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) 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. 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) 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 central role in a GRN that controls trunk and tail development through the activation of several genes encoding secreted signaling molecules. These include the NMP inducers Wnt3a and Fgf8 suggesting that Sp5/8 control the NMP niche. These studies have led to the identification of an Sp-responsive enhancer that we postulate controls the tissue-specific 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. We are now characterizing the necessity and sufficiency of the putative Wnt3a enhancer in transgenic mice and differentiating ESCs. We are also studying the role of Sp8 in the trunk-to-tail transition. Multiomics single cell analysis indicates that NMPs, caudal mesoderm, and somites that generate the caudal trunk and 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. Finally, given the role that Sp5 and 8 play in the normal development of the trunk and tail, we are asking if these genes might also function in tail regeneration in Xenopus laevis. Although still preliminary, our data suggests that Sp5/8 are required for the proper regeneration of the spinal cord and muscle.