted from Applicant's Abstract): The nature of the regulatory information that directs the development of an organism from the fertilized egg to an intact adult is one of the central problems in biology. The mammalian globin gene family is an ideal model system for the study of gene regulation: globin genes are expressed only in erythroid cells, and individual globin genes are expressed only during restricted stages of development. During development, the human red blood cell activates first an embryonic, then a fetal, and finally an adult gene. The long-term goal of this proposal is to elucidate the molecular basis of this "hemoglobin switching," through molecular, biochemical, and genetic analyses. The focus on the proposed research is the human embryonic beta-like globin epsilon, the earliest of the beta-like globin to be activated. The investigator has defined cluster of positive (PRE) and negative (NRE) cis- acting regulatory elements upstream of this gene. Two of the elements (epsilon-PRE II and V) interact synergistically to confer stage-specific expression on a minimal promoter. Binding of a nuclear factor from embryonic erythroid cells to a novel, conserved sequence motif within epsilon-PRE II is required for epsilon-PRE II function, strongly implicating this factor in the developmental regulation of epsilon gene expression. Interestingly, a post- transcriptionally modified epsilon -PRE II binding activity is observed in adult erythroid cells. The investigator proposes here to purify the epsilon- PRE II binding factor from embryonic erythroid cells and to isolate the corresponding cDNA(s), as necessary steps in understanding the mechanism by which proteins bound at epsilon-PRE's II and V cooperate to direct stage- specific expression in the embryo. Accordingly, a more detailed characterization of the function and protein binding properties of epsilon-PRE V will be undertaken. The role played by epsilon-PRE's and NRE's in the temporal control of the human epsilon-globin gene will be rigorously examined in vivo, in a transgenic mouse system. The long- term goal of these studies will be to determine the nature of the temporal information that specifies the timing of gene switching during development. The proposed research has a number of potential applications to human medicine. Many acquired and genetic abnormalities influence the pattern of hemoglobin production during human development. Not only the level of production but also the timing of specific development switches may be perturbed in a variety of ways. Interestingly, embryonic and fetal, but not adult, globin genes have been found to be activated in virtually all human erythroleukemia cell lines, even though little or no expression of these genes is detected after birth in normal erythroid cells. The deregulation expression of these genes in human erythroleukemia may reflect a defect in normal gene silencing, or aberrant activation, or both. An understanding of normal and abnormal globin gene control mechanisms is likely to have important medical implications, not only for the origins of hematopoietic malignancies, but also for the design of rational treatment strategies for a variety of other erythroid cell disorders.

Hematopoiesis and vasculogenesis in the yolk sac of the mammalian embryo are processes that begin during gastrulation and first require the induction of mesoderm. The first blood and vascular endothelial cells form when the extraembryonic mesoderm is induced to differentiate. However, little is known about the molecules involved in these processes during embryonic development. To examine the possibility that epithelial- mesenchymal interactions play an important role in yolk sac hematopoiesis and vasculogenesis in the mouse, we devised a novel transgenic embryo explant culture system. Transgenic embryos harvested prior to the formation of blood are stripped of their surrounding primitive endoderm (epithelium) and grown in collagen drop cultures alone or together with the isolated endoderm tissue. Using this system we have demonstrated that primitive (visceral) endoderm signaling is essential for activation of primitive hematopoiesis and embryonic vasculogenesis. These signals are short-range and diffusible and stage-dependent, becoming less potent around late gastrulation and eventually undetectable. Remarkably, primitive endoderm signaling can respecify hematopoiesis and vasculogenesis in tissue that is not fated to form blood or vascular cells, essentially repolarizing the A-P axis. The goal of this proposal is to elucidate the sources and molecular consequences of endodermal signaling and to identify some of the molecules involved in these critical developmental processes. In the first aim of the proposal, we will ask whether respecification of anterior ectoderm (fated to form neurectoderm) involves the induction of mesoderm first and activation of hematopoiesis and vasculogenesis second. We will determine how late in development repolarization of anterior ectoderm by primitive endoderm signals can still be achieved and how general is the ability of non-hematopoietic tissue to respond to primitive endoderm signaling. In the second aim of the proposal we will use the explant culture assay to begin to identify endodermal signaling molecules involved in induction of hematopoiesis and vascular growth. Finally, we will focus on hematopoietic development in explants by asking whether stem/progenitor cells are targets of endodermal signaling and whether definitive hematopoietic cells form in these cultures. These studies may have important implications not only for our understanding of normal development but may provide insights into mechanisms underlying human diseases originating at the level of stem/progenitor cells. They may also suggest new and more effective stem cell-based therapeutic approaches for hematopoietic and vascular diseases.

