The Role of Wnt Genes in Vertebrate Development and Cancer

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

The Yamaguchi laboratory is studying how the Wnt family of signaling molecules regulate embryonic and adult stem cells during embryogenesis and tumorigenesis. Wnt signaling has profound effects on stem cells - in the absence of a Wnt signal, stem cells often fail to self-renew, while overactivation of Wnt signaling arrests precursors in a progenitor state that can lead to tumorigenesis. Thus, understanding how Wnt signaling regulates stem cell pathways may lead to new cancer therapies and new methods for cellular reprogramming for regenerative medicine applications. My laboratory is focused on understanding gastrulation, a developmental process that converts pluripotent stem cells into the primary germ layers. Gastrulation can be considered in two distinct stages, an early gastrulation stage that is controlled by Wnt3 and leads to the formation of the head (brain), heart, and gut precursors, followed by a later gastrulation stage that is controlled by Wnt3a and generates, amongst others, the neuromesodermal progenitors (NMP) that give rise to the spinal cord and musculoskeletal progenitors of the trunk and tail. Thus, the development of the vertebrate embryo body is progressive and largely occurs in an Anterior (A)-Posterior (P) or head-tail sequence that is controlled by the ordered activation of distinct developmental programs, or gene regulatory networks (GRNs). Several Wnts are expressed in the primitive streak (PS) during gastrulation, where they elicit distinct cell fates from the pluripotent epiblast over time however the underlying mechanisms remain poorly understood. Wnts regulate target gene expression by stabilizing beta-catenin, which interacts with members of the Tcf/Lef family of DNA-binding transcription factors (TFs) to activate transcription. Although we know that the cellular response in the PS to Wnt signals occurs in an ordered and temporal fashion, how these robust and reproducible gene responses are orchestrated is not well understood. The Specific Aims of the laboratory are: 1) to characterize the Wnt-dependent GRNs that define early and late gastrulation, and to understand how the embryo transitions through these genetic programs, and 2) define the molecular mechanisms of Wnt target gene transcription. We have made significant progress in achieving our goals: In early efforts to identify the effectors of Wnt signaling in axial progenitors in vivo, we employed transcriptional profiling of mutant and wildtype (wt) embryos to identify differentially expressed Wnt3a target genes. This led to the identification of numerous candidates, including transcription factors (TF) and signaling molecules, that allowed us to begin to construct gene regulatory networks (GRNs) that predict progenitor behavior. We have had a long-standing interest in the classic T-box TF Brachyury (Tbxt) as an effector, and inducer, of Wnt3a/beta-catenin signaling that we have shown with genetic lineage tracing studies to be an early marker of the NMC. Towards our goal of understanding Wnt signaling mechanisms, we identified novel roles for the Zn-finger TFs, Sp5 and Sp8, as target genes and redundant transcriptional coactivators in the Wnt/beta-catenin signaling pathway during trunk NMC formation. Although the 4 Tcf TFs are widely considered to be the terminal effectors of the Wnt/beta-catenin signaling pathway, several other TFs including Sp5, Sp8, Tbxt and Cdx2 regulate Wnt target gene transcription during gastrulation and are themselves Wnt targets. How this diverse set of TFs might interact to regulate Wnt targets is poorly understood. We have recently shown that Sp5/8, together with Tbxt and Cdx2, establish an autoregulatory network that sustains a high Wnt/Fgf, and low retinoic acid, signaling environment in the niche. These studies led to the identification of a Wnt3a enhancer that is directly, and positively, regulated by Sp5/8, Tcf7, Tbxt and Cdx TFs and that is required for the expression of Wnt3a and the Wnt3a-Tbxt positive feedback loop. Furthermore, we have elucidated the mechanism through which Sp5/8 regulate Wnt target gene expression. We found that Sp5/8 binds to Tcf7, in a direct protein-protein interaction, to enhance Tcf7 occupancy at Wnt response elements (WREs). ChIP-seq studies showed that Sp5/8 mutant cells display reduced Tcf7 and increased Tle repressor binding at Wnt target genes suggesting that Sp5/8 regulate the exchange of repressive Tcf-Tle complexes for activating Tcf7-beta-catenin complexes. A manuscript detailing this work was recently accepted at eLife. We have also recently advanced our understanding of how the Wnt/beta-catenin pathway regulates node/organizer activity during the establishment of the LR axis. We had previously shown that Wnt3a is necessary for proper LR patterning however the underlying mechanisms were unclear. Consistent with Sp5/8 functioning as effectors of Wnt3a, we found that Sp5/8 double mutants also display heterotaxy. In a productive collaboration with Alex Joyner, we showed that Sp5/8 regulate cilia formation and gene expression in numerous ciliated tissues including the node where LR asymmetry is initiated. Remarkably, expression of Sp8 alone was sufficient to induce primary cilia in unciliated cells. This work suggests Sp5 and Sp8 as new candidates for mutation in human ciliopathies and has been recently accepted at Science. Taken together with our NMC studies, we suggest that the primitive streak (PS) secretes Wnt3a to function as a posterior organizing center - Wnt3a, acting via Sp TFs, coordinates NMC self-renewal in the caudal epiblast, with the regulation of cilia formation and LR axis determination in the underlying node. We frequently employ gastruloids, an in vitro 3D stem cell culture, to model gastrulation and trunk elongation. Using ESCs carrying mutations in Sp5 and Sp8, or that conditionally overexpress Sp5 or 8, we have found that Sp TFs promote gastruloid elongation. Interestingly, using gastruloids we have uncovered a novel role for Sp TFs in the transition from early to late gastrulation. We are also studying the role of Sp8 in the trunk-to-tail transition. Single cell multiomics analysis indicates that NMPs, caudal mesoderm, and somites that generate the tail are depleted. Interestingly, Sp8 appears to control the expression of the RA metabolizer Cyp26a1, in addition to Wnt3a, suggesting that Sp8 may establish the antiparallel gradients of Wnt3a and RA that control the differentiation of NMPs into mesoderm and spinal cord, respectively.