Stem cell therapy has the potential to dramatically change the treatment of human disease. 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 progenitor known as the neuromesodermal progenitor (NMP) 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 where it regulates the self-renewal and differentiation of NMPs however the underlying mechanisms remain poorly understood. Wnt3a regulates cellular behavior by stabilizing b-catenin, which interacts with members of the Lef/Tcf family of DNA-binding factors to transcriptionally activate target genes.
The Specific Aims of the laboratory are: 1) to understand how the fate of PS cells, and specifically NMPs, are regulated by Wnt3a. 2) to define the gene regulatory networks (GRN) that are activated by Wnt3a to control cell fate in the PS. Additionally, we plan to define the molecular mechanisms of Wnt target gene transcription. 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) We utilized cell specific markers and Tamoxifen-induced Cre-based lineage tracings to locate putative NMPs in specific germ layers of wildtype embryos. We provide functional evidence for NMP location primarily in the epithelial PS, and to a lesser degree in the ingressed PS. Lineage-tracing studies in Wnt3a/b-catenin signaling pathway mutants provide genetic evidence that trunk progenitors normally fated to enter the mesodermal germ layer can be redirected towards the neural lineage. These data, combined with previous PS lineage-tracing studies, support a model that epithelial anterior PS cells are Sox2+T+ multipotent NMPs and form the bulk of neural progenitors and paraxial mesoderm progenitors (PMPs) of the posterior trunk region. Finally, we find that Wnt3a/b-catenin signaling directs trunk progenitors towards PMP fates; however, our data also suggest that Wnt3a positively supports a progenitor state for both mesodermal and neural progenitors (Garriock et al., 2015). 2) We have generated genome-wide transcriptional profiles of wildtype (wt) and Wnt3a-/- PS and identified 729 differentially expressed genes. Although this approach has successfully identified many new Wnt3a targets, it remains a significant challenge to identify the genes that are potential effectors of Wnt3a. We have therefore generated a series of ESCs carrying Doxycycline (Dox)-inducible epitope-tagged transgenes, to screen for Wnt target genes that regulate NMP development. Since stimulation of ES cells with Wnt3a rapidly induces PSM and NMPs, we reasoned that the overexpression of transcriptional effectors of Wnt3a, in the absence of exogenous Wnt3a, should induce these cell types. These studies have led us to focus on several interesting downstream transcription factors, including Mesogenin (Msgn1) and Sp5. Msgn1: We are studying Msgn1, a bHLH transcription factor, since Msgn1-/- mutants indicate that it is required for PSM differentiation, a process known to be controlled by Wnt3a. Taking genetic and genomic approaches in mice and embryonic stem cells (ESCs), we have shown that Msgn1 alone controls PSM differentiation by directly activating the transcriptional programs that define PSM identity, epithelial- mesenchymal transition, motility and segmentation. Forced expression of Msgn1 in NMPs in vivo reduced NMP contribution to the neural tube, and dramatically expanded the unsegmented mesenchymal PSM, while also blocking somitogenesis and notochord differentiation. Expression of Msgn1 was sufficient to partially rescue PSM differentiation in Wnt3a-/- embryos, demonstrating that Msgn1 functions downstream of Wnt3a as the master regulator of PSM differentiation (Chalamalasetty et al., 2014). Sp5 and Sp8: The laboratory has also focused on the Sp1 family of Zinc-finger transcription factors. 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. We have shown that Sp5 functions redundantly with the closely related family member Sp8 and that double mutants display a phenotype remarkably similar to Wnt3a mutants (Dunty et al., 2014). Indeed, our demonstration that Wnt3a, Ctnnb1 (b-catenin), Tcf1;Lef1, and Sp5/8 define a syn-phenotype group suggests that Sp5/8 are novel transducers of this stem cell signaling pathway, and that Sp5/8 function in the self-renewal and differentiation of NMPs. By combining genetic, genomic, biochemical and molecular biology approaches, we have shown that Sp5/8 are necessary to activate Wnt target genes, but depend upon b-catenin-Tcf1/Lef1 for activity. Intriguingly, Sp5/8 bind to GC boxes in Wnt target gene enhancers and bind directly to specific components of the enhanceosome to facilitate b-catenin recruitment. Given that Sp5 is itself a Wnt target gene, we propose that Sp5 functions as a signal amplifier in a feed-forward loop to robustly enhance Wnt target gene expression. The expression of Sp5 at most, if not all, sites of Wnt expression, including Wnt-regulated adult stem and cancer cells, suggests that the function of Sp5 in the enhancer may be a universal feature of Wnt-dependent gene expression in vertebrates (Kennedy et al., 2015, submitted). 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, Lef1, Sp5 and Sp8 ChIP-Seq data. This is currently a work-in-progress. 3) We have identified a novel putative 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 not small intestine. 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 in humans. A screen for expression of embryonic Wnt target genes in adult intestine and intestinal tumors revealed that many are expressed in the intestinal stem cell compartment, and in adenomas caused by Wnt gain of function mutations. 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.
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