Catherine MacLean Developmental Biology: Professor Grabel Literature Review 2/19/13

Wnt signaling in hematopoietic stem cells

Introduction Hematopoietic stem cells (HSCs) are an extremely well-studied type of multipotent stem cells which are responsible for giving rise to progenitors of all the major blood cell types, including megakaryocyte/erythrocyte progenitors, and granulocyte/macrophage/lymphocyte progenitors, which in turn give rise to mature blood cell types (7). HSCs develop in the fetal liver and then migrate to the interior of the bones, forming colonies near surrounding stromal cells and osteoblasts (7). These areas form the microenvironment of the HSCs, which is critical to influencing their development and differentiation (7). In addition to producing blood cell progenitors, HSCs also must maintain their own renewal capabilities so that their numbers are not depleted to a level that results in pancytopenia. There must exist a population of both long and short term multipotent HSCs that is sufficient to produce blood over an entire lifespan, and enough differentiated cells to support the organisms needs. This balance between differentiation and renewal is tenuous and must be closely controlled (7). Wnt is the name given to a family of proteins first indentified in the Drosophilia and later discovered to be highly conserved across many organisms. Wnt proteins play critical roles in cell signaling cascades that ultimately upregulate transcription and expression of certain genes. By inhibiting the formation of an axin/APC/GSK3- complex, Wnt prevents degradation of catenin, which is able to enter the nucleus and stimulate Lef/Tcf transcription factors (6). This is known as the canonical Wnt pathway, while other roles for Wnt have been found in so-called non-canonical pathways. These non-canonical pathways can mediate cell-cell adhesion and signaling, and both canonical and non-canonical pathways are an area of ongoing research (6). In light of Wnts critical role in cell signaling and its role in the development of other tissues such as the small intestine, investigations of its role in HSC signaling, and development were undertaken. Results suggest that Wnt plays an active role in both differentiation and renewal of HSCs. Investigations in the mouse and human at the fetal and adult levels of development, and in both the HSCs and their downstream progenitors and mature cells have been undertaken with a variety of methods. These studies present a complex and nuanced picture in which the various Wnt proteins play specific roles in promoting renewal or differentiation and the HSCs are sensitive to the level of Wnt protein and the external influences of their microenvironment. A Role for Wnt Early studies simply sought to establish the presence of Wnt in the HSCs and their microenvironment and to demonstrate the effects of over-expression of Wnt on HSCs. Austin et al demonstrated the expression of Wnt 5a and 10b in murine fetal liver and yolk sac tissues (1). It was also shown that several members of the frizzled family, canonical Wnt receptors were present in adult hematopoietic tissues. Additionally, experiments demonstrated an enormous increase in proliferation of fetal liver HSC tissue, showing a 60 fold increase over a control for cells transfected with Wnt-10b. Cell cultures transfected with Wnt-5a also showed increased proliferation.

These results were reproduced in human fetal bone marrow and adult bone marrow and HSC lines, with the presence of Wnt-5a, 2b, and 10b demonstrated as well as increased proliferation of HSCs in cell cultures treated with Wnt. This data established that Wnt signaling was likely to play a role with HSCs, though many questions about the mechanism and the specific interactions remained unanswered. One possible route of action is the -catenin molecule, which in canonical Wnt signaling is an intermediary, but has been found to have a separate role in cell-cell adhesion and signaling. Austin et al also tested cell-cell adhesion via -catenin linkage facilitated by Wnt, and did discover increased levels in cells overproducing Wnt 5a (1). Further tests to determine the effects of this increased cell-cell adhesion were not completed. In addition to the question of mechanism, the effects of Wnt signaling on HSCs were also relevant to this line of inquiry. Importantly, studies in mice did not reveal increased completed, mature differentiation among cells treated with Wnt-5a, suggesting that the role of the protein is limited to proliferation among the more primitive varieties of cells. Cells raised in culture of Wnt-5a or 10-b tended away from becoming granulocyte progenitors, but it is marked that the level primitive cells increased relative to differentiated cells (1). Studies in humans produced similar results though demonstrated a slightly increased effect on erythroid progenitors for Wnt10b (8). In the human tissues, the levels of gene expression were not sufficient for analysis by Northern blot, despite their presence being demonstrated by other methods (8). This is not an uncommon situation for Wnt, but in this particular instance, it may point directly to the tight control on the signaling inputs to HSCs, as other results concerning over-expression of Wnt demonstrate a loss of function in HSCs (cited in 7, 3). This low level of Wnt presents a challenge for research, as it eliminates some assays from the experimental repertoire. Wnt and Self-Renewal Initial studies of the role of Wnt in HSC regulation revealed an elaborate system of effects. One of the most interesting is the role in proliferating HSCs and ensuring that a steady population is maintained in order to facilitate proper hematopoiesis. To fully investigate the selfrenewal potential, the most effective test is the ability of cells to regenerate hematopoietic function in an animal whose HSCs have been destroyed. This was achieved in mice via lethal irradiation, and the mice were then transplanted with donor stems cells from mice of several different strains. The presence of Wnt was determined to be necessary for repopulation, with Wntinhibited donor HSCs showing much lower levels of repopulation. -catenin, a downstream regulator in the Wnt pathway, was shown to be advantaegous to the self-renewal process, stimulating production of progenitor cells without differentiation (5). Further research has corroborated this critical role for Wnt. Similar transplantation assays and in in vitro analysis with inhibition or knock out of Wnt signaling also found that selfrenewal by HSCs necessitated Wnt signaling (5, 2). Critically, results suggesting the necessity of Wnt signaling were generated wit diverse experimental methods. In one case, cells in culture were incubated with a protein intended to blockthe Wnt receptor, and futher experiments were done with over expression in vivo of a protein that inhibited Wnt signaling within the HSC (5). In another example, Dkk1 was reduced in the osteoblasts, cells that make up a critical portion of the microenvironment for HSCs, suggesting a cell-cell signaling mechanism for Wnt and also

