Stem cell therapy has the potential to dramatically change the treatment of human disease. Although bone marrow stem cell transplantation is a well-known therapy for leukemia, future therapies arising from stem cell research are expected for a wide variety of diseases including other cancers, spinal cord injuries and muscle damage. The Yamaguchi laboratory is studying how the Wnt family of signaling molecules regulates the growth and development of stem cells during early embryogenesis and tumorigenesis. The laboratory is particularly interested in stem cell populations that form the neural and musculoskeletal cells of the mammalian trunk and in the gut stem cells that form the colon. Wnt signaling has profound effects on the development of these populations. In the absence of a Wnt signal, the muscles, cartilage, and bone of the trunk fail to form, as well as the colon section of the intestinal tract. Aberrant activation of Wnt signaling causes major abnormalities in musculoskeletal development and promotes colon cancer. Understanding how Wnt signaling regulates stem cell pathways may reveal new mechanisms to program stem cells to form neural and musculoskeletal cells valuable in cell based therapies or how to target effective treatments for colon cancer. We are studying a unique stem cell known as the neuromesodermal (NM) stem cell that resides in the primitive streak (PS) of the gastrulating embryo and gives rise to the spinal cord and musculoskeletal progenitors of the body. Wnt3a is expressed in the PS and functions there to maintain NM stem cells and to control their differentiation however how Wnt3a simultaneously regulates both processes remains poorly understood. Wnt3a regulates cellular behavior by stabilizing beta-catenin, which interacts with members of the Lef/Tcf family of DNA-binding factors to transcriptionally activate target genes. The goals of the laboratory are: 1) to understand how NM stem cells in vivo make cell fate decisions based on the exposure to a single growth factor, Wnt3a. 2) to understand how Wnt3a regulates NM stem cell self-renewal and differentiation by elucidating Wnt3a-dependent gene regulatory networks (GRN). 3) to identify and understand the role of gut stem cells in colon formation and tumorigenesis. We have made significant progress in achieving our goals: 1) Using sophisticated Cre-lox lineage tracing approaches, we have successfully traced the fate of NM stem cells in vivo. We have found that NM descendants destined to become neural remain in the epiblast and differentiate into the spinal cord, while cells destined for the musculoskeletal system ingress through the PS, undergo an epithelial-mesenchymal transition (EMT), and differentiate into the presomitic mesoderm (PSM). By performing lineage tracing experiments in Wnt3a-/- mutants we have shown that Wnt3a predisposes NM stem cells to undergo an EMT and form a musculoskeletal progenitor while absence of Wnt3a forces these cells to enter the neural lineage. 2) We have generated genome-wide transcriptional profiles of wildtype (wt) and Wnt3a-/- PS and identified 729 differentially expressed genes, including several previously characterized Wnt target genes. Bioinformatics analysis coupled with a comprehensive in situ hybridization screen to examine their expression in vivo identified several important new targets. Although this approach has successfully identified many new Wnt3a targets, it remains a significant challenge to identify the genes that possess functional Wnt3a-like activity. We have therefore generated a series of ESCs carrying Doxycycline (Dox)-inducible epitope-tagged transgenes, to screen for Wnt target genes that regulate NM stem cell development. Since stimulation of ES cells with Wnt3a rapidly induces PSM and NM stem cells, we reasoned that the overexpression of transcriptional effectors of Wnt3a, in the absence of exogenous Wnt3a, should induce these cells. These studies have led us to focus on two particularly interesting downstream transcription factors, Mesogenin (Msgn1) and Sp5. We are studying Msgn1, a bHLH transcription factor, since Msgn1-/- mutants indicate that Msgn1 is required for PSM differentiation. We have discovered that Msgn1 is a direct Wnt3a target gene and is sufficient to drive ESCs to undergo EMT, migration and differentiation into PSM. Using genome-wide microarrays and ChIP-Seq approaches we have