(1) Runx1 in hair follicle development, stem cell activation, and cancer
Runx1 in stem cell activation and hair follicle homeostasis
Our laboratory is focusing on understanding the dynamic behavior of adult tissue stem cells and the basic science mechanisms governing these processes. We want to establish how perturbation of these processes by mutations, systemic imbalances, injury, or aging leads to disease. One approach in our laboratory is to genetically perturb gene function in mouse skin and investigate the effect on adult stem cells and the potential link with disease.
Aml1/Runx1 controls developmental aspects of several tissues, is a master regulator of blood stem cells, and plays a role in leukemia. However, it has been unclear whether it functions in tissue stem cells other than blood. We have investigated the role of Runx1 in mouse hair follicle stem cells by conditional ablation in epithelial cells. Runx1 disruption affects hair follicle stem cell activation, but not their maintenance, proliferation or differentiation potential. Adult mutant mice exhibit impaired de novo production of hair shafts and all temporary hair cell lineages, owing to a prolonged quiescent phase of the first hair cycle. The lag of stem cell activity is reversed by skin injury. Our work suggests a degree of functional overlap in Runx1 regulation of blood and hair follicle stem cells at an equivalent time point in the development of these two tissues.Aml1/Runx1 controls developmental aspects of several tissues, is a master regulator of blood stem cells, and plays a role in leukemia. However, it is unclear whether it functions in tissue stem cells other than blood. Here, we have investigated the role of Runx1 in mouse hair follicle stem cells by conditional ablation in epithelial cells. Runx1 disruption affects hair follicle stem cell activation, but not their maintenance, proliferation or differentiation potential. Adult mutant mice exhibit impaired de novo production of hair shafts and all temporary hair cell lineages, owing to a prolonged quiescent phase of the first hair cycle. The lag of stem cell activity is reversed by skin injury. Our work suggests a degree of functional overlap in Runx1 regulation of blood and hair follicle stem cells at an equivalent time point in the development of these two tissues (Osorio, et al; Development, 2008).
B-Galactoside knocked into Runx1 gene locus indicates expression of Runx1 in the skin in the zone of the hair follicle where stem cells are first activated.
Runx1 knockout follicles remained blocked in quiescence for an extended period of time
Hair follicles in knockout mice spend a prolonged time in the telogen (quiescent) phase, indicating lack of hair follicle stem cell activation in these mice for the period of our experiments. Stem cells remained blocked in G0 phase of the cell cycle with lack of proliferation and differentiation.
Runx1 in epithelial cancer
What is the significance of Runx1 modulation of bulge cells proliferation in epithelial skin? Runx1-/- hair follicles eventually enter anagen, and can undergo repeated stimulated hair cycles. Any effects of Runx1 knockout on bulge cell proliferation do not become grossly manifested in normal development. The link of bulge cells proliferation with tumorigenesis has been a recurring theme in our field. Moreover, Runx1 works as an oncogene in blood stem cells, and is part of a family of genes involved in different types of malignancies. All these considerations led us to begin to examine the effect of a 2-step carcinogenesis treatment on skin of epithelial Runx1 knockout mice. This procedure involves inducing mutations with an initial application of 7.2-dimethylbenzanthracene (DMBA) followed by proliferation promotion with repeated applications of 12-O-tetradecanoylphorbol-13-acetate (TPA). Usually initiation occurs at a single time point, in telogen, since the stage of initiation can induce a difference in tumorigenesis.
In the future we have plans to take our mouse work a step further, into human skin. To that end we began making our own reagents (antibody) to detect the AML1 protein in tissues and we are also testing a number of commercially available antibodies. We have initiated a collaboration with Dr. Ralf Paus in Germany, Dr. Jonathan Vogel at NIH and Dr. Agniezka Kobielack at USC to obtain human samples for investigating the expression of AML1 not only in normal human skin, but also in epithelial skin tumors. In addition, we are planning to verify if mutations in AML1 correlates with increased frequency of human cancers in collaboration with Dr. Andrew Clark at Cornell University. We are just beginning to unravel the connection of AML1 with skin cancer.
Another research group (Dr. A.M. Bowcock at Univ. of St. Louis, Missouri) published a study in Nature Genetics (2003) suggesting a link in perturbation of AML1/RUNX1 function with another human skin disease, namely psoriasis. Deletion of AML1/RUNX1 in mice in our laboratory, indicated a temporary scaling of the skin in young mice (unpublished data). Although suggestive, this phenotype has not persisted beyond several days. This might not be surprising, since it is known that psoriasis is a complex trait disease, which involves multiple genetic and environmental factors. Deleting Runx1 was not sufficient for modeling this disease in mouse skin, but provides an additional link in the puzzle. For example, the phenotype of mice with RUNX1 deletion is identical with that of STAT3 deletion. These factors are thought to play synergistic roles in regulating gene expression of some target genes. Moreover, STAT3 has been clearly implicated in inducing a psoriasis-like phenotype by over-expression in mice (J. DiGiovanni and collegues, Nat. Medicine, 2004). Our work in addressing the basic mechanisms that underline human disease will serve as basis in future for more clinically oriented scientists to design drugs for targeting these specific genes for future therapies.
(2) Dynamics of hair follicle stem cells during normal tissue homeostasis
Hair follicle stem cells are infrequently dividing cells
Regulation of stem cell (SC) proliferation is central to tissue homoeostasis, injury repair, and cancer development. Accumulation of replication errors in SCs is limited by either infrequent division and/or by chromosome sorting to retain preferentially the oldest ‘immortal’ DNA strand. The frequency of SC divisions and the chromosome-sorting phenomenon are difficult to examine accurately with existing methods. To address this question, we developed a strategy to count divisions of hair follicle (HF) SCs over time, and provide the first quantitative proliferation history of a tissue SC during its normal homoeostasis. We uncovered an unexpectedly high cellular turnover in the SC compartment in one round of activation. Our study provides quantitative data in support of the long-standing infrequent SC division model, and shows that HF SCs do not retain the older DNA strands or sort their chromosome. This new ability to count divisions in vivo has relevance for obtaining basic knowledge of tissue kinetics.