Hematopoiesis and vasculogenesis in the yolk sac of the mammalian embryo are processes that begin during gastrulation and first require the induction of mesoderm. The first blood and vascular endothelial cells form when the extraembryonic mesoderm is induced to differentiate. However, little is known about the molecules involved in these processes during embryonic development. To examine the possibility that epithelial- mesenchymal interactions play an important role in yolk sac hematopoiesis and vasculogenesis in the mouse, we devised a novel transgenic embryo explant culture system. Transgenic embryos harvested prior to the formation of blood are stripped of their surrounding primitive endoderm (epithelium) and grown in collagen drop cultures alone or together with the isolated endoderm tissue. Using this system we have demonstrated that primitive (visceral) endoderm signaling is essential for activation of primitive hematopoiesis and embryonic vasculogenesis. These signals are short-range and diffusible and stage-dependent, becoming less potent around late gastrulation and eventually undetectable. Remarkably, primitive endoderm signaling can respecify hematopoiesis and vasculogenesis in tissue that is not fated to form blood or vascular cells, essentially repolarizing the A-P axis. The goal of this proposal is to elucidate the sources and molecular consequences of endodermal signaling and to identify some of the molecules involved in these critical developmental processes. In the first aim of the proposal, we will ask whether respecification of anterior ectoderm (fated to form neurectoderm) involves the induction of mesoderm first and activation of hematopoiesis and vasculogenesis second. We will determine how late in development repolarization of anterior ectoderm by primitive endoderm signals can still be achieved and how general is the ability of non-hematopoietic tissue to respond to primitive endoderm signaling. In the second aim of the proposal we will use the explant culture assay to begin to identify endodermal signaling molecules involved in induction of hematopoiesis and vascular growth. Finally, we will focus on hematopoietic development in explants by asking whether stem/progenitor cells are targets of endodermal signaling and whether definitive hematopoietic cells form in these cultures. These studies may have important implications not only for our understanding of normal development but may provide insights into mechanisms underlying human diseases originating at the level of stem/progenitor cells. They may also suggest new and more effective stem cell-based therapeutic approaches for hematopoietic and vascular diseases.

DESCRIPTION (provided by applicant): The formation of specialized cell types from multipotential stem cells is a fundamental mystery of mammalian development. Primitive hematopoietic and endothelial cells form during the first differentiation events following implantation of the mouse embryo. An accumulating body of evidence indicates that hematopoietic and endothelial cells arise from a common progenitor, the hemangioblast, and very recently it has been demonstrated that blast colony-forming cells capable of giving rise to both lineages form not only in differentiating cultures of embryonic stem (ES) cells but also in late gastrulation stage mouse embryos. Little is known about the molecules involved in these processes. Using a novel transgenic embryo explant culture system we have shown that molecules secreted from an outer layer of primitive (visceral) endoderm (VE) are essential for activation of hematopoiesis and vasculogenesis in mesoderm. We have identified two distinct endodermal signals, bone morphogenetic protein-2 (Bmp-2) and Indian hedgehog (Ihh), each of which alone is sufficient to induce hematopoiesis and vasculogenesis and can reprogram the fate of the anterior embryo from prospective neurectoderm to hematopoietic and endothelial lineages. Another BMP family member, Bmp-4, has been shown by gene targeting to play an important early role in mesodermal patterning. Bmp-4 is expressed not in VE but in parts of the embryo adjacent to primitive endoderm; we have shown that this gene is also activated by endodermal signals. The proposed studies focus on the regulation of stem/progenitor cell commitment, characterization of the precursor(s) for hematopoietic and endothelial cells in the embryo, and analysis of their function in vivo. First, we will use embryo explant cultures and analysis of compound Bmp2/Ihh and Bmp4/Ihh mutants to elucidate the mechanism by which Bmp-2 and Bmp-4 function to activate hemato-vascular development. Second, we will directly examine the responsiveness of hematopoietic and angioblastic/endothelial stem/progenitor cells to recombinant BMPs and will determine whether their formation is compromised by Bmp-2 or Bmp-4 deficiency. Third, we will use mouse transplantation models to evaluate the functional activity and transplantation potential of wild type and Bmp2 or Bmp4 null mutant embryonic hematopoietic stem/progenitor cells cultured in the presence and absence of BMPs. These studies may have important implications for our understanding of normal hemato-vascular development and may suggest new stem cell-based therapies for hematopoietic and vascular diseases.

