Maintenance and Differentiation of Stem Cells
in Development and Disease
Projects and Principal Investigators
Section A: Mechanisms of Stem Cell Self-Renewal
Section B: Cell-cell interactions in the stem cell niche
Stem cell recruitment in Hydra pattern formation
We study the role of stem cells in the regeneration process of hydra, a member of the ancient group of cnidarians exhibiting a unique regeneration capacity. Based on a comprehensive (phospho-) proteome and transcriptome approch using a SILAC hydra carried out in the first funding period, we established that the initial phase of regeneration, the injury response, is followed by a large net upregulation of proteins and an activation of Wnt-Dkk and Activin/ Nodal signaling. Building on these results, we will now elucidate how positional information is transmitted from the proteome level during injury response to the transcriptome level resulting in stem cell behavior. These studies will include functional approaches including whole animal regeneration in microfludic devices, as well as analyses of stem-cell specific gene regulatory networks.
Understanding Drosophila intestinal stem cell-derived tumors
Drosophila intestinal stem cells (ISCs) can be prevented from differentiating by blocking Notch signaling, and can grow as tumors outside their intestinal niche, in the fly's circulatory system, provided that Ras signaling is also activated. This project will use these stem cell-derived tumors to identify critical niche-derived growth factors and elucidate mechanisms of ISC self-renewal. The ability of Ras to confer niche- independent ISC growth will be deconstructed by genetic substitution of Ras in tumor transplantation assays. Host requirements for niche- independent ISC growth are determined, this information will be used to develop an in vitro ISC culture system. This system will than be used to live-image signaling interactions between ISCs, their progeny, and niche cells during ISC self-renewal and differentiation. Live imaging of the stem cell lineage should allow us to resolve outstanding questions about how asymmetry is established following ISC division, and how stem cell pool size is regulated.
Transcriptional control of stemness in the ciliary marginal zone of the vertebrate retina
Vertebrate neural stem cells have retained the life-long proliferative potential, which faciliates their differentiation into all cell types of the CNS. Fish and amphibia in addition show continous, life-long growth of the central nervous system. We have shown that the fish retina contains a stem cell niche, the ciliary marginal zone (CMZ), with constitutively multipotent retinal stem cells that give rise to all cell types in the growing and likely also regenerating retina. We have identified these stem cells to express the transcription factor Rx2, a regulatory hub that integrates activating and repressing regulatory input. We plan to address how Rx2 molecularly confers identity to the retinal stem cells and how it defines the bipartite RSC niche. For that we will decipher the downstream network that controls the establishment and dynamic maintenance of the CMZ in vivo by combining advanced genetics, bioinformatics and imaging approaches.
The role of the Wnt secretory factor Evi in stem cell maintenance and differentiation
Wnt signaling pathways are of paramount importance during development and stem cell maintenance. While much effort has been spent on understanding the signaling cascades downstream of the receptors, the physiological consequences of modulating autocrine Wnt production has received less attention. As part of this project, we aim to dissect Wnt regulatory functions in mouse embryonic stem cells and MSCs using the Wnt cargo receptor Evi/WIs as a tool to create "Wnt-silent" stem cells. Using rescue experiments with distinct canonical or non-canonical Wnt proteins we will analyze their signaling and transcriptional circuits in stem cells and their role in stem cell proliferation and differentiation. We will further create in vivo models to study the reversible loss-of pan-Wnt production during adult homeostasis.
Metabolic heterogeneity among fish stem cells in an intact niche
The recent development of tools to study the lineage from single cells in vivo has changed our view on how individual stem cells behave within a population, and revealed an immense heterogeneity regarding their symmetric or asymmetric divisions, proliferative capacity and differentiation potential. Why would two neighbour stem cells behave so different? In this proposal we will exploit the simple composition of fish neuromasts, which contain three main cell types and around a hundred cells in total, to perform a systematic and quantitative 4D analysis of lineage progression and variability among stem cells. I will use established (and develop new) metabolic reporteres in vivo, to link the metabolic state of a stem cell to its future clonal progression in their intact niche. The same tools will be used in a parallel and complimentary approach under establisehd paradigms of organ regeneration and stem cell quiescence.