Tetracycline inducible mice allow pulse-chase using a histone H2B-GFP transgene which upon repression of mRNA expression will dilute by 2-fold at each division.
Divisions can be quantified from fluorescence activated cell sorting (FACS) profiles. Using this new technology we found that hair follicle stem cells divide only ~10-70 times during an entire mouse life. These patterns of divisions are consistent with a model in which stem cells divide infrequently to prevent accumulation of mutations during replication and reduce the risk of cancer (Waghmare et al, EMBO 2008).
Hair follicle stem cells daughters undergo symmetric fate decision during normal tissue homeostasis
In homeostasis of adult vertebrate tissues, stem cells are thought to self-renew by infrequent and asymmetric divisions that generate another stem cell daughter and a progenitor daughter cell committed to differentiate. This model is based largely on in vivo invertebrate or in vitro mammal studies. We examined the dynamic behavior of adult hair follicle stem cells in their normal setting by employing mice with repressible H2B-GFP expression to track cell divisions and Cre-inducible mice to perform long-term single-cell lineage tracing.
Inefficient activation of the Cre recombinase allows marking of single cells in the hair follicle stem cell compartment the bulge, when mice are crossed with a lac Z( B-galatosidase) reporter mouse known as Rosa26R. To image shows skin of mice injected with tamoxifen to activate Cre. Control skin from transgenic mice not injected with tamoxifen is shown at the bottom.
Using the H2B-GFP and the LacZ systems we provide direct evidence for the infrequent stem cell division model in intact tissue. Moreover, we find that differentiation of progenitor cells occurs at different times and tissue locations than self-renewal of stem cells. Distinct fates of differentiation or self-renewal are assigned to individual cells in a temporal-spatial manner. We propose that large clusters of tissue stem cells behave as populations whose maintenance involves unidirectional daughter-cell-fate decisions (Zhang et al, Cell Stem Cell 2009).
We speculate that the spatial-temporal segregation of self-renewal and differentiation may ensure protection of the SC niche from penetration of cell differentiation tissue signals.
In the future we plan to investigate what are the genetic and epigenetic controls of fate decisions in the hair follicle stem cell niche.
(3) Link between infrequently dividing cells and hair follicle stem cells
This project stems from our interest in elucidating the molecular mechanisms involved in somatic stem cell self-renewal and fate choice, in normal tissue development and homeostasis. Stem cells hold great promises for future therapies of numerous deadly diseases, yet their basic properties are not well understood. Progress in the field has been hampered by the difficulty to identify tissue stem cells in the absence of specific biochemical markers. Increasing evidence suggested that stem cells are infrequently dividing cells, that could be identified in many mouse tissues as the DNA label retaining cells (or LRCs). Recently we have developed a strategy applicable to many self-regenerating tissues to label LRCs with the green fluorescent protein (GFP) fused with the histone H2B-GFP. We have used the mouse skin and the hair follicle as model systems, since the relation between LRCs and skin epithelial stem cells had been investigated in most depth. In our preliminary work we tracked and isolated GFP LRCs and demonstrated that at least a fraction of these cells had a stem cell phenotype. In this project we are developing strategies to examine in more detail the relation between the most quiescent LRCs and stem cells and uncover a potential heterogenetity within the currently characterized stem cell pool. We dissect LRCs in individual sub-populations, investigate their stemness, their special cell cycle properties, and their gene expression makeup. To accomplish these goals we use in vivo cell tracking and in vitro cell sorting methods combined with a variety of assays which include: hair follicle regeneration and skin wounding; cell culture and skin reconstitution; cell cycle kinetics; and microarray.
First, our data will have global relevance for the isolation and characterization of putative stem cell with LRCs properties from many other tissues. Although we focus on the hair follicle, our work will lead the way for similar approaches in many systems which lack the specific means to separate stem cells apart from other cells of their tissue of residence. Second, stem cell proliferation is an important biological process that must be tightly controlled in tissues in order to regulate the balance between normal and abnormal growth. Our studies will unravel at least some of the mechanisms developed by putative stem cells to control their proliferation, an important first step in understanding such perturbation in the diseased tissues. Finally, our work will provide some of the basic knowledge which will be necessary for more efficient manipulation of skin stem cells for clinical applications such as cell transplantation on burn victims.
(4) Epigenetic control of hair follicle stem cell fate
Stem cells exist as multipotent cells with the ability to choose their fate by self-renewing or differentiating to different cell lineages. These differentiated lineages display specific programs of transcriptional activity or “molecular” signatures, which are maintained in tissues by specific epigenetic marks placed on the regulatory DNA regions of these genes. When stem cells decide to cease self-renewal and differentiate to a different cell type their genome is efficiently and rapidly remodeled because stem cells maintain a certain degree of “plasticity” in their genome. This “plasticity” is thought to be at least part established by specific characteristics of the stem cell chromatin states such as the placement of bivalent epigenetic marks (both activating methylated H3K4 and repressing histone H3K27) on specific promoters, as well as by a state of hyperdynamic exchange of the histone in and out of the nucleosome. We are interested in investigating the status of the epigenome in hair follicle stem cells and in elucidating the genetic program that might be responsible for keeping stem cells in a “plastic” or multipotent state.