found that Msgn1 does so by directly binding enhancers to activate EMT and PSM-specific gene expression. Importantly, we have used gain-of-function (GOF) transgenics to show that Msgn1 induces PSM in vivo, and that the expression of Msgn1 alone in Wnt3a-/- mutants is capable of rescuing the PSM deficit. This remarkable finding argues that Msgn1 is a master regulator of PSM differentiation. Our recent finding is highly significant since we now know how Wnt3a elicits PSM differentiation from NM stem cells - by activating the expression of the PSM master regulator Msgn1. We have also shown that Msgn1 directly activates the EMT master regulator Snail1 providing new insights into how Wnts regulate EMT and metastasis. The laboratory has also focused on the Sp1 family of Zinc-finger transcription factors since our bioinformatics analysis grouped two members, Sp5 and Sp8, in a GRN together with Msgn1 and other Wnt target genes. Sp5 is regulated by Wnt3a/beta-catenin signaling during gastrulation, and is expressed at multiple sites of Wnt activity, including normal and diseased embryonic and adult tissues, suggesting that Sp5 is a universal Wnt/beta-catenin target gene. Recent studies have shown that Sp5 functions redundantly with the closely related family member Sp8. Double mutants display a phenotype remarkably similar to Wnt3a mutants indicating that Sp5/Sp8 likely function in the self-renewal of NM stem cells. We are currently elucidating the underlying mechanisms. Finally, to construct the GRN that transduces Wnt3a activity in the PS, we have undertaken a large-scale RNA-Seq project to define the transcriptomes of control, Wnt3a, Lef1, Msgn1, and Sp5/Sp8 loss and gain-of-function mutants and ESCs and are currently integrating this data with Msgn1 (and soon Sp5) ChIP-Seq data. Although very preliminary, our work has already led to a more refined classification of genes downstream of Wnt3a as likely direct, or indirect, target genes. 3) We have identified a novel colon stem cell (CoSC) population that uses Wnt3a to signal directional growth to elongate the colon portion of the gut from the small intestine region. Surprisingly, Wnt3a has a specific and potent activity to stimulate growth of CoSC but does not stimulate substantial growth of small intestinal progenitors. This is particularly interesting since most human colon cancers are caused by altered Wnt signaling. Our proposal that Wnt3a could be the major growth stimulator for the colon region of the intestine could shed light on why aberrant Wnt signaling primarily causes colon cancer. Since gain of function mutations in the Wnt pathway cause colorectal cancer, we screened our embryonic Wnt target genes for expression in the murine adult intestine and in intestinal tumors. Remarkably, many of our genes are expressed in the stem cell compartment of the GI tract, and are highly expressed in adenomas caused by Wnt gain of function mutations. These results demonstrate that our Wnt target genes are expressed in unrelated embryonic and adult stem cell compartments suggesting that their expression may be associated with 'stemness'. We have found that Sp5 alone is not required for spontaneous adenoma formation in the Apcmin mouse model of human intestinal cancer, suggesting that Sp5 may also have redundant functions during intestinal tumorigenesis. We are currently addressing the potential redundancy of Sp5 and Sp8 in intestinal cancer stem cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010345-14
Application #
8763084
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
14
Fiscal Year
2013
Total Cost
$1,010,124
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Dunty Jr, William C; Kennedy, Mark W L; Chalamalasetty, Ravindra B et al. (2014) Transcriptional profiling of Wnt3a mutants identifies Sp transcription factors as essential effectors of the Wnt/*-catenin pathway in neuromesodermal stem cells. PLoS One 9:e87018
Biris, Kristin K; Yamaguchi, Terry P (2014) Two-color in situ hybridization of whole-mount mouse embryos. Methods Mol Biol 1092:17-30
Yamaguchi, Terry P (2008) Genetics of Wnt signaling during early mammalian development. Methods Mol Biol 468:287-305
Dunty Jr, William C; Biris, Kristin K; Chalamalasetty, Ravindra B et al. (2008) Wnt3a/beta-catenin signaling controls posterior body development by coordinating mesoderm formation and segmentation. Development 135:85-94