demonstrating the importance of environment during early development of HSCs, as HSCs raised in the presence of Dkk1 were not able to regenerate even when transplanted into wild type mice (2). This suggests a definite role for Wnt signaling in the self-renewal process but does not establish a definitive relationship and does not point specifically to a Wnt protein needed for the self-renewal process. Through generation of Wnt3a deficient mice, it was shown, through secondary transplantation, that self-renewal did not occur properly in the absence of Wnt3a (4). Fetal liver cells were also studied and the absence of Wnt3a was associated with much lower levels of HSC proliferation (4). This level of specificity is incredibly important for potential clinical applications of Wnt signaling in HSCs, and also allows for a more detailed picture of the processes that surround the HSCs. Wnt and differentiation Much of the experimental evidence discussed herein relates to the renewal of HSCs rather than their differentiation, as the majority of the Wnt proteins studied had far greater effects on HSC renewal and the proliferation of progenitor cells than the differentiation of the cells. However, results discussed this far generally concerned only the canonical Wnt pathway, and some studies suggest that non-canonical action of Wnt may influence differentiation (6). So while Wnt-5a and -10b have only displayed a limited role in early progenitors, this is an excellent and ongoing avenue of further research (1,8). Wnt 3a was shown to exhibit some function in differentiation with cells deficient in the protein displaying affected myeloid progenitors and affected immature thymocytes (4). Other investigations may reveal more about role of Wnt in HSC differentiation and the downstream processes of hematopoeisis. One context in which the role of Wnt and differentiation has been studied rather extensively is in cancer. Recent research has implicated Wnt in both chronic and acute myeloid leukemias (6). It is posited that Wnts role in self-renewal will also have the same effect on the cancerous progenitor cells, giving the cancer its malignant nature (6). This is an exciting avenue of research as it provides the possibility of targeted therapies designed to eliminate the cancerous nature of cells at an early stage of development. However, caution must be exercised given that the role of Wnt in cancerous cells is so closely related to that in normal cell populations, as normal HSCs may be vulnerable to therapies developed. Unifying Results While there is consensus in results generated in many of the studies cited here, there has been controversy within this field. Some studies of over-expression of the Wnt proteins have in fact found HSC exhaustion, not proliferation, to be the result (7). Experimental approaches vary widely, which may account for the issues observed. Additionally, in some cases the role of a given Wnt protein may be redundant or working in concert with other proteins, so a given experimental approached intending elimination does not achieve the desired effect. Another line of inquiry is the level of Wnt protein present and the effect this can have on HSCs. An in-depth study comparing five strains of mice engineered to produce five different levels of Wnt signaling, lent credence to this idea. At very high levels of Wnt signaling both HSC self renewal and differentiation were impaired, possibly due to stem cell exhaustion. With high-intermediate levels, self-renewal was still impaired, but interestingly, T-cell differentiation was improved, a novel finding, especially given that this process takes place in a separate location in the thymus. Low-intermediate levels once again showed impaired self-renewal but