DESCRIPTION (provided by applicant): The first organ system to form in the developing mouse embryo is the cardiovascular system. Defects within the heart and vasculature are largely responsible for embryonic lethality in utero. Within the developing vascular network of the yolk sac as well as in certain intra embryonic regions, endothelial and hematopoietic cells arise in close spatial and temporal association and are thought to derive from a common mesodermal progenitor, the "hemangioblast." Recent work has indicated that hemangioblasts may also have smooth muscle cell potential. Regulation of hemangioblast and embryonic hematopoietic stem cell formation and cell fate specification are still not well understood. This proposal focuses on the characterization and functional analysis of specific subsets of mesodermal cells that give rise to hematopoietic, vascular endothelial, and smooth muscle cells during development. First, we will evaluate the hematopoietic potential and functional activity in vitro and in vivo of prospectively identified mesodermal stem/progenitorcell populations from differentiating embryonic stem cells (embryoid bodies) and from early mouse embryos. Cells will be isolated by flow cytometry on the basis of their expression of primitive cell surface markers and, using a GFP reporter transgene, on the basis of their expression of mMix, a homeodomain transcriptionfactor expressed in embryoid body subsets thought to contain hemangioblasts. The sorted cells will be analyzed using several different assays for stem/progenitor cells in culture and transplantation models in the mouse. Second, in analogous studies, we will evaluate the endothelial and smooth muscle potential and functional activity in vitro and in vivo of prospectively identified mesodermal stem/progenitor cell populations. Third, we will evaluate the role of the Mix homeodomain protein on the developmental potential and functional activity in vitro and in vivo of embryoid body derived mesodermal stem/progenitor cell populations. The origin of embryonic hematopoietic / vascular stem cells and their relationship to stem cells of the adult are unknown. With the increasing focus on regenerative medicine and interest in potential therapeutic applications of human embryonic and adult stem cells, the characterization of mesodermal stem/progenitor cell populations takes on high significance.

This is a revised competitive renewal of a grant to study activation of hematopoiesis in the mouse embryo. During the previous funding period, we used a novel transgenic embryo explant culture system to show that epithelial-mesenchymal interactions play an important role in yolk sac hematopoiesis and vascular development in the mouse. Diffusible signals from visceral endoderm mediate these interactions and can reprogram hematopoiesis in a tissue (anterior epiblast) that is not fated to form blood cells. We identified two hematopoietic-inducing VE signals, Indian hedgehog (Ihh) and Bone Morphogenetic Protein (BMP)-2, and found that they upregulate Bmp4 in explant culture. In the frog, the paired-type homeodomain transcription factor XMix.1 is induced by BMP4. Its ectopic expression in whole embryos transforms dorsal mesoderm to a ventral fate, resulting in formation of large numbers of blood cells. We cloned a mouse relative (mMix or Mixl) of the Xenopus and zebrafish Mix/Bix gene family and have generated a number of unique genetic models for analysis of its function during development. The single mouse Mix gene is expressed in the posterior VE prior to gastrulation and later in the primitive streak and nascent mesoderm in the gastrulating embryo. Although previous studies in the mouse embryo have pointed to a critical role for mMix in gastrulation, its function in the development of mesodermal derivatives remains unclear. Hematopoietic defects have been identified in differentiating embryonic stem (ES) cells in which mMix was genetically inactivated. We have recently discovered that conditional induction of mMix in ES cell-derived embryoid bodies results in acceleration of the mesodermal developmental program. A major finding to emerge from this work is that increased numbers of mesodermal, hemangioblastic, and hematopoietic progenitors form in response to premature activation of mMix. We hypothesize that mouse MX functions early in the recruitment and/or expansion of mesodermal progenitors to the hemangioblastic and hematopoietic lineages. In this application, we will: (1) identify the critical points in the mesoderm developmental program when mMix can regulate formation of hematopoietic stem/progenitor cells;(2) assess the developmental potentials of mMix- expressing cells and better define the surface antigens of the mesodermal progenitors for the hematopoietic lineage;(3) examine the consequences of deleting mMix function in mesodermal progenitors for the hematopoietic lineage in vivo. Characterization of mesodermal stem/progenitor cell populations and elucidation of the common as well as the distinguishing features of embryonic versus adult hematopoietic and vascular development will be of fundamental importance and may also advance our ability to modulate the self-renewal/differentiationof stem cells for therapeutic purposes. K.mPERFORMANCE