Projects and Grants
Our research program combines multiple complementary approaches to study the mechanisms by which cell fate decisions are regulated in stem and progenitor cells, with a focus on hematopoiesis and cardio-vascular development. We used a novel explant culture assay to show that signaling interactions between endoderm and mesoderm play a role in hematopoietic and vascular induction in the early mouse embryo. One such signal, Indian hedgehog (Ihh), may function, in part, through upregulation of Bone Morphogenetic Protein 4 (BMP4). We are analyzing the hematopoietic and vascular defects in mice carrying mutations in genes encoding components of the Hh and BMP signaling pathways. The activities of small molecule Hh agonists and antagonists will be tested in mouse and human stem/progenitor cell systems, with a view toward translation to clinical medicine. A related project involves the mouse and human homeodomain transcription factor, Mix. Genetic approaches have implicated this protein in the formation and/or patterning of mesodermal and endodermal tissues during development. We are using genetically manipulated mice and embryonic stem (ES) cells to elucidate the roles of Mix in mammalian development. Biochemical approaches are being used to study the transcriptional activity of Mix. Gene expression profiling has been used to identify potential target genes. In ongoing collaborations with M. Dickinson at Baylor and K. Hadjantonakis at Sloan-Kettering, we are imaging the development of the hematopoietic and vascular systems in real time, using laser-scanning confocal microscopy.
The overall goal of our research is to decipher mechanisms that serve to establish and regulate mammalian erythropoiesis. As a result, differentiating embryonic stem cells in culture are being used as one powerful approach to address these issues in diverse ways: to identify extracellular molecules and illuminate the intracellular pathway they use to play a directive role in erythroid gene expression; to functionally test the cis-acting sequences that control one of these downstream targets known as EKLF, itself a critical erythroid transcription factor; to establish gain-of-function studies that address lineage determination mechanisms during developing and differentiation; and to gain further insight into globin switching mechanisms and identify ways to alter the normal pattern of expression.
Our primary interest focuses on the mechanisms that lead to the specification of the progenitor cells of individual mesodermal tissues from pluripotent mesodermal precursors during early embryonic development. In particular, we are studying combinatorial signaling and transcriptional processes that regulate the progressive commitment of cells during the acquisition of heart and muscle fates. We are using the genetic model organism Drosophila to identify new genes and developmental mechanisms that control these processes of early organogenesis and of the development of certain stem cell-like cells during muscle development. We anticipate that the known evolutionarily conservation of genes and processes between Drosophila and vertebrate species will allow us to transfer our acquired knowledge to the studies of related processes of cardiac and muscle cell specification, as well as stem cell differentiation, in mammalian systems.
The trafficking of hematopoietic stem cells (HSCs) is critical during development and underlies modern clinical stem cell transplantation. In my laboratory, we are interested in understanding the molecular mechanisms that mediate HSC homing to and egress from their bone marrow niches. Please consult our recent publications and our laboratory web site for detailed information.
Dr. Germano's laboratory research focuses on the treatment of malignant gliomas, one of the most challenging issues in neuroscience. Gene therapy strategies currently in clinical trials use viral vectors to deliver therapeutic transgenes directly to normal and tumor cells within the central nervous system. Viral vectors, however, have a number of theoretical and practical limitations. Embryonic stem cells (ES) are totipotent cells with unlimited proliferative capacity, and, unlike other cell types, can be permanently and precisely genetically modified without the use of viral vectors. In collaboration with Dr. Keller, Dr. Germano and her team are currently investigating how these cells can be used as vectors to carry genes into the central nervous system for adjuvant treatment of brain tumors.
A highly regulated balance between quiescence and proliferation is required in order for hematopoietic stem cells to respond to daily demands and to maintain life-long production of blood cells. Very little is known about molecular mechanisms that control this balance. Deregulation of processes that control hematopoietic stem and progenitor cell fate decision leads to human leukemias and other hematological disorders. My laboratory is interested in elucidating the regulation of signaling pathways and transcriptional programs that control hematopoietic stem and progenitor cell fate. We are particularly focused in the potential role of the PI3-kinase/AKT signaling pathway and its target FOXO, a family of forkhead transcription factors, in stem and progenitor cell behavior. This signaling pathway is known to regulate cell cycling, apoptosis, metabolism, adhesion, differentiation, and oxidative stress in various cell types and its deregulation has been implicated in many cancers. By combining cellular, molecular and transgenic animal models we are addressing the role of PI3-kinase/AKT/FOXO in the normal regulation of hematopoietic stem and progenitor cells in vivo and using the embryonic stem cell system we are investigating the molecular mechanisms whereby this pathway is controlled in vitro. These studies should shed light on signaling networks that regulate stem cell fate and their susceptibilities to oncogenesis.