Studying lung stem cells in tissue maintenance, repair, and cancer
Cellular and molecular mechanisms of normal lung stem cell homeostasis and the regulation of their malignant counterparts in lung cancer are poorly understood. It is unclear if a single multipotent lung epithelial stem cell exists, or if different stem cell populations drive the regeneration of the epithelium in different areas of the lung. We will address the "one-versus-multiple-stem-cell(s)" question via unbiased lineage tracing. In addition, we aim to identify lung epithelial stem cell activity, ascribe this activity to a marker defined cell type, and understand regulatory pathways within this cell type using a genetic injury mouse model. Finally, we will determine the role of co-dependencies in mutant Kras-induced lung tumor formation and cancer stem cell function.
Clonal heterogeneity in leukemic and ‘pre-leukemic’ stem cells from patients with AML
Clonal heterogeneity occurs in many cancers, including Acute Myeloid Leukemia (AML). In cases of relapse, chemotherapy has triggered clonal selection with minor or evolved sub-clones driving resurgent disease. A better understanding of the underlying clonal architecture, the extent of genetic heterogeneity in leukemic stem cells (LSC) and its response to therapy will elucidate mechanisms of therapy escape and possibly provide novel princples for drug targeting, In diagnostic AML samples normal hematopoietic stem cells (HSC) carrying disease specific mutations have been reported, but their contribution to relapse remains so far elusive. The goals of this project are: (1) to define the clonal architecture of AML during the course of therapy and in leukemia propagating cells; (2) to define the relevance of leukemia driving mutations in HSC derived from patients with AML.
The role of centrosomes in asymmetric cell division of stem cells
Many stem cells divide asymmetrically and thereby generate mother and daughter cells with distinct cell fates. The positioning of the two centrosomes, which define the spindle poles of the mitotic spindle, is critical for asymmetric cell division, as the spindle determines and/ or cellular components depend on the correct orientation of the mitotic spindle accordingly to the cell polarity axis. Several lines of evidence suggest that the two centrosomes of a cell are inherently distinct from one another. This property seems to be crititcal for spindle orientation and the asymmetric outcome of stem cells. Here, we will investigate how centrosome asymmetry is established, regulated and monitored in normal and cancer stem cells.
Towards a mechanistic framework of plant stem cell control
Using our findings from the first funding period as a springboard, we will now define a regulatory framework for plant stem cell control, which underlies the balance between proliferation and differentiation in the continously active shoot apical meristem. To this end we will follow an interdisciplinary approach build on experimentation and mathematical modeling. On the one hand, we will decipher the mechanisms underlying communication between shoot stem cells and their niche using the WUSCHEL and HECATE1 stem cell regulators as a paradigm for non-cell autonomous transcription factor activities. While WUSCHEL protein itself is able to move from niche cells into stem cells, HECATE1 cell autonomously activates mobile downstream factors in stem cells, allowing us to study diverse mechanisms of cell-cell communication within the apical stem cell niche. On the other hand, we will use shared transcriptional and epigenetic targets of WUSCHEL and HECATE1 as a rich resource to identify and characterize novel and important players in the plant stem cell control network. Building on data from the first and second funding period, we will develop a mathematical model of plant stem cell control with predictive functions.
Developmental control of the Drosophila male stem cell niche
In the context of the SFB 873 we have shown that the Hox genes abd-A and Abd-B play critical roles in balancing stem cell maintenance and differentiation in the Drosophila male stem cell niche, as well as in positioning the niche and ensuring its integrity. Furthermore, we have identified testis- specific Abd-A and Abd-B chromatin binding regions and will now use this resource to elucidate the cis-regulatory logic of Hox activity in the testis, as well as the function of Hox targets, which translate this important regulatory input to appropiate cell behavior. In addition to many known stem cell regulators, we find many less characterized genes among the identified Hox realizators, opening new avenues to study stem cell control by developmental master regulators at the mechanistic level.