SITE(S) (organization, city, state) Mount Sinai School of Medicine, New York, NY PHS 398 (Rev. 04/06) Page 2 Form Page 2 Principal Investigator/Program Director (Last, First, Middle): BARON, Margaret H. KEY PERSONNEL. See instructions. Use continuation pages as neededto provide the required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Role on Project Margaret H. Baron, MD PhD BARONM01 Mt. Sinai School of Med. P.I. Stuart T. Fraser, PhD SFRASER Mt. Sinai School of Med. Investigator OTHER SIGNIFICANT CONTRIBUTORS Name Organization Role on Project Michael Kyba, PhD U. Texas-Southwestern Med. Cent. Collaborator Anna-Katerina Hadjantonakis Mem. Sloan-Kettering Cancer Cent. Consultant/Collaborator Stuart Sealfon, MD Mount Sinai School of Medicine Consultant/Collaborator Human Embryonic Stem Cells E3 No Q Yes If the proposed project Involves human embryonic stem cells, list below the registration number of the specific cell llne(s) from the following list: http://stemcells.nih.gov/reaistrv/index.asp. Usecontinuationpages as needed. If a specific line cannot be referenced at this time, include a statement that one from the Registry will be used. Cell Line Disclosure Permission Statement. Applicable to SBIR/STTR Only. See SBIR/STTR instructions. d Yes d No PHS 398 (Rev. 04/06) Page 3. Form Page 2-continued Number the following pages consecutively throughout the application. Do not use suffixes such as 4a, 4b. Principal Investigator/Program Director (Last, First, Middle): BARON, Margaret H. The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description,

DESCRIPTION (provided by applicant): EryP are the first differentiated cell type to form in the mammalian embryo and play a vital role in oxygen delivery and in generating shear forces necessary for normal vascular development. Despite their abundance and indispensable functions, the development and maturation of EryP remain poorly defined. Large, nucleated EryP arise within the blood islands of the yolk sac beginning ~E7.5 and begin to circulate around E9.5, when connections between the yolk sac and embryonic vasculature mature. Several days later, small cells of the definitive erythroid lineage (EryD) begin to differentiate within the fetal liver and enter the circulation, so that the two lineages are not easily distinguished. During the previous funding period, we developed transgenic mouse systems that allow the tagging and tracking of EryP and their nuclei throughout gestation. Major findings to emerge from this work were that EryP progress through previously unrecognized stages leading to their maturation, that they are a stable population present throughout gestation and do not gradually disappear, and that they accumulate transiently within the erythroblastic islands (EBIs) of the fetal liver (FL). Concomitant with EryP migration into the FL, a dramatic increase in adhesion molecule expression occurs along with significantly increased ability to bind fetal liver macrophages (FLMs). The ability of EryP to bind to FLMs is developmentally regulated, maximal during the window of time when they are found within the fetal liver, and partly dependent on VCAM-1. Large numbers of extruded EryP nuclei are found within the fetal liver at the time the first enucleated EryP are detected in the blood. EryP nuclei can be identified within FLMs after co- culture and in the native fetal liver, in vivo, suggesting that they are cleared and degraded by macrophages. After enucleation, the ability of circulating EryP to adhere to macrophages is lost and their numbers in the FL decline. We hypothesize that the fetal liver is a developmental niche for the maturation of primitive erythroblasts and that terminal steps in EryP maturation, including enucleation, occur in the EBIs of the fetal liver and involve adhesive interactions with macrophages. The fetal liver is just developing as EryP begin to circulate, around E9.5. Our observations therefore suggest a simple solution to the puzzling question of why enucleation of EryP is not detected until days after their appearance: terminal maturation, including nuclear extrusion, occurs in the fetal liver, which does not form until midgestation. The tools we have developed during the previous funding period will allow us to study the biology of primitive erythropoiesis at a resolution not previously possible. We propose to (1) determine whether macrophages provide a microenvironment for EryP maturation within the fetal liver;(2) evaluate the roles of integrins and their receptors in the maturation of primitive erythroblasts;and (3) investigate molecular events underlying the final stages of erythroid maturation. PUBLIC HEALTH RELEVANCE: Characterization of progenitor cell populations and elucidation of the common as well as the distinguishing features of embryonic versus adult erythroid development will be a prerequisite for the directed differentiation of human ES cells, HSCs or hematopoietic progenitors for therapeutic purposes in patients and for the efficient production of pure populations of red blood cells for transfusion. Pathways involved in erythroid development in the embryo may be dysregulated in leukemias and myelodysplastic disorders. The proposed studies should therefore be of broad biomedical significance.