The use of neural stem cells holds great promise for repairing neural circuits damaged by injury or disease. Successful repair requires an understanding of how growing axons recognize their correct target neurons. This is important because the consequences of inappropriate axon-targeting can be severe and maladaptive. Dr. Huntley's laboratory studies molecular mechanisms of synaptic circuit formation, targeting and plasticity. Current focus is on the role of the cadherin family of cell adhesion molecules and extracellular proteases in orchestrating connection formation during development and in forms of synaptic plasticity thought to underlie learning and memory, and in enabling maladaptive intraspinal sprouting of nocieptive primary afferent sensory axons in models of neuropathic pain.
The focus of my research is to understand the molecular and cellular nature of the undifferentiated stem cell "states", and how such states are altered during a change in cell fate. The underlying rationale for our studies is that the complement of gene-products and their inter-relationships that exist in stem cells accounts for their remarkable abilities to balance self-renewal and differentiation decision processes. We study both adult and embryonic stem (ES) cells, primarily from the mouse, but also from the human. As a first step, we have comprehensively identified most, if not all gene-products that are expressed in highly purified hematopoietic stem cell (HSC). We performed similar analyses in mouse ES cells. Such molecular "signatures" provide parts-lists that are available to the stem cells. The challenge has been to functionally address the roles that these molecules play in mediating the biological properties of HSC and ES cells. Further, we would like to understand how these molecular components are "wired" into regulatory signaling and transcriptional networks. To explore these issues we have utilized a number of global gene-expression perturbation technologies, such as inhibitory short hairpin RNA (shRNA). We have successfully down regulated the expression levels of candidate regulatory molecules in both HSC and ES cells. A number of these play crucial regulatory roles in processes such as self-renewal, proliferation, and differentiation. We have further developed strategies that allow the analyses of cell-fate change dynamics at multiple biochemical and molecular levels in response to defined and precisely controlled changes in the expression levels of key regulatory molecules. These strategies have provided the first in-depth view of how a cell-fate decision actually occurs at the transcriptional, post-transcriptional, translational, and post-translational levels. An important aspect of our overall efforts is computational and quantitative analyses. We anticipate that our approach will yield a systems biology level description and understanding of stem cell decision processes. This in turn, will have profound implications in future efforts focused on applying basic stem cell research in translational as well as clinical contexts.
The main interest of our laboratory is to study the role of the TSH (thyroid stimulating hormone) signaling pathway in thyroid development and disease. TSH and its receptor TSHR are key regulators of thyroid cell functions during embryogenesis and in the adult. TSH resistance is one of the causes of congenital hypothyroidism - the most common inborn endocrine disorder. One in every 3500 newborns is affected by the condition, which is due primarily to developmental defects leading to an absent, ectopic or hypoplastic thyroid gland. There are two areas of research in our laboratory. One part of the laboratory studies how the pluripotent embryonic stem cells respond to external signals to give rise to thyrocytes. The second area of research in the laboratory is the characterization of the early events involved in the establishment and maturation of embryonic thyroid gland. We utilize a broad range of techniques encompassing cell, molecular and developmental biology. We also employ transgenic and knockout technologies in mice. In addition, our studies may pave the way for the use of human embryonic stem cells as a model system to study thyroid development, and potentially the role of abnormal cell development in the genesis of thyroid disease.
One of the major focus of the lab has been on Langerhans cells (LCs). LCs are the only DC of the epidermis and are localized in the basal and suprabasal layers of the epidermis, where they represent the first hematopoietic barrier with the enviroment. LCs are well equipped to ingest foreign antigens that breach the skin mucosa. LCs are also found in skin lmphatics in steady state conditions in both animals and human and it is postulated that LCs migrate during steady state conditions to maintain or induce peripheral tolerance to skin antigens, which may be critical for the prevention of skin autoimmune disease. We have recently found that in contrast to other DC populations, LCS self-renew in quiescent neonatal and adult skin, throughout life, and are replaced by circulating precursors only in injured skin. These results suggest that fetal skin educates and programs the development of LCs via signals that are lost in mature and quiescent adult skin. The project aims to explore when the seeding of LCs occur in the skin during development and to identify a skin LC stem or precursor cell that maintains the LC pool during steady state conditions. Another aspect of this project aims at identifying a LC precursor present in the blood or BM that mediate LC replacement during skin injury and exploring the mechanisms important for LC recruitment to inflamed skin. We would also like to determine the mechanisms that govern LC homeostasis in human skin.