Role of polarized Wnt signalling in embryonic cell fate regulation
LRP6 is a Wnt coreceptor which plays a key role in transducing b-catenin signaling, notably in stem cells. The Wnt co-receptor Lrp6 is asymmetrically localized to the basolateral membrane in pluripotent ectodermal blastomeres in Xenopus embryos. Lrp6 asymmetry is controlled by Wnt/PCP signaling, indicating that this pathway not only regulates planar- but also apicobasal cell polarity. Following asymmetric cell division Lrp6 preferentially sorts to the deep ectodermal cell layer and becomes depleted in the epithelial cell layer. This is accompanied by elevated Wnt/b-catenin signaling in deep cells, which in turn promotes their differentiation into ciliated cells. These results indicate that coordinated Wnt/PCP and Wnt/b-catenin signaling convert apicobasal polarity information to specify ectodermal cell fate. We now study the regulation of LRP6 in embryonic polarity by examining the signaling pathways and downstream targets involved in Xenopus and mouse.
Molecular control of cellular heterogeneity within the hematopoietic stem cell compartment
Dormant hematopoietic stem cells (dHSC) harbor the highest self-renewal activity and generate active HSCs (aHSCs), followed by a series of multipotent progenitors (MPP) that differentiate into lineage-committed progenitors and finally mature cells. During the last funding period we determined the role of PTEN and Myc in HSC self-renewal and revealed a new Myc target Granzyme B as a novel regulator of HSC function. Most importantly for the coming funding period, we have performed a global quanitative transcriptome (RNA-seq) and proteome analysis of the seven major stem/ progenitor cell types present in the bone marrow. We will now use these data sets to identify the first cell surface marker set for dHSCs necessary to elucidate their role during normal and stressed hematopoiesis. Moreover, we will explore the unexpected recently revealed cellular heterogeneity within the stem cell compartment and develop strategies to prospectively isolate novel stem cell types with lineage-restricted potential. These linage- restricted stem cells (LiR-Scs) are thought to be present at the helm of the hematopoietic hierarchy next to conventional HSCs. Finally, we will genetically explore the role of MEG3 in vivo, a highly differentially expressed IncRNAs in HSCs with tumor suppressor function.
Endothelial cell fate and angiocrine control of stem cell function
Project B06 has two specific goals related to the fate of endothelial cells and to paracrine (angiocrine) stem cell-related effector functions of endothelial cells. First, we will study if physiological and pathologically challenged endothelial cell turnover occurs from differentiated cells or from stem cell compartments. Second, we will study paracrine (angiocrine) effects of endothelial cells in the vascular bone marrow niche on the maintenance and differentiation of hematopoietic stem cells. Both experimental approaches are pursued in well-defined conditional genetic mouse models and will therefore yield definite findings.
Quantifying Dynamic Functions between Marrow Niche and Normal Hematopoietic Stem Cell versus Leukemia Stem Cell
The ultimate goal if the project is to define and quantify the differences in mechanisms of binding between normal hematopoietic (HSC) versus leukemia stem cells (LSC) with the bone marrow niche. For this purpose, novel in vitro model systems and innovative physical technologies have been established in the first funding period. In continuation, we will utilize the "membrane-based niche model" to quantify the forces required to maintain the HSC versus LSC in the niche, as well as the mechanisms and molecules that induce mobilization and migration of HSC as compared to LSC. As a biologically relevant and dynamic niche model, we will develop tunable polymeric substrates whose mechanical properties can be dynamically switched by external stimuli. In contrast to commonly used chemically cross-linked gels, our dynamic niche models will permit the mechanical control of mesenchymal stromal cell functions and thus compartmentalization of stem cells.
Mathematical modeling of stem cell renewal and differentiation
Mathematical modeling is a powerful technique to address key questions and paradigms in diverse model systems and to provide quantitative insights into cell kinetics, fate determination and development of cell populations. The focus of this project will be in modeling, analysis and simulation of dynamics of stem and progenitor cells during development, tissue regeneration, and cancer. Mathematical models will be developed and validated reiteratively, based on data provided by collaborating experimentalists. This will not only contribute to a better understanding of mechanisms of fate determination, clonal evolution and prediction of short- and long-term dynamics of stem cells, in response to various signaling factors, but will also allow for a comparative analysis of design principles in divergent stem cell systems.