DESCRIPTION (provided by applicant): The objective of this new proposal is to use an interactive, collaborative, multidisciplinary approach to train talented young physician-scientists (MD and MD PhD) or PhD scientists for a successful career in investigative hematology research. The rationale for developing this program is our belief that Mount Sinai School of Medicine offers diverse scientific opportunities in an outstanding research environment, that we have a faculty that is dedicated to training scientists and physician-scientists for a career in academic hematology, stem cell biology, and regenerative medicine, and that we wish to have an impact on the education of future investigative hematologists. Faculty has been integrated into this program from various departments and institutes and from the Rockefeller University. Candidates for this training grant will be expected to devote a minimum of two years to training in basic or translational hematology. The trainees will select a mentor from one of the eight broad areas encompassed by this program: normal and malignant hematopoiesis, developmental hematopoiesis, adult and embryonic stem cell research, analysis of signaling pathways in hematologic processes, cell adhesion and migration within hematopoietic tissues and the vasculature, normal coagulation and inherited clotting disorders, immune cell development and function, and cell and gene therapy and transplantation. Throughout the training period, trainees will devote at least 90% of their time to research. Trainees will focus on an individualized research project under the guidance of one or more faculty preceptors. In addition, they will participate in weekly laboratory meetings, spend a portion of their time attending specific divisional, departmental and institutional research-oriented conferences, and will enroll in didactic, graduate level courses during the first and second years of training. Along with the enormous growth in the research portfolio of the Division of Hematology and Medical Oncology and the recruitment of new faculty over the past decade, we are now in a very strong position to provide an exceptional training opportunity that should be a magnet for the many outstanding trainees who wish to pursue a career in academic hematology research in New York City.

DESCRIPTION (provided by applicant): This is a competitive renewal for the Medical Scientist Training Program (MSTP) at Mount Sinai School of Medicine (MSSM) whose mission is to educate future physician-scientists in a rigorous integrated joint degree program in an environment that promotes cutting-edge biomedical research. These trainees will become independent investigators and leaders who will apply basic science discoveries to improved healthcare in all communities. During the past five years, MSSM has continued to increase in ranking as one of the nation's top translational biomedical research institutions. We implemented a Strategic Plan with an Institute organization cutting across departments that strengthened our translational goals. A major centerpiece of the organization is a new translational research building, The Center for Science and Medicine, that will increase our research capacity by 30% and the recruitment of ~60 new faculty. This institutional translational research growth along with prominent and experienced scientists with major NIH funding who serve as excellent mentors has been paralleled with a significant increase in the number of highly qualified applicants to our MST program and increased success of our currents matriculants with publications in highly ranked journals and increased numbers of individual training grants. In addition, we are increasing efforts to enhance the diversity of our student body as data has shown that participation in our program significantly enhances their development and success. As such, we are asking for an increase in the number of MSTP trainees supported by the NIH to 28. The MSTP has a new Director, Dr. Yasmin Hurd, who together with previous Directors in the program, Drs. Lisa Satlin and Terry Krulwich, who now serve as Associate Directors, have managed a seamless transition in leadership. Ongoing changes in the Graduate School curriculum, including 8 Multidisciplinary Training Areas aligned to the new Institutions, plus the continuous emphasis on integrating clinical exposure during the PhD phase and the medical school curriculum that allows graduate school courses to be taken in conjunction with the preclinical medical school coursework provides significant flexibility for our trainees and truly promotes integrative biomedical research and clinical training. Altogether, our program provides a strong foundation to build successful careers as physician-scientists.