More recently, we found that in addition to LCs, dermal DCs fail to equilibrate between parabiotic mice that shared the same blood supply for six months, complete mixing of dermal macrophages and that dermal DCs are able to proliferate in situ, in both human and mice skin, altogether, these results suggest that some DC populations to have evolved to become autonomous and able to maintain themselves locally. This DC homeostatic behavior may be specific to the skin, as lymphoid organ DCs have also been shown to proliferate locally to maintain DC homeostasis in situ. Therefore, my laboratory will try to identify other tissue DC precursors and characterize the mechanisms that regulate the development of these DC precursors in earl and adult life.
Moore, Kateri D.V.M.
There is a cellular milieu that surrounds and supports the blood forming or hematopoietic stem cell. Throughout adult life these stem cells are present within the bone marrow and are thought to be located in apposition to the endosteal surface of the bone. Stromal cellular elements provide a unique microenvironmental space or niche that mediates the extrinsic molecular signals involved in the balance of self-renewal and commitment decisions of stem cells. My work is focused on defining the most primitive stem cell microenvironmental niche at the cellular and molecular level. Our previous studies have shown that a cell line, AFT024, can maintain competitive repopulating stem cell activity that is qualitatively and quantitatively identical to that present in freshly purified cells. We suggest that the AFT024 stromal cell line provides a unique and positively acting molecular milieu that maintains self-renewing stem cells. To isolate the components of this milieu we have constructed a subtracted cDNA library enriched for molecules preferentially expressed in this supporting line. We have assembled a biological process oriented Web-based interface, the Stromal Cell Database (StroCDB). Candidate genes from this database are being tested in gain and loss of function studies and are being used to develop mouse models. These genetically modified mice provide model systems to both manipulate and visualize stem cells and their niches in vivo under normal homeostasis and/or after systemic and/or specific microenvironmental perturbation. In addition, we are investigating the "molecular cross-talk" that occurs when stem cells and stromal cells interact under a variety of conditions. This research will provide invaluable insights into the intrinsic and extrinsic mechanisms that balance the self-renewal and differentiation of hematopoietic stem cells and perhaps of all stem cells.
The central nervous system forms in vertebrate embryos as a result of inductive interactions between competent ectoderm and a special dorsal signaling center, known in amphibians as the Spemann organizer. Complex interactions between the products encoded by these genes result in the differentiation of neurons and glial cells, organized in a specific mediolateral and anteroposterior pattern. Several signaling pathways, triggered by bone morphogenetic proteins, fibroblast growth factors and Wnt proteins were implicated in early neural development in vertebrates.
In Drosophila neuroblasts, cell fates are regulated by asymmetric distribution of molecular determinants and the direction of the mitotic spindle. In vertebrate embryos and mammalian cells the significance of asymmetric division and spindle orientation for cell type specification is less well studied. We would like to understand the role for mitotic spindle orientation and asymmetric cell division of NPCs in neuronal cell fate determination.
Our studies demonstrate that Lgl is required for cell polarity and asymmetric cell division during primary neurogenesis in Xenopus ectoderm, and its localization may be controlled by Wnt signaling (Dollar et al., 2005, and not shown). As neural stem cells may undergo similar asymmetric divisions, we hypothesize that the role of Lgl as an essential regulator of neuronal differentiation is preserved in these cells. We study the subcellular localization of Lgl and Par proteins in cells undergoing neural differentiation and assess how modulation of their levels or localization would influence neural tissue differentiation. The knowledge of molecular mechanisms regulating neuronal differentiation should have implications on stem cell research and regenerative medicine.
Additionally, our studies show that Frodo, a new signaling protein, is required for the specification of neural tissue. Frodo is a founding member of a family of related proteins that operate in multiple signaling pathways (Brott and Sokol, 2005). Using antisense morpholino oligonucleotides (MO), an efficient in vivo method of inactivating gene products, we have demonstrated an essential role for Frodo in neural development (Hikasa and Sokol, 2004). As Frodo is a component of the Wnt signaling pathway, we use transgenic mice and frog embryos to investigate a role for Wnt signaling in asymmetric cell divisions during neurogenesis.