also showed enhanced myeloid differentiation (3). This parallels some earlier results and given the lack of a secondary transplantation assay in other studies, it is possible that the self-renewal was impaired in that case as well (1). Increased Wnt did show increased proliferation, even at mild levels of Wnt signaling elevation, which suggests that proliferation and the ability to selfrenew are not synonymous (3). This type of analysis is beneficial in its ability to parse conflicting data sets, and it shows the extremely narrow range within which HSCs are regulated. Increasingly more targeted investigations will reveal whether the interaction of various Wnt proteins can cause unexpected effects due to redundancy or competition, and potentially establish specific gradations of Wnt signaling that promote certain behavior in HSCs. Conclusion It is clear that Wnt signaling plays a feature role in many aspects of the HSC. From its critical role in self-renewal to its role in cancer progenitor cells, it has been shown to be a critical regulator of the life processes of HSCs. The role of Wnt signaling in HSCs is an area of on-going research due to the incredible intricacy of the mechanisms of control exerted by Wnt, and the varying roles of different proteins in the family. Very recent work has attempted to unify the somewhat contradictory results of earlier studies, to provide a detailed schema for the role that Wnt plays. While more needs to be done to explain conflicting data and to increase the specificity of analysis, the current body of work has illuminated a key regulatory mechanism in a critical cell type. As one of very few types of adult multipotent stem cells in the human body, with a critical role in life functions, HSCs are of utmost importance. These investigations have major implications for improving treatments for a wide range of blood cancers in which Wnt signaling has been implicated, and to providing improved treatment options to those with HSC failure and patients undergoing bone marrow transplantation. A full understanding of the role of Wnt might make possible the stimulation of HSC renewal to repopulate a patients bone marrow, which would constitute a great step forward in medicine.

References 1. Austin, Timothy W., Gregg P. Solar, Francis C. Ziegler,Linda Liem, and William Matthews. A Role for the Wnt Gene Family in Hematopoiesis: Expansion of Multilineage Progenitor Cells. Blood 1997 89:3624-3635 2. Fleming, Heather E. Viktor Janzen, Cristina Lo Celso, Jun Guo, Kathleen M Leahy, Henry M Kronenberg, and David T Scadden. Wnt signaling in the niche enforces hematopoietic stem cell quiescence and is necessary to preserve self-renewal in vivo. Cell Stem Cell. 2008 March 6; 2(3): 274283. 3. Luis, Tiago C., Brigitta A.E. Naber, Paul P.C. Roozen, Martijn H. Brugman, Edwin F.E. de Haas, Mehrnaz Ghazvini, Willem E. Fibbe, Jacques J.M. van Dongen, Riccardo Fodde, Frank J.T. Staal Canonical Wnt Signaling Regulates Hematopoiesis in a Dosage-Dependent Fashion. Cell Stem Cell 2011 9:345356 4. Luis, Tiago C., Floor Weerkamp, Brigitta A. E. Naber, Miranda R. M. Baert, Edwin F. E. de Haas, Tatjana Nikolic, Sjanneke Heuvelmans,Ronald R. De Krijger, Jacques J. M. van

Dongen, and Frank J. T. Staal Wnt3a deficiency irreversibly impairs hematopoietic stem cell self-renewal and leads to defects in progenitor cell differentiation. Blood 2009 113:546554; published ahead of print October 2, 2009 5. Reya, Tannishtha, Andrew W. Duncan, Laurie Ailles, Jos Domen, David C. Scherer, Karl Willert, Lindsay Hintz, Roel Nusse, Irving L. Weissman. A role for Wnt signaling in selfrenewal of haematopoietic stem cells. Nature May 2003 423(6938):409-414 6. (review) Reya, Tannishtha, Hans Clevers. Wnt signaling in stem cells and cancer. Nature April 2005 434(7035):843-850 7. (review) Lara Rossi, Kuanyin K. Lin, Nathan C. Boles, Liubin Yang, Katherine Y. King, Mira Jeong, Allison Mayle, Margaret A. Goodell Less Is More: Unveiling the Functional Core of Hematopoietic Stem Cells through Knockout Mice. Cell Stem Cell 2012 11(3):302-317 8. Van Den Berg, David J., Arun K. Sharma, Edward Bruno, and Ron Hoffman Role of Members of the Wnt Gene Family in Human Hematopoiesis. Blood 1998 92:31893202