? DESCRIPTION (provided by applicant): Red blood cell progenitors undergo self-renewing divisions prior to the commitment switch to erythroid differentiation but the pathways that regulate erythroid progenitor growth are still largely unknown. In a computational search for genes expressed in definitive (adult) but not primitive (embryonic) red cell lineages, we identifie the nuclear receptor transcription factor VDR, which is activated by binding to its ligand, vitamin D3 (1,25(OH)2D3). Real-time RT-PCR analysis indicated that Vdr is expressed in definitive erythroid progenitors from mouse fetal liver and bone marrow and is downregulated during erythroid maturation. Structural studies have shown that VDR activation by the vitamin D3 ligand results in significant conformational changes that stabilize the protein and induce its translocation into the nucleus, where it recruits coregulatory complexes. The VDR signaling pathway has been studied mostly in bone. The regulation of erythropoiesis by this pathway has been essentially unexplored; published studies were performed almost entirely in leukemic cell lines (not normal primary cells). We find that vitamin D3 stimulates the growth of erythroid progenitors (BFU-E and CFU-E) from mouse fetal liver and bone marrow. Not only the numbers but also the size of the BFU-E colonies is increased when the VDR pathway is activated. The CD71low subset of c-Kit+ fetal liver progenitors, which contains BFU-E, is the most sensitive to activation of VDR. Progenitors cultured with vitamin D3 differentiate normally. Vitamin D3 can partially substitute for dexamethasone (a glucocorticoid) in progenitor cultures, suggesting a possible role in stress erythropoiesis. We hypothesized that VDR activates or represses genes in developing erythroid progenitors in combination with erythroid transcription factors (TFs) such as Gata, Klf1/Eklf, and Scl/tal and that DNA binding sites for VDR may cluster with binding sites for erythroid TFs. A computational approach based on this hypothesis was used to identify candidate VDR target genes in erythroid progenitors. We have begun to confirm the vitamin D3 responsiveness of some of these genes using real-time RT-PCR. The candidates include genes known to function in erythropoiesis (c-Myc, Gata2) and others that were not (Calcium binding kinase Camk1d; N-Myc; Mlx-interacting protein, a bHLH transcription factor; Grtp1, GTPase Rab activator). We hypothesize that the VDR pathway controls erythroid progenitor cell proliferation and/or survival, at least in part through transcriptional regulation of target genes. The goal of this project is to elucidate the molecular mechanisms by which vitamin D regulates red blood cell development.

ABSTRACT: The pathways that regulate the formation and differentiation of erythroid progenitors to red blood cells are incompletely understood. We found that the vitamin D receptor (Vdr) nuclear hormone transcription factor gene is expressed in fetal and adult stages but not at the embryonic stage of development and is downregulated during maturation. VDR activation by its ligand vitamin D3 results in conformational changes that stabilize the protein and induce its translocation into the nucleus, where it recruits coregulatory complexes. The VDR signaling pathway has been studied mostly in bone but has been largely unexplored in erythropoiesis: published studies were performed almost entirely in leukemic cell lines (not normal primary cells). Activation of Vdr signaling by the vitamin D3 agonist calcitriol increased the outgrowth of EryD colonies from fetal liver and adult bone marrow, maintained progenitor potential, and delayed erythroid maturation. The stimulation in growth of erythroid progenitors resulted in a large increase in the numbers of mature red blood cells. The early (CD71lo/neg) but not the late (CD71hi) EryD progenitor subset of Linneg cKit+ cells was responsive to calcitriol, independently of its calcemic effects. Activation of VDR could partially substitute for and synergize with the stress glucocorticoid dexamethasone in enhancing progenitor proliferation compared to either ligand alone, suggesting a role in stress erythropoiesis. This possibility is supported by our finding that an erythroid specific deletion in Vdr that interferes with DNA binding results in a reticulocytosis that occurs earlier and is more pronounced than in control animals in response to stress. RNA inhibition of Vdr expression abrogated the stimulation of early erythroid progenitor growth by calcitriol. These findings suggest that Vdr has a cell-intrinsic function in early erythroid progenitors. Activation of Vdr by calcitriol blocked the upregulation of erythroid transcription factor genes Gata1, Fog1 and Klf1. Intriguingly, circadian rhythm genes are upregulated by activation of Vdr and the glucocorticoid receptor Gr and oscillations in expression of the clock gene Per1 are promoted in erythroid progenitors. The clock gene Bmal1 is required for the proliferative response to dexamethasone. Therefore, the overarching hypothesis of this proposal is that Vdr and Gr regulate erythroid progenitors in part by modulating clock gene expression and have partially redundant functions. This application will use animal and cell culture models to explore the modulation of circadian clock gene expression by Gr and Vdr in erythroid progenitors and functional relationships between these two nuclear hormone receptor TFs. These studies may lead to the identification of novel molecular targets in erythroid progenitors that can be exploited to develop new therapies for anemias and other red cell disorders. The ability to modulate ex vivo expansion or differentiation of RBC progentors in new ways would have clear clinical utility.