A tight balance between the self-renewal of hematopoietic stem cells (HSC) and their differentiation into specific blood cell lineages is critical for the production of normal numbers of blood cells throughout our life span. Defining the signaling pathways and transcriptional machinery regulating these events is essential to understand the control of lineage commitment within the hematopoietic system and ultimately to enable the manipulation of these decisions in HSC both in culture and in vivo. My laboratory has focused on the analysis of quantitative genetic variation in the hematopoietic stem cell compartment of the mouse to define regulatory pathways that are relevant in vivo. Using this strategy, we have identified transforming growth factor-beta2 (TGF-b2) as a positive regulator of the cycling activity of HSC. Furthermore, this approach allows us to analyze the organismal consequences of genetic variation in the stem cell kinetics. These include the lethality of cytotoxic agents and the progression of age-related changes in the hematopoietic system, in particular in T cell development.
Venugopalan, Nair Ph.D.
Our research focuses on the molecular mechanisms of midbrain dopamine neuron development, survival and death. Degeneration of midbrain dopamine neurons is thought to play key roles in Parkinson's disease. We study dopaminergic neuronal development and differentiating of embryonic stem cells in order to define the molecular genetic programs that enable survival of dopamine neurons in experimental and therapeutic models. Understanding the signaling mechanisms contributing to the development and demise of embryonic stem cell derived dopamine neurons may lead to clinical applications that can treat neuronal loss in Parkinson's disease.
Weinstein, Daniel Ph.D.
All tissues in the animal derive from the three primary germ layers: ectoderm endoderm, and mesoderm. Endoderm derivatives contribute to the organs of the gut, while ectodermal derivatives form the epidermis and central nervous system. The mesodermal germ layer plays a pivot role in organizing the vertebrate body axes, and itself gives rise to the muscular, skeletal, and circulatory systems. My laboratory's effort focuses on elucidating the mechanisms underlying germ layer formation and patterning in the frog, xenopus laevis. Our present studies are concerned primarily with the mechanisms that restrict inappropriate mesoderm and endoderm formation during early development. Recent work in our lab suggests that several members of the Fox family of transcription factors function as mesendodermal antagonists during Xenopus gastrulation. Work in the laboratory is focused on characterizing the mechanisms underlying Fox mediated germ layer suppression, using a combination of molecular, biochemical, and embryological approaches.
The mouse ES/EB syatem is used to specify mesodermal lineages and to derive specific renal epithelial progenitors by optimization of tissue culture, activin incubation and FACS sorting conditions. To date, this has enabled the isolation and enrichment of proximal tubule and collecting duct progenitor populations as assessed by RT-PCR marker analysis in vitor. Lineage tracing and double immunofluorescence after injection into fetal kidneys in organ culture as well as newborn mouse kidneys in vivo is used to assess functional incorporation into mesenchymally-derived proximal tubule and ureteric bud-derived collecting tubular epithelia respectively. Studies are underway to purify these cell populations; assess their full differentiation potential in vitro and in vivo and to test their efficiacy to repair and restore function to mouse models of renal injury, that left untreated lead to renal failure.
My laboratory has a long-standing interest in the biology of the limbal/corneal epithelium. Limbal damage from various etiologies causing the irreversible stem cell loss results in the functional failure of the stem cell-free epithelium overlying and protecting the critical corneal domain. The ensuing scarring and neovascularization of the otherwise transparent cornea leads to severe loss in visual acuity including complete blindness. To develop the basic cellular knowledge needed to advance ocular reconstructive procedures, we have recently isolated the limbal stem cell and are studying its molecular make-up, and functional characteristics. Cell culture studies seek to identify condition for maximal replication of this cell with preservation of undifferentiated properties. Microarray studies are currently being pursued to determine its gene expression patterns.
An application of the genome-targeted transgene delivery system is in murine and human embryonic stem cells. Differentiation-specifying genes expressed under inducible promoters might be delivered to the ES cells that will direct their development into specific tissue cell types at the appropriate time frame, and immune-modulating genes under tissue specific promoters might be delivered to abrogate immune responses in allogeneic hosts against the transplanted grafts that were derived from ES cells. Active collaborative research studies are being conducted with Drs. Gordon Keller and Jonathan Bromberg in the Black Family Stem Cell Institute