Mechanisms of regulation of neurogenesis in the microenvironment of adult brain
Neural progenitors within the adult hippocampus can self-renew and differentiate into neurons or astrocytes. Yet, the molecular and cellular mechanisms that govern these steps and how time influences these decisions remain unclear. Understanding the principles governing the cellular dynamics in the adult hippocampus during homeostasis in the young and old brain is the major goal of this subproject. To achieve this goal we will analyze neurogenesis at the population and clonal level in wt animals and genetic mouse models combining increased wnt (Dkk1-deficient mice) and decreased CD95 (CD95L-deficient mice) activity. Obtained data will be used for implementation of a mathematical model of adult neurogenesis done in collaboration with the lab of Anna Marciniak-Czochra (B08) that will be crucial to explain the observed changes in cellular dynamics. In addition, we want to characterize the intrinsic molecular players involved in neuronal differentiation of adult neuronal progenitors with a major focus in translational control.
Fate mapping of hematopoietic stem cell activity under steady state and challenges
Major progress has been made in deciphering the phenotypes of hematopoietic stem cell (HSC) which allow their prospective isolation. However, studies into HSC biology are up to now mostly based on the transplantation of donor HSC into myeloablated recipients (both experimentally and clinically), hence, there is very little information on the functions of HSC in their natural bone marrow context. Genetic fate mapping strategies have been developed in the past years that facilitate non-invasive tracing of cellular developmental pathways in vivo. To gain experimental access to HSC functions in situ, we have developed new knock-in mice that can now be used to describe the flow within the hematopoietic system from HSC via intermediate multipotent progenitors to lineage committed cells. In this project, we plan to use these models to analyze the HSC functions under normal steady state conditions, and upon intrinsic and extrinsic challenges.
Spatio-temporal Specificity of Cambium Stem Cell Signaling
Lateral growth of plant shoots and roots is essential for the formation of wood and of large plant bodies, and thus for the creation of biomass on earth. The process depends on the tissue-forming properties of a group of stem cells called the cambium, the activity of which leads to the production of secondary vascular tissue (wood and bast). Here, we want to leverage the unique features of the cambium and establish general principles of stem cell niche organization in plants and beyond by revealing spatiotemporal interfaces of two hormonal signaling pathways fundamental for cambium activity: auxin and cytokinin signaling.
Administration and coordination of the SFB873
The spokesperson's office in charge of all central coordinating activities and organizes seminars, retreats, symposia, prepares and documents the meetings of the steering committee (SC) and is in charge of executing decisions of the SC and the assembly of principal investigators. It is in charge of all financial affairs of th SFB. The coordinator's office handles all internal and external communiciation of the SFB. In essence, the coordinator's office is in charge of all central administrative duties, but its most important task is to establish an atmosphere within the SFB that is conducive to scientific exchange and collaborations between the individual projects for a maximum of scientific synergy and collaborative added value.
Flow Cytometry Core Unit
The mission of the Z02-project is:
1) To provide the consortium with pure and homogenous stem cell or progenitor preparations of outstanding quality as starting material for all subsequent experiments by state-of-the-art cell sorting equipment and qualified personnel as part of the consortium.
2) To develop innovative protocols for the preparation of stem or progentior cells from organisms for which flow cytometry-technology has not yet been standardized (e.g. Hydra, Drosophila, Arabidopsis, Medaka).
Advanced Light Microscopy
The Nikon Imaging Center is a core unit that offers serive in light microscopy within the SFB 873.
Bioin formatic analysis and comparative data mining
This core project will support research groups of the CRC with bioinformatic methods for the analysis of genome-wide data sets. In addition, it will perform a comparative analysis of genome-wide data sets to gain insights into common and divergent networks in stem cell control.
The project scientist is Eugen Rempel