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 regulates 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 aberrant activation of Wnt signaling arrests precursors in a progenitor state and can cause cancer. 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 particularly interested in axial stem cell populations that generate the mammalian trunk. We are primarily studying a unique bipotential progenitor known as the neuromesodermal progenitor (NMP) that arises in the primitive streak (PS) of the gastrulating embryo and gives rise to the spinal cord and musculoskeletal progenitors of the trunk and tail. We are also studying a distinct axial progenitor population that generates the colon. Several Wnts, most notably Wnt3a, are expressed in the PS where they specify cell fates from the pluripotent epiblast however the underlying mechanisms remain poorly understood. Wnts regulate cellular behavior by stabilizing beta-catenin, which interacts with members of the Lef/Tcf family of DNA-binding transcription factors (TFs) to activate the transcription of target genes. 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 define the Wnt-dependent gene regulatory networks (GRN) that control NMP development, 2) to characterize colon progenitors and define the role that Wnt signaling plays in colon formation, 3) define the molecular mechanisms of Wnt target gene transcription. We have made significant progress in achieving our goals: 1) To understand the Wnt3a-dependent gene regulatory network (GRN) that controls NMP development, we previously transcriptionally profiled Wnt3a-/- embryos to identify target genes. Functional studies of these Wnt target genes have led us to focus on several interesting downstream TFs, including the NMP determinant T/Bra, Mesogenin (Msgn1) a master regulator of presomitic mesoderm (PSM) fate, the Zn-finger TFs Sp5, Sp8, and Zfp703, as well as the transcriptional repressor Nkx1.2. Our work places T/Bra at the top of a transcriptional hierarchy that controls trunk and tail development through the activation of Sp5, Msgn1 and Tbx6 and the repression of the neural determinant Sox2. Although Tbox TFs have been studied for years, surprisingly little is known about the molecular mechanisms of T/Bra activity. Towards this goal, we have generated an NMP library and performed a yeast 2-hybrid assay using the T-box domain of T/Bra to identify novel protein partners of T/Bra. We have identified several interesting chromatin modifiers that appear to interact directly with T/Bra, and we are currently characterizing their activity. Not only is it important to understand how stem cells self-renew but a major problem for stem cell biologists to resolve is how stem cell homeostasis is achieved. How is stem cell self-renewal balanced by differentiation? Nkx1.2 (NK1 homeobox2 TF) is an early marker of the NMP that continues to be transiently expressed in early neural progenitors. This pattern of expression suggests a role for Nkx1.2 in the progression of NMPs to a neural fate. The results of our LOF and GOF experiments in ESCs, coupled with RNAseq and ChIPseq, indicate that Nkx1.2 does not function in NMPs to activate a neural fate but instead represses NMP differentiation. Nkx1.2 represses genes that control the differentiation of NMPs to mesoderm (Tbx6, Msgn1, Mesp1), neural (Cyp26a1, which degrades neural differentiation-promoting RA), epiblast and cardiac fates. Our results suggest that Nkx1.2 expands the NMP and neural progenitor pools by preventing their adoption of alternative cell fates. A manuscript detailing this work is in preparation. 2) We have found that Wnt3a is expressed in a DV gradient in the caudal embryo that defines novel dorsal and ventral domains in the hindgut. As the hindgut produces the colon, we hypothesized that the dorsal hindgut is a unique Wnt-responsive colon progenitor population. Analysis of the GI tracts of Wnt3a-/- mutants revealed that anterior regions were normal, but the colon was absent. Colon defects arose from changes in early hindgut morphogenesis and a subsequent failure to extend the hindgut caused, at least in part, by reduced proliferation. An identical colon agenesis phenotype was observed in Sp5/8 DKO and conditional b-catenin mutants, providing strong genetic evidence that a Wnt/b-catenin/Sp5/8 pathway is essential for colon formation. GOF experiments led to a broad expansion of the hindgut and mesoderm while depleting the epiblast suggesting that Wnt3a levels are important for the coordination of posterior morphogenesis - excessive Wnt signaling alters the relative proportions of the three germ layers. To determine if the dorsal hindgut is a colon progenitor population, we performed a genetic lineage tracing of the dorsal and ventral domains and found to our surprise that the ventrolateral hindgut, and not the dorsal hindgut, is the predominant source of colon progenitors. The lack of Wnt activity in the ventral hindgut suggests that Wnt/b-catenin signaling must be prohibited there for proper hindgut development to proceed. Indeed, ventral ectopic activation of b-catenin led to a remarkable colon expansion and the appearance of large polyp-like nodules. This phenotype was only observed in the colon despite activation throughout the A-P extent of the intestinal epithelium, leading to speculation that the selective growth of the colon was due to the involvement of additional posteriorly localized cofactors such as Bmps. Indeed, we found that Bmp signaling is reduced in the Wnt3a-/- dorsal hindgut. Similar colon hyperplasia phenotypes were observed when the tumor suppressor Apc was selectively deleted from the ventral hindgut. These results suggest that a DV gradient of Wnt signaling in the hindgut is crucial for proper growth and development of the embryonic colon (Garriock et al., 2020). 3) In an effort to unravel the mechanisms of Wnt target gene transcription, the laboratory has focused on the Sp family of Zinc-finger transcription factors. We have previously shown that Sp5 and Sp8 function in the Wnt/b-catenin signaling pathway by binding to GC-rich promoter DNA and to Tcf/Lef TFs to facilitate b-catenin recruitment to a subset of Wnt target genes (Dunty et al., 2014; Kennedy et al., 2016). We have recently found that NMPs fail to self-renew in Sp5/8 DKO and that this is likely due to a role for these TFs in cell survival. Sp5/8 appear to promote cell survival by regulating the expression of signaling genes including Nodal, Fgf4, 8, 17 and Wnt3a in the node and PS. These studies have led to the identification of an Sp-responsive enhancer that controls the activation of Wnt3a, thereby initiating the NMP genetic program. Our results suggest that the node and PS constitute a niche for NMPs in the epiblast and that Wnt3a functions in a positive feedback loop through Sp5/8 to establish the NMP niche. A manuscript detailing this work is in preparation. Finally, we have published a paper in the past year that represents the culmination of a project on Daam proteins an *TRUNCATED*